郑丰5号α-醇溶蛋白基因的克隆与序列分析

摘要

The α-gliadins are the important components of seed strorage proteins , and their composition and content play a major role in determining the processing quality of wheat .Using PCR technology ,total 32 novel genes ( Designated as ZF5A-1 -ZF5A-32;GenBank No:JX828280 -JX828311 ) were cloned from the wheat cultivar Zhengfeng 5,including 17 full-ORFα-gliadin genes(Designated as ZF5A-1-ZF5A-17) and 15 pseudogenes.The sequence analysis of deduced amino acids showed that 10 had the typical structural characteristics of α-gliadin genes reported previously,and 7(ZF5A-1,ZF5A-3,ZF5A-6,ZF5A-9,ZF5A-10,ZF5A-11,ZF5A-15)possessed an additional cysteine residue in the unique domain Ⅱ.Based on the occurrence of the four major T cell immunogenic peptides and glutamine residues in the glutamine repeats ,ZF5A-7 and ZF5A-12 could be assigned to chromosome 6A,ZF5A-4,ZF5A-13,ZF5A-14 and ZF5A-17 to chromosome 6B,and other 11 genes to chromosome 6D.The sec-ondary structure predication of 17 cloned genes and other four genes in public database demonstrated that the num-bers and distributions of α-helix and β-strand were variable in different mature α-gliadins.Generally,5 α-helixes ( H1 -H5 ) usually presented in the two glutamine repeats and two unique domains for majority of α-gliadins,but the putative proteins of ZF5A-17 lost the α-helix(H2),ZF5A-2 and ZF5A-8 had an additional α-helix in the unique domain Ⅰ(HE1),and GQ891685 and ZF5A-15 had an additional α-helix in the second glutamine repeat(HE2).In addition,most of the genes(61.11%)had a β-strand(E)in the C-terminal unique domain Ⅱ.High variations in numbers of cysteine residue ,α-helixes and β-strands in the α-gliadins of Zhengfeng 5 might be strongly related to its good rheological properties of dough .%α-醇溶蛋白是小麦籽粒贮藏蛋白的重要组分，其组成与含量对小麦加工品质具有重要影响。利用PCR从郑丰5号基因组中克隆α-醇溶蛋白基因，并对其序列进行分析。经克隆共获得32个α-醇溶蛋白新基因（ ZF5 A-1～ZF5A-32，GenBank注册序列号为JX828280～JX828311），其中15个为假基因，17个（ZF5A-1～ZF5A-17）具有完整开放阅读框。17个α-醇溶蛋白新基因中，除ZF5A-1、ZF5A-3、ZF5A-6、ZF5A-9、ZF5A-10、ZF5A-11、ZF5A-15编码的蛋白在特征Ⅱ区含有1个额外的半胱氨酸（ C）外，其他10个基因编码的蛋白均具有α-醇溶蛋白的典型结构。根据推断氨基酸序列中4种主要T细胞优势多肽的分布及多聚谷氨酰胺区的长度，推测ZF5A-7和ZF5A-12可能定位于6A染色体，ZF5A-4、ZF5A-13、ZF5A-14和ZF5A-17可能定位于6B染色体，而ZF5A-1～ZF5A-3、ZF5A-5、ZF5A-6、ZF5A-8～ZF5A-11、ZF5A-15和ZF5A-16可能定位于6D染色体。17个新克隆α-醇溶蛋白基因及4个已知α-醇溶蛋白基因编码的蛋白的二级结构预测结果表明：α-螺旋、β-折叠的位置和核心序列是相对保守的，但不同蛋白α-螺旋和β-折叠的数量以及参与形成同一保守区域α-螺旋和β-折叠的氨基酸残基数却并不相同。克隆的17个α-醇溶蛋白基因中，除ZF5A-17编码的蛋白缺少α-螺旋（ H2）、ZF5A-2、ZF5A-8编码的蛋白在特征区Ⅰ均存在1个额外的α-螺旋（ HE1）、GQ891685和ZF5A-15编码的蛋白在多聚谷氨酰胺Ⅱ区存在1个额外的α-螺旋（ HE2）外，5个保守的α-螺旋（ H1～H5）恒定出现在其他基因的2个谷氨酰胺重复区和特征区中；此外，在C-末端特征区大部分基因（61．11％）还形成1个β-折叠结构（ E）。郑丰5号中具有较多额外半胱氨酸、α-螺旋和β-折叠的α-醇溶蛋白基因，可能与其良好的加工品质密切相关。