Molecular characterization of the celiac disease epitope domains in alpha-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.)

摘要

Nineteen novel full-ORF alpha-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical alpha-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced alpha-gliadins demonstrated that 14 of these contained 1-5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 alpha-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all alpha-gliadins had high content of beta-strands and most of the alpha-helixes and beta-strands were present in two unique domains. Phylogenetic analysis demonstrated that alpha-gliadin genes had a high homology with gamma-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five alpha-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the alpha-gliadin genes can express in a high level under the control of T-7 promoter.