Cloning And Sequence Analysis Of Novel α-gliadin Genes From Zhengmai004

The α-gliadins were not only strongly correlated to the elasticity of gluten but also the mostactive proteins in triggering CD because many immunodominant epitopes were encoded by α-gliadins.Common wheat cultivar Zhengmai004was one of the major cultivars planted in a large scale in Henanprovince for its good quality of weaken gluten. In order to have a relative complete overview of its CDtoxicity and reveal the molecular basis of its good quality, one pair of primers were designed and the novelα-gliadins genes were cloned and analyzed. The results were just as follows:(1) Total43(named as Z4A-1~Z4A-43) novel α-gliadin genes were amplified and cloned usinga PCR-based strategy from common wheat cultivars Zhengmai004, including twenty-two(Z4A-1~Z4A-22)novel full-ORF which could coding for protein sequences with286~312amino acids and twenty-onepseudogenes. Comparative analysis the deduced amino acids showed that all genes isolated had typicalstructural characters of α-gliadin genes reported previously except that4genes(Z4A-7, Z4A-14, Z4A-17,Z4A-20）possessed an additional cysteine residue in the unique domain II while another gene(Z4A-15) lostthe second conserved cysteine residue in the unique domain I. The highest homology among the clonedgenes and other typical genes in GenBank was99%, indicating that all the cloned genes are the newmembers of α-gliadin genes family. So all the43sequences were submitted to the public database with theGenBank accession number of HM120221，HM120222，JX828270，JN831402~JN831406andKC715889-KC715923.(2) Based on the occurrence of the four major celiac disease toxic epitopes,8,6and8geneswere assigned to the chromosome6A,6B and6D and the total number of celiac disease toxic epitopes was16,0and23, respectively. In addition, of the5above mentioned genes with odd number of cysteineresidues,4genes(Z4A-7, Z4A-14, Z4A-15, Z4A-20) were located on chromosome6D except that Z4A-17was assigned to chromosome6B, suggesting that not only the α-gliadin genes derived from D genome wasmost deleterious but also was strongly correlated to the gluten quality.(3) Phylogenetic analysis on the deduced amino acid sequences among22cloned genes andother92genes from the several diploid wheat species representing the ancestral A, B, and D genomes ofthe hexaploid bread wheat further confirmed that α-gliadin sequences were distinguished according to the genome of origin on the basis of sequence similarity and in most cases the assignments of α-gliadin genesbased on the distribution of four major T cell peptides were reliable.(4) Secondary structure predictions demonstrated that positions and the core sequences ofα-helix and β-strand in α-gliadin were relatively conservative, but the numbers of α-helix and β-strand andlength of each α-helix and β-strand in the same position were variable in different proteins. In generally,five α-helixes (H1~H5) were always presented in the two polyglutamine and unique domains of all thegenes and70%of them also had a β-strand (E) in the C-terminal unique domains though1~2extraα-helixes would be presented in other minority genes. However, high proportion(10/22) of genes with1~2additional α-helixes were detected, which further indicated that α-gliadin genes in Zhengmai004might bestrongly correlating to its good elasticity of gluten.