Study on the molecular breeding of transgenic plants resistant to bacterial diseases

1)猕猴桃溃疡病（丁香假单胞菌）转基因抗性植物的建立。猕猴桃真菌产生相毒素，是猕猴桃溃疡病的一种致病菌。相毒素抑制参与精氨酸生物合成的鸟氨酸氨基甲酰转移酶（OCTase）。phaseolicola通过产生毒素不敏感的OCTase来抵抗phaseolotox。我们克隆了对相毒素不敏感的OCTase基因（argK），由于OCTase位于植物的叶绿体中，该基因被融合到烟草RuBisCO小亚基的叶绿体转运肽上。将构建的质粒pMY6通过农业侵染法导入猕猴桃植株。筛选卡那霉素后，获得了50个抗性植株。对所有无性系进行了抗性检测，获得了7个具有相毒素抗性的转基因无性系。在OCTase酶分析中，转基因无性系表达成功，且在猕猴桃植株的叶绿体中有更多的uct易位。2)葡萄伯克氏菌毒素合成相关基因表达机制用转座子（Tn）诱变野生型的葡萄球菌，其中一个突变体B.glumae <overhead double bar>毒素合成和致病性不足。用野生菌株的基因组文库转化后，b.s glumae <overhead double bar>的毒素生物合成和致病性与cosmid pNP147互补。结果表明，弓形黄素是造成葡萄球菌致病性的重要因子。pNP147的亚克隆得到了一个2.7kb的互补片段。该片段的ORF序列与E.coil的IS2核苷酸序列高度相似。另一方面，突变体<overhead double bar>在Tn插入位点下游3'处没有IS2样序列，而野生菌株在相应位置有IS2序列。这些结果表明，IS2可能在调节toxofl Less中起重要作用

1) Creation of transgenic plants resistant to kiwifruit canker diseasePseudomonas syringae pv. actinidiae produces the toxin phaseolotoxin, a pathoge ic factor of kiwifruit canker. Phaseolotoxin inhibits ornithine carbamoyltransferase (OCTase) involved in arginine biosynthesis, but Ps. pv. phaseolicola is resistant to phaseolotox in by producing the toxin-insensitive OCTase. We clonedthe gene encoding the phaseolotoxin-insensitive OCTase gene (argK) , and the gene was fused to the chloroplast transit peptide of RuBisCO small subunit of tobacco because OCTase is located in the chioroplast of plants. The constructed plasmid pMY6 was introduced to kiwifruit plants by agro-infection method. After sele tion of kanamycin, fifty clones were obtained as the resistant plants. In all the clones, toxin resistance was examined, and 7 transgenic clones were obtained as phaseolotoxin-resistant plants. In OCTase enzyme assay, the transgenic clones showed that the transgene was expressed, and the prod … More uct was translocated into the chloroplasts of kiwifruit plants.2) Mechanism of gene expression related to toxin biosynthesis in Burkholderia gumaeThe wild type of B.glumae was mutagenized by transposon(Tn), and one of the mutants, B.glumae <overhead double bar>, was deficient in toxin biosynthesis and pathogenicity. Whentransformed with the genomic library of the wild strain, the toxin biosynthesis and pathogenicity of B.glumae <overhead double bar> was complemented with the cosmid pNP147. Thisshowed that toxoflavin was an essential factor for pathogenicity of B.glumae. The subcloning of pNP147 gave a complemented fragment of 2.7kb DNA.An ORF of the fragment showed a high similarity to IS2 of E.coil in the nucleotide sequence. On the other hand, the mutant <overhead double bar> had no the IS2-like sequence near at 3'-downstream of Tn insertion site, although the wild strains had the IS2 sequence at the corresponding site. These results suggest that IS2 may be important for the regulation of toxofl Less