Study on the Creation of Disease-resistant Transgenic Plants Based on the Pathogenicity of phytopathogens

1)由紫丁香假单胞菌产生的毒素。引起猕猴桃溃烂病的猕猴桃病菌被鉴定为一种相锁毒素，并从该细菌中克隆了耐受相锁毒素的毒素不敏感酶(ROCTase)基因(Arg K)。在植物中，OCTase位于叶绿体中。因此，将argK基因与烟草Rubisco小亚基(SS)的转运肽(TP)基因融合，将该基因转入叶绿体。通过农杆菌介导法将融合基因导入猕猴桃植株。通过卡那霉素抗性和相溶毒素抗性筛选转基因植株，当所选转基因植株被喷雾接种紫丁香叶枯萎病菌时，虽然对照植株症状较多，但转基因植株症状较少，细菌增殖受到抑制。因此，这些转基因植株不仅对藻毒素具有耐受性，而且对ca…也具有抗性。丁香假单胞菌引起的更多坏死病。猕猴桃黑斑病是由培养基中产生有毒物质的同种核盘菌引起的。培养滤液对杂草等植物的种子萌发和根发育均有抑制作用，表明该毒素是非寄主特异性的。因此，通过溶剂萃取、柱层析、高效液相色谱分离纯化了高果树培养滤液中的植物毒性物质。3)以携带四环素(TC)抗性基因的转座子(TN)为诱变子，以pUCD623为载体，通过转座子(TN)诱变，获得了水稻苗腐病和谷腐病病原菌Burkholderia glumae的突变株。对产生的抗TC菌落进行了毒素产生的损失和对水稻幼苗缺乏致病性的筛选。对基因组DNA上单拷贝TN插入突变体的分析表明，TN插入附近的基因组区域具有与REPA基因和PAR A、B基因高度同源的基因，这些基因与染色体或质粒的复制或分布有关。此外，对缺乏致病性的天然突变体的分析表明，PAR A和B缺失，而rep A缺失。因此，推测在接连的培养过程中，Glumae基因组DNA的一部分丢失或转移，DNA的缺失与B.glumae的致病性丧失有关。较少

1) A toxin that produced by Pseudomonas syringae pv. Actinidiae, which causes canker disease of kiwifruit plants, was identified as a phaseolotoxin, and the gene (arg K) for toxin-insensitive OCTase (ROCTase) tolerant to phaseolotoxin was cloned from the bacterium itself. In plants, OCTase is located in chloroplasts. Thus, the argK gene was fused with a transit peptide (TP) gene of Rubisco small subunit (SS) from tobacco plant to transfer the gene into chloroplasts. The fused gene was introduced to kiwifruit plants by Agrobacterium-mediated transformation. Transgenic plants were selected by kanamycin-resistance and by phaseolotoxin tolerance, When the selected transgenic plants were spray-inoculated by P. syringae pv, actinidiae, the transgenics exhibited few symptoms although control plants developed a lot of symptoms, and the bacterial multiplication was suppressed in the transgenic plants. Thus, these transgenic plants were not only tolerant to phaseolotoxin but also resistant to ca … More nker disease by P. syringae pv. Actinidiae.2) Dollar spot disease of bentgrass is caused by Sclerotinia homoeocarpa that produces some toxic substances in the culture medium. The culture filtrate inhibited the seed germination and root development of bentgrass and other plants, suggesting that this phytotoxin is host non-specific. Thus, the phytotoxic substances from the culture filtrate of S. homoeocarpa were purified by solvent extraction, column chromatography, and liquid chromatography. The chemical structures of the toxins were determined as diterpenes by Mass and NMR spectrometry.3) Mutants of Burkholderia glumae, the causal agent of rice seedling rot and grain rot diseases, were isolated by transposon (Tn) mutagenesis with Tn 4431 harboring tetracycline (Tc) resistance gene, which was carried on the suicide vector pUCD623. The resulting Tc-resistant colonies were screened for the loss of toxin production, and also for the lack of pathogenicity to rice seedlings. >From analysis of the mutants with a single copy of Tn insertion on the genomic DNA, it was shown that the genomic region neighboring the insertion of Tn has the genes highly homologous to repA gene and par A and B genes, which are related to replication or distribution of chromosome or plasmids. Also, analysis of the natural mutants with lack of pathogencity showed loss of par A and B, but with rep A. Thus, it was suggested that a part of the genomic DNA of B. glumae was lost or transferred during the successive incubation in a medium, and the DNA deletion has to do with the loss of pathogenicity in B. glumae. Less