矮化大豆GmEXPA5基因的克隆与表达分析

摘要

以矮化大豆HK808的顶芽cDNA为模板,根据植物α-膨胀素基因保守区设计简并引物,并克隆保守区片段,结合RACE技术克隆得到一个新的膨胀素全长基因,将其命名为GmEXPA5(登录号为JN207916.1),该基因全长1233bp,其中开放阅读框636bp,编码211个氨基酸;推导的氨基酸序列含有两个膨胀素保守域domain1(ex-pansin EG45)和domain2(expansin CBD),无明显的信号肽序列,属于α膨胀素亚家族.构建pEASY-Blunt E1 -GmEXPA5重组质粒,获得稳定的原核表达体系,IPTG诱导后SDS-PAGE结果表明所表达蛋白与预期蛋白大小一致.实时荧光定量PCR(real-time PCR)表达分析表明GmEXPA5基因在矮化大豆HK808与野生型大豆东农42两种材料的不同器官中均有表达,而在野生型茎尖及茎部的表达量显著高于矮化大豆,推测该基因与大豆矮化性状的形成有一定的关系.%Using cDNA of shoot apical meristem in dwarf soybean-HK808 as template,degenerated primers were designed accoding to conserved domain of α-expansin gene of plant,a new full-length gene named GmEX-PA5(accession number is JN207916.1)was isolated by RT-PCR and rapid amplification of cDNA ends(RACE). The sequence analysis showed that GmEXPA5 had a full length of 1 233 bp containing the open reading frame(636 bp),which encoded 211 amino acid with two conserved domain,domain1(expansin EG45)and domain2(expansin CBD). Without obvious signal peptide,GmEXPA5 could be from α-expansin subfamily by alignment analysis of deduced amino acid. The pEASY-Blunt E1-GmEXPA5 recombinant plasmid was constructed and the stable pro-karyotic expression system was obtained. After induced by IPTG,the analysis of SDS-PAGE gel electrophoresis showed that the relative molecular weight of the induced protein was 26 kD,which was consistent with its theoreti-cal value. Real-time quantitative PCR analysis revealed that GmEXPA5 was expressed in all different organs but the expression levels in wild type was significantly higher than that of HK808 in shoot tips and stems. It suggested that GmEXPA5 play an unique role in the formation of dwarf phenotype.