Characterization Of α-gliadins In Wheat And Related Species And Molecular Cloning Of Their Coding Genes

The gliadins are the major components of wheat storage proteins. It is well known that the composition and content of the gliadins play important roles in determining bread-making quality. Previous investigations showed that the related species of hexaploid wheat, such as Aegilops tauschii (DD, 2n=2x=14) possessed extensive storage protein variations, which could provide potential elite gene resources for wheat quality improvement. In this study, some specific α-gliadins from Aegilops tauschii and Triticum aestivum L. were detected by reversed-phase high performance liquid chromatographic (RP-HPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The specific PCR primers were used to amplify, clone and sequence their encoding genes, and homologous analysis of storage protein gene family was carried out. Some of these α-gliadin genes cloned were further characterized by expressing in Escherichia coli. The main results were as the followings:1. Separation and identification of α-gliadins in Aegilops tauschii and common wheatThe components of α-gliadins in Aegilops tauschii accessions T15, T26, T43 and in common wheat cultivars Zhongyou 9507, Gaocheng 8901 were detected by RP-HPLC. Further characterization and the accurate molecular weights of these gliadins were determined by MALDI-TOF-MS. It was found that the components of α-gliadins in common wheat cultivars were much more complex than in Aegilops tauschii. There were about four α-gliadin fractions in T15, three in T43 and six in T26. But Zhongyou 9507 as well as Gaocheng 8901 contained about 20 different α-gliadins, suggesting that hexaploid wheat had higher polymorphism in gliadin compositions.2. Cloning, characterization and homologous analysis of α-gliadin genesGenomic DNA of Aegilops tauschii accessions T15, T43 and T26, and cDNA of common wheat cultivars Zhongyou 9507 and Gaocheng 8901 were used as templates, and a pair of AS-PCR primers for α-gliadin genes was used to amplify the coding regions of α-gliadin genes. Single strongly amplified band with about 850bp from all accessions were obtained, and then the amplified products were ligated into a pGEM-T Easy vector (Promega) and sequenced by primer walking. 19 complete code nucleotide sequences were obtained, including one in T15, one in T43, two in T26, eight in Zhongyou 9507, and seven in Gaocheng 8901. They were all named and deposited in GenBank with the accession number from EF561270 to EF561288.All of these sequences contained no introns and ends at a stop codon TGA. The deduced amino acid sequences had typical characters of α-gliadin, including six domains in structure: signal of 20 amino acid residues, N-terminal repetitive domain, polyglutamine domain I, unique domain I, polyglutamine domain II and unique domain II. Furthermore, some of the deduced molecular weights of mature proteins were similar to the accurate molecular weights determined by MALDI-TOF-MS, suggesting that these α-gliadin commonents identified were well correspond to their coding genes cloned. Amino acid sequence analysis showed that they demonstrated a high similarity with other α-gliadin genes previously cloned, but the unique features were present. Particularly, Gli-At3 and Gli-Z7 contained an extra cysteine residue in domain II, which might affect the pattern of disulfide bond formation, and therefore both genes might be important in determining bread-making quality. We also analyzed the cloned genes distribution on the chromosomes of hexaploid bread wheat, Gli-G2 and GH-Z3 assigned to chromosome 6A; Gli-G1,Gli-G6,Gli-Z2,GH-Z4 and GH-Z8 assigned to chromosome 6B; Gli-G3, Gli-G4, Gli-G5, Gli-G7, Gli-Z1, Gli-Z5 and GH-Z7 assigned to chromosome 6D.3. Expression of cloned genes in E. coliA pair of AS-PCR primers was designed without signal to amplify some of genes cloned. The amplified products were ligated into expression vector pGEX-4T-2 and then transformed into E. coli strain BL-21 (DE3) plysS. After induced by isopropyl-β-D-thiogalactoside (IPTG), cells were harvested, and then the expressed proteins were extracted and identified by SDS-PAGE. All 5 α-gliadin genes were expressed successfully in E. coli. This indicated that these genes can express under the control of the functional promoter.