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D . degree in Entomology (A CM NAM Dr. Mariam B. Sticklen Major professor D... é /‘»¢/w’ MSU is an Affirmative Action/Equal Opportunity Institution 042771 PLACE II RETURN BOXtonmmmbehockMflomyunoord. TO AVOID FINES mum on or baton data duo. DATE DUE DATE DUE DATE DUE MSU I. An Affirm-tin Adar/E“ Opportunity Institution W1 TRANSFORMATION STUDIES ON RICE (ORYZA SATIVA L.) USING SYNTHETIC BACILLUS THURINGIENSIS CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES FOR INSECT RESISTANCE BY Reynaldo V. Ebora AN ABSTRACT OF A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1995 ABSTRACT TRANSFORMATION STUDIES ON RICE (ORYZA SATIVA L.) USING SYNTHETIC BACILLUS THURINGIENSIS CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES FOR INSECT RESISTANCE BY Reynaldo V. Ebora A simple technique modified from INSTA—Prepn‘protocol was developed for the rapid isolation of plasmid DNA from bacterial cultures producing plasmids of comparable quality to those isolated by commercial product. Seven plasmids containing the synthetic Bacillus thuringiensis cryIA(b) driven by different promoters were constructed. The constitutive promoters of cauliflower mosaic virus 358 and rice actinl, and the tissue specific promoter of maize phosphoenol pyruvate carboxylase were transcriptionally fused to the synthetic cryIA(b). Three of the constructs also contained the potato proteinase inhibitor II (pin2) driven by its wound inducible promoter. Fujisaka 5 rice was co-transformed by microprojectile bombardment of embryogenic calli and immature embryos with the different plasmid constructs and plasmids containing the selectable marker bar which confers resistance to glufosinate ammonium. Southern blot analysis showed that several of the plants have the bar, but no hybridization at high molecular weight undigested DNA was noted. All the plants died when Sprayed with 1.0% Ignite (20 % glufosinate ammonium). Three plants (117, 132 and 187) putatively transgenic for synthetic cryIA(b) were identified by Southern analysis. Second generation of these plants were positive for synthetic cryIA(b) by PCR. Two plants from 187 and one from 117 were positive in western analysis. Basmati 370 and Fujisaka 5 transformed with pDX101, a plasmid containing the pin2 driven by rice Act? promoter were analyzed. First generation plants were positive in PCR, slot and Southern blots analyses, but no hybridization at high molecular weight was noted. Bioassay of these putatively transgenic plants against Ostrinia furnacalis, Spodoptera frugiperda and Nilaparvata lugens showed insignificant difference from the untransformed plants, although positive results in western blot was previously obtained. Southern blot analysis of DNA isolated from the ratoons of first generation and from the second generation plants gave negative results although some samples were PCR positive. Results showed that the plants might be chimeric and that the gene was not stably integrated. DEDICATION Dedicated to my parents, Toribio and Belen Ebora, who inculcated me with the value of education and pursuit of knowledge. To my wife Madie, whose love, moral support, encouragement and personal sacrifices help me to succeed in my endeavors. To my daughters Karen and Kalene, who are my inspirations. iv ACKNOWLEDGMENTS I would like to extend my appreciation and gratitude to Dr. Mariam B. Sticklen, my major professor, for her patience, encouragement, valuable advice and personal concern to me and my family. Special and cordial thanks to Dr. Mark Whalon, for his advice and support throughout my Ph.D. program and for treating me as a colleague and a friend. I am very thankful to my guidance committee members: Dr. Joseph Saunders, Dr. Leah Bauer and Dr. Alex Raikhel for valuable suggestions in the preparation of this manuscript. My gratitude and appreciation to Dr. Gary Toenniessen and The Rockefeller Foundation for providing my doctoral fellowship, without their financial support this study would not be possible. I appreciate the administrators and staff of BIOTECH — University of the Philippines at Los Bafios for allowing me to be on study leave to pursue my Ph.D. degree. I am also very thankful to the following for all their valuable help and contributions: Dr. Eliseo P. Cadapan, Dr. Bernardo P. Gabriel, Dr. Venus J. Calilung and Dr. B. Merle Shepard for endorsing my application to the Rockefeller Foundation and Michigan State V University. Dr. Ray Wu for providing the rice Act7 promoter and other plasmid constructs. Dr. Michael Koziel for the CryIA(b) antibody and plasmid constructs containing the synthetic B. thuringiensis cryIA(b). Mrs. Neerja Hajela and Dr. Ravindra Hajela who are always available to help and give me valuable advice. Dr. Neelam R. Yadav, for valuable suggestions in the tissue culture of rice and for being a good friend and colleague. Dr. Frank Davis, Dr. Douglas Landis, Dr. Sutrisno, Mr. Marcus Lee and Mr. Andi Trisyono for providing the insect cultures. Mrs. Jihua Liu, Nasim Abdolrhamani and JoAnn Palma for the help in tissue culture and insect bioassay. Cheribeth Tan and Letlet Carpio—Altamirano for some help in the dissection of rice immature embryos. My colleagues and friends in the lab., Herman, Wati, Don, Heng, Shibo, Sun, Alex, Neerja, Nasim, Dean, Sharif, Keith, Lan-Ying and An—Liu for the camaraderie. Don Warkentin, for the interesting discussions in interpretation of research data. All members of the MSU Filipino Club, for making East Lansing "a home away from home" for us. vi TABLE OF CONTENTS Page LIST OF TABLES .......................................... xii LIST OF FIGURE .......................................... xiv GENERAL INTRODUCTION ..................................... 1 CHAPTER I. DEVELOPMENT OF SIMPLE TECHNIQUES FOR THE RAPID ISOLATION OF PLASMID DNA FROM BACTERIAL CULTURES...................... ...... 14 SUMMARY .................................................. 15 INTRODUCTION ............................................. 15 MATERIALS AND METHODS .................................... 17 RESULTS .................................................. 19 DISCUSSION ............................................... 19 CHAPTER II. CONSTRUCTION OF DIFFERENT PLASMIDS FOR THE EXPRESSION OF SYNTHETIC BACTLLUS THURINGIENSIS CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES IN RICE AND OTHER MONOCOTS .............. 25 SUMMARY .................................................. 26 INTRODUCTION ............................................. 26 MATERIALS AND METHODS .................................... 31 Plasmid Construction and Molecular Cloning ............ 31 RESULTS .................................................. 39 pRES7293 (7.25 kb) .................................... 39 vii pRES7793 (8.56 kb) .................................... 4o pRES7193 (14.63 kb) ................................... 4o pRES7493 (8.82 kb) .................................... 40 pRES7393 (10.1 kb) .................................... 44 pREs107 (7.67 kb) .................................... 46 pRES108 (6.23 kb) .................................... 48 pHSE201 (13.18 kb) ................................... 51 pHSE202 (14.49 kb) ................................... 51 DISCUSSION.. .............................................. 54 CHAPTER III TRANSFORMATION STUDIES ON FUJISAKA 5 RICE USING SYNTHETIC BACILLUS THURINGIENSIS CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES FOR INSECT RESISTANCE ................. 63 SUMMARY .................................................. 64 INTRODUCTION ............................................. 65 MATERIALS AND METHODS .................................... 68 Surface Sterilization of Mature and Immature Rice Seeds .......................................... 68 Determination of Appropriate Concentration of Glufosinate Ammonium for Selection of Transformed Calli ............................................... 7O Transformation, Selection and Regeneration ............. 71 Rice DNA Isolation ..................................... 73 Screening of Putatively Transgenic Plants .............. 74 Southern Blot Analysis of Genomic DNA .................. 75 Western Blot Analysis .................................. 77 Bioassay of Putatively Transgenic Rice Plants viii Against European corn borer .......................... 78 RESULTS .................................................. 78 Surface Sterilization of Mature and Immature Rice Seeds ........................................... 78 Selection of Transformed Calli ....................... 79 Molecular Analyses of Putatively Transgenic Plants .............................................. 83 PCR and Western Analyses of Second Generation Putatively Transgenic Plants ........................ 98 Bioassay of Putatively Transgenic Rice Plants Against 0. nubilalis ............................... 101 DISCUSSION .............................................. 103 Surface Sterilization of Mature and Immature Rice Seeds ......................................... 103 Selection of Transformed Calli ........................ 105 Molecular Analyses of Putatively Transgenic Plants ............................................. 108 CHAPTER IV. MOLECULAR ANALYSIS AND BIOASSAY OF BASMATI 370 AND FUJISAKA 5 RICE PUTATIVELY TRANSFORMED WITH POTATO PROTEINASE INHIBITOR II GENE FOR INSECT RESISTANCE ........................... 117 SUMMARY ................................................. 118 INTRODUCTION ............................................ 118 MATERIALS AND METHODS ................................... 120 Transformation, Selection and Regeneration ............ 120 DNA Isolation and Purification ........................ 120 ix PCR Analysis .......................................... 120 Slot and Southern Analysis ............................ 123 Insect Bioassay ....................................... 124 Preference of S. frugiperda ........................ 125 Food consumption of S. frugiperda .................. 125 Bioassay against 0. nubilalis ...................... 126 Bioassay against.N. lugens ......................... 126 RESULTS ................................................. 127 DISCUSSION .............................................. 135 GENERAL CONCLUSION AND RECOMMENDATION ................... 148 LITERATURE CITED ........................................ 150 Table 2.1. Table 3.1. Table 3.2. Table 3.3. Table 3.4. Table 4.1. LIST OF TABLES Characteristics of different plasmid constructs containing the synthetic B. thuringiensis cryIA(b) driven by different promoters and pin2 driven by pin2 promoter for monocot transformation .................... 57 Number of surviving cell clusters from immature embryos of Fujisaka 5 after various lenght of incubation time (days) in callus inducing medium containing different concentrations of glufosinate ammonium ...................................... 80 Summary of different transformation experiments, number of regenerated plants and putatively transgenic rice plants identified by PCR and slot blot analyses ...... 84 Fujisaka 5 rice plants putatively transformed by plasmid constructs containing the synthetic cryIA(b) driven by different promoters showing hybridization at high molecular weight undigested DNA, indicative of gene integration to the chromosome as identified by Southern blot hybridization. These plants need to be further characterized up to the second generation to determine stable transformation ............ 99 Survival of European corn borer larvae after 4 days of feeding on stems of second generation untransformed and putatively transgenic rice plants as alternative host .......................... 104 Mean number (f SD) of second instar fall armyworm present on putatively transgenic and untransformed Basmati 370 leaves after 24 h in a preference test .................... 136 xi Table Table Table Table Table Table . Mean weight (mg i SD) of fall armyworm larvae after 72 h of feeding on putatively transgenic and untransformed Basmati 370 leaves ....................................... 137 Mean frass weight (mg f SD) from thirty second instar fall armyworms after 72 h of feeding on leaves of putatively transgenic and untransformed Basmati 370 ..... 137 Mean percentage of survival (t SD) of second instar European corn borer after 4 days of feeding on leaves and stems of putatively transgenic and untransformed Basmati 370 .................................. 138 . Mean weight (mg f SD) of second instar European corn borer after 4 days of feeding on leaves and stems of putatively transgenic and untransformed Basmati 370 ..... 138 . Mean frass weight (mg i SD) of second instar European corn borer after 4 days of feeding on leaves and stems of putatively transgenic and untransformed Basmati 370 ..... 139 . Mean amount of honeydew protein (mg/ml 1 SD) produced by individual brown planthopper after 48 h of feeding on putatively transgenic and untransformed Basmati 370 ..... 139 xii Figure 1.1. Figure 1.2. Figure 2.1. Figure 2.2. Figure 2.3. LIST OF FIGURES Restriction enzyme analysis of different plasmid DNA isolated from transformed E. coli cells using INSTA—Prepn'and modified protocols. A total of 14 ul of isolated DNA was digested with 10 units of EcoRI for one hour at 37 °C. Undigested plasmid DNA was loaded side by side to compare the quality of the sample. (A - INSTA—Prepw'protocol, B — Modified INSTA-Prepm protocol without gel barrier, C — One extraction each of PCIA and CI, D — One extraction of of PCIA only, E — Modified INSTA-PrepTM protocol using unbuffered liquefied phenol.; 1 — pGL2 (4.4 kb), 2 - pCIB4418 (5.8 kb), 3 — pRES7393 (10.0 kb), 4 — pRES107 (7.7kb), U — undigested, R - digested with EcoRI, L - BstEII digested Lambda DNA as molecular weight marker ........ 20 Restriction enzyme analysis of different clones containing the plasmid vector pUC isolated from transformed E. coli JM101 using modified procedure C. The isolated DNA was subjected to RNase treatment (20 ug/ml) for 1 h at 37°C prior to restriction enzyme digestion. No detectable amount of RNA was seen in the gel. (L - BstEII digested Lambda DNA as molecular weight marker, E — EcoRI, B — BamHI & EcoRI, Numbers 1 to 5 are clone designations) ....... 21 Nucleotide sequence of synthetic B. thuringiensis cryIA(b) and its corresponding amino acids. (Courtesy Dr. Michael Koziel, CIBA—GEIGY) .................................. 32 Restriction map of pCIB4418, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35$ promoter and terminator .......... 34 Restriction map of pCIB4421, containing the synthetic B. thuringiensis cryIA(b) driven by tissue—specific maize phosphoenol pyruvate carboxylase (PEPC) promoter and 35S terminator ............................... 36 xiii Figure 2.4. Figure Figure Figure Figure Figure Figure Figure 2.5. 2.11. Restriction map of pDM302, containing the bar coding sequence driven by rice Actin1 (Act7) promoter and nos terminator. The Act7 promoter was used to express the synthetic B. thuringiensis cryIA(b) in other plasmid constructs ........................... 37 Restriction map of pTWa, containing the potato proteinase inhibitor II (pin2) coding sequence driven by pin2 promoter with rice Act1 intron1, and pinZ terminator. In a separate expression cassette, the selectable marker bar is driven by CaMV 35$ promoter and nos terminator. The pinZ expression casette was used together with synthetic cryIA(b) to construct other plasmids ..................................... 38 Restriction map of pRES7293, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice ActI and CaMV 35S promoter. ................................... 41 Restriction map of pRES7793, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice Act? and maize PEPC promoter ..................................... 42 Restriction map of pRES7193, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35S promoter and terminator, pin2 driven by pin2 promoter with rice Act1 intron1, and pinZ terminator. There are two copies of the synthetic cryIA(b) expression cassette per plasmid molecule ..... 43 Restriction map of pRES7493, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35$ promoter and terminator, and pin2 driven by pin2 promoter with rice Act7 intron1, and pinZ terminator in two separate expression cassettes ................ 45 Restriction map of pRES7393, containing the synthetic B. thuringiensis cryIA(b) driven by PEPC promoter and CaMV 358 terminator, pinZ driven by pinZ promoter with rice Act1 intron1, and pin2 terminator in two separate expression cassettes ........ 47 Restriction map of pRES107, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice Act7 promoter and CaMV 35S terminator ......................... 49 xiv Figure 2.12. Figure 2.13. Figure 2.14. Restriction map of pRES108, containing the synthetic B. thuringiensis cryIA(b) driven by rice Act1 promoter and CaMV 35$ terminator .................................. 