簇毛麦HMW-GS及其启动子基因的克隆与序列分析

Cloning and Sequence Analysis of the HMW-GS Gene and Its Promoter from Dasypyrum villosum

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.

A HMW-GS gene VHG-2 ,GenBank accession No. is FJ600492 ,and its promoter sequence VHGp- 1 ,GenBank accession No. is FJ600489, was isolated and cloned from the genome of D. villosum. VHGp-1, sequence length of 1 099 bp, contains E-box, N-box, G-box, HMW glutenin specific 38 bp enhancer and TA- TA-box,which were promoter role regulatory elements of the typical HMW-GS gene. It indicated that VH- Gp-1 is a HMW-GS promoter gene of D. villosum. VHG-2,sequence length of 1 572 bp,has a single complete open reading frame （ORF） or coding region, which can encode 498 amino acids. The deduced amino acid sequence of this coding region contains the signal peptides （9.1 residues）,N-terminal region （105 residues） ,the central repeat region （330 residues）, and C-terminal region （42 residues）. In repeat domains, hexapeptide （PGQGQQ） and nonapeptide （GYYPTSP/LQQ） were mainly repeating unit. The sequence predicts six cysteine residues, five within N-terminal and one within C-terminal domains,and 3^rd and the 4^th cysteine is neighboring. These structure characteristics are typical in y-type HMW glutenin subunits, and suggested that VHG-2 is a y-type HMW-GS gene of D. villosum. Phylogenetic analysis showed that the promoter VHGP-1 was closely related with the D-hordein promoter gene of H genome in H. chilense. The HMW-GS gene VHG-2 has a relatively close homology with y-type HMW-GS gene of Agropyron elongatum and Pseudoroegneria stipifolia.