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利用2对特异引物,从簇毛麦(Dasypyrum villosum)基因组中分离克隆出一个簇毛麦HMW-GS基因VHG-2(GenBank登录号为FJ600492)及其启动子序列VHGp-1(GenBank登录号为FJ600489).VHGp-1序列长度为1099bp,从5′至3′方向依次有E-box、N-box、G-box、HMW谷蛋白特异38bp增强子和TATA-box等典型的HMW-GS基因启动子作用调控元件,说明VHGp-1为簇毛麦HMW-GS的启动子基因.VHG-2序列长度为1572bp,具有单一完整的、可编码498个氨基酸的开放阅读框(ORF),该ORF推导的氨基酸序列结构分析表明,编码区依次包含由21个氨基酸残基组成的信号肽、105个氨基酸残基组成的N-末端区、330个氨基酸残基组成的中部重复区和42个氨基酸残基组成的C-末端区;中部重复区主要重复单元为6肽(PQQGQQ)和9肽(GYYPTSP/LQQ);有6个半胱氨酸残基(Cys),其中5个分布在N-末端区,1个分布在C-末端区,第3、4个相邻.这些特征与报道的y-型HMW-GS多肽结构基本一致,说明VHG-2是簇毛麦的y-型HMW-GS基因.系统进化分析表明,簇毛麦HMW-GS启动子序列(VHGP-1)与智利大麦(H.chilense)H基因组的D-hordein基因、拟鹅观草和阿拉善鹅观草St基因组的HMW-GS基因的启动子具有比较近的同源关系,簇毛麦HMW-GS基因(VHG-2)与冰草、拟鹅观草和中间偃麦草的y-型HMW-GS基因具有较近的同源关系.
Two pairs of specific primers were used to isolate and clone a HMW-GS VHG-2 gene (GenBank accession No. FJ600492) and its promoter sequence VHGp-1 (GenBank accession No. FJ600489) .VHGp-1 has a length of 1099bp and typical HMW-GS genes such as E-box, N-box, G-box, HMW gluten-specific 38bp enhancer and TATA-box from 5 ’to 3’ Which shows that VHGp-1 is the promoter of HMW-GS in V. napus.VHG-2 has a length of 1572bp and has a single intact 498 amino acid open reading frame (ORF) The deduced amino acid sequence structure analysis showed that the coding region consisted of a signal peptide consisting of 21 amino acid residues, an N-terminal region consisting of 105 amino acid residues, a middle repeat region consisting of 330 amino acid residues and a 42 amino acid residue (PQQGQQ) and 9 (GYYPTSP / LQQ); six cysteine residues (Cys), of which five were located at the N-terminal One is located in the C-terminal region and the third and the fourth are adjacent to each other.These features are consistent with the reported structure of the y-type HMW-GS polypeptide , Indicating that VHG-2 is the y-type HMW-GS gene of H. villosa. Phylogenetic analysis showed that the VHW-GS promoter sequence (VHGP-1) of H. villosa and H.chilense H The homology of HMW-GS gene of -hordein gene, Stichopus japonicus and Strasbrain Alaskans Stalk genome was relatively close. The HMW-GS gene (VHG-2) The y-type HMW-GS gene of C. intermedius and Thinopyrum intermedium has close homology.