Studies On Kasugamycin Sensitivity Gene(ksga) Of Bradyrhizobium Japonicum

In this study ,there are two aspects:On one hand, the TnSgusAS insertion mutagenesis of chromosome of Bradyrhizobium japonicum 22-10 were obtained by TnSgusAS arbitary mutagenesis, and six kasugamycin resistant mutants have been screened. And oligonucleotied primers were designed according to the sequence of Tn5, then used PCR to identify with TnSgusAS insertion of chromosome. The result shows that there is no TnS insertion in chromosome, and implicates that kasugamycin resistant mutant is autegenic mutation.On the other hand, the sequence of Methyltransferase gene (ksgA) which dimethylates adjacent adenines near the 3 ' end of 16S rRNA about Agrobacterium tumefaciens; Sinorhizobium meliloti and Mesorhizobium loti were downloaded from NCBI Gene Bank Blast, and primers were designed in their conserved regions by alignment analysis to amplify fragment of ksgA gene from total DNA of B.japonicum22-10. DNA sequence shows that the segment consist of 520bp. NCBI Gene Blast analysis shows that this 520bp segment share high homology with known ksgA gene of Sinorhizobium meliloti. Then used Tail-PCR method to amplify sequence flanking the 520bp fragment, and obtained a 1598bp fragment which contain a complete open reading frame (ORF) and consist of 858bp named B.jksgA gene. Blastx analysis shows that B.jksgA gene share 69% % 69% and 65% homology at the amino acid level compared with reported ksgA gene of S.meliloti M.lito and A.tumefaciens.According to 1598bp DNA sequence which contain B.jksgA gene, primers were designed to amplify sequence flanking B.jksgA ORF, which left flanking sequence is 395bp, and right is 345bp. At same time used ORF ofKm resistance gene to replace the ORF of ksgA gene, then B.jksgA gene deletion constructs (LksgA) have been obtained. The LksgA construct was ligated into multipurpose broad host rang cloning vector pIJ3200, and recombinant plasmid pIJ32LksgA was constructed. Recombinant plasmid was transformed Bradyrhizobium japonicum 22-10 with electroporation method, then double homogenous exchange occurred in with incompatible plasmid pPHUI. And obtained four B.jksgA gene replacement mutants through selecting Rif resistance, Km resistance and Kasugamycin resistance. PCR result shows that the ksgA gene of four mutant is just replaced by LksgA construct. It has proved that B.jksgA gene is the kasugamycin sensitivity gene(ksgA) of B.japonicum22-10.