Composition And Locus Analysis Of Gliadin Gene In T.urartu

Wheat is one of the most important crops in China, and more attentions are paid to wheat quality improvement in recent years. Gliadins account for 50% 60% of the storage proteins in wheat grain and play a major role for viscoelasticity of dough that affects the processing quality of wheat. However, Gliadins display high polymorphic protein complexes and are encoded by a multi-gene family, so it is difficult to determine the number and expression patterns of wheat gliadin genes. In this paper, we systematically studied the gliadin genes of T. urartu PI428198（G1812） which has draft genome sequence at the genomic, transcriptional and protein levels.Taking the DNA of tender leaves from G1812 at different growth times and c DNA of its seeds at 5DPA, 10 DPA, 15 DPA, 20 DPA and 25 DPA as the template for PCR amplification, we got a total of 28 gliadin genes, including 23 α-gliadin genes（12 with complete ORF）, 3 γ-gliadin genes（all with complete ORF） and 2 ω-gliadin gene（,one with complete ORF）. At the same time, using the method of genomic prediction from the genome of G1812, we also predicted 9 gliadin genes, which had been got in PCR cloning method. The method of RNA-Seq was used to analyze the expression patterns of these 28 gliadin genes. Most of the expressed genes displayed a similar expression trend, namely, up- to down-regulated expression from flowering to maturity. In the samples of grains at 5 DPA and flag leaves at 10 DPA, these genes were hardly detected, but their expression was rapidly up-regulated from 5 to 10 DPA, and reached their peaks at 15 DPA, then decreased gradually at 20 and 25 DPA. The accumulation of these expressed gliadin genes were further identified by proteomic methods. 16 protein spots that had relatively high expression were found, including 12 gliadin proteins, 3 low molecular weight glutenin and 1 Avenin-3 protein. The 12 gliadin protein spots were matched with the corresponding genes identified. The phylogenetic and evolutionary analysis demonstrated that almost all the gliadin genes could find their homologs in tetraploid or hexaploid wheat, consistent with the fact that T. urartu is the A-genome progenitor species.Three primer pairs were designed in the conserved regions of gliadin genes, and were used to screen the G1812 BAC library, from which 128 positive clones were identified and isolated. On the basis of the restriction patterns of the BAC clones and the end sequences of positive clone, we selected 24 positive clones to sequence by the three-generation sequencing technology. Contig₁471₁455₁454 sequence had 24 ORF, and 10 ORF can find the functional annotation of proteins, of which there were 2 Omega-gliadin. Omega-2-gliadin was a complete gene, but the similarity of Gli-27 and Gli-28 was low. This Contig sequence also had a duplication with the length of about 23.6kb. The Contig₁₁484₁485₁486₁487₂ sequence contained 25 ORF, of which 8 ORF could find the functional annotation of proteins, including 3 Alpha gliadins. Although Alpha-2-gliadin and Gli-2 had a SNP difference, their deduced amino acid sequences were same.