Study On Transferring Metallothionein Gene Of Mice Into Populus×euramericana Cv.’Nanlin895’

Heavy metal pollution is one of the main problems which will affects environmental quality and ecological stability, the pollution not only affects the quality of air and water, the crop growth, but also causes serious harms to the health of human and animals through the food chain. The treatment is very difficult because of the widespread heavy metal pollution and its own non-degradable characteristics. In this study, Metallothioein(MT) which combined with heavy metal only in mammalian animals was used as target gene, and highly biological capacity, rapid growth of plants (poplar of nanlin895) had been selected as the target gene transfer plants, the target gene was integrated into the plant genome by using plant genetic engineering technology to apply transgenic poplar within MT gene for the enrichment of harmful heavy metals in contaminated soils,so we can achieve the purpose that the restoration of habitat from the source control,the reduction of heavy metals into the food chain, as well as the greater control of animals and humans within the occurrence of heavy metal poisoning.During the experiment, plantlets of nanlin895were used as accepted materials, a mice metallothionein (MT) gene plant expression vector pCAMBIA2301-MTwhich transfering by Agrobacterium-mediated was constructed, the preliminary study of plant genetic transformation has been expanded, for the eventual stability of genetic and gene expression in transgenic poplar which had gotten the MT gene. So we can use MT to bind heavy metal efficiently,and it also will provide a basis for the enrichment of heavy metals in contaminated soils.1、The successful construction of plant expression vectors:the MT gene box which contains35S-35S promoter, AMV, Tnos and MT gene was cloned to the plant expression vector pCAMBIA2301, the new recombinant plasmid contained nptll, gus and the MT gene box could make the poplar acquire kanamycin resistance ability and express Gus which we can check out by X-Gluc dyeing on the way of agrobacterium mediated transformation.2、The determination of antibiotics that are effective in eliminating agrobacterium tumefaciens:an assay was performed to identify the antibiotics that are most effective against Agrobacterium tumefaciens strains EHA105, and to determine if these antibiotics inhibited poplar disc, we tested four antibiotics:Carbenicillin, Ampicillin, Cefotaxime and Timentin.Cefotaxime was the most active antibiotic against EHA105. cefotaxime with the concentration of200mg/L had little or no effect on poplar disc development.3、The determination of agrobacterium mediated vacuum infiltrated transformation: the best transforming condition has been drawn from the results of poplar disc’s Gus dyeing:pre-culture time was3days, agrobacterium concentration was OD6000.5, vacuum infiltration infection time was5minutes, co-cultivation temperature was28℃, co-culture time was2days, two positive plants have been gotten by PCR identification.4、The expression of gus in poplar of nanlin895:after the agrobacterium EHA105mediated transformation of nanlin895, the gus gene on pCAMBIA2301-MT which has been obtained by EHA105successfully expressed in leaf disc, it could be checked out by the dyeing of X-Gluc.