| 李 万，杨明明，高 翔，董 剑，赵万春.西农538 LMW-GS基因的克隆、原核表达及功能鉴定[J].麦类作物学报,2017,(4):445 |
| 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.) |
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| DOI：10.7606/j.issn.1009-1041.2017.04.03 |
| 中文关键词: 小麦 LMW-GS基因 原核表达 微量掺粉试验 |
| 英文关键词:Wheat LMW-GS gene Prokaryotic expression Farinograph |
| 基金项目:国家现代农业产业技术体系研究项目（CARS-3-2-47）；“十二五”农村领域国家科技计划课题（2011AA100501） |
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| 中文摘要: |
| 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915 bp，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。 |
| 英文摘要: |
| 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 to Glu-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-GS originated from Xinong 538 had a negative effect on dough quality properties. |
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