Cloning Of HcMT From Halostachys Caspica And Genetic Transformation In Arobidopsis Thaliana

Phytoremediation technology is to chlorophyte to absorb, transfer,and degrade pollutants. Thus reducing the heavy metal esidues in the soil, and makeit into a can be an environment-friendly and a cost-effective remediation technologyof recycled and environmentally friendly material. In recentyears, about solving a problem that most of the phytoremediation hyperaccumulatorsare slow-growth, small-aboveground biomass， researchers at home andabroad through a lot of experimental researchs have found that efficient expression offoreign genes in plants can improve plant absorption, transport and the ability todegrade metals. Therefore through the technique of genetic engineering to improvethe heavy metals tolerance of chlorophytes, thereby increasing the accumulation ofmetals in plants is the most effective way of phytoremediation of contaminated soil.Metallothioneins(MTs) are low-molecular-weight, cysteine-rich proteinsinvolved in heavy metal resistance that bind to heavy metals. At the present time,there are a lot of researches theories of mammal MT has been introduced into plants,However, transformation of Halostachys caspica MT gene into plants has not beenreported so far.This study according to the metallothionein’S EST sequences of Halostachyscaspica, a specific primers were designed, metallothionein gene was cloned fromHalostachys caspica with RT-PCR(as named HcMT), in order to research its influenceon plant heavy metal tolerance. This gene is411bp in length, the ORF is237bp(Contains a start codon), a82bp5’-UTR and92bp3’-UTR. It encodes66aminoacids which contains14cysteines. The predicted molecular weight of HcMT is7.70kD, and an isoelectric point of5.05. Bioinformatics analysis indicated that MT genefrom the Halostachys caspica is completely in line with the typical MT2proteinfeatures, the genes and other plant MT2have high homology and this protein sharedabout69%amino acid sequence similarity with them. It can be seen from the phylogenetic tree that this protein shared93%amino acid sequence similarity withthat of Salicornia brachiata. Protein family Prediction results showed that MT proteinin this experiment belongs to the MTII protein family.The results of semi-quantitative RT-PCR and the real-time PCR showed thatHcMTshowed up-regulated expression patterns under100mM CuSO4、ZnSO4、CdCl2stress, the expression of the gene was induced by heavy metal stress. These resultssuggest that HcMT gene is able to respond to heavy metal stress like CuSO4, ZnSO4and CdCl2.To stuy identify the subcellular location of HcMT gene in Halostachys caspica,we successfully constructed the subcellular socalization vectorpCAMBIA1302-HcMT that the HcMT gene was recombined with the GFP, and thisprovide a basis for the successive study and its application. In the meantime, weconstructed the plant expression recombinant vector pCAMBIA1301.1-HcMT andtransformed Arabidopsis thaliana with the HcMT gene by Agrobacterium tidbitsimpregnation method, resistant seedlings were obtained through antibiotics screeningto the research on its role in transgenic plant andalso lay the groundwork for the research of heavy metal tolerance mechanismsand application of the HcMT gene.