Molecular Cloning And Sequence Analysis Of LMW-GS And Alpha-, Gamma-gliadin Genes From Common Wheat And Aegilops Tauchii Native To China

Gluten proteins not only play a determining role in wheat quality but also are the triggers to induce thedevelopment of celiac disease (CD). Screening desirable quality related genes from common wheatcultivars with elite quality or from other valuable Triticeae species may accordingly aid in exploringpotential beneficial gene resources both for quality improvement and CD prevention. Composition ofHMW GS has become the main basis of parental selection in quality breeding, it is the key to screendesirable LMW-GS, α-, γ-gliadin genes for wheat quality improvement in our country. Yumai34,Zhengmai9023, Zhengmai004, and Zhengfeng5are the four major common wheat cultivars sown on alarge scale in the Huang-Huai Wheat region of China for theirs elite quality. As the D-genome donor ofcommon wheat, it was revealed that Aegilops tauchii native to China displayed distinctive characters thatwere absent in other sublineages and was regarded as the unique and valuable genetic resources for wheatimprovement. Therefore, in order to screen desirable LMW-GS, α-, γ-gliadin genes with specific profiles,disclose the molecular basis for theirs elite quality of the four major common wheat cultivars and have acomplete overview on the role of Chinese Aegilops tauchii accessions in wheat improvement, based on thegenetic diversity revealed by ISSR, the LMW-GS, α-, γ-gliadin genes from the four major common wheatcultivars and2to7representitive accessions of Chinese Aegilops tauchii were cloned and characterizedusing a PCR-based method. Moreover, to shed light on the effects of different loci on gluten quality and thestructure–function relationships of a single LMW-GS, α-, γ-gliadin gene, the deduced amino acidsequences and secondary structures among the sequences cloned in this work and other representativesequences in the public database were comparative analyzed in a large scale. In addition, to reveal thestructure-function relationship in the transcriptional level and provide a basis for the functional analysis invitro, the prokaryotic expression in E. coli and transcriptional expression at about15days post-anthesis ofseveral genes differing in the length of repetitive domain and number of cysteine residues from Yumai34were investigated by SDS-PAGE, Western blotting and qRT-PCR. The main results are as follows:1. Total of638novel LMW-GS, α-, γ-gliadin genes were obtained from the four wheat commoncultivars and several Aegilops tauchii accessions, all of them were submitted to GenBank with theaccessions numbers JN831382to JN831435, JN849083to JN849096, JX828193to JX828401, KC715868to KC716082, KF278601to KF278638, KF412579to KF412621, KF880532to KF880594. Of them，total76genes with specific profiles in the length of repetitive domain and the number of cysteine residues and2valuable Chinese Aegilops tauchii accessions low in T cell peptides were screened.2. Phylogenic analysis based on the amino acid sequences among the cloned genes and othersequences in public database demonstrated that the majority of the α-gliadin genes with an extra cysteineresidue in the C-terminal unique domain was derived from Aegilops tauchii or located on6D chromosomeof common wheat cultivars whereas the majority of the γ-gliadin genes owning an extra cysteine residue inthe N-terminal repetitive domain, was mainly separated from common wheat cultivars with strong glutenand the potential diploid donors of B genome of common wheat. Although only a few LMW-GS genes with an extra cysteine residue were detected in public database, the significant effects on gluten quality ofone LWM-GS derived from D genome of common wheat with an extra cysteine residue had been verifiedby functional analysis in vitro. Therefore, it was strongly suggested that as it had been demonstrated thatGlu-D1locus contributes most to gluten quality, Gli-D2, Gli-B1, and Glu-D3loci also appeared to makespecific contributions to gluten quality.3. Secondary structure prediction and comparative analysis showed that the number of α-helix andβ-strand in each protein and the amino acid residues involved in each conserved α-helix and β-strand werealways variable in different proteins although the positions and core sequences in each α-helix and β-strandwere relatively conserved. Two unique domain of α-gliadins, polygultamine domain IV and C-terminalnon-repetitive V of γ-gliadins, C-terminal I and C-terminal III of LMW-GS usually formed more α-helicesand β-strands and were respectively the the most important regions for the function of α-, γ-gliadins andLMW-GS. What’more, by comparative analysis the number of the α-helices and β-strands in differentLMW-GS representative different alleles or different haplotypes in each locus, it was strongly suggestedthat secondary structure prediction could provide insights into the structure-function relationship of a singleLMW-GS and α-, γ-gliadins, and LMW-GS encoded by Glu-D3and Glu-A3locus respectively contributedmost and least to gluten quality among the three loci.4. Prokaryotic expression in E. coli and transcriptional analysis revealed by qRT-PCR of7genesderived from common wheat cultivar Yumai34and differed in the length of repetitive domain and numberof cysteine residues showed that high-level expression of LMW-GS, α-, γ-gliadin in vitro was still difficult.While the transcriptional level of LMW-GS and α-gliadin genes with an extra cysteine residue or owningmore α-helices and β-strands were higher than that of the typical genes.Therefore, it was suggested thatLMW-GS and α-gliadins were the two major proteins.5. Analysis of the5major T cell immunogenic peptides occurred in α-, γ-gliadins derived from thefour common wheat cultivars showed that the four cultivars had the full potential to trigger CD, but thefindings of the11exceptional genes with low or even non-toxic peptides derived from Triticuvummonococcum and Aegilops tauchii in the phylogenic tree further confirmed the possibility to produce wheatvarieties low in T cell epitopes.6. Genetic diversity revealed by ISSR of77Aegilops tauchii accessions native to China and themolecular cloning and sequence analysis of LMW-GS，α-，γ-gliadin genes from2to7representtiveaccessions further confirmed that Chinese Aegilops tauchii accessions in Huanghuai wheat region, mostlikely the accessions clustered in Group II and Group III of ISSR dendrogram, at least the two accessionsT006and SC-1used in the current work, were distinctive and precious genetic resources for wheat qualityimprovement and CD prevention.