Preliminary Research On Responses To Pb And Zn Stress Of Carex Putuoshan Phytochelatin Synthase

Plants have a series of mechanisms to responsive to heavy metal stress, and Phytochelations(PCs) is one of them. It plays an important role in stress response of heavy metal. PCs is not a direct product of gene, but catalytic synthesis of glutathione as substrate by Phytochelatin synthase(PCS). In the research, taking the Pb hyperaccumulation Carex putuoshan as the material and Carex Evergold as a control, a novel carex Phytochelatin Synthase gene CpPCS was obtained. And we conducted a survey of the Carex putuoshan response mode under heavy metal stress, especially the temporal expression of CpPCS, the CpPCS structure, phylogenetic analysis, and the prediction of protein 3D structure. The molecular mechanism of Carex putuoshan under heavy metal stress was partly investigated, and we would provide a basis for further utilize in plants. The main results are as follows:(1) Real-time quantitative PCR analysis indicated that CpPCS(Carex putuoshan) and CePCS(Carex Evergold) gene expressed in the root and leaf. The CpPCS and CePCS gene were up-regulated by Pb and Zn treatments. The expression pattern of CpPCS was positively correlated with the super-enrichment characteristics of Carex putuoshan. And the CpPCS and CePCS expression in root were higher than that of leaf under the same condition. And the expression of CpPCS was higher than that of CePCS under the same condition. The expression level of PCS gene in leaves of Carex putuoshan reached maximum in 36 hours after combined stress, about 12 times of blank control group and 4 times of Carex Evergold under same treatment. CpPCS protease activity center is also 8 times the CePCS protein. Therefore, CpPCS catalyzed synthesis of PCs binding heavy metal ions is much higher than the amount of CePCS. This is the enzyme activity key characteristics of Carex putuoshan hyperaccumulate lead.(2) Based on RACE cloning, a carex PCS gene was obtained, named CpPCS. The full length of cDNA was 1461 bps, and it encoded 486 amino acids with molecular weight of 53.86 kD and pI value of 6.12. The predicted fat coefficient was 87.65. The unstable factor was 47.56. which was classified as unstable protein. Subcellular localization predicted that CpPCS protein was localized in the nucleus. The NCBI blast found that the PCS of Carex putuoshan and Zea mays, Phragmites australis, Allium sativum, Nicotiana glauca, Boehmeria nivea amino acid sequence similarity were 64%,63%,63%,63%,62% respectively. Two typical phytochelatin subfamily domains constitute CpPCS protein, which includes three adjacent Cys-Cys elements in the C-terminal region. The average hydrophilicity of CpPCS protein was predicted to be -0.094, so it was a hydrophilic protein. CpPCS protein has two transmembrane regions. It was found that the four proteins have a similar space frame by comparing with the three dimensional structure of BjPCSl, PvPCS, and AtPCS protein, they were estimated to make similar functions in living organisms. Phylogenetic analysis of PCS proteins from different species showed that it had the closest relationship with the Cvnodon dactvlon and Phrasmites australis.