Study On Wheat Transformation Of LMW-GS Gene Ee34and Molecular Characterization Of SRL/TRQ-type ω-gliadin Genes

Agropyron elongatum (StSt,2x), one of the most important wild resources for common wheat (Titicum aestivum L.) improvement, has been proved that the material contains the potential quality-associate gluten genes. One LMW-GS gene Ee34with seven cysteine residues (6conserved and one free) from A. elongatum was performed a flour supplementation test. The results showed that the dough strength of bread wheat flour was significantly increased by the presence of this LMW-GS subunit. In order to obtain the genetically modified wheat with high flour quality, Ee34was used to construct the plant expression vector p3301-Ubi-Ee34and then transformed into the wheat cultivar Jimai2] by using Agrobacterium-mediated transformation method. The positive transgenic plants were obtained by molecular identification and genetic analysis. Seeds of transgenic progeny were examined by SDS-PAGE. The results indicated some transgenic plants displayed a specific band in the LMW-GS region. Meanwhile, a pair of primers was designed specific to Ee34as molecular marker. It will advance the theoretical and applied research of wheat quality breeding.Gliadins are the main storage proteins in the seed endosperm of wheat. At present, a number of α/β-and γ-gliadin genes have been cloned from common wheat, but only several ω-gliadin genes have been reported, especially SRL-/TRQ-type co-gliadin genes. In order to amplify the entire coding region of SRL-/TRQ-type ω-gliadin genes via genomic PCR from a somatic hybrid introgression line and its two parents JN177,a bread wheat genotype (Triticum aestivum L.) and a tall wheatgrass (Agropyron elongatum, StStStStEeEeEbEbExEx,10x), a pair of degenerate PCR primers were designed based on published data. Cloning and sequencing of the resulting amplicons identified7distinct sequences only from A. elongatum, ranging in length from837to951bp. And the full-length sequences were not obtained from Ⅱ-12and the parent JN177. Of seven, three sequences were SRL-type ω-gliadin genes, the remaining four were TRQ-type ω-gliadin genes, of which one included double signal peptides. The deduced peptides of these sequences contained common signal peptides, N-and C-terminal conserved regions and long repetitive regions. The repeat motif pattern in the seven variants was mainly based on FPQ2-5, which was the same as that of B-genome ω-gliadin. A phylogenetic analysis revealed that these sequences had a high degree of similarity with those of B-genome co-gliadins in wheat. Also, five genes were isolated from endosperm of A. elongatum via RT-PCR analysis, and the sequence alignment showed they were identical with five out of the seven via genomic amplification. These sequences will promote the structure and evolution research of ω-gliadin genes.