Clone And Function Analysis Of Two Copper-Binding Protein Gene In Oryza Sativa

People paid close attention to soil pollution by heavy metals.Copper is a microelement on growth of plant.It takes part in a variety of enzymatic reactions as a cofactor.However,excess copper causes toxic to plants.Plants gradually obtain a series of important mechanism to adapt to metal stress.Protein disulfide isomerase and E2 ubiquitin conjugating enzyme SPM2 were identified as Cu-binding protein in rice.We focus on the function of these two Cu-binding protein in rice under copper(Cu)and cadium(Cd)stress.According to the two sequence(Genbank accession AAX85991.1,ABA99827.1),we designed a pair of degenerate oligonucleotide primers used to amplify OsPDIL1-1 and OsSpm2 from Oryza sativa by RT-PCR.The OsPDIL1-1 contains 1539 bp coding sequence which encode a putative peptide of 512 amino acids.The OsSpm2 contains 441 bp coding sequence which encode a putative peptide of 146 amino acids.Their bioinformatics analysis showed that the acid sequence encoded by OsPDILl-1 and OsSpm2 are highly conserved.Meanwhile,both of them have high homology with Monocotyledons.On this basis,tissue expression and induced expression by heavy metals of OsPDIL1-1 and OsSpm2 in the members of rice were analyzed by real-time quantitative PCR.The tissue expression analysis found that OsPDIL1-1 and OsSpm2 was expressed in the roots,shoots and leaves.OsPDILl-1 expression was higher in the roots and lower level in the shoots,the lowest expression in leaves.OsSpm2 expression was higher in the roots and at a low expression level in the shoots and leaves.The induced expression analysis by copper and cadmium show that heavy metals induced the expression of OsPDIL1-1 and OsSpm2 in the roots.But there were different influences on time variation.The expression of PDIL1-1 was sustainable increased in roots in the presence of 10 pmol L-1 CuSO4 or 50 μmol L-1 CdCl2 in 24 hours.OsSpm2 was very sensitive to Cd and Cu toxicity in the roots.The expression of OsSpm2 increased within a short period of time,but the expression gradually recovered with time goes on.Moreover,we found that the pretreatment of DMTU and DPI can significantly decreased the expression of OSPDILJ-1 in the roots under heavy metals stress.In order to study the growth of transgenic E.coli under heavy metals stress,we cloned OSPDIL1-1 into the prokaryotic expression vector pET-30a.The recombinant plasmid pET-30a-OsPDIL1-1 were transformed into E.coli BL21 and then induced to express by IPTG The growth of recombinants were better than that of controls under various stress.The results showed that adaptability of recombinants were enhanced by OSPDIL1-1,which let them gain tolerance to Cu and Cd.