Molecular Identification Of α-,γ-gliadin Genes Of Advanced-Generation Derived From Common Wheat Crossed By Wild Emmer Wheat

Glutenin and gliadin are the main components of wheat storage protein, and they constituted the main ingredients of gluten. Processing quality of wheat flour was determined by the types, quantity and scale of glutenin and gliadin, which affected the quality of gluten. The gliadin of wheat grain is about 40%-50%of the amount in storage proteins and it made the gluten have plasticizer、viscosity and extensibility. And gliadin was also the main triggers to induce the development of celiac disease (CD). Therefore, to improve wheat quality by screening desirable gliadin genes and to reduce the incidence of CD are crucial significance for wheat genetic breeding. Triticum dicoccoides(2n=4x=28, AABB) was the donor of T.durum(2n=4x=2%, AABB) and T.aestivum(2n=6x:=42, AABBDD) and was the suitable donor of common wheat for genetic improvement, which had many super characteristics and the unique genetic variation. In this study, T. dicoccoides (D97),common wheat cultivar Chuannong (CN16), and their four hybrids HnesBAd7-209, BAd7-210, BAd7-212 and BAd7-213(>F11,2n=6x=42) were used as the experimental material. The a-,y-gliadin genes were cloned, and their nucleotide and deduced amino acid sequences were analyzed usingthe sequences reported in GenBank, and the phylogenetic trees were constructedto understand the genetic basis of T. dicoccoides to improve the gliadin of common wheat. This study provided scientific basis and direction for utilization of wild emmer to improvement of common wheat quality and CD prevention. The main results were as follows:1. Total of 248 a-gliadin genes and 82y-gliadin genes were obtained from wild emmer D97, CN16 and four hybrid lines. Except 129 a-gliadin genes and 69y-gliadin genes, the remaining genes were pseudogene because of coding region having stop codons.2. Further analysis showed that all of the cloned sequences had typical structure of a-,y-gliadin genes. In the cloneda-gliadin genes, some from CN16 and the four advanced generation hybrid lines had seven cysteine residues, becausethe serine was changed into cysteine in theunique domain Ⅱ of C-terminal due to the codon TCC-?TGC. InBAd7-210, BAd7-212 and BAd7-213,the gliadin genes with five conserved cysteine residues were obtained. Of which,the first cysteine of sequence7-212-α1-13 was mutated into tyrosine due to TGC→TAC,the third cysteine of 7-210-al-2, 7-212-α1-19 and 7-213-α1-1 mutated into arginine due to TGT→CGT; The fourth cysteine of 7-210-α2-13,7-213-α2-7 was mutated into arginine due to TGC→CGC, and the fifth cysteine of 7-212-α1-12 was mutated into arginine due to TGC→CGC.In the clonedy-gliadin genes, some ofother materials possessedspecial number like seven or nineof cysteine except those of7-213. For example,D97-y-3 and CN16-y-4 had nine cysteine residues becausethe tyrosine was changed into cysteine in C-terminal non-repetitive V due to TAT→TGT. However, in D97-γ-1、D97-γ-4、D97-γ-5、 D97-γ-6、D97-γ-7、D97-γ-12、D97-γ-14、CN16-γ-9、CN16-γ-10、CN16-γ-11、 7-209-γ-2、7-209-γ-3、7-210-γ-9、7-212-γ-5 and 7-212-γ-7 sequences, an extra cysteine residues occurred in N-terminal repetitive Ⅱ because of tyrosinemutation due to TAC→TGC. The seven cysteine residues were resulted from that in non-repetitive Ⅲ being mutated into glycinedue to TGT→GGTin 7-209-γ-13, while intoarginine due to TGT→CGT in7-212-γ-2 and 7-212-γ-8.Phylogenic analysis based on the amino acid sequences among the cloned genes and other published sequences in database demonstrated that majority of the a-gliadin genes with an extra cysteine residue in the C-terminal unique domain Ⅱ were located on D genome while majority of the y-gliadin genes owning an extra cysteine residue in the N-terminal repetitive domain Ⅱ on B genome. There were more gliadin genes with an extra cysteine residue in D97 than in CN16, which indicated wild emmer could be used for improving common wheat quality..3. Analysis of the five major T cell immunogenic peptides in α-,γ-gliadins from the six tested-materials showed that these materials had the full potential to trigger CD. Of the gliadin genes cloned from D97, the two sequences D97-α1-22 and D97-a2-14 located on 6B, had much less or close polyglutamines (PolyGlu) in the PolyGlu domaine Ⅱ in comparison with the domaine Ⅰ. But, the three sequences D97-α2-6, D97-a2-13 and D97-α2-16 on 6A, only possessed Glia-α20.And the similar gene7-209-α1-19 only with Glia-a20was observed in the BAd7-209. These results implied that the good genetic resources with less T cell epitopes might be found in wildemmer to produce high quality wheat.