Sequence Analysis Of Gliadin Genes From Three New Wheat Varieties

According to the conserved regions ofα-,γ-, andω-gliadin gene sequences, the gene-specific primers were designed to amplify the full gene coding regions from three new wheat varieties, including Chuannong 16, Liangmai 2 and Liangmai 3. The PCR amplyfied fragments were sequenced, and investigated. The results were as follows:1. Elevenα-gliadin gene sequences, includedα-CN16-6 (GenBank NO. DQ246449),α-CN16-9 (DQ246446),α-CN16-12 (DQ246447) andα-CN16-14 (DQ246448) from Chuannong 16,α-LM2-12 (DQ417343),α-LM2-14 (DQ417344) andα-LM2-17 (DQ417345) from Liangmai 2, andα-LM3-5,α-LM3-11,α-LM3-12 andα-LM3-16 from Liangmai 3, were obtained. The length of all sequence varied from 852bp to 942bp. Two sequence,α-CN16-6 andα-LM2-14, were considered as pseudogenes, because of the premature stop codons in open-reader-frame (ORF). The structure characters ofα-gliadins gene were identified in all sequences. N-terminal, included 5 amino acid residues, was confirmed and separated from the repetitive domain. The repetitive domain was deduced the same repetitive motifs PF/YPQPQ, which difference was that variable number of repetitive motif PFPQPQL was involved in different sequences. At the two polyglutamine regions, the extensive different of length among all sequences were found, and some other amino acid residues were also detected, besides glutamines. The mutations were mostly happened at the first base in codons. Mostα-gliadins contained six conserved cysteine residues in two unique domains, but the arginine was detected at the forth conserved cysteine residue position in sequenceα-CN16-6. The substitution of serine residue was observed by an additional cysteine residue at unique-Ⅱregions in sequencesα-CN16-6,α-CN16-12,α-CN16-14 andα-LM2-14. Theα-gliadins gene sequences from Chuannong 16 showed special characters, which's frequency of addition cysteine residue was higher than others sequences from Liangmai 2 and Liangmai 3.2. Sixγ-gliadin gene sequences, included the full-length of coding regions and part of flanking sequences, which were designated asγ-CN16-1 (1335bp),γ-CN16-3 (1360bp),γ-CN16-4 (1403bp),γ-CN16-5 (1359bp),γ-CN16-20 (1359bp) andγ-CN16-24 (1360bp), were obtained from Chuannong 16. Sequencesγ-CN16-1 andγ-CN16-3 were considered as pseudogene, because of the premature stop codons and small insertions/deletions leading to frameshift mutations, was also considered as pseudogene. Sequence analysis indicated that these sequences had some characteristics. The coding sequences had the same structure with knownγ-gliadins. The repetitive domain was composed of repeat units PFPQ_1-2(PQQ)_1-2, and the length of repeat units was strongly different. The difference of sequences length was observed in the polyglutamine regions. Mostγ-gliadins gene sequences contained eight conserved cysteine residues, but an additional cysteine residue was detected at repetitive domain in sequenceγ-CN16-3. Some TATA box in 5'-flanks and one potential polyadenylation signals in 3'-flanks, were also found. Multi-alignment analysis indicated that these sequences had the closest homology with previousγ-gliadins sequences from common wheat, and higher homologous (more than 80%) from closely related species of wheat, and only 50% homologous from rye (Secale sylvestre).3. One complete coding region sequence ofω-gliadin genes and four partial sequences, which were designated asω-CN16-63 (GenBank NO. EF116277),ω-CN16-3,ω-CN16-60,ω-CN16-64 andω-CN16-75, were obtained from Chuannong 16. And one complete coding region sequence and one partial sequences, which were designated asω-LM2-7 (GenBank NO. EF116278) andω-LM2-14, were also obtained from Liangmai 2. Sequence length ofω-CN16-63 andω-LM2-7 were 1080bp and 1158bp, respectively. These tow sequences were considered as pseudogenes, because of the premature stop codons. Especially, sequenceω-CN16-60 had the frameshift mutations of small insertions/deletions. Sequence analysis indicated that these complete genes and partial sequences belong to theω-2 type. In the repetitive domains, the percentage of proline was approximately 30%, and the proportion of glutamine was more than 40%. The percentage of glutamine of sequenceω-LM2-7, which was 44.8%, was the highest. The repetitive domain of sequence was composed of repeat units PFPQ_1-2(PQQ)_1-2, and there were strongly different in the length and the amino acid types of repeat units. Because there were not cysteine residues in theseω-gliadins gene sequences, the inter- and intramolecular disulfide bonds were not formed. Multi-alignment analysis suggested that these sequences had closer homology with otherω-gliadins sequences from Triticum aestivum and Aegilops tauschii, while had 70% homologous with C-hordein sequences from barley (Hordeum vulgare) andω-secalins sequences from rye (Secale cereale).