栽培一粒小麦α-,γ-,ω-醇溶蛋白基因的克隆与序列分析

Cloning and Sequence Analysis of Novel α-,γ-,ω-gliadin Genes of Triticum monococcum

为全面揭示栽培一粒小麦诱发乳糜泻（Celiacdisease，CD）的毒性和在小麦品质改良中的应用价值，根据已注册基因的保守序列分别设计2～3对特异引物，采用AS-PCR技术对栽培一粒小麦的醇溶蛋白新基因进行克隆、CD毒性肽识别和同源性分析。结果表明，从栽培一粒小麦中分别克隆得到26个具有独特编码区的α-（命名为：G忙A—1～GnA-7，GenBank登陆号为JN831382-JN831385和JX828193-JX828195）、γ-（命名为：Gli-R—J～Gli-R-6，GenBank登陆号为HM120220，JX828376～JX828380）、ω-（命名为：Gli-w-1～Gli-w-13）醇溶蛋白新基因和1个已知基因（Gli-R-7，ACJ03494）。CD毒性肽的识别分析表明，栽培一粒小麦具有较强的CD毒性，分布于醇溶蛋白的5种主要T细胞优势多肽和4种“毒性肽”除A基因组来源的”醇溶蛋白中不舍有的毒性肽Glia—α2和Glia—α外，其余多肽在克隆基因的编码蛋白中均有分布。克隆基因与已知基因的同源性分析表明，α-，γ-醇溶蛋白的推断氨基酸序列存在明显的基因组来源的差异性，而ω-醇溶蛋白基因的基因组来源差异不明显。此外，所克隆的7个α-醇溶蛋白变异相对广泛，而γ-，ω-醇溶蛋白基因的组成则相对单一，具有极高的相似度。

Triticurn monococcum is one of the important basic species and genetic resources of Tritic- urn. In order to have a complete overview of its celiac disease （CD） toxicity and estimate its potential value in wheat quality breeding, 2-3 pairs of gene-specific primers of α-,γ-,ω-gliadins were designed to amplify their coding sequences from genomic DNA, and to identify their immunologic peptides and analyze their homology with other species. Total 26 new gliadin genes with unique and typical struc- ture were obtained, including 7 with α- （named as Gli-A-1 -Gli-A-7 , GenBank No. JN831382 JN831385, JX828193 - JX828195）, 6 with γ- （named as Gli-R-1 - Gli-R-6, GenBank No. HM120220, JX828376--JX828380） and 13 with ω- （named as Gli-w-1 - Gli-w-13 ）, and 1 known γ- gliadin gene （ACJ03494）. Analysis on their CD epitodes demonstrated that Triticum monococcum had strong toxicity to CD patients because all the 5 major T cell peptides except for the petides Glia-α2 and Glia-α absent in A genome and four ＂toxic＂ structures distributed in gliadin were detected among the cloned genes. Phylogenetic analysis showed that the deduced amino acid sequences of α-, γ-gliadins could be distinguished by their origins of genome while the differences among ω-gliadins were not ob- viously. In addition, the cloned 7 α-gliadin genes had a relative higher variability than that of the cloned 7-,ω-gliadin genes.