大豆Glyma08g11030基因结构及原核表达分析

Structure Characteristics and Prokaryotic Expression Analysis of Soybean Glyma08g11030 Gene

为了进一步明确Glyma08g11030基因的功能,利用生物信息学和PCR的方法,对Glyma08g11030进行序列分析及蛋白结构域分析。生物信息学分析表明,该基因cDNA全长1419bp,开放阅读框长1362bp,编码453个氨基酸,蛋白质分子质量约为51.288ku,等电点为8.14。序列分析表明,Glyma08g11030基因组包含2个外显子和1个内含子。多序列比对结果表明,Glyma08g11030蛋白含有1个F-BOX保守域。进化树分析表明,该蛋白与木豆、菜豆、花生、豇豆、红小豆具有同源关系。为了获得纯化的Glyma08g11030蛋白,将扩增片段克隆至原核表达载体pET28a,构建重组质粒pET28a-Glyma08g11030,经过SDS-PAGE电泳检测获得了目的蛋白,结果表明,该蛋白主要以包涵体形式存在。为进一步纯化和鉴定Glyma08g11030蛋白及研究其功能奠定了基础。

In order to further clarify the function of Glyma08g11030 gene,sequence analysis and protein domain analysis of Glyma08g11030 were performed by means of bioinformatics and PCR. Bioinformatics analysis showed that the full-length of Glyma08g11030 cDNA was1 419 bp,with an open reading frame of1 362 bp,encoding 453 amino acids. The molecular weight of the protein was about 51.288 ku,and the isoelectric point was 8.14. Sequence analysis indicated that the Glyma08g11030 genome contained two exons and one intron. Multiple sequence alignment revealed that the Glyma08g11030 protein contained one F-BOX conserved domain. Phylogenetic tree analysis showed that the protein had a homologous relationship with pigeonpea,kidney bean ,peanut,cowpea and adzuki bean. In order to obtain the purified Glyma08g11030 protein,the amplified fragment was cloned into the prokaryotic expression vector pET28a,and the recombinant plasmid pET28a- Glyma08g11030 was constructed. The target protein was obtained by SDS-PAGE. The results showed that the protein mainly existed in the form of inclusion bodies. The results of this study laid the foundation for further purification and identification of Glyma08g11030 protein and investigation of its function.