大豆PM2蛋白11氨基酸结构域的耐盐功能鉴定

大豆PM2蛋白属LEA3（late embryogenesis abundant group3）蛋白.以含大豆PM2基因的重组载体为模板,PCR法扩增出可编码6拷贝11-氨基酸重复序列的基因片段PM2D.构建pET28a/PM2D重组载体,并转化大肠杆菌得到重组菌BL21/PM2D.SDS-PAGE电泳结果表明,BL21/PM2D重组菌可被诱导表达相对分子质量约为15 000的蛋白条带,与目的蛋白的理论相对分子质量（13 600）接近.利用DNASIS软件分析,PM2D缺失短肽的亲水指数为1.12.将重组菌BL21/PM2和BL21/PM2D分别涂布在含500 mmol/L NaCl和1 200 mmol/L 山梨糖的固体培养基上,计算重组菌的存活率.结果显示,两种重组菌在含500 mmol/L NaCl培养基上的存活率分别为30.8%和26.0%,均高于对照菌（BL21/pET28a）的存活率;在含1 200 mmol/L山梨糖培养基上的存活率分别为59.7%和59.3%,与对照BL21/pET28a菌株的存活率相比无明显差异.PM2D短肽长度为PM2蛋白全长的1/4,但BL21/PM2D重组菌的耐盐能力为BL21/PM2重组菌的80%左右.表明11-氨基酸重复序列区是PM2蛋白序列的一个耐盐结构域.

11-mer repeating region in soybean PM2 protein enhances salt tolerance of Escherichia coli

Soybean PM2 protein belongs to the family of group 3 LEA(late embryogenesis abundant)protein,which contains the conserved 11-mer repeating motif. Nucleotide acid fragment PM2D encoding six copies of 11-aa repeating motif(aa292 ~ 404)was amplified by PCR reaction. The prokaryotic expression plasmid of pET28a/PM2D was constructed and then transformed into Escherichia coli strain to create recombinant BL21/PM2D. The strains containing empty vector of pET28a and full-length PM2 gene were obtained as control,respectively. Total protein was isolated from the lysis of recombinant strain induced by IPTG. The result of SDS-PAGE analysis showed that a specific protein band of 15 000 appeared on the gel and it was close to the theoretic molecular weight(13 6 00)of PM2D polypeptide. This indicated that PM2D polypeptide could be expressed in recombinant BL21/PM2D. The hydrophobicity index of PM2D polypeptide was calculated to be 1.12 by DNASIS software. On the medium supplemented with 500 mmol/L NaCl,the survival ratio of recombinant BL21/PM2 and BL21/PM2D was 30.8% and 26.0%,respectively,which were higher than 11.4% of BL21/pET28a. While on the medium containing 1 200 mmol/L sorbitol,the survival ratio of recombinant BL21/PM2 and BL21/PM2D was 59.7% and 59.3%,respectively,showing no obvious difference compared to 54.9% of BL21/pET28a as control. PM2D polypeptide is only a quarter of PM2 protein in length,but the survival ratio of BL21/PM2D can reach to about 80% of BL21/PM2. All these results indicate that 11-mer repeating region is a functional region with salt-tolerance in PM2(LEA3)protein.