利用酵母表达体系研究大豆SALI3-2蛋白功能

以含有大豆Sali3-2cDNA的重组载体为模版,扩增出SALI3-2蛋白及其缺失信号肽的SIcDNA片段,构建酵母表达载体pYES2/S、pYES2/SI以及可表达SALI3-2-His6的pYES2/SH.空载体pYES2/ct和重组载体在转化酿酒酵母INVSC1后得到相应的酵母重组子INV/CK(对照菌)、INV/S、INV/SI和INV/SH.Westernblot实验结果表明,SALI3-2蛋白在酵母INVSC1中可被半乳糖诱导表达.对INV/CK、转基因的重组菌INV/S、INV/SI在无胁迫、含1.5mol/LNaCl或山梨醇培养基中的生长情况进行分析.结果表明,大豆SALI3-2蛋白及其缺失多肽SI的表达对无胁迫条件下酵母的生长没有影响,而在NaCl胁迫下可明显提高酵母转化子的耐盐能力.信号肽的缺失使SALI3-2蛋白的耐盐能力有所下降,可见信号肽对该蛋白耐盐功能的发挥有重要作用.另外,SALI3-2蛋白及其缺失多肽SI的表达不能提高酵母重组子的抗渗透能力,表明SALI3-2蛋白对细胞不具有明显的渗透胁迫调节作用.

Functional analysis of soybean SALI3-2 in yeast

Soybean SALI3-2 protein is a member of the BURP-domain family. In this study,the full-length cDNA of SALI3-2 protein and its signal sequence-deleted fragment were cloned into the yeast expression plasmid pYES2/ct to construct the pYES2/S for the expression of SALI3-2,the pYES2/SH for the expression of SALI3-2-His6,and the pYES2/SI for the expression of the C-terminal of SALI3-2 without signal peptide. These constructed vectors as well as the control vector pYES2/ct were respectively transformed into yeast INVSC1 to create recombinants INV/S,INV/SH,INV/SI and the control INV/CK. Western blot analysis revealed that the expression of SALI3-2 could be induced by galactose in the recombinants harboring Sali3-2. Growth curves of all yeast recombinant were analyzed in condition of non-stress,high salinity (1.5 mol/L NaCl) and osmotic (2.2 mol/L sorbitol) stress. Results showed that the expression of SALI3-2 or its deleted peptide SI did not influence the yeast growth in the condition without any stress but could elevate the salt tolerance of yeast under 1.5 mol/L NaCl stress. Signal peptide deletion weakened the salt-tolerant function of SALI3-2. It indicats that the signal peptide is important for the function of this protein. The expression of SALI3-2 protein or its deleted peptide SI could not enhance the osmotic stress-tolerance of yeast transformants. It suggests that SALI3-2 protein can not help cells to counteract osmotic stress.