Cloning, Expression And Stress Resistance Analysis Of BjCRP1 Gene From Brassica Juncea L.

Abiotic stresses such as salt, drought, cold and freezing could produce severe damages on growth and development of plants. However, a rare group of plants still can grow normally in severe natural conditions, e.g. soil with high concentration of heavy metals, indicating that these plants have evolved corresponding resistance mechanisms to deal with the severe conditions during the long-term evolution. In the aspect of metal hyperaccumulation, the prominent plant behaviors include the high ability of metal ions absorption, transport, cellular compartmental sequestration and chelation, among which, the expression and regulation of transmembrane transporters play a key role. Therefore, identifying and characterizing new membrane protein genes for metal tansporting is of great significant for resistance molecular biology.In this study, total RNA was extracted from CdCl2 treated Brassica juncea, and BjCRP1 gene was amplified by RT-PCR. Sequence analysis showed BjCRP1 belongs to members of Ribosome-associated Membrane Protein 4 (RAMP4) family. We constructed the prokaryotic expressional vector pET32a- BjCRP1 and transferred the recombinant vector into BL21(DE3)pLysS strain. After inducing by the addition of 0.8mmol IPTG, and analysed the total protein through SDS-PAGE electrophoresis, the BjCRP1 protein was found to be expressed mainly in the supernatant, and separated at a position around 29Kda, which size is the same as expected. Using spectrophotometer we tested the salt tolerance ability of recombinant BL21 (pET32a- BjCRP1) and control BL21(pET32a).The results showed that over-expression of BjCRP1 markedly increased Cd tolerance and salt tolerance of BL21 compared with the control of empty vector transformed BL21 strain, and the critical concentration of CdCl2, NaCl resistance could up to 1.5mmol/L and 0.8mol/L. Furthermore, using semi-quantitative RT-PCR we analyzed the mRNA aboundance of BjCRP1 in the roots, stems and leaves of B. juncea seedlings, which were treated with CdCl2, ZnCl2, NaCl and PEG, respectively. The results showed that the mRNA transcriptional level of BjCRP1 was up-regulated quickly upon 0.2mmol/L CdCl2 treatment and reached its peak in the root after 4 hours and in the leaf after 4 hours. BjCRP1 also showed different kind of upregulation in response to zinc, salt and PEG stress.The results showed that BjCRP1 may take part in the physiological process of both heavy metal (Cd, Zn) and osmotic (NaCl, PEG) stress, and have a possible role in increasing plants integrate resistance to abiotic stress.Meanwhile, the eukaryotic expression vector pBI121- BjCRP1 containing CaMV 35S promoter was constructed and transformed into Arabidopsis thaliana by Agrobacterium- mediated flower dip method. Presently, we have got the T0 transformed A. thaliana seeds. This provides a basis for further characterizing the role of BjCRP1 gene in the plant development and resistance to abiotic stresses.