| Project/Area Number |
05556056
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | Meiji University |
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Principal Investigator |
YONEYAMA Katsuyoshi Meiji Univ., Dep.of Agriculture, Professor, 理工学部, 助教授 (50110060)
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| Co-Investigator(Kenkyū-buntansha) |
ANZAI Hiroyuki Meiji Seika Ltd.Pharmaceut.Res.Cr, Researcher, 農畜薬研究所, 研究員
YAMAGUCHI Isamu Riken, Bioscience, Head Researcher, 主任研究員 (20087589)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥16,100,000 (Direct Cost: ¥16,100,000)
Fiscal Year 1994: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1993: ¥10,300,000 (Direct Cost: ¥10,300,000)
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| Keywords | transgenic plant / disease resistance / phaseolotoxin / P.syringae pv.phaseolicola / P.syringae pv.actinidiae / P.glumae / toxoflavin / OCTase / イネもに枯細菌病菌 / 毒素耐性 / 病原性 / OTCase / Pseudomonas / 毒素耐性遺伝子 |
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| Research Abstract |
Attempt was made to create the transgenic plants resistant to the bacterial diseases caused by Pseudomonas syringae pv.phaseolicola, P.syringae pv.actinidiae and P.glumae.
In P.syringae pv.phaseolicola, the toxin phaseolotoxin produced by this bacterium was isolated and purified. Using the toxin, we tried to clone the phaseorotoxin-resistant gene from the producer itself, and succeeded to obtain a phaseorotoxin-insensitive ornithine carbamoyltransferase (OCTase) gene, which is involved in the self-resistance of P.syringae pv.phaseolicola to phaseorotoxin.
A 5' part DNA of the OCTase gene was amplified by PCR method, and fused with the transitpeptide gene of tobacco Rubisco small subunit that produced by PCR amplification. The fused DNA again was enzymatically linked with a 3' part of OCTase gene to complete the OCTase gene with a transit peptide gene. The chimeric OCTase gene was replaced with beta-glucuronidase gene of the plant vector pBI 121 to construct the plasmid pMY6. After transf
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erred into Agrobacterium tumefaciens LBA4404, the fused gene was introduced into tobacco plants by leaf disk method. The transgenic tobacco plants obtained showed strong resistance to phaseorotoxin, and also remarkably suppressed the proliferation of P.syringae pv.phaseolicola inoculated by injection method.
In the case of P.syringae pv.actinidiae, the toxin was isolated and partially purified from the culture filtrate. This toxin was found to be very similar to phaseorotoxin in the facts that it produces yellow halos on the leaves of bean and kiwi fruit plants, and has a growth-inhibitory activity to Esherichia coli which is complemented by supplement of arginine or citruline in the minimal medium. At present, therefore, kiwi fruit plants were in the course of gene introduction using pMY6.13EA05 : Another experiment was made to demonstrate the correlation between the pathogenicity and toxin production in P.glumae. A wild type of the bacterium was introduced by the transposon Tn4431 to inactivate the toxin production. The toxin-nonproducing mutant obtained was recovered in the pathogenicity with the cosmid genomic library, which constructed from the genomic DNA fragments of the wild strain. This recovered pathogenic bacterium produced the toxin toxoflavin again. It was clear that toxin production plays an important role in the pathogenicity of P.glumae. Less
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