Tolerance And Accumulation Of Copper And Cadmium In Transgenic Plants Over-Expressing Exogenous Metallothioneins(EHMT1, BCMTS)

Heavy metals pollution in soil and water has become a global environmental problem, organisms grew in the environment polluted by heavy metals developed some mechanisms against the toxic effects of heavy metals, one of the mechanisms is the synthesis of some ligands such as organic acids, phytochelatins (PCs), metallothionein (MTs), which could chelate heavy metal ions, forming non-toxic complexes, thereby reducing the toxic effects of heavy metals. In this study, we optimized the regeneration system of Brassica campestris ssp. chinensis Makino. EhMT1 cloned from Elsholtzia haichowensis was transferred to B. campestris, B. campestris expressed EhMT1 gene were more tolerant to Cd and Cu. At the same time, we cloned BcMT1 and BcMT2 from B. campestris and transferred the two genes into Arabidopsis and tobacco, we successfully obtained transgenic plants, and a preliminary analysis of MT genes function was studied.Experimental results showed that wild-type and transgenic B. campestris were not different in MS medium, but when exposed to 200 ?M CdCl2, roots length of wild type had severe inhibition, while inhibition of transgenic plants was significantly lower than wild type, and the transplants had a large number of adventitious roots. in treatment of 100 ?M CuSO4, the roots of wild type stopped growing, while transplants were also had shorter root elongation, but a lot of adventitious roots happened, which is one of the tolerance performance of transgenic plants than wild-type. In 200 ?M CdCl2 treatment, there was no difference of Cd concentration in shoot and root when treated for the first 1 and 3 days between the transgenic and wild type, transgenic plants had higher Cd concentration in shoot and root than wild-type on the 7th day of treatment. In 100 ?M CuSO4 treatment, Cu concentration in root of transgenic plants was not different in each time period compared with wild type, but Cu concentration in shoot was higher than wild type.In this study, we cloned BcMT1 and BcMT2 genes from B. campestris, the open reading frames were 139bp and 245bp seperately. To study the response of BcMT1 and BcMT2 expression when exposed to different abiotic stresses,5-day-old seedlings were exposed to 200 ?M CdCl2,100 ?M CuSO4, 10 ?M ABA, and 100 mM NaCl, and the transcription level was examined by semi-quantitative RT-PCR. BcMT1 and BcMT2 displayed different expression patterns under abiotic stresses. BcMT1 and BcMT2 expression in both leaf and root was induced after 1 h by 200 ?M CdCl2 treatment. BcMT1 and BcMT2 expression was markedly increased in leaf treated with NaCl and ABA. In the presence of 100 ?M CuSO4, BcMT1 expression but not BcMT2 was induced at 24h-treatment. In addition, after 24h-treatments with different concentrations of ZnSO4, K2CrO4 and CUSO4, expression of BcMT1 and BcMT2 were upregulated in varying degrees. In Yeast experiments, compared with the wild type, overexpression BcMT1 and BcMT2 increased the tolerance of yeast to Cd, Cu and Cr.To examine the role of BcMTs, seeds of wild type and transgenic Arabidopsis were germinated on normal MS medium and then transferred either to fresh MS medium or to medium containing 200 ?M CdCl2 or 100 ?M CUSO4 for 7 days. The growth of BcMTs transgenic Arobidopsis was indistinguishable from wild type and the line with empy vector (pBI121) without excessive Cd or Cu. We observed typical symptoms of heavy metal toxicity, including root growth inhibition, shoot growth retardation, which were more pronounced in wild type and the line with empy vector than transgenic seedlings.We measured the concentrations of Cd and Cu in wild type and transgenic Arabidpsis which were transformed BcMT1 and BcMT2 after Cd and Cu treatment for 7 days. In the presence of Cd, no significant difference in Cd concentration was observed between wild type and transgenic lines. However, when exposed to Cu, the transgenic line BcMT1-1, BcMT1-2, and BcMT2-1 increased Cu concentration in shoots. In the analysis of the metal transporter protein expression,24 hours after Cu treatment, in transgenic line BcMT1-1, the expression of COP1 and ZIP4 was significantly increased. In BcMT2-1 lines, expression of COP1 and COP5 was increased, but ZIP4 expression did not change significantly, the higer accumulation of Cu may be related to the metal transporter protein.Treatment with 200?M CdCl2 or 100 ?M CuSO4 for 24 h led to an obvious accumulation of H2O2 and O2·- in the leaves of wild type. Productions of H2O2 and O2·- were considerably lower in the leaves of transgenic Arabidopsis expressing BcMT1 or BcMT2 compared with those in Wild type. To investigate whether the increased tolerance to heavy metals in BcMTs-transgenic Arabidopsis may be due to the elimination of ROS by BcMTs, directly or indirectly, the expression levels of four antioxidant genes (GPX3, CAT2, Cu/Zn-SOD3, POD) were examined by semi-quantitative RT-PCR in wild type and transgenic Arabidopsis exposed to Cd and Cu stress. Under Cu stress, when the seedlings were treated for 24h, the expression of POD and CAT2 in transgenic Arabidopsis was increased than untreated and wildtype.We generated two lines of BcMT2-transgenic tobacco.When exposed to 200 ?M CdCl2 or 100 ?M CuSO4, root length of transgenic Tabbcco TBcMT2-6 and TBcMT2-8 expressing BcMT2 had a smaller inhibition compared with wild type. when exposed to 100 ?M CdCl2,20 ?M CuSO4 and 70 ?M K2CrO4 for 7 days, wild-type and transgenic tobacco did not have difference in Cu and Cr concentration, however, Cd concentration in transgenic tabbcco was higher than wild type both in shoot and root.