郑麦366γ-醇溶蛋白基因的克隆、系统进化和乳糜泻毒性分析

Cloning,Phylogenic Analysis and the Toxicity for Celiac Disease Patients of γ-Gliadins Genes from Common Wheat(Triticum aestivum L.)Cultivar Zhengmai 366

采用基因组PCR法,从强筋优质小麦品种郑麦366中克隆得到12个具有独特编码区的γ-醇溶蛋白新基因(命名为ZH366R-1-ZH366R-12,GenBank注册序列号为JN849083-JN849089和JX828391-JX828395),其中,ZH366R-5、ZH366R-8、ZH366R-11和ZH366R-12有完整的开放阅读框,除ZH366R-8存在一个额外的半胱氨酸(C)外,其他3个基因均具有γ-醇溶蛋白的典型分子特征,含有8个保守的半胱氨酸残基.克隆基因与42个来源于强筋优质小麦品种或代表普通小麦起源的二倍体祖先供体种的γ-醇溶蛋白的聚类分析和已知免疫多肽的分布分析表明,γ-醇溶蛋白在整个推断氨基酸序列和主要免疫肽的分布上均存在一定的基因组特异性.在重复区II含有1个额外半胱氨酸残基的γ-醇溶蛋白通常是由Gli-B1位点编码的,且在优质强筋小麦品种中均有分布,推测Gli-B1位点编码的γ-醇溶蛋白可能对面筋品质有独特贡献,而由Gli-D1位点编码的γ-醇溶蛋白,通常在重复区II分布有较多种类和数量的免疫多肽,乳糜泻(CD)毒性最强.

Total 12 unique clones（designated as ZH366R-1-ZH366R-12,GenBank No.JN849083-JN849089 and JX828391-JX828395）,including 4novel full-ORFγ-gliadins genes（ZH366R-5,ZH366R-8,ZH366R-11 and ZH366R-12）and 8pseudogenes,were amplified from common wheat cultivar Zhengmai 366 using a PCR-based strategy.Comparative analysis on the deduced amino acids sequences revealed that the other 3full-ORF genes had the typical structural characters ofγ-gliadins genes except that ZH366R-8 possessed an additional cysteine residue in the repetitive domain II.Phylogenetic analysis on the deduced amino acid sequences and identification of the known immunogenic peptides among the 4novel genes and other 42 genes in public database from several common wheat cultivars with strength gluten and the diploid ancestral donors representing the origin of common wheat showed thatγ-gliadins had obvious genomic differences in the molecular characteristics and distribution of the immunogenic peptides.The genes with an extra cysteine residue in the repetitive domain II mostly encoded by Gli-B1 locus occurred in the common wheat cultivars with high strength gluten quality while the genes encoded by Gli-D1 locus generally occurred in more kinds and quantity of immunogenic peptides.Thus,it was suggested that theγ-gliadins encoded by Gli-B1 locus might have specific contributions to gluten quality whileγ-gliadins encoded by Gli-D1 locus was more toxic for CD patients.