50 Portion of the nucleotide sequence at the site of fusion in the constructs pRES107 and pRES108. A) Nucleotide sequence at the junction of the rice Act7 transcribed region and the synthetic B. thuringiensis cryIA(b) in pRES107 and pRES108. Bold italic letters represent the nucleotide sequence generated upon filling up of the HindIII site from rice Act7 promoter by Klenow fragment. Underlined italic letters are nucleotide sequence generated upon filling up by Klenow fragment of the BamHI site from pCIB4418. Initiation codon (ATG) of synthetic cryIA(b) is indicated by bold underlined letters. N is undetermined nucleotide sequence coming from pCIB4418 preceeding the initiation codon. B). Nucleotide sequence generated upon fusion of the 3' and 5' ends of two rice Act1 promoter resulting to a tandem promoter (pRES107), both in correct orientation with respect to the synthetic cryIA(b). Underlined letters are nucleotide sequence generated upon filling up of the HindIII sites by Klenow fragment. Lower case letters in A) & B) represent the rice Act1 intron1 sequence. The slashed lines indicate intron-exon boundaries, slashed lines in bold are sites of fusion ................. 52 Restriction map of pCA-1 containing the selectable marker neomycin phosphotransferase II (npt II) gene that confers resistance to kanamycin, driven by nos promoter and terminator in pBIN19 Agrobacterium binary vector. This fragment was recovered and used to construct pHSE201 and pHSE202, both containing the synthetic B. thuringiensis cryIA(b) .................................... 53 XV Figure 2.15. Figure 2.16. Figure 3.1. Figure 3.2. Figure 3.3. Restriction map of pHSE201, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35S promoter and terminator and nptII driven by nos promoter and terminator in an Agrobacterium binary vector pBIN19 ............................... 55 Restriction map of pHSE202, containing the synthetic B. thuringiensis cryIA(b) driven by PEPC promoter and 35S terminator and nptII driven by nos promoter and terminator in an Agrobacterium binary vector pBIN19 ............................... 56 Production of putatively transgenic rice plants by particle bombardment of embryogenic calli A) Embryogenic calli produced from mature seeds (indicated by arrows). B) Bombarded calli selected for resistance to glufosinate ammonium (5.0 to 5.5 mg/l). Dark brown calli are susceptible calli killed by glufosinate ammonium and later discarded. C) Actively dividing glufosinate ammonium resistant embryogenic calli. D) Compact glufosinate ammonium resistant embryogenic calli ready for transfer to regeneration medium. E) Shoot formation from selected embryogenic calli. F) Rice plantlet with fully developed roots grown in MS medium in Magenta box. G) Fully grown rice plants in clay pots in the greenhouse. H) Fertile putatively transgenic rice plants .................................... 82 Slot blot analysis of genomic DNA from untransformed and putatively transgenic rice plants. The blots were probed with 32P labeled 1.8 kb BamHI—BstEII fragment from pCIB4418 containing the synthethic cryIA(b). Positive plants selected for further screening are marked by numbers. pCIB4418 (50 and 100 99) is positive control. DNAs isolated from untransformed rice plants are designated as NT .................. 86 Slot blot hybridization of genomic DNA from untransformed and putatively transgenic rice plants. The blot was probed with 32P labeled 1.8 kb BamHI—BstEII fragment from pCIB4418 containing the synthetic cryIA(b) xv1 Figure 3.4. Figure 3.5. and 1.5 kb HindIII—SpeI fragment from pDX101 containing the pin2 coding sequence and nos terminator. The probes (50 ng) were mixed in equal quantities. pCIB4418 (50 and 100 99) and pRES7393 (50 P9) are positive controls. pRES7393 contains both the pin2 and synthetic cryIA(b) coding sequences while pCIB4418 contains synthetic cryIA(b) only. DNAs isolated from untransformed rice plants are designated as NT. Positive plants selected for further screening are marked by numbers .............. 87 Southern blot analysis of rice genomic DNA. (A). Ethidium bromide stained DNA (25 ug/lane). (B) Autoradiogram of gel blot hybridized with 32p labeled 1.8 kb BamHI—BstEII synthetic cryIA(b) coding sequence from pCIB4418. The expected 2.1 kb fragment is observed in the BamHI—EcoRI digested DNA from plant 117. Larger (3.2 kb) and smaller (1.0 kb) bands are seen in plant 132. Positive bands are indicated by arrows. (C) Autoradiogram of gel blot hybridized with 32p labeled 0.86 kb EcoRV fragment containing the bar coding sequence and nos terminator from pDM302. The blot in (B) was washed to completely remove the cryIA(b) probe prior to hybridization. Plant 132 is positive for bar producing the 0.8 kb and 0.6 kb fragments (indicated by arrows). (D) The same autoradiogram as (C) but exposed for a longer time to show the hybridization at high molecular weight. The migration position of markers are shown to the left with sizes in kb. (0 — undigested, D — digested with BamHI and EcoRI, NT — untransformed plants, pCIB4418 — positive control. Numbers on the top of the lanes are plant designations) ..... 88 Southern blot analysis of rice genomic DNA. (A) Ethidium bromide stained DNA (25 ug/lane). (B) Autoradiogram of gel blot hybridized with 32" labeled 0.86 kb EcoRV fragment from pDM302 containing the bar coding sequence and nos terminator. Plant 79 is positive for bar producing about 0.8 kb and 0.6 kb fragments. Positive bands xvii Figure 3.6. Figure 3.7. (indicated by arrows) are also present in the undigested DNA but not at the bulk of high molecular weight DNA seen in the ethidium bromide stain. (C) Autoradiogram of gel blot hybridized with 32P labeled 1. 8 kb BamHI-BstEII fragment from pCIB4418 containing the synthetic cryIA(b). The blot in (B) was washed to completely remove the bar probe prior to hybridization. Hybridization at high molecular weight undigested DNA is seen in most samples. Positive bands for plant 65 and 79 are indicated by arrows. The migration position of markers are shown in the left with sizes in kb. (U — undigested, D - digested with BamHI and EcoRI, NT — untransformed plants, pCIB4418 - positive control. Number on the top of the lanes are plant designations) ......... 92 Southern blot analysis of rice genomic DNA. (A) Ethidium bromide— stained DNA (25 HQ/Iane). (B) Autoradiogram of gel blot hybridized with 32P labeled 1. 8 kb BamHI-BstEII synthetic cryIA(b) from pCIB4418. The expected 2.1 kb fragment is seen as a faint band in the BamHI-EcoRI digested DNA from plant 187 (indicated by arrow). The migration position of markers are shown to the left with sizes in kb. (U — undigested, D - digested with BamHI and EcoRI, NT - untransformed plants, pCIB4418 — positive control. Numbers on top of the lanes are plant designations) ................................ 95 Southern blot analysis of rice genomic DNA. (A) Ethidium bromide— stained DNA (25 ug/lane). (B) Autoradiogram of gel blot hybridized with 32P labeled 1. 8 kb BamHI-BstEII synthetic cryIA(b) from pCIB4418. A band slightly larger than the expected 2.1 kb fragment is seen in the BamHI— EcoRI digested DNA from plant 27 (indicated by arrow). (C) Autoradiogram of gel blot hybridized with 32P labeled 0. 86 kb EcoRV fragment containing the bar coding sequence and nos terminator from pDM302. The blot in (B) was washed to xviii Figure 3.8. Figure 3.9. completely remove the cryIA(b) probe prior to hybridization. Plant 127, 137 and 27 showed positive bands in the undigested DNA but not at high molecular weight (indicated by arrows). The migration position of markers are shown to the left with sizes in kb. (U — undigested, D - digested with BamHI and EcoRI, NT — untransformed plants, pCIB4418 — positive control). Number on top of the lanes are plant designations .......... 96 PCR analysis of first (R.) and second generation (R1) plants of transformants 117, 132 and 187. Positive plants are detected by the presence of the 0.8 kb amplified fragments using MB49 & MBSO, primers that are specific for synthetic cryIA(b). Upper lanes, lane 1, BstEII digested Lambda DNA; lanes 2 and 3, 187 (R0); lane 4, water control; lane 5, negative control, lanes 6—12, (R1) 187—2, 187-3, 187-4, 187—5, 187—6, 187-7, 187-8; lane 13, 117 (R0), lanes 14-21, (R.) 117-1, 117—2, 117-6, 117- 19, 117—21, 117—29, 117—43, 117—45; lane 22, 100 pg pCIB4418 (positive control). Lower lanes, lane 1, BstEII digested Lambda DNA; lane 2, 132 (R0); lane 3, negative control;lanes 4-12, (R1) 132— 3, 132-9, 132-12, 132—14, 132—16, 132- 17, 132—19, 132-32, 132—2; lane 13, water control; lanes 14-16, positive controls (100, 40 and 20 pg pCIB4418, respectively); lane 17, negative control; lane 18, 132 (R0); lane 19, 187 (R0) lane 20, positive control (40 pg PCIB4418);lane 21, 117 (R0); lane 22, water control ............................... 100 Western blot analysis of second generation (R0) putatively transgenic rice plants. The blots were hybridized with immunoaffinity purified polyclonal antibody specific for the insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki from Dr. M. Koziel (CIBA—GEIGY). Upper blot — 20 ul protein extract per lane, Lower blot — 380 pl protein extract per lane. Arrows mark the migration x1x Figure 4.1. Figure 4.2. Figure 4.3 Figure 4.4. position of the positive bands. Numbers on the top of the lanes are plant designations. NT - untransformed plants, C — cells and crystals of B. thuringiensis HD1-9, P — purified crystals ................................ 102 Restriction map of pDM104, containing the selectable marker AroA, which confers resistance to glyphosate driven by rice Act1 promoter and soybean sub unit leader (SSU) and nos terminator .............................. 121 Restriction map of pDX101, containing the potato proteinase inhibitor II (pin2) driven by rice Act1 promoter and pin2 terminator ..................... 122 Southern blot analysis of Basmati 370 and Fujisaka 5 rice genomic DNA. (A) Ethidium bromide-stained rice DNA (15 ug/lane). (B) Autoradiogram of gel blot hybridized with 32p labeled 1.5 kb HindIII-SpeI fragment from pDX101 containing the pin2 coding sequence and terminator. An approximately 3.0 kb fragment is observed in the HindIII—SpeI digested DNA from all the plants instead of the expected 1.5 kb fragment (indicated by arrow). The migration position of markers are shown to the left with sizes in kb. (NT ~ untransformed plants; X - pDX101 positive controls corresponding to 1,5 and 10 copies/genome; Numbers on the top of the lanes are plant designations. B - Basmati 370, F - Fujisaka 5) ............................. 129 Southern blot analysis of Basmati 370 and Fujisaka 5 rice genomic DNA. (A) Ethidium bromide-stained rice DNA (15 ug/lane) (B) Autoradiogram of gel blot hybridized with 321: labeled 1.5 kb HindIII—SpeI fragment from pDX101 containing the pin2 coding sequence and terminator. An approximately 3.0 kb fragments (indicated by arrow) are observed in the HindIII—SpeI digested DNA isolated from putatively transgenic plants instead of the expected 1.5 kb fragments. Hybridization at high molecular xx Figure 4.5. weight undigested DNA was observed as discrete bands of different sizes that do not much the bulk of ethidium bromide stained DNA. No hybridization was observed in the DNA isolated from the untransformed plants. The migration position of markers are shown to the left with sizes in kb. (NT — untransformed plants; X — different amount of pDX101 positive controls; S — digested with HindIII and SpeI, U — undigested; Numbers on the top of the lanes are plant designations) ............................. 131 Southern blot analysis of Basmati 370 rice genomic DNA isolated at late tillering stage (LTS) and from ratoons (RT). (A) Ethidium bromide—stained DNA (15 ug/lane) (B) Autoradiogram of gel blot hybridized with 32P labeled 1.5 kb HindIII-SpeI fragment from pDX101 containing the pin2 coding sequence and terminator. Hybridization of high molecular weight undigested DNA isolated at late tillering stage was observed but do not much the bulk of ethidium bromide—stained DNA. No hybridization was observed in the DNA isolated from the ratoons of the same plants. The migration position of markers are shown to the left with sizes in kb. (NT — untransformed plants; x - pDX101, positive controls; S — digested with HindIII and StyI; P — digested with HindIII and SpeI; U — undigested. Numbers on top of the lanes are plant designations) ............. 133 xxi GENERAL INTRODUCTION Rice (Oryza sativa L.) is one of the world's most important cereal food crops in terms of caloric intake (Niizeki, 1989) and a staple food for the majority of people in the world (Pathak & Saxena, 1980; Raina, 1989; Sobral et al., 1989; Mikkelsen & de Datta, 1991). About 143.5 million hectares of land is planted to rice, more than 90% of which is in Asia (de Datta, 1981; David, 1991). Rice is affected by different insect pests, and 20 species are of major importance (Swaminathan, 1986). These species attack all parts of the rice plant at all growth stages, and some are vectors of viral diseases (Pathak, 1969 & 1970; Yokoyama et al., 1984). Some of the major lepidopteran pests of rice are stem borers which include 17 species of Pyralidae and 3 species of Noctuidae. Of these, the striped rice borer, Chilo suppresalis (Walker), dark headed rice borer, Chilo polychrysus (Meyrick), yellow rice borer, Tryporyza incertulas (Walker), white rice borer, T. innonata (Walker) and pink borer, Sesamia inferens (Walker) are the most widely distributed and most destructive (Pathak & Saxena, 1980). T. incertulas is widespread in tropical Asia and there are no known resistant germplasms which can be readily introduced into cultivated rice (Fujimoto et al., 1993). The control of lepidopteran pests of rice is usually 2 with the use of broad—spectrum chemical insecticides. This control measure also destroys natural enemies leading to severe outbreaks of the rice brown planthopper, Nilaparvata lugens (Stél) (Heinrichs & Mochida, 1984, Kisimoto, 1984) creating a more severe problem (Rombach et al., 1989). Currently, the use of Bacillus thuringiensis (Bt) for the control of lepidopteran pests is a potentially important component of integrated pest management (IPM) systems in rice because it is specific and do not upset natural enemy complexes. Aside from causing direct mortality, Bt also causes arrested feeding on Asiatic rice borer, C. suppressalis (Rombach et al., 1989). During sporulation, lepidopteran specific Bt produces crystal protoxin (6-endotoxin) of approximately 130 to 140 kDa which is solubilized and degraded to a biologically active toxin of 40 to 70 kDa in the insect gut (Bulla et al., 1979; Bietlot et al., 1989; Choma et al., 1991; Chen et al., 1993; Ihara et al., 1993; Lu et al., 1994). After penetrating the peritrophic membrane the 6—endotoxins are known to bind with high affinity to specific receptors found on the brush border of the columnar cells lining the midgut, followed by pore formation causing an influx of potasium ion, disruption of cell homeostasis, and eventual lysis (Hofmann et al., 1988a&b; Slatin et al., 1990; Ge et al., 1991; English & Slatin, 1992; Bauer & Pankratz, 1992; Knowles & Dow, 1993; Knowles, 1994). Although receptor binding is an important step in the mechanism of toxic 3 action of the O-endotoxin, binding alone do not always cause toxicity. For instance, Wolfersberger (1990) reported that binding affinities of the toxic fragments of cryIA(b) and cryIA(c) on brush border membrane vesicles of the gypsy moth, Lymantria dispar, are inversely related to toxicity. It is known that N-acetyl galactosamine is part of the receptor in insect gut epithelia that recognizes an insecticidal protein from Bt (Knowles et al., 1991). For CryIA(c), its receptor has recently been purified from Manduca sexta and shown to be aminopeptidase N (Knight et al., 1994; Sangadela et al., 1994). After binding to the receptor, Bt toxins insert rapidly and irreversibly into the plasma membrane of the gut cell and form pores or lesions (Van Rie et al., 1989). The ability of a toxin to form a membrane pore is the determinant of its specificity (Wolfersberger, 1991; Honee & Visser, 1993). It is not yet known whether the toxin alone forms the pore in vivo, or whether the toxin and receptor together form a complex (Knowles, 1994). The formation of nonselective pores in the columnar cell apical membrane results in entry of K3 and efflux of H3, which rapidly depolarizes this membrane. Small anions can probably also enter through the toxin pore. The columnar cells containing macromolecules which cannot leak out through a 0.6—nm pore, would absorb water osmotically and thus swell burst by the process of colloid—osmotic lysis (Knowles & Ellar, 1987). This leads to breakdown of the permeability barrier of the membrane, cell lysis, disruption 4 of gut integrity and finally death of the insect from starvation and septicaemia. It was also proposed that Bt toxin kill cells by pH mediated damage (Harvey et al., 1986). The tertiary structure of a 6—endotoxin has been elucidated by X—ray crystallography of CryIIIA, a beetle toxin (Li et al., 1991). Most known Cry toxins contain five highly conserved sequence blocks (Hofte & Whiteley, 1989; Hodgman & Ellar, 1990) and alignment of the sequences of different Bt toxins (Hodgman & Ellar, 1990) reveals that these blocks make up the core of the CryIIIA structure. Knowles & Dow (1993) believe that