| 玉米Zmcen基因的克隆、表达与生物信息学分析 |
| |
| 引用本文： | 雷海英,白凤麟,刘建霞,王志军.玉米Zmcen基因的克隆、表达与生物信息学分析[J].华北农学报,2016(3):18-24. |
| |
| 作者姓名： | 雷海英 白凤麟 刘建霞 王志军 |
| |
| 作者单位： | 1. 长治学院 生物科学与技术系,山西 长治,046011;2. 山西大同大学 农学与生命科学学院,山西 大同,037009;3. 长治学院 化学系,山西 长治,046011 |
| |
| 基金项目： | 国家自然科学基金项目(21201024)；山西省自然科学基金项目(2012021009-1)；山西省科技攻关项目(20140311005-3) |
| |
| 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。
|
| 关 键 词： | Zmcen 基因克隆 表达 纯化 生物信息学 |
Cloning,Expression and Bioinformatics Analysis of Maize Zmcen Gene |
| |
| 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 meth-od,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 a-lignment analysis were conducted by the maizeGDB resources,and the ZmCen was EF-hand domain family protein. The CDS region of Zmcen gene was 5 19 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 ana-lyzed 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. |
| |
| Keywords: | Zmcen gene cloning Expression Purification Bioinformatics |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
