一年生簇毛麦α-醇溶蛋白基因的分离、原核表达与功能鉴定

醇溶蛋白是面筋的主要成分之一,对小麦品质具有重要影响。根据数据库中全长α-醇溶蛋白基因设计了1对通用引物,从5份一年生簇毛麦(Dasypyrum villosum)品系中共得到52条序列,长度在816~873 bp之间(GenBank登录号为KJ004676~KJ004727)。核酸序列分析表明,其中有8条假基因,有1条(KJ004680)缺失终止密码子。推导氨基酸序列显示,KJ004677、KJ004686、KJ004714和KJ004696含有1个额外的Cys,其中,前3条序列由于Tyr→Cys所致,而KJ004696则由于Ser→Cys突变。序列间的差异主要出现在N-端重复区和多聚谷氨酰胺I区,根据N端重复区多肽序列的差异将一年生簇毛麦α-醇溶蛋白分为5种类型。为了分析具有额外Cys的α-醇溶蛋白所具有的品质效应,选取KJ004708(具有典型的6个Cys)和KJ004714(具有1个额外的Cys)分别构建表达载体,IPTG诱导后均得到分子量约30 kD的蛋白,与理论值相符;目的条带经切胶串联质谱鉴定证明,这2个α-醇溶蛋白基因在大肠杆菌中正确表达。对表达的蛋白亚基进行纯化、复性和低温冷冻干燥,经4 g粉质仪分析表明,KJ004708和KJ004714均能改善面团的加工品质,其中具有1个额外Cys的KJ004714亚基对面粉品质的改善更为显著。

Cloning,Prokaryotic Expression and in vitro Functional Analysis of α-Gliadin Genes from Dasypyrum villosum

Gliadin, which has a great effect on wheat quality, is one of main components in gluten. According to the full lengths of α-gliadin genes deposited in NCBI database, a conserved primer pair was designed to clone α-gliadin genes in five Dasypyrum villosum lines. A total 52 sequences (816 to 873 bp in length) were isolated (GenBank accession numbers: KJ004676 to KJ004727) including eight pseudogenes and another sequence KJ004680 without stop codon. Deduced amino acid sequence anaylsis showed that KJ004677, KJ004686, and KJ004714 contain an extra Cys from the Tyr → Cys mutation, whereas, the extra Cys in KJ004696 resulted from the Ser → Cys mutation. Amino acid variation mainly occurred in N-terminal repetitive region and polyglutamine domain I. Variation in N-terminal repetitive region formed five groups in the 43 α-gliadins. To study the effects of an extra Cys on dough quality, we constructed the prokaryotic expression vectors forKJ004708 (with the typical six Cys residues) and KJ004714 (with an extra Cys) and obtained proteins of ~30 kD from Escherichia coli BL21(DE3) under the induction of isopropyl-β-D-thiogalactoside (IPTG) with the predicted molecular weight. These expressed proteins were verified by matrix-assisted laser desorption-ionization time-of-flight MALDI-TOF/TOF tandem mass spectrometry analysis. The result showed that these α-gliadins were expressed correctly in E. coli. After purification, renaturation, and freeze-drying process, the functions of the expressed proteins were tested with 4 g Farinograph. Both KJ004708 and KJ004714 had positive effects on flour quality, especially KJ004714 with an extra Cys.