摘要： 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cDNA为模板,将其构建到带有GST表达标签的原核表达载体pGEX-6p-1中,构建的重组载体pGEX-6p-ZmCen转化至大肠杆菌BL21(DE3),通过0.3mmol/LIPTG在27℃下诱导表达20h,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PreScission 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19.78kDa,等电点为4.78。采用maizeGDB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。

关键词: Zmcen基因克隆, 表达, 纯化, 生物信息学

Abstract: To study the characteristics and biological function of Zmcen gene,investigate the protein structure of Zea mays L.,the ORF of maize Zmcen gene was cloned into the expression vector pGEX-6p-1 through PCR method,and the GST recombined expression vector pGEX-6p-Zmcen was constructed.Recombinant GST-ZmCen fusion protein was expressed as soluble protein in Escherichia coli BL21 (DE3) after induction with 0.3 mmol/L IPTG at 27 ℃ for 20 h.The ZmCen protein with the GST affinity chromatography and verified the protein by 12% SDS-PAGE and Western Blotting with anti-GST antibody were purified.Fusion protein was cut out the GST-Tag by PPase and was purified.And by the method of biological information,the results showed that the Zmcen gene was located on the 7^# chromosome,and the full-length gene had 6 exons and 7 introns.The ZmCen protein domain search and alignment analysis were conducted by the maizeGDB resources,and the ZmCen was EF-hand domain family protein.The CDS region of Zmcen gene was 519 bp,encoding 172 amino acid with a molecular weight of 19.78 kDa and pI of 4.78.Using maizeGDB database searching,the expression level analysis showed that Zmcen gene expression had a higher level in cells divided vigorous of the entire maize life cycle.The evolutionary history of centrins was analyzed by means of phylogenetic analysis in plants.The centrin gene was a highly 80.21% homology among centrins of 16 different plants.The results of the research can provide a theoretical basis for exploring the unknown biological function of ZmCen protein.

Key words: Zmcen gene cloning, Expression, Purification, Bioinformatics

中图分类号: 

• S512

• Q71

• Q78