Study on the creation of transgenic plants resistant to bacteial of fungal diseases

为培育抗丁香假单胞菌病原菌的转基因植物进行了尝试。phaseolicola, p.s ringae猕猴桃属和丁香属。分离纯化了该细菌产生的相毒素Phaseolicola。利用该毒素，我们试图从其自身克隆出相毒素抗性基因，并成功获得了一个与相毒素不敏感的鸟氨酸氨基甲酰转移酶（OCTase）基因，该基因参与了丁香p.s ynringae pv的自抗性。Phaseolicola到phaseorotoxin。用PCR方法扩增出OCTase基因的5′部分DNA，并与PCR扩增得到的烟草Rubisco小亚基过肽基因融合。融合的DNA再次与OCTase基因的3'部分酶联，使OCTase基因与转运肽基因完整。将嵌合OCTase基因与植物载体pBI 121的β -葡糖醛酸酶基因置换，构建质粒pMY6。将融合基因转入农杆菌LBA4404后，通过叶片法导入烟草植株。获得的转基因烟草植株对相毒素具有较强的抗性，并能显著抑制丁香假丝菌的增殖。注射法接种Phaseolicola。对于丁香假单胞菌pv。从培养滤液中分离并部分纯化了该毒素。该毒素与相毒素非常相似，在豆类和猕猴桃植物的叶子上产生黄色晕，并对大肠杆菌具有生长抑制活性，在最小培养基中补充精氨酸或瓜氨酸。因此，目前猕猴桃植株正处于利用pMY6.13EA05进行基因导入的过程中：我们又做了一个实验来验证p.g ulumae致病性与产毒之间的相关性。通过转座子Tn4431引入一种野生型细菌来灭活毒素的产生。利用野生菌株基因组DNA片段构建的cosmid基因组文库，恢复了所获得的不产毒突变体的致病性。这种回收的致病菌又产生了毒素弓形黄素。结果表明，毒素的产生在葡萄球菌致病性中起重要作用。少

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 … More 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