it is reasonable to assume that all Cry toxins have a similar general conformation. The toxin is composed of three distinct domains (Li et al., 1991). Domain I, which is likely to be involved in membrane insertion and pore formation is a bundle of six amphipathic a-helices surrounding a central hydrophobic helix. Domain II comprises three B—sheets terminating in loops at the apex of the molecule and contains segments inferred to be the specificity determining regions (Li et al., 1991). Domain II is thus probably the receptor-binding domain (Knowles & Dow, 1993). Domain III is a B—sandwich containing the conserved C-terminal region of most Cry toxins. The safety of Bt spore—crystal preparation and the economic importance of lepidopteran pests makes 6—endotoxins particularly attractive candidates for genetic engineering (Brunke & Meeusen, 1991). As single proteins directly 5 encoded by bacterial genes, Bt 6—endotoxins are comparatively straightforward to introduce to plants (Goldburg & Tjaden, 1990). The genes for Cry proteins are already well characterized (Debabov et al., 1984; Whiteley et al., 1984; Carlton & Gonzalez, 1984; Aronson et al., 1984 & 1986; Adang et al., 1985; Thorne et al., 1986; Hofte & Whiteley, 1989) and expressed in Escherichia coli, Pseudomonas fluorescens (McPherson et al., 1988; Stone et al., 1989) and in insect cell lines using Baculovirus expression vectors (Merryweather et al., 1990). As of 1989, over 42 cry genes have been sequenced (Whiteley & Schnepf, 1986; Murray et al., 1991). In the list prepared by Yamamoto and Powell (1993), there are 20 distinct cry genes and several lines of evidence suggest that 19 of these genes specify a family of related insecticidal proteins. These cry genes have been divided into 4 major classes and several subclasses characterized by both structural similarities and insecticidal spectra of the encoded proteins. The 4 major classes of Cry proteins are Lepidoptera specific (I), Lepidoptera and Diptera specific (II), Coleoptera specific (III), and Diptera specific (IV) (Hofte & Whiteley, 1989). The O-endotoxins range in size from 27 to 140 kDa. There are some Cry proteins that do not fit conveniently into this classification scheme (Knowles, 1994) and 4 other cry genes are not yet classified (Yamamoto & Powell, 1993). For instance, CryIB family has a member (originally designated as Cry V) that kills both Lepidoptera and Coleoptera (Tailor 6 et al., 1992), while CryIIB is non—toxic to Diptera (Widner & Whiteley, 1989) and CryIIIC is 130 kDa and has cryptic coleopteran activity (Lambert et al., 1992). At least eleven distinct cry genes encoding lepidopteran—specific proteins were cloned, 4 of which were expressed at insecticidal levels in transgenic plants (Meeusen & Warren, 1989) including tomato (Fischhoff et al., 1987; Delannay et al., 1989), tobacco (Adang et al., 1987; Barton et al., 1987; Vaeck et al., 1987 & 1989; Warren et al., 1992: Carozzi et al., 1992), cotton (Umbeck et al., 1987; Perlak et al., 1990), potato (Vaeck et al., 1989; Cheng et al., 1992, Van Rie et al., 1994) and rice (Yang et al. 1989, Xie et al., 1990). To obtain useful levels of 5—endotoxin in plants, it is necessary to engineer the plant to produce the activated toxin, rather than the entire protoxin. Transgenic plants produce higher levels of mRNA and protein only when the gene fragment coding for the 6-endotoxin's amino terminal is used for transformation (Goldburg & Tjaden, 1990). Although an increased level of expression in transgenic cotton was reported when the nucleotide sequence of the truncated structural gene was modified without changing the encoded amino acid and with the use of more efficient promoter (Perlak et al., 1990), the expression levels of cry genes in transgenic plants have been generally extremely low (Adang et al., 1987; Barton et al., 1987; Fischoff et al., 1987; Vaeck et al., 1987; Cheng et al., 1992; Barton & Miller, 7 1993, Ebora et al., 1994a&d). Based on a combination of effects from DNA synthesis, promoter enhancement, and vector design, Perlak et al. (1991) reported toxin peptide concentration of 0.1% of the cell protein (Barton & Miller, 1993). The low levels of lepidopteran toxin cryIA(b) expression in plants and cells are due to RNA instability (Murray et al., 1991), inefficiency in the synthesis of gene products, or rapid degradation of either the mRNA or protein following synthesis. In cases that low amount of toxins are present, a very sensitive assay has to be established to detect bioactivity (MacIntosh et al., 1990b). However, several reports on the use of synthetic cryIA(b) genes optimized for plant transformation resulted to higher expression level and improved efficacy. Modifications to the nucleotide sequence consisted of nucleotide changes that eliminated signals for intron splicing, polyadenylation, and transcription termination. Rare codons were replaced with those that are more commonly found in plants, and sequences with the potential to form secondary structure in mRNA were eliminated (Yamamoto & Powell, 1993). Synthetic cryIA(b) was successfully introduced into japonica rice by electroporation of protoplasts and the regenerated plants have biological activity against striped stem borer and leaf folder (Fujimoto et al., 1993). Another synthetic cryIA(b) synthesized by CIBA-GEIGY was successfully introduced and expressed in elite hybrid corn plants. Transgenic corn plants expressing the synthetic cryIA(b) were effectively 8 protected from economic damage even after heavy infestation of first and second generation corn borers (Koziel et al., 1993). This synthetic cryIA(b) was used in our transformation studies under a cooperative agreement with Dr. Michael Koziel (CIBA—GEIGY). In several plant families wounding by chewing insects or other mechanical damage results in the localized and systemic accumulation of high levels of proteinase inhibitor proteins in leaves (Green & Ryan, 1972 & 1973; Brown & Ryan, 1984; Pefia—Cortes et al., 1988, 1989 & 1991; Ryan, 1990; Farmer et al., 1992) which are part of the array of defensive chemicals against insect pests (Ryan, 1973; Hilder et al., 1987; Johnson et al., 1989; Pefia-Cortes et al., 1989) and pathogens (Pautot et al., 1991). Potato and tomato plants synthesize two inhibitors of serine proteinases (Green & Ryan, 1972 & 1973) in response to wounding and insect feeding. These are designated as Inhibitor I (Mr 8100) and Inhibitor II (Mr 12300) (Bryant et al., 1976; Plunkett et al., 1982; Cleveland et al., 1987). Inhibitor I is an inhibitor of chymotrypsin that only weakly inhibits trypsin at its single reactive site whereas Inhibitor II contains two reactive sites, one of which inhibits trypsin and the other one inhibits chymotrypsin (Sanchez—Serrano et al., 1987, Johnson et al., 1989). In potato, proteinase inhibitor II (Pin2) accumulated at high levels in stolon and tubers, and in leaves damaged by insect attack or mechanical wounding (Keil et al., 1989 & 1990; Thornburg et al., 1987; 9 Kim et al., 1991). The accumulation of the inhibitor is observed not only in the damage part but also in nearby unwounded tissues. Previous studies showed that introduction of serine proteinase inhibitor gene derived from cowpea into tobacco by Agrobacterium transformation showed resistance to the tobacco budworm (Heliothis virescens) (Brunke & Meeusen, 1991). Similarly, trypsin and chymotrypsin inhibitor genes also imparted resistance to insects when introduced into tobacco. Growth reduction in the insect clearly correlated with levels of the inhibitor in plant leaves. This trait was found to be stable and heritable (Meeusen & Warren, 1989). In Manduca sexta, larval growth inhibition was also observed mainly due to trypsin inhibitory activity (Johnson et al., 1989). Besides the obvious mechanism of preventing proper digestion of ingested proteins, leading to starvation and death, more subtle effects on the balance of sulphur amino acids in the developing insects, due to high content of cysteine in most inhibitor, and their resistance to proteolysis, have been suggested (Gatehouse & Boulter, 1983; Boulter et al., 1990). Protease inhibitors may also affect other proteases vital for insect development, such as those involved in moulting (Gatehouse et al., 1991). The pin2 is driven by a single promoter that is active both in leaves upon wounding and constitutively in tubers (Keil et al., 1989; Pefia-Cortes, 1991; Sanchez—Serrano et al. 1993). It can also be induced by abscisic and jasmonic 10 acids (Hildmann et al., 1992). In transgenic rice plants, systemic induction of a potato pin2 promoter by wounding, methyl jasmonate and abscisic acid were observed by Xu et al. (1993) using the bacterial B-glucuronidase (GUS) coding region as reporter gene. Potentiation of Bt insecticidal activity by serine protease inhibitors was also previously reported. MacIntosh et al. (1990) and Boulter et al. (1990) have suggested combining protease inhibitors with Bt toxins in transgenic plants to achieve greater efficacy against insect pests. MacIntosh et al. (1990) have demonstrated significant potentiation of Bt activity by co—administering serine protease inhibitor in laboratory bioassays. A variety of serine protease inhibitors isolated from plant and animal sources were effective at low levels in enhancing Bt activity against their target insects. The potentiation of Bt activity in transgenic plants was demonstrated by the co-expression of the two proteins in plants, which was accomplished by the expression of CMTI—Btk HD—1 fusion protein in tobacco. The protease inhibitor potentiated the Bt protein activity by approximately 6 fold. Hilder et a1. (1987) also confirmed that protease inhibitor exhibits insecticidal activity by cloning the inhibitor gene directly into plants. The mechanism by which the protease inhibitors potentiate Bt insecticidal activity is unknown. MacIntosh et a1. (1990) suggested that it is unlikely that the observed potentiation is due to direct inhibition of gut juice 11 proteases since the levels of serine protease inhibitors that potentiate Bt activity are approximately 105 times below the insecticidal levels reported by Gatehouse & Boulter (1983). Furthermore, potentiation was observed with several insect species that posssess very different gut juice protease composition. Other mode of action of the protease inhibitor enhancement of Bt may involve the inhibition of specific gut membrane associated proteases that serve to inactivate Bt or inhibition of proteases that are required for activation of inactive zymogens (e.g. chitin synthase). MacIntosh et al. (1990) proposed that protease inhibitors may inhibit the degradation of membrane bound receptors, therefore increasing their half—lives and the ability to bind Bt proteins. Although insect resistance against Bt 6—endotoxin was reported (McGaughey, 1985 a&b; McGaughey & Johnson, 1987; McGaughey & Beeman, 1988; Van Rie et al., 1990; Sims & Stone, 1991; Whalon et al., 1992; Tabashnik et al., 1995, Rahardja and Whalon, 1995), it is still a promising approach that can be incorporated in an IPM program both as conventional insecticide or in transgenic plants if a proper resistance management plan is implemented. For instance, Justin et al. (1989) showed that treatment with Bt increased the susceptibility of Heliothis armigera and Spodoptera litura to chemical insecticides. It is suggested that suitable introduction of Bt in an IPM program, may delay the process of development of insecticide resistance in insect 12 pests. Tabashnik et al. (1994) has shown a reversal of resistance to Bt in diamondback moth, Plutella xylostella and indicated that the usefulness of transgenic plants may be prolonged effectively by crop rotation. However, Tabashnik et al. (1991) also previously reported that susceptibility to Bt is not quickly restored when treatments are discontinued and thus, rotations of Bt with other insecticides with different modes of action may not be a viable strategy. It is suggested that the use of other strategies such as biological controls, host free periods, cultural controls, novel insecticides, and plant resistance is the only means for limiting the use of Bt toxins and the development of resistance to them. The combination of breeding plants harboring different genes that reduce their sensitivity to pests may provide great advantages both in making agriculture more efficient and economical and in reducing its negative impact on the environment (Logemann & Schell, 1993). Although significant progress was made in the application of gene transfer techniques in rice (Uchimiya et al., 1986; Gasser & Fraley, 1989; McElroy et al., 1990 & 1991) using particle gun or biolistic device, electroporation and polyethylene glycol treatment of protoplasts (Uchimiya et al., 1986; Dekeyser et al., 1990; Peng et al., 1990), the production of transgenic plants is still a time consuming and demanding task (Cao et al., 1991). The production of transgenic cereals is still rather 13 difficult because the regeneration and transformation methods are far from routine (Marx, 1987; Vasil, 1988 & 1990). Our studies focused on the transformation of Fujisaka 5 and Basmati 370 for insect resistance using synthetic cryIA(b) and pin2. We made several plasmid constructs containing both the synthetic cryIA(b) and pin2 driven by rice actin1 (ActI), phosphoenol pyruvate carboxylase (PEPC), cauliflower mosaic virus 35S (CaMV 35$), potato proteinase inhibitor II (pin2) promoters and combinations, for rice transformation studies. Plasmid constructs containing pin2 and rice Act7 promoter were provided by Dr. Ray Wu (Cornell University) under a cooperative agreement. The general objectives of this study are to: 1) construct plasmids containing synthetic cryIA(b) driven by rice Act7 5' regulatory sequence and other promoters. 2) use the different plasmid constructs containing the synthetic cryIA(b) and pin2 for the transformation studies on rice immature embryo and seed derived calli. 3) detect the presence of the introduced synthetic cryIA(b) in the rice genome by polymerase chain reaction (PCR) and analyze gene integration by Southern blot hybridization. 4) examine the expression of synthetic cryIA(b) in transformed rice plants by western analysis and 5) assess the efficacy of putatively transgenic plants against lepidopteran pests. CHAPTER I DEVELOPMENT OF SIMPLE TECHNIQUES FOR THE RAPID ISOLATION OF PLASMID DNA FROM BACTERIAL CULTURES 14 15 SUMMARY A simple technique modified from INSTA-PrepTM protocol without the use of gel barrier was developed and found to be as efficient as the commercial product in isolating plasmids of various sizes from different strains of E. coli. The quality of the plasmids in terms of digestibility by restriction enzymes is comparable to those isolated by the INSTA—PrepTM kit. The procedure eliminates the use of gel barrier and involves one extraction each of phenol: chloroformzisoamyl alcohol (50:49:1) and chloroformzisoamyl alcohol (49:1), respectively. The plasmids isolated by the modified procedure can be subjected to RNase treatment and ethanol precipitation to obtain more purified samples. INTRODUCTION In cloning experiments, small scale isolation of plasmid DNA is usually required in screening large number of clones and identifying the desired transformants. Several standardized molecular biology techniques are used to successfully isolate plasmid DNA of various sizes from different strains of EScherichia coli. One of the most commonly used method is the alkaline lysis procedure (Birnboim & Doly, 1979; Birnboim, 1983; Sambrook et al., 1989), which also involves ethanol or isopropanol precipitation. This procedure usually requires preparation of several types of reagents and at least half day to complete. 16 Several rapid isolation protocols were developed and marketed by different companies like the INSTA-Prep“‘(5 Prime 9 3 Prime, Boulder, CO), Promega Wizard (Promega, Inc. Madison, WI) and QIAprep spin 20 (QIAGEN Inc., Chatsworth, CA) which dramatically reduce the processing time and simplify the various extraction steps in the conventional procedure. These products usually give satisfactory and reproducible results. They are also reasonably priced considering the convenience and the amount of time saved on the preparation of reagents. However, these products are usually not available in developing countries like the Philippines, where ordering of special reagents and kits usually takes several weeks or months. For this reason, several procedures modified from the INSTA—Prepn'protocol were tested so that we can prepare and use our own reagents. The INSTA—PrepTM kit uses a special type of gel that migrates to form a tight barrier between the heavier, organic phase (phenolzchloroform:isoamyl alcohol or chloroformzisoamyl alcohol) and the lighter aqueous phase during centrifugation. The organic phase and the interface material are sequestered below the INSTA—PrepTM gel, effectively removing them from the aqueous upper phase, which contains the isolated plasmid DNA (INSTA—Prep“‘ Protocol, 1995). Since most of the components of the kit are commonly used reagents, we modified the protocol and prepared our own reagents. The experiments reported here were done with the help of Mrs. Neerja Hajela. 