论文部分内容阅读
为研究玉米Zmcen基因的特征及生物学功能,并对其编码蛋白结构与功能进行分析。以玉米c DNA为模板,将其构建到带有GST表达标签的原核表达载体p GEX-6p-1中,构建的重组载体p GEX-6p-ZmCen转化至大肠杆菌BL21(DE3),通过0.3 mmol/L IPTG在27℃下诱导表达20 h,获得了可溶性的GST融合蛋白。采用GST亲和层析柱对重组蛋白进行纯化,经12%SDS-PAGE电泳和GST标签抗体Western Blotting检测,鉴定为含有GST-Tag的融合蛋白,纯化的蛋白经Pre Scission Protease(PPase)过夜酶切切除GST标签,得到较纯的ZmCen蛋白;同时利用生物信息学的方法,解析ZmCen蛋白质的结构特征。结果表明,该基因定位于玉米第7#染色体上,全长基因含有6个外显子和7个内含子,519 bp的开放阅读框,编码172个氨基酸,分子量约为19.78 k Da,等电点为4.78。采用maize GDB数据库对玉米整个生命周期的表达水平进行分析表明,细胞分裂旺盛的部位,Zmcen基因的表达量较高。对16种植物进行系统发育树分析显示,Zmcen基因与水稻、小麦、拟南芥等的centrin基因同源性高达80.21%,该结果为探索Zmcen蛋白的未知生物学功能提供了线索。
In order to study the characteristics and biological functions of Zmcen, the structure and function of the encoded protein were analyzed. The recombinant plasmid pGEX-6p-ZmCen was transformed into E.coli BL21 (DE3) by using c DNA of maize as a template and was constructed into prokaryotic expression vector pGEX-6p-1 with GST expression tag. / L IPTG induced expression at 27 ℃ for 20 h, a soluble GST fusion protein was obtained. The recombinant protein was purified by GST affinity chromatography and was identified as GST-Tag fusion protein by 12% SDS-PAGE electrophoresis and Western Blotting with GST tag. The purified protein was purified with PreScission Protease (PPase) overnight enzyme The GST tag was excised and the pure ZmCen protein was obtained. At the same time, the structural characteristics of ZmCen protein were analyzed by bioinformatics method. The results showed that the gene was located on chromosome 7 of maize. The full length gene contained 6 exons and 7 introns, and contained a 519 bp open reading frame encoding 172 amino acids with a molecular weight of 19.78 kDa. The power point is 4.78. The maize GDB database was used to analyze the expression level of maize in the whole life cycle. It showed that Zmcen gene was highly expressed in the cells with strong cell division. Phylogenetic tree analysis of 16 species of plants showed that centrin gene homology between Zmcen gene and rice, wheat and Arabidopsis was as high as 80.21%. This result provides a clue to explore the unknown biological function of Zmcen protein.