1991 Fiscal Year Final Research Report Summary
formation of transqenic plant resistant to bacterial infection containing insect antibacterial protein genes
| Project/Area Number |
01890004
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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 |
広領域
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| Research Institution | University of Tokyo |
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Principal Investigator |
NATORI Shunji Univ. of Tokyo., Pharmaceu. Sci. Prof., 薬学部, 教授 (50012662)
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| Co-Investigator(Kenkyū-buntansha) |
KOMARI Toshihiko Nippon Tabaco, Institute of Plant, 遺伝育種研究所, 研究員
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| Project Period (FY) |
1989 – 1991
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| Keywords | transgenic plant / sarcotoxin I / sarcotoxin II / resistant plant / Sarcophage peregrina / antifungal protein / tobacco / cDNA |
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| Research Abstract |
We have been isolating antibacterial proteins from the hemolymph of injured larvae of Sarcophaga peregrina (flesh fly). So far we have purified three protein named sarcotoxin I, II and sapecin. Of these, sarcotoxin I is effective to Gram-negative bacteria and sapecin is effective to Gram-positive bacteria. cDNAs for these proteins have been isolated and complete nucleotide sequences were determined. We have also purified an anti-fungal protein from the same hemolymph. This protein was toxic to Candida albicans and various yeast strains.
The purpose of this project was to integrate the genes (cDNAs) for these antimicrobial proteins into the clomosomes of tobacco and to test if the plant resistant to bacterial infection is established.
We first tried this experiment using cDNA for sarcotoxin I. This is a small protein consisting of 39 amino acid residues. We integrated this cDNA to a plasmid named pGA643 having multi-cloning sites, and then introduced this plasmid to tobacco leaves using A
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grobacterium LAB 4404. As this plasmid contained 35S RNA promoter, it is possible to express the sarcotoxin I gene in the plant. We isolated cells from tobacco leaves and established callus. From callus we developed transgenic plants. We extracted RNA from the leaves of transgenic plants, and tested if mRNA for sarocotoxin I was present or not by Northern blot hybridization. Clearly, we detected a positive signal, indicating that integrated sarcotoxin I gene was expressed. However, we could not detect neither antibacterial activity nor sarcotoxin I itself in the crude extract of the leaves. It is likely that plant cells cannot translate sarcotoxin I mRNA. We tried similar experiment using sarcotoxin II cDNA, but the results were the same as those of sarcotoxin I cDNA. Namely, we could not detect protein although we could detect mRNA.
It seems to be important to investigate why plant cells cannot translate mRNA of insect protein. We are going to test this using in vitro translation system. Less
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