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作物学报 ›› 2011, Vol. 37 ›› Issue (01): 79-86.doi: 10.3724/SP.J.1006.2011.00079

• 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 γ-醇溶蛋白基因的克隆、原核表达与功能鉴定

王明霞1,高翔1,2,*,陈其皎1,2,*,董剑1,2,赵万春1,2,李艳亮1,李敏1   

  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 / 陕西省小麦新品种培育工程研究中心，陕西杨凌 712100
  • 收稿日期:2010-05-19 修回日期:2010-08-02 出版日期:2011-01-12 网络出版日期:2010-10-09
  • 基金资助:

    本研究由国家自然科学基金青年科学基金(30900896), 陕西省“13115”科技创新工程重大项目(2007ZDKG-01), 现代农业产业技术体系建设专项(NYCYTX-001)和西北农林科技大学基本科研业务费青年项目(QN2009007)资助。

Cloning, Prokaryotic Expression, and Functional Testing of a γ-Gliadin Gene from Wheat Cultivar Shaan 253

WANG Ming-Xia1,GAO Xiang1,2,*,CHEN Qi-Jiao1,2,*,DONG Jian1,2,ZHAO Wan-Chun1,2,LI Yan-Liang1,LI Min1   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100; 2 Wheat Engineering Research Center of Shaanxi Province / New varieties cultivation of wheat Engineering Research Center of Shaanxi Province; Yangling 712100, China
  • Received:2010-05-19 Revised:2010-08-02 Published:2011-01-12 Published online:2010-10-09

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被引次数

6

摘要： 利用设计合成的特异γ-醇溶蛋白基因引物，采用PCR方法从小麦品种陕253克隆获得一个γ-醇溶蛋白基因(GenBank登录号GQ857626)。序列分析表明，该基因编码产物的II区由于碱基转换产生一个额外的半胱氨酸残基。构建了GQ857626的原核表达载体并转入表达菌株E. coli Rosetta gami B(DE3)，IPTG诱导其成功表达。使用HisTrap HP组氨酸标记亲和层析柱纯化该基因的表达产物，并使用4 g粉质仪分析其功能。结果显示，将此纯化蛋白通过氧化还原反应整合到基础面粉后，面团的形成时间缩短，稳定时间减少，弱化度增加，导致主要的粉质指数明显下降，说明该亚基对面团流变学性质整体表现不利。

关键词: 普通小麦, γ-醇溶蛋白, 原核表达, 粉质参数

Abstract: The γ-gliadin gene (GenBank accession number GQ857626) was isolated from a wheat (Triticum aestivum) cultivar with a hight quality Shaan 253, using inductuion a set of genome specific primers. The deduced amino acid sequence of GQ857626 exhibited an additional cysteine residue in the region II of mature γ-gliadin subunits compared to the previous report. A prokaryotic expression vector was constructed to express GQ857626 in the host bacterium Escherichia coli Rosetta-gami B (DE3). The SDS-PAGE and Western blot analysis confirmed that the fusion protein was expressed by induction of 0.1 mmol L-1 isopropyl-β-D-thiogalactoside (IPTG), and its actual molecular weight was about 50 kD nearly equal to the predicted. After separation and purification using HisTrap HP affinity chromatography, the function of fusion protein was tested by means of micro 4 g farinograph. When the subunit of GQ857626 was integrated into the control flour through oxidation-reduction reaction, many important farinograph parameters changed drastically, such as development time, dough stabilizing time, degree of softening. Although the gluten strength of the control flour could be improved in some degree, the subunit of GQ857626 showed a negative effect on the rheological properties of wheat flour.

Key words: Wheat, γ-gliadin, Prokaryotic expression, Farinograph parameter

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