作　　者： ; ; ; ; ; ;

机构地区： 西北农林科技大学农学院

出　　处： 《作物学报》 2014年第4期600-610,共11页

摘　　要： 利用设计的HMW-GS特异引物,从簇毛麦TA10220中克隆到6条HMW-GS基因序列(1.0～1.7 kb,GenBank登录号为KF887414～KF887419),比普通小麦HMW-GS基因序列小,其中KF887414～KF887417为能正常表达的基因,KF887418和KF887419是编码区出现终止信号的假基因。综合推导氨基酸的序列分析以及基于编码蛋白全序列、N-端和C-端域的进化树分析,认为KF887414属于y型HMW-GS,KF887415～KF887419的氨基酸序列同时具有x和y型的结构特征。将具有完整编码区的4个基因在宿主菌Escherichia coli Rosetta-gami B(DE3)中经IPTG诱导表达,通过SDS-PAGE分析表达的蛋白质和种子提取的HMW-GS,经Western blot进一步检测表达产物,证明蛋白质表达成功。通过His-Trap HP柱纯化表达蛋白,回收产物经SDS-PAGE分析后,低温冷冻干燥备用。将冷冻干燥的回收产物加入中国春基础面粉中,利用微量掺粉试验通过测定粉质参数来鉴定簇毛麦来源的HMW-GS对面粉品质的影响,结果表明,簇毛麦来源的4个HMW-GS对于面粉加工品质具有显著的正向效应。 Dasypyrum villosum carrying many novel HMW-GS alleles is an important genetic resource for wheat protein im- provement. In this study, we isolated six HMW-GS genes from D. villosum TA10220 （1.0-1.7 kb, GenBank accession numbers： KF887414-KF887419）, which were substantially smaller than those from common wheat, using a pair of specific primers. An in-frame stop codon was found in the coding sequences of KF887418 and KF887419 and thus these genes might be pseudogenes. The comprehensive analysis of deduced amino acid sequence, and phylogenetic and evolutionary analyses of full sequence, N- and C-terminal domains revealed that KF887414 was closely related to y-type HMW-GS, but KF887415-KF887419 had struc- tural characteristics of both x- and y-types. DNA fragments of KF887414-KF887417 were subcloned into the pEASY-E2 expres- sion vector and expressed in Escherichia coli Rosetta-gami B（DE3） cell under IPTG induction. The four genes were successfully expressed in E. coli system according to SDS-PAGE analysis （both the expressed protein and HMW-GS isolated from seed） and westem-blotting assay. The fusion protein was purified and recovered by His-Trap affinity chromatography and low temperature cryodesiccation, and then integrated into the control flour by using a 4 g Micro-dough LAB Farinograph. Results showed that the four HMW-GSs originated from D. villosum had positive effects on dough quality property.

关 键 词： 簇毛麦 高分子量谷蛋白亚基 原核表达 掺粉实验

领　　域： [农业科学]