17 MATERIALS AND METHODS Plasmid isolation procedures were modified from the INSTA—PrepTM protocol (5 Prime -> 3 Prime, Inc.R Boulder, CO) and their efficiency was compared. For the standard INSTA— PrepTM protocol, the plasmid DNAs were isolated using the following procedure supplied by the manufacturer (Tarczynski et al., 1994): E. 0011 containing the plasmid was cultured overnight in 15 ml of LB broth with ampicillin (100 ug/ml) overnight at 37°C with vigorous shaking at 200 rpm and used for all the treatments. For the standard INSTA—PrepTM procedure, 1.5 ml of the culture was transferred to sterile Eppendorf tube and the cells were precipitated by centrifugation at 12,000 rpm for 10 seconds. The supernatant was poured off and the remaining liquid was removed by standing the tube upside down in several layers of folded Kimwipes. The pellet was resuspended in 50 ul TE (10 mM Tris—HCl, 1 mM EDTA, pH 8.0) by vortex mixing. Three hundred ul of phenolzchloroform: isoamyl alcohol (PCIA, 50:49:1, volume by volume) was added to the tube and then mixed by gentle inversion six times. When the cells were lyzed and a white fluffy material appeared, the mixture was transferred to a pre—spun INSTA— PrepTM tube containing the gel barrier and then centrifuged at 10,000 rpm for 30 seconds, leading to the separation of the two phases. Three hundred ul of chloroformzisoamyl alcohol (CI, 49:1 volume by volume) was then added and mixed by repeated gentle inversion of the tube. The mixture was 18 again centrifuged at 10,000 rpm for 30 seconds to separate the organic and aqueous phases by the gel barrier. The aqueous phase containing the plasmid DNA on the top layer was recovered and transferred to a new tube. The whole procedure takes about 2 to 3 minutes to completely process a sample. Four different procedures were tested which are modified and derived from the INSTA-PrepTM protocol and their efficiency was compared. Plasmids ofdifferent sizes and host cells were used in the study. The plasmids were pGL2 (4.4 kb) in DHSa, pCIB4418 (5.8 kb) in JM101, pRES7393 (10.0 kb) in SURE, and pRES107 (7.7 kb) in SURE (Stratagene Cloning Systems, La Jolla, CA). The following procedures were tested: A. Standard INSTA—Prepm protocol and reagents. B. INSTA—PrepTM protocol and reagents but without the use of gel barrier. C. Modified INSTA—PrepTH protocol with one extraction each of home made PCIA and CI, respectively and without the use of gel barrier. Liquefied phenol was used. D. Same as procedure C but using buffered liquefied phenol. Phenol was extracted twice with 1.0 M Tris (pH 8.0), then with 0.1 M Tris (pH 8.0) and 0.2% B—mercaptoethanol until the pH of the aqueous phase is 7.6. E. Modified INSTA-PrepTM protocol with only one extraction of PCIA and without the use of gel barrier. Fourteen pl of the aqueous phase containing the 19 isolated plasmids were digested with 10 units each of BamHI and EcoRI for 1 h at 37 °C and then loaded in a 0.8% agarose gel using INSTA—PrepTM loading buffer. The samples were loaded side by side with the undigested plasmid and electrophoresed at 100 V for 2 hours. The amount and quality of the plasmid in terms of digestibility by restriction enzyme, and the amount of RNA present were compared. RESULTS The results showed that the modified procedures worked well and the quality of the DNA is comparable to those isolated by INSTA—PrepTM kit. The plasmids are well digested and there is no detectable amount of RNA present in the gel (Figure 1.1) when the INSTA—Prepn'loading buffer containing RNase was used for all the samples. The plasmid isolated using procedure C and then subjected to RNase treatment (20 ug/ml) for 1 hour at 37°C prior to restriction enzyme digestion also produced good quality DNA. No detectable amount of RNA was noted in this sample when subjected to electrophoresis and the gel was stained with ethidium bromide (Figure 1.2). DISCUSSION All the modified procedures tested produced plasmids that are of comparable quality to those isolated by the commercial product. The results showed that the use of the gel barrier can be eliminated and still isolate a good U R u RIJR U R u R u R u n U a u n U n L A3 83 c3 03 ES A4 34 c4 04 54 URUHURURURURURURURURL Figure 1.1. Restriction enzyme analysis of different plasmid DNA isolated from transformed E. coli cells using INSTA- PrepTM and modified protocols. A total of 14ul of isolated DNA was digested with 10 units of EcoRI for one hour at 37°C. Undigested plasmid DNA was loaded side by side to compare the quality of the sample. (A — INSTA—Prepm protocol, B — Modified INSTA—Prepm protocol without gel barrier, C - One extraction each with PCIA and CI respectivelfi, D — One extraction of PCIA only, E — Modified INSTA—Prep protocol using unbuffered liquefied phenol.; 1 — pGL2 (4.4 kb), 2 — pCIB4418 (5.8 kb), 3 — pRES7393 (10.0 kb), 4 — pRES107 (7.7 kb); U — undigested, R - digested with EcoRI. 21 Figure 1.2. Restriction enzyme analysis of different clones containing the plasmid vector pUC isolated from transformed E. coli JM101 using modified procedure C. The isolated DNA was subjected to RNase treatment (20 ug/ml) for 1 h at 37°C prior to restriction enzyme digestion. No detectable amount of RNA was seen in the gel. (L — BstEII digested Lambda DNA as molecular weight marker, E — EcoRI, B — BamHI & EcoRI, Numbers 1 to 5 are clone designations) 22 quality plasmid DNA without comprimising the ease and speed of processing. The modified procedures have the following advantages as compared to the INSTA-Prepm procedure: 1) It does not require the use of INSTA—PrepTM gel barrier. 2) It is about half the cost of the kit. 3) Modified procedure B and E require shorter processing time and less steps. The second extraction with CI can be done without transferring the aqueous phase previously extracted with PCIA to a new tube. However, transferring it to a new tube prior to the second extraction is more efficient since it will not dilute the final concentration of the reagents. From the ethidium bromide stained gel, it can be clearly seen that the quality of the plasmids isolated using the modified procedures is comparable to those isolated by standard INSTA—Prepw'protocol and reagents. High amount of DNA was obtained in all the plasmids except for pCIB4418 in JM101 which gave low yield for all the procedure tested even in standard INSTA—PrepTM protocol. This result was due to the fact that in this particular experiment, only small amount of cells was obtained in the 1.5 ml overnight culture. In the routine plasmid isolation the use of cells recovered from 3.0 ml culture consistently produce good plasmid yield using any of the modified procedures. It should be noted that in this study all of the plasmid tested have high copy number which might have contributed to the 23 successful results. It is highly possible that the modified procedures will also work in low copy number plasmids except that more cells will be needed. The standard INSTA—PrepTM kit was reported to be also working well with low copy number plasmid (Tarczynski et al., 1994). The initial amount of TE buffer or sterile distilled water and the volume of PCIA and CI should be adjusted accordingly in order to assure proper cell lysis. In our experience, when large amount of cells is used, it is usually better to subject the lysate to an additional PCIA or CI extraction and then transfer the aqueous phase to new tube in each extraction step in order to maximize the removal of protein. It was also noted that the volume of the aqueous phase tend to reduce in each step and that the concentration of the plasmid DNA can be adjusted by the addition of TE (pH 8.0) or sterile distilled water in the final extract. The plasmid isolated by any of these modified procedures can also be subjected to RNase treatment (20 ug/ml) for 1 hour at 37°C prior to restriction enzyme digestion. No detectable amount of RNA was noted in this sample when subjected to electrophoresis and the gel was stained with ethidium—bromide. Another option is to do the RNase treatment during restriction enzyme digestion. RNase is active in almost all of the commonly used restriction enzyme buffers. The isolated plasmid DNA resuspended in TE buffer or sterile distilled water can also be subjected to conventional phenol:chloroform:isoamyl alcohol (25:24:1, 24 volume by volume) extraction if higher degree of plasmid purity is desired. Ethanol or isopropanol precipitation can also be done. For rapid isolation and screening of clones for restriction digestion any of the modified procedures can be used. In our case, we prefer the modified procedure C in our routine small scale plasmid isolation, which enables us to screen a large number of colonies for a short period of time. The results using this procedure are reproducible and consistent. CHAPTER II CONSTRUCTION OF DIFFERENT PLASMIDS FOR THE EXPRESSION OF SYNTHETIC BACILLUS THURINGIENSIS CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES IN RICE AND OTHER MONOCOTS 25 26 SUMMARY Seven plasmids containing the synthetic Bacillus thuringiensis cryIA(b) driven by different promoters were constructed from fragments coming from plasmids pCIB4418, pCIB4421, pTW—a and pDM302. The constitutive promoters of cauliflower mosaic virus 358 (35$ 5') and rice actin1 (Act? 5'), and the tissue specific promoter of maize phosphoenol pyruvate carboxylase (PEPC 5'), were used to express the synthetic cryIA(b). All the constructs are transcriptional fusions and have the following promoters for synthetic cryIA(b): pRES7193 - 35S 5' (two expression cassettes per plasmid), pRES7293 - tandem Act7 5'-358 5', pRES7493 — 35S 5', pRES7793 — tandem Actl 5'—PEPC 5', pRES7393 — PEPC 5', pRES107 — tandem Act7 5', and pRES108 — Act1 5'. The cryIA(b) expression cassette in all the constructs has PEPC intron #9 and 358 terminator. pRES7193, pRES7393, and pRES7493 also contain the potato proteinase inhibitor II (pin2) driven by its wound inducible promoter and terminator. The plasmid size ranges from 6.23 to 14.63 kb and can be used as closed circular or linear DNA fragments in transformation experiments. INTRODUCTION One of the important aspects to be considered in plant transformation studies is the use of appropriate DNA constructs that will facilitate the expression of the introduced genes. The success of plant transformation is partly dependent on the vector design (Gruber & Crosby, 27 1993). Plant vectors used for free DNA delivery typically consist of an expression cassette that contains a promoter region, transcription initiation site and a portion of 5' nontranslated leader of the promoter of interest joined to a synthetic multilinker followed by polyadenylation signal (Morrish et al. 1993). For genetic modifications of rice, access to functionally and well characterized promoters and other regulatory sequences is essential (Hensgens et al., 1993). Promoter strength is critical in allowing high level transcription of selected coding sequences in plant cells (Morrish et al., 1993). We made several plasmid constructs, driven by different promoters (CaMV 35$, PEPC, PinII, Rice Act1) that can be used for co-transformation, which was efficiently used to introduce nonselectable genes into rice. In co—transformation, a plasmid carrying the nonselectable gene and another carrying the selectable marker gene are mixed and used for transformation as contrasted to transformation of two linked genes on the same plasmid (Lyznik et al., 1989). Co—transformation using direct gene transfer system is an established technique designed to introduce nonselectable gene markers into plant genomes (Peng et al., 1990). One advantage of this method is that construction of a plasmid carrying both the selectable marker and the nonselectable gene is not required, thus, it is convenient for generating plants carrying multiple genes (Kyozuka & Shimamoto, 1993). The original plasmid constructs containing the synthetic cryIA(b) was obtained from Dr. 28 Michael Koziel (CIBA-GIEGY Agricultural Biotechnology Research Unit, Research Triangle Park, NC) while those containing the rice Actin1 (Act7) promoter and potato proteinase inhibitor II (pin2) promoter and coding sequence were provided by Dr. Ray Wu (Cornell University). The use of these constructs is under cooperative agreement with Dr. Mariam B. Sticklen. The cauliflower mosaic virus 355 promoter (Guilley et al., 1982) was succesfully used in dicots, but was reported to be much less effective in monocots (Fromm et al., 1985; Hauptmann et al., 1987; Zhang et al., 1991; Last et al., 1991; Taylor et al., 1993). McElroy et al. (1990) demonstrated that this promoter has low activity in transformed rice cells. However, the 358 promoter was effectively used by Koziel et al. (1993) and Fujimoto et al. (1993) to express the synthetic cryIA(b) in corn and rice, respectively. Similarly, Kyozuka & Shimamoto (1993) showed by quantitative and histochemical analysis of 358 promoter activity in transgenic rice plants that it is expressed well in many tissues, including root, leaf, flower and seed. Phosphoenolpyruvate carboxylase (PEPC) is one of the two carboxylases of C4 plants which function in photosynthesis and is expressed in green tissues. PEPC is localized in meSOphyll cells, and the other ribulose biphosphate carboxylase (RuBPC) is found in bundle sheath cells. On the other hand, cg plants like rice, only have RuBPC which is localized in mesophyll cells. The PEPC 29 promoter of corn, a.C§ plant was successfuly used by Matsuoka et al. (1993) to express the GUS gene in rice. The expression of the genes in rice was almost exclusively in mesophyll cells of various organs such as leaf blade, leaf sheaths, rachis and glume with no or very little activity observed in other cells. The regulatory system which directs cell specific and light inducible expression of PEPC promoter was shown to be working in rice. PEPC promoter was also used to express the synthetic cryIA(b) in corn (Koziel et al., 1993). The 5' region of the rice actin (Actl) gene is another efficient promoter for regulating the constitutive expression of foreign gene in transgenic rice. Study using Act7 5' region fused to GUS gene showed that tissue from the transgenic rice plants have a level of GUS protein that represents as much as 3.0% of total soluble protein (Zhang et al., 1991). It is also expressed throughout the vegetative and gametophytic tissue of transgenic rice plants. It was observed to be much more efficient than other promoters in expressing foreign genes in monocots. This promoter contains a number of Act1 transcribed elements, including the first exon (non—coding), an intron contained within the mRNA 5' untranslated leader (intron1) and a portion of the second exon of the Act7 gene (McElroy et al., 1990 & 1991). Hensgens et al. (1993) believed that it is preferable to use homologous promoters rather than heterologous promoters for rice genetic engineering. 