Molecular Cloning Of ω-gliadin Genes And Study On The Expression Of α-gliadin Gene Family By RNAi

The wheat gliadins are mainly responsible for viscosity of dough. To date, a number ofα-andγ-gliadin genes have been cloned from common wheat, but only severalω-gliadin genes have been published on GenBank. A pair of degenerate PCR primers that targeted the entire coding sequence of the Triticeae co-gliadin genes was used to amplify the DNA of a somatic hybrid introgression line and its two parents Jinan177, a bread wheat genotype(Triticum aestivum L.) and a tall wheatgrass (Agropyron elongatum, 10x). Cloning and sequencing of the resulting amplicons identified 16 distinct sequences, ranging in length from 924 to 1146 bp. Six of the sequences represented pseudogenes. The sequences shared a high degree of homology to knownω-gliadin sequences. The primary structure of the deduced polypeptides comprised a short N-terminal and C-terminal conserved domain, and a long repetitive and variable domain. A phylogenetic analysis produced three major clades:the first contained five tall wheatgrass parent sequences and one introgression line sequence, suggesting the transfer of at least one tall wheatgrassω-gliadin gene into the introgression line; the second group clustered three introgression line sequences with six Jinan 177 sequences, both of these groups were populated by ARQ/E-typeω-gliadins; the third group comprised three bread wheat SRL-typeω-gliadins. The co-gliadin of one of the introgression lines in group 2 has a cysteine residue in its C-terminal domain which may allow it to be involved in the determination of end-use quality. We present that somatic hybridisation of bread wheat could introgress wild and produce novelω-gliadin genes in the introgression lines.Coeliac disease is a chronic enteropathy caused by intolerance to gluten proteins. There are many toxic peptides that can result in celiac disease in a-gliadins. In order to inhibit/decrease the expression of a-gliadin gene family, conserved fragments of all a-gliadin genes released were found by sequence aligned and one sequence from 11-12 possessed conserved fragments was selected to design the RNAi trigger sequences. Then they were used to construct the RNA interference eukaryotic expression vectors p3301-Ubi-AR12 and p3301-Ubi-AR34. As followed, both of them were transformed into the wheat strain respectively by using Agrobacterium-mediated transformation method. Thirty positive transgenic plants were obtained by PCR screening. Seeds of six plants presented apparently silencing of a-gliadins examined by acid-PAGE and band scan analysis compared with control. In order to investigate whether the composition and expression patterns of HMW-GS and LMW-GS were influenced by RNAi, SDS-PAGE profile was examined to analyse these positive lines in which present a-gliadins silencing. It was shown that the composition and expression patterns of LMW-GS were changed but HMW-GS not. It will provide useful information in further functional study of wheat gluten by RNAi.