小麦品种陕253γ-醇溶蛋白基因的克隆、原核表达与功能鉴定

摘要

The γ-gliadin gene (GenBank accession No.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.%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登录号为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.