30 Homologous promoters are regulatory sequences already present in rice while heterologous promoters are coming from other sources. Another useful regulatory sequence that was used for the production of transgenic plants is the promoter of potato proteinase inhibitor II (pin2). This promoter is well characterized (Keil et al., 1986) and found to be wound inducible and activated by insect feeding (Sanchez—Serrano et al., 1987; Thornburg et al., 1987; Pefia—Cortes et al., 1988, 1989 & 1991; Johnson et al., 1989; Ryan, 1990; Kim et al., 1991). The pin2 promoter is approximately 900 bases from the 5' region of the pin2 coding sequence. An important characteristic of heterologous gene expression in monocot cells is the enhancing effect of the presence of an intron between the promoter and the 5' end of the coding region of the gene (Chibbar et al., 1993). In maize, intron-mediated enhancement of heterologous gene expression was also reported (Mascarenhas et al., 1990; Luchrsen & Walbot, 1991). McElroy et al. (1991) found that addition of Act7 intron1 to the transcription unit of a GUS reporter gene under the control of 35S promoter stimulated GUS activity more than 10—fold in transformed rice cells. The mode of action by which introns increase expression is not well understood but appear to be an effect on mRNA transport or stability, possibly involving the efficiency with which the particular pre—mRNAs are spliced (Waugh & Brown, 1991). An enhancement of the expression of Bt 31 proteins in transgenic plants was also observed when the small subunit leader and transit peptide of Arabidopsis thaliana were added in the constructs (Wong et al. 1992). The use of a tandem promoter to express foreign genes in plants was also previously reported. Barnes (1990) and Cheng et al. (1992) have used nos and 35S tandem promoter to express the firefly luciferase codons and cryIA(c) from B. thuringiensis HD-73 in tobacco and potato, respectively. Tandem promoters were also used by Hain et al. (1985) to transform plant protoplasts. Kay et al. (1987) also reported that duplication of 355 promoter sequence creates a strong enhancer for plant genes. Similarly, Omirulleh et al. (1993) reported higher activity of chimeric promoter with double 35$ enhancer element in protoplast derived cells and transgenic plants in maize. The isolation and use of a dual promoter from the Ti plasmid of Agrobacterium tumefaciens was also reported (Velten et al., 1984). The plasmid constructs that we made were used in various co—transformation experiments. We have used several combinations of the plasmids taking into consideration the type of promoter for the different resistance genes. MATERIALS AND METHODS Plasmid Construction and Molecular Cloning Plasmids containing the synthetic cryIA(b) (Figure 2.1) driven by Cauliflower Mosaic Virus 35S promoter (pCIB4418, Figure 2.2) and phosphoenol pyruvate carboxylase (PEPC) 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 32 ATGGACAACA ACCCCAACAT CAACGAGTGC MetAspAsn AsnProAsn IleAsnGluCys GTGGAGGTGC TGGGCGGCGA GCGCATCGAG ValGluVal LeuGlyGly GluArgIleGlu AGCCTGACCC AGTTCCTGCT GAGCGAGTTC SerLeuThr GlnPheLeu LeuSerGluPhe GTGGACATCA TCTGGGGCAT CTTCGGCCCC ValAslee IleTrpGly IlePheGlyPro GAGCAGCTGA TCAACCAGCG CATCGAGGAG GluGlnLeu IleAsnGln ArgIleGluGlu GAGGGCCTGA GCAACCTGTA CCAAATCTAC GluGlyLeu SerAsnLeu TyrGlnIleTyr CCCACCAACC CCGCCCTGCG CGAGGAGATG ProThrAsn ProAlaLeu ArgGluGluHet CTGACCACCG CCATCCCCCT GTTCGCCGTG LeuThrThr AlaIlePro LeuPheAlaVal TACGTGCAGG CCGCCAACCT GCACCTGAGC TeralGln AlaAlaAsn LeuHisLeuSer CGCTGGGGCT TCGACGCCGC CACCATCAAC ArgTrpGly PheAspAla AlaThrIleAsn GGCAACTACA CCGACCACGC CGTGCGCTGG GlyAsnTyr ThrAspHis AlaValArgTrp CCCGACAGCC GCGACTGGAT CAGGTACAAC ProAspSer ArgAspTrp IleArgTyrAsn CTGGACATCG TGAGCCTGTT CCCCAACTAC LeuAslee ValSerLeu PheProAsnTyr AGCCAGCTGA CCCGCGAGAT TTACACCAAC SerGlnLeu ThrArgGlu IleTerhrAsn CGCGGCAGCG CCCAGGCCAT CGAGGGCAGC ArgGlySer AlaGlnGly IleGluGlySer AACAGCATCA CCATCTACAC CGACGCCCAC AsnSerIle ThrIleTyr ThrAspAlaHis ATCATGGCCA GCCCCGTCGG CTTCAGCGGC IleMetAla SerProVal GlyPheSerGly ATGGGCAACG CTGCACCTCA GCAGCGCATC MetGlyAsn AlaAlaPro GlnGlnArgIle ATCCCCTACA ACTGCCTGAG CAACCCCGAG IleProTyr AanysLeu SerAsnProGlu ACCGGCTACA CCCCCATCGA CATCAGCCTG ThrGlyTyr ThrProIle AsleeSerLeu GTGCCCGGCG CCGGCTTCGT GCTGGGCCTG ValProGly AlaGlyPhe ValLeuGlyLeu AGCCAGTGGG ACGCCTTCCT GGTGCAGATC SerGlnTrp AspAlaPhe LeuValGlnIle TTCGCCCGCA ACCAGGCCAT CAGCCGCCTG PheAlaArg AsnGlnAla IleSerArgLeu GCCGAGAGCT TCCGCGAGTG GGAGGCCGAC AlaGluSer PheArgGlu TrpGluAlaAsp CGCATCCAGT TCAACGACAT GAACAGCGCC ArgIleGln PheAsnAsp MetAsnSerAla CAGAACTACC AGGTGCCCCT GCTGAGCGTG GlnAsnTyr GanalPro LeuLeuSerVal GTGCTGCGCG ACGTCAGCGT GTTCGGCCAG ValLeuArg AspValSer ValPheGlyGln AGCCGCTACA ACGACCTGAC CCGCCTGATC SerArgTyr AsnAspLeu ThrArgLeuIle TACAACACCG GCCTGGAGCG CGTGTGGGGT TyrAsnThr GlyLeuGIu ArgValTrpGly CAGTTCCGCC GCGAGCTGAC CCTGACCGTG GlnPheArg ArgGluLeu ThrLeuThrVa] GACAGCCGCA CCTACCCCAT CCGCACCGTG AspSerArg ThrTerro IleArgThrVal CCCGTGCTGG AGAACTTCGA CGGCAGCTTC ProValLeu GluAsnPhe AspGlySerPhe ATCCGCAGCC CCCACCTGAT GGACATCCTG IleArgSer ProHisLeu MetAsleeLeu CGCGGCGAGT ACTACTGGAG CGGCCACCAG ArgGlyGlu Tererrp SerGlyHisGln CCCGAGTTCA CCTTCCCCCT GTACGGCACC ProGluPhe ThrPhePro LeuTyrGlyThr GTGGCACAGC TGGGCCAGGG AGTGTACCGC ValAlaGln LeuGlyGln GlyValTyrArg 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 y 1801 1861 1921 33 ACCCTGAGCA GCACCCTGTA CCGTCGACCT ThrLeuSer SerThrLeu TyrArgArgPro AGCGTGCTGG ACGGCACCGA GTTCGCCTAC SerValLeu AspGlyThr GluPheAlaTyr TACCGCAAGA GCGGCACCGT GGACAGCCTG TyrArgLys SerGlyThr ValAspSerLeu CCACCTCGAC AGGGCTTCAG CCACCGTCTG ProProArg GlnGlyPhe SerHisArgLeu AGCAACAGCA GCGTGAGCAT CATCCGTGCA SerAsnSer SerValSer IleIleArgAla GAGTTCAACA ACATCATCCC CAGCAGCCAG GluPheAsn AsnIleIle ProSerSerGln AACCTGGGCA GCGGCACCAG CGTGGTGAAG AsnLeuGly SerGlyThr SerValValLys CGCCGCACCA GCCCCGGCCA GATCAGCACC ArgArgThr SerProGly GlnIleSerThr CAGCGCTACC GCGTCCGCAT CCGCTACGCC GlnArgTyr ArgValArg IleArgTyrAla ATCGACGGCC GCCCCATCAA CCAGGGCAAC IleAspGly ArgProIle AsnGlnGlyAsn CTGCAGAGCG GCAGCTTCCG CACCGTGGGC LeuGlnSer GlySerPhe ArgThrValGly AGCAGCGTGT TCACCCTGAG CGCCCACGTG SerSerVal PheThrLeu SerAlaHisVal CGCATCGAGT TCGTGCCCGC CGAGGTGACC ArgIleGlu PheValPro AlaGluValThr CAGAAGGCCG TGAACGAGCT GTTCACCAGC GlnLysAla ValAsnGlu LeuPheThrSer ACCGACTACC ACATCGATCA GGTGTAG ThrAspTyr HisIleAsp Ganal--— TTCAACATCG GCATCAACAA CCAGCAGCTG PheAsnIle GlyIleAsn AsnGlnGlnLeu GGCACCAGCA GCAACCTGCC CAGCGCCGTG GlyThrSer SerAsnLeu ProSerAlaVal GACGAGATCC CCCCTCAGAA CAACAACGTG AspGluIle ProProGln AsnAsnAanal AGCCACGTGA GCATGTTCCG CAGTGGCTTC SerHisVal SerMetPhe ArgSerGlyPhe CCTATGTTCA GCTGGATTCA CCGCAGTGCC ProMetPhe SerTrlee HisArgSerAla ATCACCCAGA TCCCCCTGAC CAAGAGCACC IleThrGln IleProLeu ThrLysSerThr GGCCCCGGCT TCACCGGCGG CGACATCCTG GlyProGly PheThrGly GlyAsleeLeu CTGCGCGTGA ACATCACCGC CCCCCTGAGC LeuArgVal AsnIleThr AlaProLeuSer AGCACCACCA ACCTGCAGTT CCACACCAGC SerThrThr AsnLeuGln PheHisThrSer TTCAGCGCCA CCATGAGCAG CGGCAGCAAC PheSerAla ThrMetSer SerGlySerAsn TTCACCACCC CCTTCAACTT CAGCAACGGC PheThrThr ProPheAsn PheSerAsnGly TTCAACAGCG GCAACGAGGT GTACATCGAC PheAsnSer GlyAsnGlu ValTyrIleAsp TTCGAGGCCG AGTACGACCT GGAGAGGGCT PheGluAla GluTyrAsp LeuGluArgAla AGCAACCAGA TCGGCCTGAA GACCGACGTG SerAsnGln IleGlyLeu LysThrAspVal Figure 2.1. Nucleotide sequence of synthetic Bacillus thurlngiensis cryIA(b) and its corresponding amino acids. (Courtesy of Dr. Michael Koziel, CIBA—GEIGY). 34 Hind II (4.79) NspI-II (4.43)~ CaMV 35$ 5' ’5‘;on (5.67) #881111“ (5.81) ApaLI (4.11 ATG (.0001) pCIB4418 0N1 (0.38) (5.82 kb) Synthetic cryIA(b) \NcoI (1.02) PvuI (3.17 CaMV 35$ 3' PEPC Intron9 EcoRI (2.15) 5811 (1.10) Kpnl (2.14 BglII (1.96 Sacl (l.' 5) \9511 (1.60) TAG (1.95) PstI (1.68) BstEII (1.82) Figure 2.2. Restriction map of pCIB4418, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35$ promoter and terminator. 35 promoter (pCIB4421, Figure 2.3) were obtained from Dr. Michael Koziel (CIBA—GEIGY). These plasmids were used to make other constructs containing the rice Actin1 (Act?) 5' region from pDM302 (Figure 2.4) which contains the Act? transcribed elements, including the first exon (non—coding) and an intron contained within the mRNA 5' untranslated leader (intron1) and a portion of the second exon (McElroy et al., 1991). A 3.0 kb fragment from pTW—a (Figure 2.5) containing the pin2 coding sequence driven by the wound inducible pin2 promoter with rice Act? intron and pin2 terminator was ligated to plasmids pCIB4418 and pCIB4421 to produce plasmids with both cryIA(b) and pin2 driven by different promoters and terminators in the same replicon. Plasmids pDM302 and pTW—a were provided by Dr. Ray Wu (Cornell Univeristy) under a cooperative agreement with Dr. Mariam B. Sticklen. Restriction mapping of each plasmid was done to confirm the restriction sites prior to the construction of other plasmids. The rice Act? promoter was isolated from pDM302 by digesting the plasmid with HindIII and fragments were separated by electrophoresis in 0.8% agarose gel. The 1.44 kb promoter was isolated either by electroelution, with the use of NA45 membrane (Schleicher & Schuell, Inc. Keene, NH) or GeneClean II kit (B10101 Inc., La Jolla, CA). Transformation was done by electroporation using the Electro Cell Manipulator 600, BTX Electroporation System (BTX, San Diego, CA) or by CaCl-MgCl treatment of competent cells 36 HindIII (4 .79) NspHI 4.43) ,..-;;r:.”””°’v§\ «as! {flififi \Col (5 .76) _.".;n.~'~13:§::-- 0R V (6 .02 ) p C l B 4421 \ PvuI (3.17) x (7.12 kb) 5.: EC RI § stEII (6.90) 0 e N . (2'15) Synthetic cryIA(b) amHI (7‘11) Kpnl (2.14 CaMV 358 3' BglII (1.96 PEPC Intron9 ATG ('0001) 5301 “-95 ON] (0.38) TAG(195 8511311 (1.82) pa (1 60) NcoI (1.02) Figure 2.3. Restriction map of pCIB4421, containing the synthetic B. thuringiensis cryIA(b) driven by tissue specific maize phosphoenol pyruvate carboxylase (PEPC) promoter and 353 terminator. 37 Rice Act? Exont Rice Act? Intront Rice Act1 Exon2 Figure 2.4. Restriction map of pDM302, containing the bar coding sequence driven by rice Actin1 (Act?) promoter and nos terminator. The Act? promoter was used to express the synthetic B. thuringiensis cryIA(b) in other plasmid constructs. 38 HindI I (5.9) oooooooooo ....... oooooooooooooooooo ....... nnnnnnnnnnn ..... ooooooooo .- a ....... ccccccc on. oooooo a. ..... ‘- ..... o. ..... u. ..... ..... u a ccccc - pUC19 pTW-a (7.4 kb) Nos3' bar CaMV 35$ 5' Figure 2.5. Restriction map of pTWa, containing the potato proteinase inhibitor II (pin2) coding sequence driven by pin2 promoter with rice Act? intron1, and pin2 terminator. In a separate expression cassette, the selectable marker bar is driven by CaMV 358 promoter and nos terminator. The pin2 expression casette was used together with synthetic cryIA(b) to construct other plasmids. 39 modified from Mandel & Higa (1970) and Hanahan (1983). For DNA manipulations, the procedures used were either standard (Sambrook et al., 1982) or our modified techniques. The constructs were transformed into SURER (Stratagene Cloning Systems, La Jolla, CA), DH5a and JM101 and plated on ampicillin plates (100 ug/ml). The transformants were screened by isolating the plasmid DNA using the modified alkaline lysis procedure (Birnboim & Doly, 1979), INSTA— PREPTM kit (5 Prime 4 3 Prime, Inc, Boulder, CO) or our modified procedure C (Chapter I). Digestion of the plasmid DNA was carried out using the conditions specified by the restriction enzyme manufacturer (Boeringer Mannheim Biochemicals, Indianapolis, IN). Stock cultures of confirmed clones were preserved in 50% glycerol at —80°C. RESULTS pRES7293 (7.25 kb) The recovered 1.44 kb rice Act? promoter was ligated to the linearized pCIB4418 previously digested with HindIII and dephosphorylated by treatment with bacterial alkaline phosphatase to prevent self ligation. The desired clone was identified by partial restriction mapping using different restriction enzymes both as single or double digests. The correct orientation of the promoter with respect to the coding sequence was identified by the following enzyme digestion and the presence of the following fragments as seen on ethidium bromide—stained 0.8% agarose gel: BamHI - 40 5.89 kb and 1.37 kb; HindIII — 5.82 kb and 1.44 kb; EcoRI — 4.23 kb and 3.02 kb; BamHI—HindIII — 4.80 kb, 1.09 kb, 1.02 kb and 0.35 kb; EcoRI—EcoRV — 3.02 kb, 2.29 kb and 1.94 kb. The construct has a rice Act7—358 tandem promoter and its partial restriction map is shown in Figure 2.6. pRES7793 (8.56 kb) The 1.44 kb fragment from pDM302 containing the rice Act? promoter was ligated to HindIII digested and bacterial alkaline phosphatase treated pCIB4421 and then transformed into competent cells. The clones were characterized by partial restriction mapping and the desired clones with respect to the orientation of the rice Act? promoter to the cryIA(b) coding sequence was identified by the presence of the following fragment sizes after digestion with restriction enzymes: HindIII — 7.12 kb and 1.44 kb; BamHI — 5.89 kb and 2.68 kb; BamHI—HindIII — 4.79 kb, 2.33 kb, 1.09 kb and 0.35 kb; EcoRV — 8.56 kb; EcoRI — 5.55 kb and 3.02 kb. The partial restriction map is shown in Figure 2.7. pRES7193 (14.63 kb) and pRES7493 (8.82 kb) The 3.0 kb fragment containing the pin2 coding sequence driven by pin2 promoter with rice Act? intron and pin2 terminator from pTW—a was cloned to the HindIII digested and dephosporylated pCIB4418. Upon restriction mapping, it was noted that one of the clones designated as pRES7193 (Figure 2.8) has two intact copies of the linearized pCIB4418 41 HindIII (4.79) m Nsle (4.43) \ ; ' ad (568) Rice .4ch Exonl 9’96” : amHI (5.88) ApaLI (411) Rice Act] Innonl ’49". all pUC Rice Act] Exon2 indIII (6.23) p R 537293 (7.25 kb) CaMV 35$ 5' PvuI (3.17 EcoRI (2.15 Kpnl (2.14 —-ECORV (7.11) CaMV 35$ 3' : amHI (7.25) 4‘ .III BglIl(1.96 \‘3 PEPC Eur: C Wb) “G 10001) yn 1c ry Sac1(l.95 Eco I(0.38) TAG(1.95) 38151 (1.82) Pst (1.60) Ncol(l.02) PStI (1.68) SalI (1.10) Figure 2.6. Restriction map of pRES7293, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice Act? and CaMV 35S promoter. 42 HindIII (4.79) I N81) (4 .4 3) ...... ;,..,¥.;io,jgt;:§:§:§:§.’z (5 . l 7 ) .................. ...... acI ......... (5.68) A 211.1 (4.1 p Rice Acti Exont (3531818511 - I Rice Act1 lntron1 6;.“ HindIII (6.23) Rice Act1 Exon2 \ R \ Pvul (3.17 § pRES7793 p x o EcoR (8.56 kb) 3. col (7.10) (2.15) ’3 R! KpnI (2.14 § ean 353 3' S” \EcoRV (7.46) 381“ “-96 PEPC Intron9 :51” Sad (1.95 § TAG (1.9 y S" BstEII (1.82) Synthetic cryIA(b) PstI (1.68) “E" PstI (1.60) (8.33) Ncol (1.02) Am (000') Figure 2.7. Restriction map of pRES7793, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice Act? and maize PEPC promoter. 43 Hindll (13.75) TG pin2 3' %/ (”"2 CaMV ass 5' . ice Act1 lntron1 Synthetic cryIA(b) BamHI (14.63) ATG (0.0001) A BamHI (12.10 INcol (1.02) 'stEII (1.82) AG SacI (1.95) glII (1.96) Kpnl (2.14) coRI (2.15) HIndIII (10.59 PEPC Introng CaMV 358 3' pRES7193 (14.63 kb) ’ 111 (3.17) pUC cry/A (b) ~pnlpin EcoRI (7.96 sPHI (4.43) sad (7‘76 indIII (4.79) TAG 8511511 (7.63) a” (4'8” Ncol (6.83) ATG EcoRV (5.67) Bmflfl(i&) Figure 2.8. Restriction map of pRES7193, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35S promoter and terminator, pin2 driven by pin2 promoter with rice Act? intron1, and pin2 terminator. There are two copies of the synthetic cryIA(b) expression cassette per plasmid molecule. 44 ligated together plus the 3.0 kb HindIII fragment from pTW—a. The origin of replication of the two fragments are oriented in the same direction which enables it to replicate in the host cells. Digestion of the plasmid with different restriction enzymes resulted to the following fragments: HindIII - 5.82 kb (two different fragments of the same size) and 3.00 kb; BamHI - 5.89 kb, 5.81 kb, 1.50 kb, 1.02 kb, 0.30 kb and 0.10 kb; BamHI—HindIII — 4.79 kb (two different fragments of the same size), 1.50 kb, 1.10 kb, 1.02 kb (two different fragments of the same size), 0.30 kb, and 0.10 kb; EcoRI—EcoRV - 6.51 kb, 3.51 kb and 2.30 kb (two different fragments of the same size); EcoRV — 8.81 kb and 5.81 kb; EcoRI — 8.81 kb and 5.81 kb. The construct contains the pin2 and two copies of synthetic cryIA(b) sequences in the same replicon but driven by different promoters and terminators. The synthetic cryIA(b) is driven by 35S promoter while pin2 is driven by pin2 promoter with rice Act? intron. Another clone, pRES7493, derived from this experiment contains only one each of pin2 and synthetic cryIA(b) expression cassettes (Figure 2.9). The generated fragments upon restriction mapping is almost the same as pRES7193 except that it only has the 8.82 kb fragment upon digestion of EcoRI or EcoRV while EcoRI-EcoRV double digestion gave 6.52 and 2.30 kb fragments. pRES7393 (10.0 kb) The 3.0 kb HindIII fragment from pTW—a containing the 45 SI'I BamHI (7.79 ‘IIIIIIII pin2 3' Eco ’ V (8.67) :amHI (8.81) - TG (0.0001) CaMV 35$ 5' 0N1 (0.38) Synthetic cryIA(b) Rice Act1 lntron1 Ncol(l.02) XbaI (5.84) pRES7493 3110.10) (8.8 kb) . a (1.60) 'stl (1.68) PstI Hin III 4 79 PEPC lntron9 51511 (1.82) d ‘ ‘ ) CaMV 358 3' A6095) SacI(L95) NspHI (4.43) BglII (1.96) ApaI-I (4.11) Kpn (2.14) Pvul (3.17) EcoRI (2.15) Figure 2.9. Restriction map of pRES7493, containing_the synthetic B. thuringiensis cryIA(b) driven by CaMV 35S promoter and terminator, and pin2 driven by pin2 promoter with rice Act1 intron1, and pin2 terminator in two separate expression cassettes. 46 pin2 coding sequence driven by pin2 promoter with rice Act? intron and pin2 terminator was ligated to the HindIII digested and dephosphorylated pCIB4421. The correct construct was confirmed for the presence of the following fragment sizes upon digestion with different restriction enzymes: HindIII — 7.12 kb and 3.00 kb; BamHI - 6.20 kb, 2.30 kb, 1.50 kb; BamHI-EcoRI - 4.05 kb, 2.30 kb, 2.15 kb and 1.50 kb; BamHI—HindIII — 4.80 kb, 2.30 kb, 1.40 kb, 1.50 kb; EcoRI — 10.0 kb; EcoRV - 10.0 kb; EcoRI-EcoRV — 6.75 kb and 3.23 kb. The construct contains both the pin2 and synthethic cryIA(b) in the same replicon but driven by different promoters and terminators. The synthetic cryIA(b) was driven by PEPC promoter while pin2 was driven by pin2 promoter with rice Act? intron. Partial restriction map of the construct is shown in Figure 2.10. pRES107 (7.67 kb) The 1.44 kb HindIII fragment from pDM302 containing the rice Act? promoter and intron was recovered from agarose gel and ligated to blunt—ended pCIB4418. pCIB4418 was digested with BamHI and the 4.79 kb fragment containing the cryIA(b) coding sequence, PEPC intron #9, 35S terminator and pUC vector was recovered by GeneClean II kit (Bi0101 Inc., La Jolla, CA) from 0.8% agarose gel. The recessed ends in both fragments were filled in by Klenow fragment. This approach resulted to a construct containing a tandem rice Act? promoter both in correct orientation with respect to the 47 SP“ Xbal HindIII (7.79) 'IIIIIIIII'I -:-§'§'E':'EI:.§.;:E.E.:::m NCOI (8.76) 0' ooooo In ooooo o S BamHI (6.29 / Xb81(5.84 /Rice Act? lntron1 § é- °amHI (10.1) g pin2 5' PRES-I393 TG (0.0001) \ HindIII (4.79 5 °NI (0'38) Synthetic cryIA(b) ‘ C01 (1.02) NspHI (4.43) all (1.10) P I l. pUC PEPC lntron9 St ( 60) ApaLI (4.11) v s 3, stI (1.68) Ga” 35 511311 (1.82) Pvul (3.17) ' 9"}: AG (1.95) Ele (2-15 KpnI (2.14) SacI (1.95) BglII (1.96) containing the Figure 2.10. Restriction map of pRES7393, synthetic B. thuringiensis cryIA(b) driven by PEPC promoter and CaMV 35$ terminator, pin2 driven by pin2 promoter with and pin2 terminator in two separate rice Act? intron1, expression cassettes. 