The Study Of Genes In 263-H9 Response To Salt Stress

Transposon tagging is one of the effective technology for genes function analysis under several stress conditions such as salt stress,heat shock and so on, and mTn3 Transposon tagging is the most popular and successive one.Using the pools of the yeast genomic library with mini-transposon to transform the haplaid Saccharomyces cerevisiae strain W303-1A, we got many mutants sensitive to high salt stress totally. By the rescue plasmid, the insertion sites of the transposon in mutants were sought out, and those insertions affected 31genes.The mutant named 263-H9 were studied more. The insertion site of mTn3 transposon in 263-H9 was in the intergenic region between GIP2 and YER053C-A. 263-H9 showed hypersensitivity under several stress conditions. Unlike 263-H9, the GIP2A or YER053C-AA strains named DW-101 or DW-102 remained strong resistance to salt shock. DW-100, a W303-1A strain in which Kanr was inserted into the same site as the transposon of 263-H9, still remained the resistance. But DW103, a 263-H9 strain in which the transposon was replaced by Kan, showed osmolarity-hypersensitive phenotype. Our data indicated that the osmolarity-hypersensitive phenotype of 263-H9 had no relationship with the only transposon in the mutant.The diploid W2 constructed by 263-H9 and DW-100 showed strong resistance under several stress; it proved that the mutation site in 263-H9 was recessive. Using the pHR81 yeast chromosome library to transform 263-H9, we got one plasmid named p2-001. This plasmid can recover the hypersensitive phenotype of 263-H9.By sequencing, the yeast chromosome fragment was obtained. It contains the entire PBS2 and the TRKl fragment. The Pbs2p MAPKK can be activated by Ssk2p and Stel lp MAPKKKs, which are activated only by osmotic stress. The Pbs2p MAPKK has a long N-terminal non-kinase region that presumably contains a number of functional subdomains. RSD-III (residues 283-353) is essential for Pbs2p mediated signal transduction. Deletion of RSD-III from Pbs2p resulted in a complete loss of its ability to transmit signals to Hog1p, even in the presence of all three intact MAPKKKs (Ssk2,Ssk22 and Ste11).Our data indicated that the mutation site of 263-H9 was at the 936bp in the PBS2 and 5 bases was lost. The mutation results in the false construction of Pbs2p. So HOG signal pathway was blocked and 263-H9 showed osmolarity-hypersensitive phenotype. More study of 263-H9 was going on.