Clone And Functional Analysis Of A Selenium-Resistant Gene In Arabidopsis

Se is an important trace element, which can prevent disease, delay aging, be resistant to the damage caused by a variety of external stress, but it is toxic at higher level. Therefore, it is very important for theoretical and practical value to study of Se tolerance mechanism and find out the real key factor of plants tolerance to Se. In this study, we screened a selenium-resistant mutant vse2 by chemical inducible activation tagging T-DNA insertion lines mutants, and the mutant vse2 was resistant to certain concentration of sodium selenite. We cloned VSE2 gene by using Tail-PCR, and studied its function. The results are as follows:1. The mutant vse2 was isolated from XVE activation labelling sub-mutant library. In 30μM Na2SeO3 and 10μMβ-estradiol+30μM Na2SeO3 cultivating conditions, the mutant vse2 has more significant resistance to Se stress than wild type. These means that the Se tolerance of mutant vse2 has nothing to do with P-estradiol.2. Using Tail-PCR, we cloned VSE2 gene, the gene is TPS22 (Atlg33750), which encoded a terpenoid cyclases/protein prenyltransferases superfamily protein. T-DNA is inserted into the first intron downstream (29bp) of the ATG start codon of the TPS22 gene. The analysis of the amino acid sequence alignment revealed that the protein encoded by TPS22 gene had a high similarity with Arabidopsis lyrata.3. Through the analysis of TPS22 gene expression patterns, the expression level of TPS22 gene decreased in mutant vse2, which is knocked down. And TPS22 gene expression level is different in all tissues, highest in the root while lowest in the flower。4. The content of selenium in roots and leaves of mutant vse2 is found to be significantly higher than in that of wild type. Meanwhile, the expression level of PHT1;1 gene is lower in mutant vse2, indicating that the less content of selenium in mutant vse2 maybe related to the lower level of transporter.5. SMT gene expression was higher in the mutant vse2, speculating that the selenocysteine of mutant vse2 change as seleno-methyl-cysteine, reduced the toxicity of selenium.6. The activity of GPX and the expression level of GPX1 gene are higher than WT under Se stress. These showed that the scavenging reactive oxygen species activity of the mutant vse2 was improved.7. When added with BSO in the culture medium under Se stress, tolerance to Se of mutant mutant vse2 disappeared. And the content of GSH and the expression level of GSH1 gene are higher than WT only under selenium stress. This results suggest that rPS22-mediated Se tolerance is partially GSH dependent.8. To further detect the content of CTK and the expression level of IPT and TPS25, we found that CTK content increased under selenium stress, and the expression level of IPT, TPS25 gene increased as well. We speculate that the increase of CTK content might be due to the increase of IPT, TPS25 gene.9. The mutant vse2 is sensitive to drought, mannitol and low temperature stress.Taken together, the molecular mechanism of Se tolerance mediated by TPS22 gene might be relative to these three results. The first, the expression level of PHT1;1 gene is lower in mutant, and the content of Se entering in mutant vse2 is less, which reduce the injury of excessive selenium on plant.In addition, the higher expression level of SMT and GPX gene reduced the insertion of error selenoprotein. At the same time, GPX activity increased the ability of scavenging H2O2 in mutant vse2, which increased Se tolerance. The second, TPS22-mediated Se resistance is partially GSH dependent. The last, the decrease of TPS22 gene expression level resulted that higher expression of isopentenyl transferase and terpene synthase genes, enhanced cytokinin content, improved the mutant anti stress ability.In addition, the TPS22 gene is knocked dowm, which makes the mutant vse2 sensitive to drought, low temperature and osmotic stress.