西农538 LMW-GS基因的克隆、原核表达及功能鉴定

Isolation,Prokaryotic Expression and Functional Analysis of LMW-GS from Xinong 538 (Triticum aestivum L.)

为了探索普通小麦品种西农538的LMW-GS对面粉加工品质的影响,根据NCBI中已公布的LMW-GS基因序列,设计了一对特异性引物,从西农538基因组DNA中克隆出LMW-GS基因后,对其进行原核表达及掺粉试验。序列分析表明,克隆得到的LMW-GS基因序列(GenBank登录号为KX452081)有单一完整的开放阅读框,编码区长915bp,无内含子。同源性比对及进化树分析发现,该基因属于Glu-D3、Type V(Group 10)、m型、C组LMW-GS基因。SDS-PAGE和Western blot分析表明,该基因原核表达成功。微量掺粉试验表明,诱导表达的蛋白对小麦面粉加工品质有负效应。

Low-molecular-weight glutenin subunits （LMW-GS） play an important role in determining the processing quality of wheat （Triticum aestivum L. ） flour. In this study,we isolated a LMW-GS gene （915 bp,GenBank accession number. KX452081） from wheat cultivar Xinong 538, using a pair of specific primers. The comprehensive analysis of deduced amino acid sequence, and phylogenetic and evolutionary analyses of full sequence,N- and C-terminal domains revealed that KX452081 was closely related toGlu-D3 loci,C-group,Type V （Group 10） and m-type LMW-GS. The target DNA fragments were subcloned into the pEASY-Blunt E1 expression vector and expressed in Escherichia coli Trans B （DE3） cell under IPTG induction. The gene was successfully expressed in E. coli system according to SDS-PAGE analysis and western-blotting assay. The fusion protein was purified and recovered by His-Trap affinity chromatography, and then integrated into the control flour by using a 4 g Micro- dough LAB Farinograph. Results showed that the LMW-GN originated from Xinong 538 had a nega- tive effect on dough quality properties.