节节麦-黑麦双二倍体α醇溶蛋白基因的克隆与序列分析

以人工合成节节麦-黑麦双二倍体基因组DNA为模板,用小麦种子醇溶蛋白保守引物进行PCR扩增,经克隆测序获得了843 ～ 897 bp共15个新的DNA序列(GenBank登录号为:JQ029719,JQ046392～JQ046405),分别编码280 ～298个氨基酸.序列比对结果表明,它们具有α-醇溶蛋白基因的典型结构特点,是α-醇溶蛋白基因家系成员,其中有两个序列为同义突变.利用14个新氨基酸序列与乳糜泻( celiac disease)病人毒性抗原相关序列的比对,发现有8个序列的Glia-α-2和Glia-α-9型抗原序列产生缺失和替换.与来自粗山羊草属和黑麦属的α-醇溶蛋白基因的编码氨基酸建立系统树,结果表明,14个DNA序列编码的氨基酸序列与粗山羊草属的相关序列聚在一起.

Cloning and Sequence Analysis of oα-gliadin Gene from Aegilops tauschii × Secale cereale Amphiploids

Fifteen novel α-gliadin genes from Aegilops tauschii×Secale cereale amphiploids were amplified and cloned using conserved primers specific to wheat α-gliadin genes.A total of 15 sequences were obtained with lengths ranging from 843 to 897 bp,containing ORFs encoding peptides with 280 to 298 amino acids and GenBank accession numbers JQ029719,JQ046392 to JQ046405,respectively.A BLAST search showed that these sequences belonged to typical structural characters of α-gliadin genes reported previously.Comparative analysis of homologous proteins showed that they were members of the α-gliadin super-family,of which two sequences were synonymous mutations.Searching for four different T cell stimulatory epitopes for celiac disease patients,it was found that eight of fifteen sequences did not contain the Glia-α-2 and Glia-α-9 epitope typical feature.The phylogenetic trees based on the amino acids sequence of Secale and Aegilops indicated that the cloned gliadin genes clustered in the clade of Aegilops α-gliadin gene.