小麦品种陕253低分子量谷蛋白亚基基因的克隆及原核表达

Cloning and Prokaryotic Expression of a Low Molecular Weight Glutenin Gene from Wheat Variety Shaan 253

利用LMW-GS特异引物,从强筋小麦品种陕253中克隆了1个1498bp的片段(GenBank登录号为FJ172533),该片段包含全长为912bp的低分子量谷蛋白亚基的完整编码序列。经比较推导氨基酸序列的同源性,发现该基因属于Glu-D3位点编码低分子量谷蛋白亚基的基因,编码产物N-端具有LMW-m型低分子量谷蛋白亚基的典型特征,系统演化分析也支持这一结果。构建了该基因的表达载体pET32a-GluD3-S253,在宿主菌E.coli Rosetta-gami B(DE3)中经IPTG诱导表达融合蛋白。SDS-PAGE和Western blot检测表达产物,证实融合蛋白表达成功。

Low-molecular-weight glutenin subunit （LMW-GS） plays an important role in determination of flour viscoelastic properties in wheat （Triticum aestivum L.）. LMW-GS has large polymorphism and variation in molecular size, thus, it is difficult to be isolated using one-dimensional electrophoresis. Shaan 253 is a wheat variety with characteristics of high yield and early maturity, especially, with elite high-molecular-weight glutenin subunits, such as 5＋10 on ID, 14＋15 and 20 on 1B, and 1 on 1A. The purpose of this study was to understand the contribution of LMW-GS to the processing quality in Shaan 253. Using a pair of specific primers of LMW-GS and pMD19-T vector, one DNA fragment of 1 498 bp （GenBank accession No. FJ172533） was obtained from Shaan 253. The fragment contained the complete coding sequence of 912 bp and encoded 304 amino acid residues. According to sequence analysis, this gene was involved in Glu-D3 loci, and had high similarities to other known LMW-GS genes with the highest identity of 99.34%. Deduced amino acid sequence showed there were typical features of LMW-m type in N-terminal region, which was confirmed by the phylogenetic analysis. The expression vector of pET32a-GluD3-S253 was constructed and transformed into the host bacteria Escherichia coli Rosetta-gami B （DE3）. The expression product was testified using SDS-PAGE and Western-blot, indicating that the fusion protein was successfully expressed.