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作物学报 ›› 2009, Vol. 35 ›› Issue (4): 672-678.doi: 10.3724/SP.J.1006.2009.00672

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦品种“陕253”低分子量谷蛋白亚基基因的克隆及原核表达

吴丹1，高翔12*，于旭1，董剑12，赵万春12，陈其皎12，庞红喜12，李哲清12   

  1. 1西北农林科技大学农学院，陕西杨凌712100；2陕西省小麦工程技术研究中心，陕西杨凌712100
  • 收稿日期:2008-09-08 修回日期:2008-12-12 出版日期:2009-04-12 网络出版日期:2009-02-16
  • 通讯作者: 高翔
  • 基金资助:

    本研究由陕西省“13115”科技创新工程重大项目（2007ZDKG-01）；农业部小麦现代产业技术体系建设陕西小麦综合试验重大项目（NYCYTX-001）资助。

Cloning and Prokaryotic Expression of a Low Molecular Weight Glutenin Gene from Wheat Variety"Shaan 253"

WU Dan1,GAO Xiang12*,YU Xu1,DONG Jian12,ZHAO Wan-Chun12,CHEN Qi-Jiao12,PANG Hong-Xi12,LI Zhe-Qing12   

  1. 1College of Agronomy,Northwest A&F University,Yangling 712100,China;2Wheat Engineering Research Center in Shaanxi Province,Yangling 712100,China
  • Received:2008-09-08 Revised:2008-12-12 Published:2009-04-12 Published online:2009-02-16
  • Contact: GAO Xiang

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2154

被引次数

15

摘要：

利用LMW-GS特异引物, 从强筋小麦品种“陕253”中克隆了1个1 498 bp的片段(GenBank登录号为FJ172533)，该片段包含全长为912 bp的低分子量谷蛋白亚基的完整编码序列。经比较推导氨基酸序列的同源性，发现该基因属于Glu-D3位点编码低分子量谷蛋白亚基的基因，编码产物N-端具有LMW-m型低分子量谷蛋白亚基的典型特征，系统演化分析也支持这一结果。构建了该基因的表达载体pET32a-GluD3-S253，在宿主菌E. coli Rosetta-gami B (DE3)中经IPTG诱导表达融合蛋白。SDS-PAGE和Western blot检测表达产物，证实融合蛋白表达成功。

关键词: 小麦, 低分子量谷蛋白亚基, 基因克隆, 融合蛋白, 原核表达

Abstract:

Low-molecular-weight glutenin subunits (LMW-GS) play an important role in the determination of flour viscoelastic properties in wheat (Triticum aestivum L.). LMW-GS has large polymorphism and variation in molecular size, thus, it is difficult to be isolated using one-dimensional electrophoresis. Shaan 253 is a wheat variety with characteristics of high yield and early maturity, especially, with elite high-molecular-weight glutenin subunits, such as 5+10 on 1D, 14+15 and 20 on 1B, and 1 on 1A. The purpose of this study was to understand the contribution of LMW-GS to the processing quality in Shaan 253. Using a pair of specific primers of LMW-GS and pMD19-T vector, one DNA fragment of 1 498 bp (GenBank accession No. FJ172533) was obtained from Shaan 253. The fragment contained the complete coding sequence of 912 bp and encoded 304 amino acid residues. According to sequence analysis, this gene was involved in Glu-D3 loci, and had high similarities to other known LMW-GS genes with the highest identity of 99.34%. Deduced amino acid sequence showed there were typical features of LMW-m type in N-terminal region, which was confirmed by the phylogenetic analysis. The expression vector of pET32a-GluD3-S253 was constructed and transformed into the host bacteria Escherichia coli Rosetta-gami B (DE3). The expression product was testified using SDS-PAGE and Western-blot, indicating that the fusion protein was successfully expressed.

Key words: Wheat, Low-molecular-weight glutenin subunits(LMW-GS), Gene cloning, Fusion protein, Prokaryotic expression

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