48 cryIA(b) coding sequence. Restriction mapping showed the presence of the following fragments: EcoRI — 3.21 kb, 3.02 kb and 1.44 kb; PstI — 3.11 kb, 3.04 kb, 1.44 kb and 0.08 kb; BamHI - 6.23 kb and 1.44 kb; BamHI—EcoRI — 3.02 kb, 2.50 kb, 0.73 kb and 0.71 kb (two fragments of the same size), EcoRI—PstI - 2.66 kb, 2.64 kb, 1.06 kb, 0.47 kb, 0.38 kb (two fragments of the same size) and 0.08 kb; BamHI—BglII — 6.23 kb and 1.44 kb. The partial restriction map is shown in Figure 2.11. pRE8108 (6.23 kb) A construct containing the synthetic cryIA(b) driven by rice Act? promoter was made from pRES107 by digesting it with BamHI and recovering the bigger fragment. In pRES107, the two BamHI sites are within the internal region of the tandem rice Act? promoter (Figure 2.11) and removal of the 1.44 kb BamHI fragment and subsequent ligation of the larger 6.23 kb fragment conserved the intact sequence of the rice Act? promoter. Restriction mapping of the construct showed the presence of the following fragments: EcoRI— 3.21 kb and 3.02 kb; PstI — 3.11 kb, 3.04 kb and 0.08 kb; BamHI—EcoRI — 3.02 kb, 2.50 kb and 0.71 kb; EcoRI—PstI — 2.66 kb, 2.64 kb, 0.47 kb, 0.38 kb and 0.08 kb, confirming the plasmid structure. The partial restriction map of the construct is shown in Figure 2.12. pRES107 and pRE8108 are both transcriptional fusions aand the nucleotide sequence at the junction of the rice Act? 49 SphI (4. 9) ’stI _.._. EcoRI (5.17) ‘ ‘ Rice Act? 5' NspHI (4.43) ‘ “=_ . aCI (5.68) 1' I 9’ ’Il,’ :amHI (5.88) Rice Act? Exon1 9 Rice Act? lntron1 ” ApaL 4.11) ’4; ph1(6-23) Rice Act? Exon2 : pRES107 Rice Act? 5‘ g coRI (6.61) PvuI (3.17 (7.67 kb) 5'- EcoRI (2.15 Rice Act? Exont -" 7, CaMV3SS 3' ' 3‘31 (7-12) KpnI (“'14 PEPC lntron9 Rice Act? lntron1 BglII (1'96 Rice Act? Exon2 \BamI-II (7 32 Sad (1.95 TAG (1.95 Synthetic cryIA(b) ACCI(7-67) 381511 (1.8 ATG (0.0001) Pst (1.68) EcoNI(0.38) P511 ( ~60) NCOI (1.02) SalI (1.10) Figure 2.11. Restriction map of pRES107, containing the synthetic B. thuringiensis cryIA(b) driven by tandem rice Act? promoter and CaMV 35$ terminator. 50 SphI 4.79) Nsle (4.43)\ ' 'stI ApaLI (4.11 acI (5.68) 1'; ' A t? E n1 ’I R“ C m ’4, BamHI(5.88) Rice Act? lntron1 ’3 pUC . (a ch(6.23) Rice Act? Exon2 ‘ "' pRES108 TG(0.0001) . (6'23 kb) coNI(O.38) Synthetic cryIA(b) Pvul (3.17 CaMV358 3' Ned (1.02) EcoRI (2.15 _ PEPC lntron9 all (1.10) Kpn1(2.14 13g x5“ (1.60) BglII (1.96 Sacl 1.9 PstI (1.68) B51 11 (1.82) TAG (1.95) synthetic B. Figure 2.12. Restriction map of pRES108, promoter and CaMV 35S terminator. containing the thuringiensis cryIA(b) driven by rice Act? 51 gene transcribed region is shown in Figure 2.13A, while the sequence generated upon fusion of the tandem rice Act? promoter is shown in Figure 2.13B. The entire open reading frame of the synthetic cryIA(b) and its amino acid sequence is conserved and there is no unwanted initiation codon. Agrobacterium Vectors Two plasmid constructs originally made for Agrobacterium transformation of dicots can also be used for monocot transformation using appropriate Agrobacterium strains. Construction of these plasmids was done with the help of Mrs. Neerja Hajela. pHSE201 (13.18 kb) and pHSEZOZ (14.49 kb) The entire 3.18 kb HindIII—EcoRI fragment from pCIB4418 containing the 35S promoter-synthetic cryIA(b)—PEPC intron #9 and 35S terminator was separated from the other fragment in 0.8% agarose gel and recovered by GeneClean II kit. pCA—1 (Figure 2.14) was digested with HindIII and EcoRI and the 10.0 kb fragment containing the pBIN19 vector, nos promoter— nptII—nos terminator cassette was recovered. The two fragments were ligated, transformed into DHSa and plated in LB medium with kanamycin (50 ug/l). pHSE202 was constructed by ligating the 4.49 kb HindIII—EcoRI fragment from pCIB4418 containing the PEPC promoter—synthetic cryIA(b)—PEPC intron #9—35S terminator expression cassette to the 10.0 kb fragment from pCA-1. Since both are directional cloning, the right clones were selected for the presence of the 3.18 kb 52 .A 5'———ttgtag/GTAGACGATAAGCTYGATCCNNNNNATGGACAACA——-3' 3'—-—aacatc/CATCTGCTATTCGA/cancGNNNNNTACCTGTTGT———5' IB 5'—-—ttgtag/GTAGACGATAAGCT/AGCTT—-—3' 3'———aacatc/CATCTGCTATTCGA/TCGAA--—5' Figure 2.13. Portion of the nucleotide sequence at the site of fusion in the constructs pRES107 and pRES108. A) Nucleotide sequence at the junction of the rice Act? transcribed region and the synthetic cryIA(b) in pRES107 and pRES108. Bold—italic letters represent the nucleotide sequence generated upon filling up of the HindIII site from rice Act? promoter by Klenow fragment. Underlined italic letters are nucleotide sequence generated upon filling up by Klenow fragment of the BamHI site from pCIB4418. Initiation codon (ATG) of synthetic cryIA(b) is indicated by bold underlined letters. N are undetermined nucleotide sequence coming from pCIB4418 preceeding the initiation codon. B) Nucleotide sequence generated upon fusion of the 3' end and the 5' end of two rice Act? promoter resulting to a tandem promoter (pRES107), both in correct orientation with respect to the synthetic cryIA(b). Underlined letters are nucleotide sequence generated upon filling up of the HindIII sites by Klenow fragment. Lower case letters in A) & B) represent the rice Act? intron1 sequence. The slashed lines indicate intron-exon boundaries, slashed lines in bold are sites of the fusion. 53 oooooooooooooooooo uuuuuu ooooooooooo ..... ooooooooo o o ooooooo o. oooooo cccccc u ...... .- ooooo ..... o- ..... o ..... 00 ..... (16.5 kb) Eco ' ‘. ' (Drawn not to scale) Figure 2.14. Restriction map of pCA—1 containing the selectable marker neomycin phosphotransferase II (npt II) gene that confers resistance to kanamycin, driven by nos promoter and terminator in pBIN19 Agrobacterium binary vector. This fragment was recovered and used to construct pHSEZO1 and pHSE202, both containing the synthetic B. thuringiensis cryIA(b). 54 (pHSE201) or 4.49 kb (pHSE202) insert after HindIII—EcoRI digestion. The partial restriction maps of the plasmids are shown in Figures 2.15 & 2.16. The characteristics of the different plasmid constructs are shown in Table 2.1. DISCUSSION All the expression cassettes were subcloned in pUC vector which has high copy number in E. coli, facilitating the isolation of large amount of DNA for microprojectile bombardment. All of the constructs are transcriptional fusion and the coding sequence of synthetic cryIA(b) and pin2 are driven by separate promoters and terminators. Although translational fusion of the two genes will allow its simultaneous expression in transformed cells, we preferred to have it in separate expression cassetes since it is not certain if the protein product of fused pin2 and synthetic cryI(b) will be biologically active against target insects. This particular aspect should be further evaluated by bioassay. In all the constructs, the original sequence context surrounding the translation initiation site from pCIB4418 and pCIB4421 was conserved. This was done because it has already been optimized and shown to be expressed well in corn, a monocot (Koziel et al., 1993). The presence of a G immediately after the initiation codon (ATG) in the synthetic cryIA(b) is a part of a eukaryotic consensus sequence and the consensus sequence of ACAATGG for plant transcripts (McElroy et al., 1991). 55 pHSE201 (13.18 kb) CaMV3SS 5' PEPC lntron9 CfiMV353 3' "" ..- .-. .- - " °5tix1:5>'.<3ompozx~m >513§$3£6iw ' " .4. "" " 28:15:". MW " "-v Figure 2.15. Restriction map of pHSE201, containing the synthetic B. thuringiensis cryIA(b) driven by CaMV 35$ promoter and terminator and nptII driven by nos promoter and terminator in an Agrobacterium binary vector pBIN19. 56 pHSE202 pBIN19 (14.49 kb) Figure 2.16. Restriction map of pHSE202, containing the synthetic B. thuringiensis cryIA(b) driven by PEPC promoter and 358 terminator and nptII driven by nos promoter and terminator in an Agrobacterium binary vector pBIN19. 57 Table 2.1. Characteristics of different plasmid constructs containing the synthetic B. thuringiensis cryIA(b) driven by different promoters and pin2 driven by its promoter for monocot transformation. Plasmid/ Promoter Coding Sequence Terminator (Size)a pCIB4418 353 5' cryIA(b) PEPC Int.9,35S 3' (5.82 kb) pCIB4421 PEPC 5' cryIA(b) PEPC Int.9,3SS 3" (7.12 kb) pRES7193 pin2 5' pin2 pin2 3' (14.63 kb) Act7 Int.1 358 5' cryIA(b) PEPC Int.9,35S 3' (two copies of this cassette per plasmid molecule) pRES7293 Act? 5'-3sss' cryIA(b) PEPC Int.9,3SS 3' (7.25 kb) pRES7493 pin2 5' pin2 pin2 3' (8.82 kb) Act7 Int.1 35$ 5' cryIA(b) PEPC Int.9,358 3 pRES7793 Act? 5'—PEPC 5' cryIA(b) PEPC Int.9,3ss 3' (8.56 kb) pRES7393 pin2 5' pin2 pin2 3' (10.1 kb) Act7 Int.1 PEPC 5 ' cryIA(b) PEPC Int.9,358 3' pRES107 Act? 5'-Act? 5' cryIA(b) PEPC Int.9,3SS 3' (7.67 kb) pRES108 Act? 5' cryIA(b) PEPC Int.9,3SS 3' (6.23 kb) Agrobacterium Vectors pHSE201 35$ 5' cryIA(b) PEPC Int.9,358 3' (13.18 kb) nos 5' nptII nos 3' pHSE202 PEPC 5' cryIA(b) PEPC Int.9,358 3' (14.49 kb) nos 5' nptII nos 3' apCIB4418 and pCIB4421 were provided by Dr. Michael Koziel (CIBA—GEIGY). The vector for pCIB and pRES constructs is pUC while it is pBIN19 for pHSE. 58 The size of the contructs designed for microprojectile bombardment and other physical methods of transformation ranges from 6.23 to 14.63 kb. Gruber and Crosby (1993) noted that most of the physical vectors for plant transformation are usually 4.0 to 7.0 kb. Plasmids as large as 9.0 and 11.0 kb were used as vectors without compromising transgene stability (Potrykus et al., 1985; Vasil et al., 1991; Gruber & Crosby, 1993). Larger vectors may be somewhat unstable, and in one case the selectable markers encoded on a 18.7 kb plasmid were not all expressed within each transformant (Gruber & Grosby, 1993). A review by Gruber & Crosby (1993) also showed that DNA conformation plays a role in the frequency of physical transformation. Both linear and supercoiled plasmids are used as vectors, and there is evidence that linear form transforms plant more efficiently (West et al. 1991). Based on the results of partial restriction mapping, all the plasmid constructs can be used as linear DNA fragment without disturbing the expression cassettes (promoter—coding sequence-terminator) by the digestion of these plasmids by the following restriction enzymes: pCIB4418 and pCIB4421 — HindIII or EcoRI to produce a linear DNA fragment, double digestion by HindIII and EcoRI will separate the entire expression cassette from the pUC vector; pRES7293, pRES7793, pRES107 and pRES108 — KpnI, to produce a linear DNA fragment; and pRES7193 and pRES7393 — HindIII, to release the pin2 and synthetic cryIA(b) into two 59 separate linear expression cassettes. It should be noted that in pRES7193 there are two expression cassettes of cryIA(b) per pin2 expression cassette in each plasmid molecule. In terms of the preparation of plasmid DNA for particle bombardment, the use of pRES7193, pRES7393 and pRES7493 has the advantage of having the genes isolated at one time and thus only a single preparation is required as compared to the separate isolation of the genes from two different constructs. The different constructs can be used in electroporation, PEG treatment of protoplast, silicon carbide-mediated transformation and particle bombardment, as closed circular or linear forms. The constructs pHSE201 and pHSE202 containing the synthetic cryIA(b) driven by 358 and PEPC promoters, respectively, subcloned to binary vector pBIN19 can be introduced to Agrobacterium strains that are capable of infecting monocot. Raineri et al. (1990) reported that strain A281, a hypervirulent strain of A. tumefaciens can infect rice. Extensive root proliferation was also observed following inoculation with the limited-host—range (LHR) strain A856. Transgenic rice tissues have been recovered from immature rice embryos using Agrobacterium but regeneration of plants was not reported from these cultures (Raineri et al. 1990). Transient transformation of rice tissue by A. tumefaciens was also reported by Liu et al. (1992). Zaghmout and Trolinder (1993) reported a simple and 60 efficient method for directly electroporating Agrobacterium plasmid DNA into wheat callus cells, a method that can also be tried on rice cells. Agrobacteriumbmediated transformation was also tried on corn (Grimsley et al., 1987; Boulton et al., 1989; Gould et al., 1991; Schlapp & Hohn, 1992; Raineri et al., 1993) with limited success. Upon further experimentation and verification, the Agrobacterium system may become a useful alternative to currently employed procedure for rice transformation (Cao et al., 1991). All of the constructs can also be used to produce transgenic plants that can be incorporated in resistance management since it has tissue specific (PEPC), wound inducible (pin2) and constitutive (rice Act? and 358) promoters. Concerns on the develOpment of insect resistance to conventional Bt spray and to transgenic plants expressing the Bt Cry genes are discussed in various papers (Gould, 1988a&b; Tabashnik et al., 1991; Van Rie et al., 1990; Sims & Stone, 1991; Gould & Anderson, 1991; McGaughey & Johnson, 1992; Sticklen, 1992; McGaughey & Whalon, 1992; Whalon & McGaughey, 1993; Marrone & MacIntosh, 1993; Tabashnik, 1994; Kennedy & Whalon, 1995). It is believed that deployment of Bt toxins in genetically engineered crop plants will intensify selection for resistance (Gould, 1988; Gould et al., 1994). There is no doubt that widespread use of transgenic plants without proper management will lead to the development of resistance in insects (Ebora & Sticklen, 61 1994c&d). Under laboratory conditions, resistance to Bt was already observed in Indianmeal moth (McGaughey & Beeman, 1988; McGaughey & Johnson, 1992), almond moth (McGaughey & Beeman, 1988), tobacco budworm (Stone et al., 1989; Sims & Stone, 1991), sunflower moth (Brewer, 1991), and Colorado potato beetle (Whalon et al. 1993, Rahardja & Whalon, 1995). A recent review by Kennedy and Whalon (1995) showed that high levels of resistance to Bt subsp. kurstaki and somewhat lower levels of resistance to Bt subsp. aizawai have been reported in field populations of diamondback moth (Tabashnik et al., 1990; Shelton & Wyman, 1992; Shelton et al., 1993; Telekar & Shelton, 1993; Tabashnik, 1994). Some of the integrated pest management strategies for limiting the development of insect resistance to Bt toxins depend upon increasing the relative fitness of resistant genotypes by providing spatial or temporal refugia from exposure to toxin (Leeper et al., 1986; Gould, 1988a; Williams et al., 1992). Spatial refugia could be created either by expressing the toxin only in specific organs of a given crop plant in which a construct with tissue specific promoter can be used for plant transformation, or by interplanting a mixture of transgenic and non—transgenic plants (Gould, 1988). Temporal refugia on the other hand, could be generated by triggering expression of the toxin in response to some specific stimulus such as a developmental signal or environmental cue. As a possible method of managing the development of 62 resistance, Williams et al. (1992) devised a system for temporarily controlling the expression of the 6—endotoxin by having a construct where the toxin gene is driven by a chemically—responsive promoter. Two strategies for expression of foreign genes in plants for insect control are the use of essentially constitutive promoters or the use of tissue specific promoters. It was also suggested by Koziel et al. (1993) that the use of a variety of resistance genes, either in linear progression, on a revolving basis, or in combinations should combat the development of resistance to any single mechanism or gene product. This system will effectively work if the genes have different mode of actions and that there is no cross resistance. It was predicted that minimizing the exposure of insect populations to lethal levels of toxin should limit the increase of resistant allele frequencies. Restriction of toxin gene expression to those plant tissues which are more susceptible to pest damage could decrease selection pressure while still providing appropriate control. The main advantage of using a tissue-specific promoter is that the toxin will be present within specific location in the plant (e.g. leaves, but not in stem,) providing toxin free plant parts for susceptible individuals to survive and breed with the resistant individuals. CHAPTER III TRANSFORMATION STUDIES ON FUJISAKA 5 RICE USING SYNTHETIC BACILLUS THURINGIENSIS’CRYIA(B) AND POTATO PROTEINASE INHIBITOR II GENES FOR INSECT RESISTANCE 63 64 SUMMARY Fujisaka 5 was co—transformed with different constructs containing the synthetic B. thuringiensis cryIA(b) and pin2, and plasmids containing the selectable marker bar which confers resistance to PPT or glufosinate ammonium by microprojectile bombardment of embryogenic calli and immature embryos. The