大豆PM2蛋白(LEA3)表达可提高酵母重组子的耐盐性

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

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INV/PM2在无胁迫、和含1.8 mol/LNaCl2、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。

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 the control. This suggests that the expression yeast. But no obvious growth improvement was medium containing high salinity as compared to of PM2 protein could confer salt tolerance in observed in a high sorbitol medium between the recombinant INV/PM2 and INV/pYES2.