Cloning, prokaryotic expression and in vitro functional analysis of α-gliadin gene from common wheat | 普通小麦中α-醇溶蛋白基因(GQ891685)的克隆、表达及品质效应鉴定

Chinese. 【目的】利用E. coli体外大量高效表达带有1个额外半胱氨酸残基的α-醇溶蛋白,经Ni+-NTA柱纯化获得目标蛋白,通过氧化-还原反应将其整合到基础面粉中,利用粉质仪研究其对面团流变学特性的影响,以确定该基因表达产物的加工品质效应。【方法】根据α-醇溶蛋白基因编码区保守序列设计引物,从小麦品种陕253中克隆到1条含α-醇溶蛋白基因编码区的目的片段,长度为1 243 bp(GenBank登录号GQ891685),构建了该基因的原核表达载体pET32a-S253-Agli,在大肠杆菌E. coli BL 21(DE3)中经IPTG诱导表达,对表达的蛋白进行亲和层析及低温冷冻干燥回收、纯化,通过微量掺粉试验,在4 g粉质仪上测定其对面团流变学特性的影响。【结果】该基因片段包含900 bp的完整编码序列及部分5′-调控元件;Uniq domainⅡ区第6位氨基酸密码子C→G的转换,导致丝氨酸Ser→半胱氨酸Cys的变化,这1额外的半胱氨酸将有可能参与形成1个分子间二硫键;用IPTG诱导表达,经SDS-PAGE及Western blotting检测,证实融合蛋白表达成功并主要以包涵体形式存在;粉质结果表明,添加S253-Agli蛋白虽然能够增加面团的带宽及机械耐力系数,却因显著缩短面团稳定时间及形成时间而对面团的粉质参数产生了总体的负面效应。【结论】具有1个额外半胱氨酸残基的α-醇溶蛋白GQ891685增强了面筋弹性,但降低了面筋强度。

English. 【Objective】 An α-gliadin gene, with an additional cysteine residue, was cloned and expressed in E. coli system. The target protein was purified by Ni+-NTA column, and then integrated into the control flour to understand the potential quality of this α-gliadin gene. 【Method】 A DNA fragment with 1 243 bp (GenBank accession GQ891685)was cloned from Shaan 253 by primers designed according to conserved domains of known α-gliadin genes. Meanwhile, the gene expression vector pET32a-S253-Agli was constructed. Fusion protein was induced in the host strain E. coli BL21(DE3)by IPTG. In order to determine its processing quality of subunit S253-Agli, the expressed product was purified and recovered by affinity chromatography and low temperature cryodesiccation, and then integrated into the control flour utilization of 4 g Micro-doughlab. 【Result】 Sequence analysis showed that the DNA fragment of this gene contained a complete 900 bp coding region and some of regulatory elements. In the deduced amino acid sequences, an extra cysteine replacement of serine at position 6, which may form an intermolecular disulfide bond, was caused by single base conversion of C/G. The fusion proteins, induced by IPTG, were confirmed by SDS-PAGE and Western-blot and mainly existed as inclusion body. The farinograph results suggested that the purified subunit S253-Agli has negative effects on flour quality because of its reduction of main parameters, including development time and stability time, although some of farinograph parameters increased significantly, such as width of curve and mixing tolerance index.【Conclusion】 It was suggested that the potential quality of α-gliadin protein encoded by gene GQ891685 increased the elasticity but decreased the strength of wheat flours.