appropriate concentration of glufosinate ammonium for the selection of transformed calli was 5.0 to 5.5 mg/l. Higher concentrations were more effective in inhibiting the growth of untransformed cells but reduced the regeneration capability of the transformed calli. A total of 316 plants were regenerated from 3400 bombarded calli and immature embryos in 28 independent experiments. Sixty five of these plants were positive for synthetic cryIA(b) by PCR, 46 of which showed positive signal in slot blot analysis. Three putatively transgenic plants (Plants 117, 132 and 187) were identified by Southern blot analysis. Second generation plants from these transformants were positive for the presence of synthetic cryIA(b) by PCR. Two plants from 187 and one from 117 were positive in western blot analysis. Southern and northern analyses, and bioassays against lepidopterous pests of rice will be done on these plants to determine whether the gene(s) are stably integrated and consistently expressed. Several other plants were also positive in Southern blot hybridization but smaller fragments were also noted in the undigested DNA, which might indicate the presence of extrachromosomal DNA. 65 Several of the plants were positive for the presence of the bar coding sequence but no hybridization at high molecular weight undigested DNA was noted. All the plants died when sprayed with 1.0% Ignite (20% glufosinate ammonium) although some survived at lower concentration of the herbicide (0.7%). INTRODUCTION Rice (Oryza sativa L.), a major cereal crop in Asia, is affected by different kind of insect pests that cause serious damage and yield loss. For lepidopteran pests, leaf folders and stem borers alone are estimated to cause an annual loss equivalent to $681 million worlwide. Furthermore, there are no known resistant germplasm which can be readily introduced into cultivated rice for yellow stem borer, which is a major pest in tropical Asia (Fujimoto et al., 1993). Chemical control of stem borers are often ineffective once they are already inside the rice stalks. Broad spectrum pesticide applications to control lepidopteran pest of rice destroy natural enemies and can cause severe outbreaks of the rice brown planthopper, Afilaparvata lugens (Stél) (Heinrichs & Mochida, 1984; Kisimoto, 1984) that transmits grassy stunt, ragged stunt, and wilted stunt virus diseases. Beneficial insects are also very important in maintaining most borer populations below economic threshold levels. Generally, egg parasitism is very high in Asian borers (Way & Bowling, 1991). For integrated 66 pest management systems, selective control agents for lepidopteran key pests that do not upset natural enemy complexes must be developed (Rombach et al., 1989), like the use of Bacillus thuringiensis (Bt) both as conventional insecticide or in transgenic plant. Bt was reported to cause arrested feeding of the Asiatic rice borer, C. suppressalis aside from direct mortality (Rombach et al., 1989). The production of transgenic rice expressing the synthetic cryIA(b) using the protoplast system was reported (Fujimoto et al., 1993). Synthetic cryIA(b) was also introduced into maize and protected the plants from first and second generation corn borers. Other insect resistance genes that were introduced to plants are proteinase inhibitors which affect the growth and development of insects belonging to different Orders. It was also reported to potentiate the activity of Bt against lepidopteran pests (McIntosh et al. 1992). The potato proteinase inhibitor II (pin2) gene is well characterized and was expressed in transgenic plants, mostly dicots. It is an inhibitor of serine proteinases (Green & Ryan, 1972), both trypsin and chymotrypsin (Johnson et al., 1989). One of the selectable marker genes used in monocot transformation is bar which confers resistance to L— Phosphinotricin (PPT) (Thompson et a1. 1987). PPT, also known as glufosinate, is an analog of glutamate which acts as a competitive inhibitor of the enzyme glutamine synthase. This enzyme is involved in assimilation of ammmonia and 67 plays a key role in the regulation of nitrogen metabolism (De Block et al., 1987). Inhibition of the enzyme causes accumulation of ammonia that causes the death of plant cells (Tachibana et al., 1986a&b). Resistance to the herbicide is conferred by phosphinothricin—N—acetyltransferase (PAT) which inactivates PPT by acetylation, using acetyl coenzymeA as a co-factor. We have undertaken transformation studies on Fujisaka 5 rice using our plasmid constructs containing the synthetic cryIA(b) and pin2 driven by different promoters. The different studies were conducted with the following objectives: 1) to develop an efficient disinfection procedure for rice immature and mature seeds. 2) to determine the appropriate concentration of glufosinate ammonium for selection of transformed rice embryogenic calli. 3) to use the different plasmid constructs containing the synthetic cryIA(b) and pin2 for the transformation of rice immature embryo and seed derived calli. 4) to detect the presence of the introduced synthetic cryIA(b) in the rice genome by polymerase chain reaction (PCR) and Southern blot analyses. 5) to examine the expression of synthetic cryIA(b) in putatively transformed rice plants by western analysis, and 6) to evaluate the efficacy of the transgenic plants against 68 lepidopteran pests of rice. MATERIALS AND METHODS Surface Sterilization of Mature and Immature Rice Seeds Seeds of Fujisaka 5 were previously obtained from the International Rice Research Institute (IRRI) (Los Banos, Philippines) and greenhouse grown plants while immature embryos were obtained from greenhouse grown plants only. Fresh greenhouse grown mature seeds were dried at 37°C for 3 to 5 days before dehusking. The seeds were laid in a corrugated rubber mat and then pressed with a rubber stopper until the husks came off. The immature seeds and dehusked mature seeds were washed with the Liqui—Nox detergent (Alconox, Inc. New York, NY) (10 drops/200 ml) with continous stirring for 2 minutes, rinsed with tap water three times and then kept in running water for about a minute. Several disinfection procedures were used since their efficiency varies with batches of seeds coming from the greenhouse and in the field. The following procedures were tested: Procedure I. Rice immature and dehusked mature seeds previously washed with detergent and rinsed with running water were soaked in 70% ethanol for a minute. The seeds were then transferred to 60% Clorox (5.25% NaOCl) and stirred for 30 minutes using a magnetic stirrer. The disinfected seeds were rinsed with sterile distilled water three times under the hood, transferred to sterile Petri 69 dish and then air dried before transferring in callus inducing media. The Petri dishes were sealed with Parafilm in order to avoid desiccation of the media and outside contamination. Procedure II. Almost the same as Procedure I except that the immature and mature rice seeds were rinsed with 70% ethanol after they were rinsed three times with sterile distilled water. The surface sterilized seeds were then dried in the laminar flow hood. Procedure III. Dehusked mature seeds or immature seeds of rice previously washed with detergent and rinsed with running water were soaked in 60% Clorox (5.25% NaOCl) with stirring for 45 minutes. The seeds were then rinsed three times with sterile distilled water under the hood and soaked in 70% ethanol for a minute, air dried and transferred to callus inducing media in the same manner as Procedure I. Twenty disinfected mature and immature seeds were plated in each Petri dish with three dishes per treatment. The plates were then incubated under light (4 fluorescent bulbs of 34 watts each) at 25—28°C and observed for the presence of microbial contaminants every other day up to 10 days. After 10 days, the immature embryos were dissected from the uncontaminated immature seeds using flame sterilized forceps and scalpel and plated onto a fresh callus inducing media. The dissected immature embryos were observed every other day and the occurence of microbial contaminants was noted. For both disinfected seeds and 70 immature embryos, the production of callus from the uncontaminated samples were noted and compared. Determination of Appropriate Concentration of Glufosinate Ammonium for Selection of Transformed Calli Embryogenic calli were initiated from immature embryos of Fujisaka 5 plated on MS medium supplemented with 500 mg/l enzymatic casein hydrolysate (Sigma, St. Louis, M0), 3.0% sucrose and 2.25 uM 6-benzylaminopurine. Cultures were incubated at 24 t 2°C in the dark and subcultured onto fresh media every 15 to 21 days for one to two months and then used in the different experiments. Different concentrations of PestanalR (93% glufosinate ammonium) (Riedel-de Haen, Germany) were prepared by mixing appropriate amount to the callus inducing media. A stock solution of glufosinate ammonium was filter sterilized and added to the autoclaved medium as it cooled down to 50 to 60°C. The following concentrations (mg/l) were tested: 0, 4.0, 5.0, 5.5, 6.0, 6.5 and 7.0. The treatments were replicated at least three times with one Petri dish per replicate, ten calli (approximately 1 cm diameter/callus) per Petri dish. The calli were previously maintained for about a month in a callus inducing media before use in the experiment. To avoid contamination and dessication of the medium the Petri dishes were sealed with Parafilm before incubation at 28°C under light. The calli were transferred to fresh medium with the same concentration of glufosinate 71 ammonium every 15 to 21 days. The development of calli or cell clusters were observed and noted in every transfer. Well developed calli or cell clusters were separated and brownish or dying calli were discarded. The number of cell microclusters or calli was noted. Comparison between treatments was done. Transformation, Selection and Regeneration Seed and immature embryo derived calli, and isolated immature embryos (about 12 to 15 days after pollination) of FujisakaS were subjected to microprojectile bombardment using the Biolisticm PDS 1000 from Du Pont (Wilmington, DE). Bombardment was done according to the procedure provided by the manufacturer with slight modifications. The following were mixed in a sterile Eppendorf tube: 45 pl tungsten (GTE Sylvania) (1.2 pm, 60 mg/ml), 5 ul DNA (iug/ul) pDM302 or pTW—a, 5 ul DNA (other plasmid containing the synthetic cryIA(b) or pin2) (1ug/ul), 50 ul 2.5 M CaCl2 and 20 ul 0.1M spermidine. The mixture was vortexed for 10 minutes and then centrifuged at 10,000 rpm for 10 sec. The supernatant was discarded and the DNA coated tungsten particles were washed with 250 pl 100% ethanol, vortexed briefly, and centrifuged at 10,000 rpm for 10 secs. The pellet was resuspended in 60 ul of 100% ETOH and 5—10 ul was used per shot. The embryogenic calli or immature embryos were shot two to three times under 25 mm Hg vacuum at a distance of 12 cm from the plate containing the calli. Co— 72 transformation experiments were done using either pDM302 or pTWa and the diffferent plasmid constructs (See Chapter II for plasmid maps). pDM302 and pTWa contain the bar gene which confers resistance to phosphinothricin (PPT) or glufosinate ammonium as selectable marker. Combination of different plasmids containing the synthetic cryIA(b) but driven by different promoters were used. Plasmids were used at different ratios (1:1, 1:3, 1:1:1:1, 1:2:1, 1:1:1, depending on treatment combinations). In pTWa, bar is driven by 358 promoter, while it is under the control of rice Act? promoter in pDM302 (Figure 2.4). The bombarded immature embryos were kept in callus inducing media overnight and then transferred to the same media supplemented with 5.0 or 5.5 mg/l glufosinate ammonium for selection. Calli were kept on selection medium for 3 to 5 months, transferred to the same medium without selection until they produced yellowish and compact structures. Selected calli were then transferred tx>1% (Chu et al., 1975) medium supplemented with 2 mg/l kinetin and 2.0% sucrose for shoot formation and regeneration. Well-formed plantlets were then transferred to MS medium in Magenta boxes until they produced enough roots and then transferred to clay pots with ready mix soil (Bacto Pro Plant Mix, Michigan Peat Co.). As soon as enough leaves were available, genomic DNA was isolated and subjected to PCR and slot blot analyses. All the plants were grown to maturity and allowed to produce seeds. 73 Rice DNA Isolation Genomic DNA of rice was isolated from fresh leaves using the CTAB procedure modified from Rogers & Bendich (1985). For small scale isolation, about 100 to 200 mg of leaves was ground in mortar and pestle in liquid nitrogen. The powdered tissue was transferred in Eppendorf tube and resuspend in 1.0 ml CTAB buffer (0.1M Tris—HCl, pH 8.0, 1.4M NaCl, 0.02M EDTA, 2.0% cetyltrimethylammonium bromide) containing 1.0% B-mercaptoethanol. The samples were then incubated at 60°C for 30 minutes to 1 hour. The mixture was centrifuged at 12,000 rpm for 3 minutes and the lysate was transferred to a new tube. The lysate was allowed to cool down and equal volume of chloroformzisoamyl alcohol (24:1) was added. The mixture was then centrifuged at 10,000 rpm for 2 minutes to separate the phases. Chloroformzisoamyl alcohol extraction was done at least three times. The top layer or aqueous phase was recovered and DNA was precipitated by the addition of 0.7 to 1.0 volume of isopropanol and incubation at —20°C for at least an hour. The DNA was then pelleted at 10,000 rpm for 20 minutes and washed with 1 ml cold 70% ethanol. The pellet was dissolved in TE buffer (pH 8.0) with 20 ug/ml RNase A and incubated overnight at 37°C. The same procedure was used for large scale isolation except that the ground tissue was lyzed by incubating at 60 °C for at least 1 hour and the volume was scaled up. The amount of DNA was estimated either by running a gel and comparing the intensity of ethidium bromide stain 74 with the standard or by taking the absorbance reading at A260 and A280 of the DNA solution after extracting the RNase with phenol:chloroform: isoamyl alcohol and ethanol precipitation. Screening of Putatively Transgenic Plants Since our main interest is to produce an insect tolerant or resistant rice plant, we have screened all regenerated plants for the presence of the synthetic cryIA(b) and pin2 genes. The plants were not sprayed with herbicide prior to DNA analysis or before they produced seeds. In co—transformation experiment, it is possible that the plants are transformed only by synthetic cryIA(b), pin2 or bar, and spraying with herbicide will kill those plants that are expressing only the synthetic cryIA(b) or pin2 but not the bar gene. Seeds and leaf samples were collected from all the plants and new tillers were allowed to grow. The plants were then sprayed with 0.7% to 1.0% Ignite (20% glufosinate ammonium, Hoechst—Roussel Agri—Vet Co., Leland, MS) and 0.1% Tween 20. The plants were observed everyday for the presence of symptoms up to 2 weeks. Putatively transgenic plantlets were screened by polymerase chain reaction (PCR) and slot blot analyses. PCR was performed in a Perkin Elmer Gene Amp PCR System 9600 (Perkin—Elmer Corp. Norwalk, CT) using the thermostable Taq polymerase (Boehringer Mannheim, Biochemicals, Indianapolis, IN). The presence of the cryIA(b) was detected using a pair 75 of primers which have 100% homology to their target templates and amplify the internal 881 bp fragment of the synthetic cryIA(b). The primers have the following sequences: MB49 — 5' GCGAGCGCATCGAGACCGGCTACA 3' and MBSO — 5' GCGAGCGCATCGAGACCGGCTACA 3'. Both primers were 24 mer and used at 50 pmole/25 ul reaction volume. The PCR reaction was kept at 94°C for 5 minutes and then on 30 cycles of 94°C — 15 secs., 60°C — 15 secs, 72°C - 1 min. After 30 cycles it was held at 72°C for 5 minutes and then at 4°C until ready for loading. The PCR products were electrophoresed in 0.8% agarose gel, stained with ethidium bromide and then visualized under UV. The total amount of genomic DNA isolated from 100—200 mg of powdered leaf tissues was used in slot blot analysis. The purified DNA was resuspended in 400 pl TE (pH 8.0) and 40 ul of 3M NaOH was added and mixed. The mixture was then incubated for 0.5 to 1 h at 65°C to destroy the RNA and denature the DNA. The reaction was neutralized by the addition of 400 111 2M NH4OAc, pH 7.0. The total volume was loaded in Minifold II slot blot system (Schleicher and Schuell, Keene, NH) attached to a vacuum line to facilitate the transfer of DNA to the membrane. The membrane was air— dried and then baked at 80°C for 30 minutes in a vacuum oven . Southern Blot Analysis of Genomic DNA For Southern blot analysis, 25 to 30 ug DNA from 76 putatively transgenic and untransformed plants were digested with at least 100 to 200 units each of BamHI and EcoRI (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a total volume of 200 pl at 37°C overnight. The digested and undigested DNA were loaded in 0.8% agarose gel after reducing the volume to about 30 ul by drying in a Speed Vac and then