小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化

doi:10.3969/j.issn.1000-7091.2013.02.001

华北农学报 ›› 2013, Vol. 28 ›› Issue (2): 1-5. doi: 10.3969/j.issn.1000-7091.2013.02.001

所属专题： 小麦；生物技术；

• 论文 • 下一篇

小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化

付振艳¹, 张正斌², 王晓军¹, 徐萍²

1. 中国科学院新疆理化技术研究所, 新疆乌鲁木齐 830011;
2. 中国科学院遗传与发育生物学研究所农业资源中心, 河北石家庄 050021

收稿日期:2013-01-16 出版日期:2013-04-28
通讯作者: 张正斌(1962－)，男，陕西合阳人，研究员，博士，主要从事植物抗旱遗传改良研究。
作者简介:付振艳(1981－)，女，河南林州人，副研究员，博士，主要从事植物抗旱遗传改良研究。
基金资助:
国家“973”项目(2010CB951501);中国科学院知识创新重要方向性项目(KSCX2-EW-N-02;KSCS2-EW-J-5);中国科学院“西部博士资助项目”(XBBS200914);新疆维吾尔自治区自然基金会项目(2011211B507)

The Fusion-expression of Wheat TaNADP-ME1gene in E．coli and Purification of Fusion Protein

FU Zhen-yan¹, ZHANG Zheng-bin², WANG Xiao-jun¹, XU Ping²

1. The Xinjiang Technical Institute of Physics Chemistry, Chinese Academy of Sciences, Urumqi 830011, China;
2. Center for Agricultural Resources Research, Institute ofgenetic Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China

Received:2013-01-16 Published:2013-04-28

摘要/Abstract

摘要： NADP依赖的苹果酸酶(NADP-ME)是C4光合途径关键酶。为了确定TaNADP-ME1基因的功能,利用重组技术将前期克隆到的TaNADP-ME1基因构建到原核表达载体pET32a,双酶切和PCR鉴定阳性克隆,CaCl2法转化大肠杆菌BL21(DE3)pLysS,IPTG诱导融合蛋白表达,Ni2+-NTA琼脂糖亲和层析柱纯化融合蛋白。成功获得了重组原核表达载体pETE1,TaNADP-ME1基因在BL21(DE3)pLysS中得到了融合表达,SDS-PAGE表明,融合蛋白分子量为80 kDa,并成功纯化到融合蛋白。

关键词: 小麦, TaNADP-ME1基因, 融合表达, 蛋白纯化

Abstract: NADP-dependent malic enzyme(NADP-ME)is a key enzyme in C4photosynthesis,the objective is to construct the TaNADP-ME1gene into prokaryotic expression vector,express fusion protein in E. coli and purify the fusion protein. The TaNADP-ME1gene was constructed into expression vector pET32a by recombination technolo-gy,recombination plasmid was identified by digestion with restriction enzymes and PCR amplification,and trans-formed into BL21(DE3)pLysS by CaCl2method,fusion protein was induced by IPTG and purified by Ni2+-NTA agarose column. This research successfully acquired the recombination vector pETE1,and TaNADP-ME1gene was accurately expressed in BL21(DE3)pLysS,SDS-PAGE revealed that the molecular weight of purified fusion protein was about 80 kDa and successfully acquired the fusion protein. This study laid agood foundation for identification thegene function of TaNADP-ME1gene.

Key words: Wheat, TaNADP-ME1gene, Fusion expression, Protein purification

中图分类号:

付振艳, 张正斌, 王晓军, 徐萍. 小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化[J]. 华北农学报, 2013, 28(2): 1-5. doi: 10.3969/j.issn.1000-7091.2013.02.001.

FU Zhen-yan, ZHANG Zheng-bin, WANG Xiao-jun, XU Ping. The Fusion-expression of Wheat TaNADP-ME1gene in E．coli and Purification of Fusion Protein[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(2): 1-5. doi: 10.3969/j.issn.1000-7091.2013.02.001.

http://www.hbnxb.net/CN/Y2013/V28/I2/1

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小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化

The Fusion-expression of Wheat TaNADP-ME1gene in E．coli and Purification of Fusion Protein

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小麦TaNADP-ME1基因在大肠杆菌中的融合表达及可溶蛋白纯化

The Fusion-expression of Wheat TaNADP-ME1gene in E．coli and Purification of Fusion Protein

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