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中国农学通报

中国农学通报 ›› 2015, Vol. 31 ›› Issue (23): 53-57.doi: 10.11924/j.issn.1000-6850.casb15030104

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

杜氏盐藻MAPKK激酶基因DsMAPKKK的克隆、原核表达与纯化

柴晓杰,刘艺琼,王逸云,武天祥,刘丽颖   

  1. 大连海洋大学,大连海洋大学,大连海洋大学,大连海洋大学,大连海洋大学
  • 收稿日期:2015-03-13 修回日期:2015-05-21 接受日期:2015-06-08 出版日期:2015-08-20 发布日期:2015-08-20
  • 通讯作者: 柴晓杰
  • 基金资助:
    国家自然科学基金项目“杜氏盐藻PKC在盐胁迫信号传导中的功能研究”(31472260)；国家自然科学基金项目“盐藻中磷酸化转录因子对盐胁迫的分子响应”(30972240)。

Prokaryotic Expression, Purification and Cloning of MAPKK Kinase Gene DsMAPKKK from Dunaliella salina

  • Received:2015-03-13 Revised:2015-05-21 Accepted:2015-06-08 Online:2015-08-20 Published:2015-08-20

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摘要： 为了进一步研究MAPKK激酶的生理功能，利用RT-PCR技术扩增了DsMAPKKK的开放阅读框序列，并利用T4连接酶与质粒pGS21a连接，构建了原核表达载体pGS21a-DsMAPKKK。将该重组质粒导入大肠杆菌BL21(DE3)感受态细胞，经IPTG诱导成功表达了融合蛋白。经SDS-PAGE检测，融合蛋白在上清和包涵体中均存在。可溶性蛋白经过GST柱纯化，电泳分析结果表明，在68kD左右有单一的蛋白条带，说明融合蛋白得到有效纯化。蛋白印迹结果显示在68kD左右有明显的杂交条带，初步证明纯化的蛋白就是带有GST标签的MAPKK激酶。DsMAPKKK的成功表达与纯化，为深入探讨DsMAPKK激酶的性质及功能打下了坚实的基础。

关键词: 多年生黑麦草, 多年生黑麦草, 抗倒酯, 浸种, 出苗率, 幼苗生长, 生理特性

Abstract: In order to study the physiological function of MAPKKK in Dunaliella salina (DsMAPKKK), the open reading frame (ORF) of DsMAPKKK gene was amplified by RT-PCR. The prokaryotic expression vector pGS21a- DsMAPKKK was constructed by using T4 ligase to link the ORF of DsMAPKKK and the vector pGS21a. Then the recombinant plasmid was introduced into E. coli BL21 (DE3) and induced by IPTG. The fusion protein was expressed successfully in E. coli BL21. Detected by SDS-PAGE, the fusion protein had existed in both supernate and inclusion body. The soluble protein was purified through GST column, electrophoretic analysis showed that there was a single protein band at about 68 kD, indicating that the fusion protein had been effectively purified. The result of western blotting demonstrated that there were obvious hybridization bands at about 68 kD, preliminarily demonstrated that the purified protein was DsMAPKKK protein with a GST label. The successful expression and purification of DsMAPKKK laid a solid foundation for further exploration of the nature and function.