electrophoresed at 25 volts overnight. The DNA was transferred to S&S Nytran membrane by 10X SSPE using the procedure modified from Southern (1975) as recommended by the manufacturer (Schleicher & Schuell, Keene, NH). After the transfer, the membrane was air dried and baked at 80°C for at least 30 minutes in a vacuum oven. The membranes were then hybridized with the 3zP—labeled 1.8 kb BamHI-BstEII fragment from pCIB4418 which contains the synthetic cryIA(b) coding sequence or with 1.5 kb HindIII—SpeI fragment of pDX101 containing the pin2 coding sequence and terminator. The fragments were oligolabeled using T7QuickPrimeTM kit (Pharmacia, Sweden). The labeled fragment was subjected to gel filtration on Sephadex G-50 column to remove the unincorporated label. Prehybridization and hybridization were done at 42°C inside a plastic bag containing a total volume of 50 ml solution composed of 6X SSPE, 5X Denhardt, 0.7% SDS and 50 ug/ml denatured Herring sperm DNA, 10 g dextran sulfate and 50% formamide. After hybridization, the membrane was washed twice in a solution containing 7X SSPE and 0.1% SDS at room temperature for 15 minutes, then twice in 1X SSPE and 0.5% SDS at 37°C, 15 min. 77 and finally in 0.1x SSPE and 1.0% SDS at 65 to 68°C for one hour. Autoradiography was performed using Kodak X—Omat AR-S film exposed at —80°C for 2 to 14 days with a Lightning Plus intensifying screen (DuPont, Wilmington, DE) and developed in an automatic film developer. Western Blot Analysis Leaf samples from second generation putatively transgenic plants 187 and 117 were analyzed by Western blotting using procedures modified from Burnette (1981) and Towbin et al. (1979). Leaf sample (approximately 200 mg) was ground in liquid nitrogen, resuspend in 400 pl 2X SDS sample buffer (Laemli, 1970) containing 14mM B-mercaptoethanol and 100mM phenylmethylsulfonyl fluoride and then incubated at 95°C for 10 min. In the first blot, 20 ul of the extract was loaded onto a SDS—polyacrylamide gel (10% running, 6% stacking), electrophoresed at 200 volts and electroblotted on a 0.45 pm nitocellulose membrane (Schleicher & Schuell, Keene, NH) The presence of CryIA(b) protein was visualized by incubating the membrane with a 500 fold diluted goat anti—CryIA(b) primary antibody provided by Dr. M. Koziel (CIBA—GEIGY), followed by alkaline phosphatase conjugated anti—goat IgG (10,000 fold dilution, Sigma, St. Louis, MO). The membrane was then incubated with the substrates nitro blue tetrazolium and 5-bromo—4—chloro—3—indolyl phosphate. Purified CryIA(b) protein and unpurified cells and crystals of B. thuringiensis HD1—9 grown at room temperature, were 78 used as positive controls. B. thuringiensis HD1-9 produces CryIA(b) protein only, when grown at room temperature due to the small deletion in the -COOH terminal portion of CryIA(b) that makes it temperature labile relative to CryIA(a) and CryIA(c). Protein molecular weights were determined by comparison with Rainbown'coloured protein molecular weight markers (Amersham International Inc. Buckinghamshire, England). Bioassay of Putatively Transgenic Rice Plants Against European Corn Borer The capacity of Ostrinia nubilalis to use putatively transgenic and untransformed rice as alternative hosts was investigated. Second generation putatively transgenic plants from plant 132, 117 and 187 were tested. PCR positive and PCR negative plants were used. PCR negative plants are segregating progenies that do not contain the synthetic cryIA(b). Rice stems from one month old tiller was divided into 4.0 cm length and 2 pieces were placed in a 50 mm x 9 mm Petri dish with tight lid, previously lined with moistened filter paper. Eight 9 day old larvae previously reared on artificial diet were introduced into each Petri dish and incubated at room temperature for 4 days and then the number of surviving larvae was counted. RESULTS Surface Sterilization of Mature and Immature Rice Seeds 79 No significant difference was noted on the efficiency of different procedures for the surface sterilization of rice seeds as determined by the number of contaminated samples after various length of incubation time (data not shown). The disinfection procedures were equally effective in removing bacterial but not very effective for fungal contaminants that are commonly infecting the grains of field grown rice. Selection of Transformed Calli The effective concentration that inhibit the growth of calli was 7.0 g/l glufosinate ammonium (Table 3.1). It is possible that higher concentrations will produce better inhibition of untransformed calli. Calli maintained in medium with 5.0, 5.5 and 6.0 mg/l glufosinate ammonium were able to produce some resistant cell clusters that were capable of multiplying although not as fast as the normal calli grown in medium without any selection. However, in separate transformation experiments using 6.0 mg/l glufosinate ammonium, the calli that survived the selection had difficulty in regeneration. Instead of forming shoot, they just continue to divide and produce embryogenic calli that are loose and whitish (Figure 3.1C). With these results, 5.0 and 5.5 mg/l glufosinate ammonium were used for selection. Putatively transformed calli kept on these media for 5 months have started to regenerate about 4—6 months after transferring to regeneration medium without any 80 Table 3.1. Number of surviving calli and cell clusters from immature embryos of Fujisaka 5 after various lenght of incubation time in callus inducing medium containing different concentrations of glufosinate ammonium.a Concentration Number of Days (mg/L) 0 15 36 51 72 No. of Callib No. of Cell Microclusters° 0 10 10 10.0 Entire calli growing 4.5 10 10 10.0 12.7 12.7 5.0 10 10 7.3 4.3 4.3 5.5 10 10 8.0 4.0 4.0 6.0 10 10 5.0 1.3 1.3 6 5 1O 10 3 7 1 O 1.0 7 0 10 10 2 7 0 6 0.6 a Averaged data from 3 replicates, 10 calli per replicate. Calli were transferred to fresh media containing glufosinate ammonium every fifteen to twenty one days. ° Calli were discarded when it turned dark brown. ° The number of microclusters observed at 72 days is the same as at 51 days, but continue to grow and increased in Size. 81 Figure 3.1. Production of putatively transgenic rice plants by particle bombardment of embryogenic calli. A) Embryogenic calli produced from mature seeds (indicated by arrow). B) Bombarded calli selected for resistance to glufosinate ammonium (5.0 to 5.5 mg/l). Dark brown calli are susceptible calli killed by glufosinate ammonium and later discarded. C) Actively dividing glufosinate ammonium resistant embryogenic calli. D) Compact glufosinate ammonium resistant embryogenic calli ready for transfer to regeneration medium. E) Shoot formation from selected embryogenic calli. F) Fully developed plantlet growing in MS medium in Magenta box. G) Putatively transgenic rice plants grown in clay pots in the greenhouse. H) Fertile putatively transgenic rice plants. 82 83 glufosinate ammonium. All the regenerated plants were fertile and almost all were phenotypically similar to seed grown plants. Molecular Analyses of Putatively Transgenic Plants The summary of different transformation experiments is shown in Table 3.2. A total of 316 plants were regenerated from 3400 bombarded calli and immature embryos in 28 independent experiments. Sixty five of these plants are PCR positive, 46 of which showed positive signal in slot blot analysis. Genomic DNA from several plants hybridized well with the‘”P labeled cryIA(b) in slot blot (Figure 3.2). The same results were obtained when other slot blots were probed with equal quantity of 32P labeled cryIA(b) and pin2 (Figure 3.3). Putatively transgenic plants transformed with plasmids containing both pin2 and synthetic cryIA(b) showed more intense bands. For the positive controls, pRES7393 produced a more intense band than pCIB4418 at 50 pg. pRES7393 contains both the cryIA(b) and pin2 while pCIB4418 has cryIA(b) only. Southern analyses of the putatively transgenic plants are shown in Figures 3.4 to 3.7. In Figure 3.4B, digestion of total genomic DNA from plant 117 with BamHI and EcoRI showed the expected 2.1 kb fragment. Plant 117 was regenerated from embryogenic calli bombarded with plasmids pCIB4418 and pRES108. Hybridization at high molecular weight 84 Table 3.2. Summary of the different transformation experiments, number of plants regenerated and putatively transgenic rice plants identified by PCR and slot blot analyses. Expt. Plasmid No. of Calli No. of No. of Putatively No.a Bombarded Plants Transformed Plants Regenerated Slot Blot PCR 1 4418 62 4 1 1 2 4418 87 6 o 2 3 7393 113 2 0 0 4 7293 77 3 0 1 5 4418 30 8 0 0 6 7393 246 30 5 8 7 4418 200 35 13 9 8 4418 108 9 4 7 9 4418 54 9 0 0 10b 7293 39 4 1 0 11 107 160 5 0 0 12 107 100 4 0 0. 7393 13 107 100 16 1 3 4418 14 4421 100 52 7 7 107 4418 15 108 110 13 1 1 16 4418 110 24 1 1 7393 85 Table 3.2. continued Expt. Plasmid No. of Calli No. of No. of Putatively No.a Bombarded Plants Transformed Plants Regenerated Slot Blot PCR 17 108 100 10 0 3 4418 ' 18 108 100 20 4 3 19 4418 100 2 1 0 7393 20 4418 100 8 2 1 108 21 4418 80 9 5 8 108 22b 108 80 5 o 2 23 108 100 2 0 0 24 108 100 10 ND 0 25° 4418 241 6 ND 0 26° 4421 214 6 ND 2 27° 107 236 8 ND 3 28° 108 253 6 ND 2 TOTAL 3400 316 46 65 a All treatments except with superscript were done by co—transformation with pDM302. ° Co—transformation with pTW—a. ° Co—transformation with pGL2. ND = Not Done. 86 Figure 3.2. Slot blot analysis of genomic DNA isolated from untransformed and putatively transgenic rice plants. The blots were probed with 32P labeled 1.8 kb BamHI—BstEII fragment from pCIB4418 containing the synthethic cryIA(b). Positive plants selected for further screening are marked by numbers. pCIB4418 (50 and 100 pg) is positive control. DNAs isolated from untransformed rice plants are designated as NT. 87 Figure 3.3. Slot blot hybridization of genomic DNA isolated from untransformed and putatively transgenic rice plants. The blots were probed with 32p labeled 1.8 kb BamHI—BstEII fragment from pCIB4418 containing the synthetic cryIA(b) coding sequence and 1.5 kb HindIII—SpeI fragment from pDX101 containing the pin2 coding sequence and nos terminator. The probes (50 ng each) were mixed in equal quantities. pCIB4418 (50 and 100 pg) and pRES7393 (50 pg) are positive controls. pRES7393 contains both the pin2 and cryIA(b) coding sequences while pCIB4418 contains cryIA(b) only. DNAs isolated from untransformed rice plants are negative controls designated as NT. 88 Figure 3.4. Southern blot analysis of rice genomic DNA. (A) Ethidium bromide stained rice DNA (25 Eg/lane). (B) Autoradiogram of gel blot hybridized with P labeled 1.8 kb BamHI—BstEII synthetic cryIA(b) coding sequence from pCIB4418. The expected 2.1 kb fragment is observed in the BamHI-EcoRI digested DNA from plant 117. Larger (3.2 kb) and smaller (1.0 kb) bands are seen in plant 132. Positive bands are indicated by arrows. (C) Autoradiogram of gel blot hybridized with 329 labeled 0.86 kb EcoRV fragment containing the bar coding sequence and nos terminator from pDM302. The blot in (B) was washed to completely remove the cryIA(b) probe prior to hybridization. Plant 132 is positive for bar producing the 0.8 kb and 0.6 kb fragments (indicated by arrows). (D) The same autoradiogram as C) but exposed for a longer time to show the hybridization at high molecular weight. The migration position of markers are shown to the left with sizes in kb. (U - undigested, D — digested with BamHI and EcoRI, NT — untransformed plants, pCIB4418 — positive control. Numbers on the top of the lanes are plant designations). 89 90 DNA was observed as smear (Figure 3.43). For plant 132, regenerated from calli treated with plasmids pCIB4418, pCIB4421 and pRES108 all containing the synthetic cryIA(b) driven by 358, PEPC and rice Act? promoters, respectively, the BamHI-EcoRI digested genomic DNA produced two distinct bands (3.6 kb and 1.1 kb) after hybridization with the'aP labeled cryIA(b) probe (Figure 3.4B). Hybridization at high molecular weight in the undigested DNA was also observed but its position does not correspond to the bulk of the ethidium bromide stained DNA (Figure 3.4A). The two strongly hybridizing bands comigrated with the 8.4 kb and 4.8 kb fragments from BstEII digested Lambda DNA. When the blot was stripped with cryIA(b) probe and hybridized with the 32p labeled 0.8 kb EcoRV fragment from pDM302 containing the entire bar coding sequence and nos terminator, positive signals were observed only in plant 132 (Figure 3.4C). Two distinct bands were observed in the high molecular weight undigested DNA that are in exactly the same position as the positive bands that hybridized with the'aP labeled synthetic cryIA(b) probe. No positive control for bar was provided in this blot since it was originally intended for cryIA(b) probing. Two distinct bands were also observed in the BamHI-EcoRI digested DNA which are approximately 0.8 kb and 0.6 kb, respectively. However, although bar was detected, the plant showed no resistance when sprayed with 1.0% Ignite (200 g/l glufosinate ammonium, 91 Hoechst—Roussel Agri—Vet Co. Leland, MS.) and 0.1% Tween 20. The plant showed yellowing symptoms as early as 2 days after spraying and was necrotic after a week. The sprayed plant died after about 2 weeks. Southern analysis of genomic DNA isolated from plant 65 and 79 regenerated from embryogenic calli treated with plasmids pRES108 and pCIB4418, respectively is shown in Figure 3.5. For plant 65, a BamHI—EcoRI fragment which comigrated with the 2.1 kb BamHI-EcoRI fragment from pCIB4418 hybridized to the cryIA(b) probe indicating the presence of a full length cryIA(b) coding sequence. Hybridization of the cryIA(b) probe to the high molecular weight undigested DNA was also observed indicative of gene integration into the plant chromosome (Figure 3.5C). This pattern match the ethidium bromide stained DNA (Figure 3.5A). The same result was obtained from this plant using lower amount of DNA (15 #9) for blotting. Distinct bands in undigested DNA from plant 79 and 65 that comigrated with BstEII digested lambda DNA at about 5.6 kb and 2.5 kb, respectively were also noted. No hybridization was observed in the undigested and restricted genomic DNA from untransformed rice plants. Hybridization of the same blot with 32P labeled 0.8 kb fragment from pDM302 containing the bar coding sequence and nos terminator gave positive signals only for plant 79 (Figure 3.58). Similar to plant 132, two distinct bands of about 0.8 kb and 0.6 kb were seen in the BamHI—EcoRI 92 Figure 3.5. Southern blot analysis of rice genomic DNA. A) Ethidium bromide stained rice DNA (25 ug/lane). B) Autoradiogram of gel blot hybridized with 32p labeled 0.86 kb EcoRV fragment from pDM302 containing the bar coding sequence and nos terminator. Plant 79 is positive for bar producing about 0.8 kb and 0.6 kb fragments. Positive bands (indicated by arrows) are also present in the undigested DNA but not at the bulk of high molecular weight DNA as seen in the ethidium bromide stain. C) Autoradiogram of gel blot hybridized with 32P labeled 1.8 Kb BamHI—BstEII fragment from pCIB4418 containing the synthethic cryIA(b). The blot in B) was washed to completely remove the cryIA(b) probe prior to hybridization. Hybridization at the high molecular weight undigested DNA is seen in most samples. Positive bands for plants 65 and 79 are indicated by arrows. The migration position of markers are shown to the left with with sizes in kb. (U - undigested, D — digested with Bam HI and EcoRI, NT — untransformed plants, pCIB4418 — positive control. Numbers on the top of the lanes are plant designations). ing 79 s a bulk ized hid [81's €913 Tmrftomazvm :mmmmp—p l\ O 94 digested DNA sample. A strongly hybridizing band which comigrated with the 8.4 kb fragment of BstEII digested Lambda DNA was observed in the undigested genomic DNA from plant 79 (Figure 3.5B). This band do not match with the bulk of the ethidium bromide—stained DNA (Figure 3.5A) and the gene was probably not stably integrated into the plant genome. Like plant 132, this plant died two weeks after spraying with 1.0% Ignite although some tillers survived when first sprayed with lower concentration (0.7%) of the herbicide. Southern analysis of another batch of putatively transgenic plants is shown in Figure 3.6. All of the previously slot blot positive plants hybridized with the‘aP labeled cryIA(b) in the undigested genomic DNA. However, the expected band was noted only in plant 187 as a faint band at 2.1 kb. It can be noted that the band in the 40 pg pCIB4418 positive control was also faint and that no distinct band was observed on 20 pg pCIB4418. Apparently, the probe has low sensitivity and might require higher amount of labeled fragments and radioactivity to achieve a more intense signal. Several other plants also showed hybridization at high molecular weight undigested DNA, indicative of integration of the synthetic cryIA(b) in the chromosome (Figure 3.7). Plant 27 showed a distinct band in the BamHI-EcoRI digested genomic DNA which is slightly larger than the expected 2.1 kb fragment (Figure 3.7B). 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