Cloning And Functional Analyze Of A Cadmium Responsive Gene In Arabidopsis

Cadmium pollution in soil would be transferred to organs and tissues through the food chain, causing a serious threat to human health. Phytoremediation of heavy metal pollution is one of the effective methods relying on the understanding of molecular mechanisms of plant response to cadmium stress. Therefore, cloning of cadmium-stress responsive genes is very necessary. In this study, cadmium chloride was used as screening index upon Arabidopsis mutant library, and a cadmium sensitive mutant cdml (cadmium-sensitive mutant 1) was isolated. The results are as follows:1. Using Tail-PCR method, we cloned the PRT3 gene from the cdml mutant and found that the T-DNA element was inserted in promoter region (384 bp) of PTR3 gene, Bioinformatics analysis showed that the gene encodes a transporter protein belonging to MFS superfamily.2. Under CdCl2 stress, the cdml mutant showed sensitive to Cd stress compared with the wild type. This finding provides a theoretical basis for further study on the mechanism of PTR3 in heavy metal stress response.3. The full-length PRT3 coding sequence was amplified from cDNA of Arabidopsis thaliana (Col-0) and cloned into the pBI121 vector, build for PTR3 overexpression vector. The resulting construct was transformed into Agrobacterium strain GV3101 for transforming into the Col-0 plants by the floral dip method. The transgenic lines were isolated by antibiotic screening and genetic identification.4. Under CdCl2 stress, PTR3 overexpressioning lines showed enhanced tolerance to Cd stress compared with the wild type. These findings provide a theoretical basis for further study on the mechanism of PTR3 in heavy metal stress response.5. In order to analyze the biological role of PRT3 in lead tolerance, transcript levels of candidate genes were analyzed by qRT-PCR. It was found that the expressions of PDR8、 GSH1、 GSH2、 GR1、 GR2、PCS1、 PCS2、 ABCC1、 ABCC2 showed significant differences, but transcript levels of ATM3、 ACBP1 in cdml mutant and PTR3 over-expression lines didn’t show significant difference in comparison with wild type plants. The results suggest that synthesis of GSH might be regulated by PTR3 in plant response to Cd stress.6. Under CdCl2 stress condition, cadmium contents of the cdml mutant in root and shoot were significantly reduced in comparison with wild-type plants, but the cadmium contents of the PTR3 over-expression in root were significantly increased in comparison with wild-type plants. In order to further analyze the regulation mechanism of PTR3 gene in cadmium stress response, GSH contents of different materials were quantified. We found that in compared with wild type plants no matter under normal growth condition or CdCl2 stress, GSH contents was significantly reduced in cdml mutant while increased in PTR3 overexpression lines, demonstrating that expression of PTR3 gene might be related to GSH synthesis.To sum up, all these results suggest that reduced PTR3 gene expression was caused by T-DNA insertion in cdml mutant and therefore inhibited GSH synthesis, thereby leading to sensitivity of mutant plants to cadmium stress. Based on these results, we conclude that PTR3 gene regulates Cd tolerance in Arabidopsis by affecting GSH and PCS synthesis.