Cloning And Analysis Of Genes Related To Salt Tolerance From Sinorhizobium Meliloti 042BM

Sinorhizobium meliloti 042BM was isolated from root nodule of alfalfa(Medicago saliva) from Xinjiang Region, and can grow in the presence of 0.6mol/L NaCl in FY minimal medium. The collection of 3000 random Tn5-1063a insertion mutants was screened. Two salt sensitive mutants were obtained and named as 042BM-X1 and 042BM-X2.The test of salt tolerance showed that they cannot survive in the presence of 0.4mol/L and 0.5 mol/L NaCl in solid and liquid FY medium, respectively. With Southern blot analysis, they were found to contain a single Tn5 insertion in their genome. It suggested the salt-sensitive phenotype was a result of Tn5 insertion.Sequences flanking Tn5 in the mutants were cloned by self-ligation. Because each transposon contains an origin of replication functioned in E.coli, but not in Rhizobium. Sequences flanking Tn5-1063a can be recovered from the genome of mutant by excision,self-ligation and transfer to E.coli.The total DNA of mutant was excised with EcoRI,which cut the genome frequently but not cut the transposon.After sequencing the self-ligated transpon, DNA fragment flanking Tn5 was obtained.The result showed 042BM-X1 contains a Tn5 insertion in the gene Smc00190, which function was unknown and was demonstrated to be related to salt tolerance by this study,and the gene was named as rst-0x1. Strain 042BM-X2 was found to contain a single Tn5 insertion in the gene Smc02682, which showing a high degree of similarity with the Rhizobium tropici gshB gene, encoding the enzyme glutathione synthetase.In our experiment, this gene was demonstrated to be related to salt tolerance.so it is named as rst-0x2.DNA fragments containing rst-0x1 and rst-0x2 as well as their promoters were abtained by PCR.The results of sequencing and BLAST analysis suggested these fragments shares 99% identity with Sinorhizobium meliloti 1021. Function of rst-Oxl was unknown, while rst-0x2 shares 81% identity with gshB gene of Rhizobium tropici, which encoding glutathione synthetase. Glutathione paritcally restores the salt-tolerant ability of 042BM-X2, and we hypothesized that glutathione plays an important role in the cells of Sinorhizobium meliloti 042BM by affecting its ability to regulate intracellular K+ levels.