Functional Analysis And Application Study Of BjCET1,a Member Of CE Family From Brassical Juncea L.

In recent years,the scale of urbanization,industrialization and agriculture in China has been expanding,the discharge of industrial "three wastes" and the excessive use of chemical fertilizer have been increasing the heavy metal pollution of soil,which has a serious toxic effect on animals and plants.How to effectively control and repair the heavy metal pollution of soil has become one of the research hotspots.At present,phytoremediation technology has some advantages in operation,economy and technology,among which Brassica juncea L.,as one of the main model plants of phytoremediation technology,has the ability of enrichment or super enrichment of many heavy metal ions such as Cd、Zn、Pb、Se、Au,etc.It is of great scientific significance and practical value to explore and analyze the molecular mechanism of heavy metal accumuation/hyperaccumulation for the development of artificial metal-hyperaccumulators for phytoremediation of heavy metal contaminated soil and improvement of the content of essential trace elements in human diet.The protein members of CE(cation efflux)family are believed to be related to the transport and enrichment of Cd、Zn、Ni、Co or Fe in plants.The BjCET1 gene of the family was cloned from the cDNA pool of Brassica juncea L under cadmium stress,and its promoter BjCETIP was cloned by genome walking.In this study,BjCET1P and BjCET1 transgenic Arabidopsis thaliana plants of T3 generation were obtained by Agrobacterium infection method.The functional mechanism of BjCET1P promoter and BjCET1 gene and remediation potential of polluted soil were studied by heavy metal stress analysis,yeast functional complementation,Gus staining,non-invasive micro test technique(NMT),etc.The results are as follows:1.GUS staining of the BjCET1P drived gus transgenic Arabidopsis thaliana confirmed that BjCET1P was a constitutive strong promoter,its expression was localized in apical and vascular tissues..2.Yeast functional complementation experiments results showed that the expression of BjCET1 in yeast could significantly improve the tolerance of yeast to Zn、Cd、Co、Ni especially Zn stress;ICP-MS showed that the expression of BjCET1 significantly reduced the Zn content of yeast cells,indicating that the expression of BjCET1 improved the ability of yeast cells to pump out Zn.3.Using confocal laser scanning microscopy(confocal),we observed the roots of tobacco plants with the fusion expression of BjCETl-GFP protein.Through the experiments of plasmolysis,we verified that the main expression of BjCET1 protein in the roots was on the cytoplasmic membrane.4.Using NMT technique,we tested the effect of BjCET1P2-drived BjCET1 gene expression on the Cd2+ion flux in the roots of Arabidopsis thaliana under Cd stress.The results showed that BjCETl expression significantly increased the net influx of Cd2+in the Arabdosis roots under Cd stress conditions,The physiological mechanism of overexpression of BjCETl for increasing Cd concentration in plants.5.The heavy metal stress analysis showed that the BjCET1P2-drived BjCET1 expression significantly increased the ability of A.thaliana plants’ tolerance to Cd,Cu,Hg,Mn and Zn stress,and the fresh weight and growth of the transgenic plants were growing significantly better than that of the controls.Preliminary analysis of the plant Cd content showed that the Cd accumulation in the above-ground parts of the BjCET1 transgenic plants was significantly higher than that of the controls,indicating the potential of the gene and promoter for phytoremediation of Cd polluted soil.