大豆PM2蛋白(LEA3)表达可提高酵母重组子的耐盐性

大豆PM2蛋白属LEA3(late embryogenesis abundant group3)蛋白.本实验以含大豆PM2基因的重组载体pET28a/PM2为模板,PCR法构建酵母表达载体pYES2/PM2,并转化酵母细胞得到重组菌INV/PM2.SDS-PAGE电泳结果表明,INV/PM2重组菌可被诱导表达分子量约为50 kDa的蛋白条带,与目的蛋白的理论分子量(50 kDa)接近.利用液相色谱-电喷雾质谱对酵母重组子表达的重组蛋白进行分析鉴定.分别测定未转基因重组菌INV/pYES2和转PM2基因的重组菌烈V/PM2在无胁迫、和含1.8 mol/LNaCl、2 mol/L山梨糖培养基中的生长曲线.结果表明大豆PM2蛋白的表达对酵母正常生长没有影响.在含高盐的培养基里,转PM2基因酵母INV/PM2的延滞期短于对照菌.这表明PM2蛋白的表达可以提高酵母细胞的耐盐能力.但是在含山梨糖的高渗培养基里,两种菌的生长情况无明显差异.

EXPRESSION OF SOYBEAN MP2 PROTEIN(LEA3)CONFERS SALT TOLERANCE IN SACCHAROMYCES CEREVISIAE

Soybean PM2 protein belongs to the family of group 3 LEA(late embryogenesis abundant)proteins.Nucleotide acid fragment of PM2 gene was amplified by PCR reaction using the plasmid pET28a/PM2 as the template.The yeast expression plasmid of pYES2/PM2 was constructed and then transformed into yeast to create recombinant INV/PM2.The results of SDS-PAGE analysis showed that a specific protein band of 50 kDa was expressed in the recombinant yeast of INV/PM2.The protein was close to the theoretic molecular weight of PM2 protein and was identified as PM2 protein by LC-ESI-MS assay.The growth curves of recombinant INV/PM2 and INV/pYES2 with an empty vector were generated under the non-stress,high salinity(1.8mol/L NaCl) or osmotic(2mol/L sorbitol) conditions,respectively.The growth rates of the recombinants with PM2 protein under the non-stress condition indicated that the expression of PM2 protein is not deleterious to the growth of yeast.The yeast cells expressing PM2 protein showed shorter lag period when transferred to a medium containing high salinity as compared to the control.This suggests that the expression of PM2 protein could confer salt tolerance in yeast.But no obvious growth improvement was observed in a high sorbitol medium between the recombinant INV/PM2 and INV/pYES2.