The Remediation And Utilization Of Heavy Metal Contaminated Soil And Characteristics Of Cd Stress Responsed Genes In Ramie(Boehtriia Nivea (L.) Gaud.)

Soil contamination by heavy metals is a threaten to economic, agriculture and people health. Phytoremediation has the characteristic of lower cost, eco-friendly, which has been a reseach hotpot recently. Ramie(Boehmeria nivea (L.) Gaud.) is a China unique fibre crop which has the characteristic of fast growth speed, high biomass and strong adaptable ability. It has high economic and ecological value to screen ramie varieties suitable for phytoremediation. And it is important to research the tolerence mechanism of ramie to Cd by cloning the Cd responsed genes followed by expression and function analysis.In this study, the remediation and utilization of soil contaminated by Cd and Pb with7different varieties of ramie were analysed by field experiments. The remediation effect of ramie-Pteris vittata L. intercropping mode to soil contamination by Pb and As were investigated by field experiments. The full length cDNA of ascorbate peroxidase gene (BnAPX1), glutathione reductase gene (BnGR1) and phytochelatin synthase gene (BnPCSl) were cloned. The tissue specific and induced expression of3genes were studied. The prokaryotic and plant expression vector of BnAPXl, BnGR1and BnPCS1were constructed respectively, then BnPCSl transgenosis Arabidopsis thaliana was obtained and the gene function analysis was done.The main results are as following:1. All ramie varieties growed well on the Cd-Pb contaminated soil, and the yield of Zhongzhu No.l was significantly higher than that of other varieties. Cd concentration in husk and Pb concentration in root was higher than other organs. The Cd extraction efficiency of Zhongzhu No.1was2.95%, which was significantly higher than that of other varieties. It can get the highest effect of remediation and economic at the same time.2. Considering the economic efficiency, ramie and P. vittata intercropped in normal density(5brushes/m2) was a efficacious planting method for Pb-As contaminated soil remediation with the highest effect of remediation and economic at the same time.The bioamass of ramie shoot and economic yield per unit area were significantly reduced in low ramie-P. vittata intercropping density, Ramie-P. vittata intercropping can significantly improve the concentration and accumulation of Pb in ramie. The concentration and accumulation of Pb and As in P. vittata which intercropped with ramie was significantly lower than that in P. vittata monocropped. The As extraction efficiency of P. vittata monocropped was significantly higher than that of other treatments.3. The full-length of BnAPX1gene was1201bp with a ORF of870bp, which encoded a31.4kD protein with one heme binding site, one substrate binding site and one K+binding site, respectively. The full-length of BnGR1gene was1977bp with a ORF of1494bp, which encoded a53.7kD protein with one pyridine nucleotide-class I disulfide oxidoreductase active site, one NADP binding site, one FAD binding sites, two GSSG binding sites and a special cytoplasmic domain of GR, respectively. The full-length of BnPCSl gene was1956bp with a ORF of1512bp, which encoded a53.7kD protein with a typical domain(C358C359XXXC363XXC366) of PCS protein and three essential amino acids(Cys56、His162and Asp180).4. The tissue-specific analysis showed that the BnAPX1、BnGRl and BnPCS1genes were expressed in root, stem, shoot tip, young and mature leaves with different expression level. The expression level of BnAPX1gene was the highest in young leaves and the lowest in stem, respectively. The expression level of BnGR1and BnPCS1genes were both the highest in mature blades and the lowest in stem, respectively. BnAPX1and BnGRl genes both responsed to Cd stress and were up-regulated by ABA and SA treatments. BnPCS1gene responsed to Cd stress and was up-regulated by ABA treatment.5. Prokaryotic expression vectors of BnAPX1、 BnGR1and BnPCS1genes were constructed, named as pQE-N1-BnAPX1, pGEX-4T-BnGRl and pGEX-4T-BnPCS1respectively, which were successfully expressed in E.coli BL21, respectively. The molecular weight of the induced proteins of3genes were about31.4kD,53.7kD and56kD, respectively, which were consistented with the expected molecular weight. Then the plant expression vectors of BnAPX1, BnGR1and BnPCS1genes were constructed and transformed into agrobacterium tumefaciens, respectively. We obtained transgenic Arabidopsis thaliana of BnPCS1gene whose Cd tolerance was higher than that of WT Arabidopsis thaliana.