| Along with the country’s industrialization and urbanization process of accelerated,heavy metal contaminated soils in urban is becoming increasingly serious.Heavy mental pollution can pose a serious threat to the landscape,the growth environment of plants and the residents’ quality of life.Plants have developed a number of defense mechanisms for in order to deal with heavy metal stress.Phytochelatins(PCs),a small molecular peptide,which are produced under catalyzation of Phytochelatin synthase(PCS),play the part of phytoremediation as its ability of detoxifying and chelating heavy metal ion such as cadmium,copper and lead in plants.Chrysanthemum indicum is a kind of perennial plant of composited,which has strong cold and drought tolerance.Meanwhile,because C.indicum has the capability of absordability,accumulation and remediation of heavy metal contaminated soil,it is considered to be a potential material for phytoremediation.At present,the related researches of C.indicum were mainly in the aspects of introduction and cultivation,anti-inflammatory,antioxidant and extract components,etc.However,there are few studies on the improvement of resistant varieties genes of C.indicum by molecular breeding technology.Therefore,intensive research on cadmium responsive genes of C.indicum is of great significance to improve C.indicum resistant varieties,as well as provides excellent plant materials for phytoremediation.In this study,We cloned and studied the function of Ci PCS and its promoter,The main results are as follows:1.The phytochelatin synthase gene(Ci PCS)was cloned from C.indicum.Bioinformatics analysis of Ci PCS showed that its ORF was 1761 bp in length,encoding 586 amino acids,which contained phytochelatin domain and phytochelatin＿C conserved domain.The protein structure was dominated by alpha helix and random coils.Concluded from the phylogenetic tree,we found that Ci PCS protein had the highest homology with Artemisia annua.The expression of Ci PCS were analyzed through real-time quantitative PCR,the results showed that Ci PCS were up-regulated in the roots and leaves in C.indicum under cadmium stress,so Ci PCS was involved in the cadmium stress response.2.A 1021 bp promoter sequence of Ci PCS was cloned by Genome Walking.Cisregulatory element analysis showed that the promoter region had light response elements such as Sp1 and box 4;plant hormone-like response elements such as jasmonic acid,auxin and gibberellin,etc;circadian rhythm regulation related elements and MYB binding sites.3.Four different segments(P,P1,P2,P3)with 5’-end deletion were constructed into PBI121-GUS expression vector.The constructed vectors were used to transform Nicotiana benthamiana by injection and stained with GUS.GUS histochemical staining results showed that the 5’-end deleted segments of the promoter were all active,and the key elements of Ci PCS promoter activity were located in the region of-558～-807 bp.4.A plant expression vector p BI121-Ci PCS-GFP was constructed and transformed into Arabidopsis thaliana by inflorescence transfection,and five transgenic strains were obtained.The analysis of cadmium tolerance of transgenic plants showed that heterologous overexpression of Ci PCS could improve Arabidopsis thaliana cadmium tolerance at the seed and seeding stage,such as promoting seed germination and root length elongation.The transgenic lines treated with cadmium could increase the activities of SOD,POD and CAT,and reduce the accumulation of MDA.5.By agrobacterium-mediated leaf disc method,C.indicum was transformed to obtain overexpression transgenic strains,and five positive strains were obtained through resistance screening and transcription level detection.The results of real-time quantitative PCR showed that the expression of Ci PCS in the leaves was significantly higher in the overexpression transgenic strains compared with the control(p<0.01),indicating that the Ci PCS had been integrated into the genome of C.indicum... |
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