小麦<em>TaGAPC</em>定点突变体基因载体构建及其原核表达

doi:10.7668/hbnxb.2016.01.008

华北农学报 ›› 2016, Vol. 31 ›› Issue (1): 46-50. doi: 10.7668/hbnxb.2016.01.008

所属专题： 小麦；生物技术；

小麦TaGAPC定点突变体基因载体构建及其原核表达

郭子平¹, 翟清华¹, 曾令锋¹, 邓西平², 杨淑慎¹

1. 西北农林科技大学生命科学学院, 陕西杨凌 712100;
2. 西北农林科技大学黄土高原土壤侵蚀与旱地农业国家重点实验室, 陕西杨凌 712100

收稿日期:2015-11-27 出版日期:2016-02-28
通讯作者: 杨淑慎(1956-),女,陕西黄陵人,教授,博士,主要从事植物抗逆生理、植物细胞培养工程方面的研究。
作者简介:郭子平(1989-),男,河南商丘人,在读硕士,主要从事植物逆境细胞分子生物学研究。
基金资助:
国家自然科学基金项目(31271625);黄土高原土壤侵蚀与旱地农业国家重点实验室专项(10502)

Construction of TaGAPC Site-directed Mutagenesis Gene Vector and Prokaryotic Expression Analysis from Wheat

GUO Ziping¹, ZHAI Qinghua¹, ZENG Lingfeng¹, DENG Xiping², YANG Shushen¹

1. College of Life Sciences, Northwest A & F University, Yangling 712100, China;
2. State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau, Northwest A & F University, Yangling 712100, China

Received:2015-11-27 Published:2016-02-28

摘要/Abstract

摘要： 为了研究催化活性半胱氨酸Cys¹⁵⁴、Cys¹⁵⁸残基对小麦细胞质甘油醛-3-磷酸脱氢酶(TaGAPC)蛋白酶活性的影响,利用重叠延伸PCR法分别将这2个位点的半胱氨酸突变成丝氨酸,并分别连入pET28a(+)原核表达载体。在25℃条件下, TaGAPC及其定点基因TaCys154S/TaCys158S经0.25 mmol/L IPTG诱导后高效表达,SDS-PAGE电泳检测结果显示,目标重组蛋白均为可溶性且大小约为40 kDa,与预测结果一致。在此基础上,经超声波破菌及镍柱纯化获得3种纯化重组蛋白。这为后续TaGAPC及其定点突变体蛋白酶活性测定及活性位点分析提供试验材料。

关键词: TaGAPC, 重叠延伸PCR, 定点突变, 原核表达, 蛋白纯化

Abstract: In order to elucidate the roles of catalytic active Cys¹⁵⁴,Cys¹⁵⁸ in Triticum aestivum L.cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (TaGAPC),these two cysteins were site-directed mutated into serine by overlap-extension PCR.Then recombinant prokaryotic expression vectors of TaGAPC and its mutants TaCys154S/TaCys158S were constructed and transformed into E.coli BL21.The recombinant protein were induced by 0.25 mmol/L IPTG at 25℃ and subsequently purified by Ni²⁺-IDA column.These researches could lay the foundation for the enzyme activity assay of TaGAPC and its mutants TaCys154S/TaCys158S and cysteine active sites.

Key words: TaGAPC, Overlap-extension PCR, Site-directed mutagenesis, Prokaryotic expression, Protein purification

中图分类号:

Q78
S512.03

郭子平, 翟清华, 曾令锋, 邓西平, 杨淑慎. 小麦TaGAPC定点突变体基因载体构建及其原核表达[J]. 华北农学报, 2016, 31(1): 46-50. doi: 10.7668/hbnxb.2016.01.008.

GUO Ziping, ZHAI Qinghua, ZENG Lingfeng, DENG Xiping, YANG Shushen. Construction of TaGAPC Site-directed Mutagenesis Gene Vector and Prokaryotic Expression Analysis from Wheat[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(1): 46-50. doi: 10.7668/hbnxb.2016.01.008.

http://www.hbnxb.net/CN/Y2016/V31/I1/46

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