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

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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