| Project/Area Number |
63860008
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
植物保護
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| Research Institution | The Akita Prefectural College of Agriculture |
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Principal Investigator |
HIROETSU Wabiko Biotech. Inst. Dept. of Genet. Associate Prof., 附属生物工学研究所, 助教授 (10191842)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Bacillus thuringiensis / protoxin gene / crystal protein / Agrobacterium / transgenic tobacco / biocontrol / deletion mutation / Badllus thuringiensis / プロトキシン蛋白 / 形質転換植物 / プロトキシン / 欠失変異 / Tiプラスミドベクター / 植物形質転換 |
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| Research Abstract |
A bacterium Bacillus thuringiensis produces insecticidal protoxin protein. The purpose of this research is to generate transgenic plants which are resistant to insect and to understand the structure-function relationship of the protein.
1. The toxin gene was cloned in pGA580 which is a Ti-plasmid-derived binary vector. Agrobacterium carrying the recombinant clone was infected onto Tabacco plant and kanamycin resistant plants were selected. The regenerated transgenic plants possessed toxin DNA and expressed toxin MRNA, which were determined by Southern, and Northern blot analysis. Unfortunately, so far we are unable to detect toxicity of the plants against insect larvae.
2. In order to understand the importance of the carboxy-terminal end of the toxin molecule, we generated sequential deletions by introducing stop codon from 3' end of the toxin gene. Insect bioassay indicated that the gene possessing 607 codon was toxic, whereas 605 codon-gene was nontoxic. The toxin gene in which 3'-half of the protoxin is deleted, was placed inder strong promoter, preca of E. coli. When the promoter was induced, the protein produced abundantly forming inclusion body. This inclusion body was alkali-insoluble and non-toxic. Since low level expression of the same gene conferred toxcity, the result suggests that 3'-half region is involned in solubilization in alkali.
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