Clone And Functional Analysis Of Selenium-resistant Gene VSE1in Arabidopsis

To locate and express selenium metabolic key enzyme gene in main crops to adjust selenium level of the agricultural products to meet the need of human health ingestion selenium has important theoretical and practical value, using molecular biology means to separate selenium resistance genes and transgenic technology to cultivate more selenium resistant plants is development trend of in the future.Forward genetics, through spontaneous mutation or artificial mutation to the genome of individual organisms or cells to look for relevant phenotype or trait change, and then found the corresponding mutant gene from the individual organisms or cells of these specific traits change, and revealed function. In this study, we screened a selenium-resistant mutant named vsel by chemical inducible Activation Tagging T-DNA Insertion Lines mutants, and vsel mutant was resistant to certain concentration of sodium selenite by the identification. But there were no significant differences in growth and development beween the vsel mutants and the wild type under normal growth condition, On that basis, we cloned the VSE1gene by using Tail-PCR, and studied the function under selenium stress.1. vsel mutant screened from XVE activation label sub-mutant library. In5mg/L Na2SeO3and10μMβ-estradiol+5mg/L Na2SeO3cultivating conditions vsel mutant had more significant resistance to selenium stess than wild type. Genetic analysis displayed that vsel mutant resistant to Se was regulated in the manner of a single recessive gene. In addition, vsel mutant showed obvious tolerance to H2O2stress, the results implied tolerance pathway of vsel mutant to selenium stess may be related to antioxidant system.2. The VSE1gene (AT1G07890) in Arabidopsis was cloned by Tail-PCR technology. VSE1gene encoded a cytosolic ascorbate peroxidase(cAPX), which was one of the important enzymes in plants and was important factor in AsA-GSH cycle to eliminate H2O2, especially in chloroplast. The analysis of the amino acid sequence alignment revealed the protein encoded by VSE1gene had a high similarity with Brassica oleracea.3.Through the analysis of VSE1gene expression patterns, VSE1gene was knocked out in vsel mutants and VSE1gene expression level was different in all tissues, highest in the siliques while lowest in the rosettles. By the determination of selenium content in tissues, selenium content in roots of vsel mutants was found to be significantly higher than in roots of wild type. The expression levels of four selenium stress-related genes AtGPX,AtHMT,AtGSH and AtATM were analysed, the expression of AtGPX and AtGSH in vsel mutants was higher than wild type, but there was no difference in the expression of AtHMT and AtATM. vsel mutants showed enhanced tolerance to Se stress compared with the wild type,but phenotype of vsel mutants resistant to selenite dissolved in0.2mM BSO+5mg/L Na2SeO3cultivating conditions, indicating that tolerance of selenium of vsel mutants may be related to GSH pathway and the increasing expression of AtGPX and AtGSH. Besides, the response of the vsel mutants to H2O2stress and DAB histochemical assay of H2O2showed tolerance of VSE1gene knocking out mutants to selenium stess may be related to antioxidant system. All these results suggest the VSE1gene is involved in the regulation of selenium stress tolerance through activating of AtGPX, AtGSH, chelating Se and scavenging of ROS.