Molecular Cloning Of High Molecular Weight Glutenin Subunits Genes From Aegilops Searsii And Aegilops Bicornis

High molecular weight glutenin subunit (HMW-GS) of bread wheat (7! aestiviun L.) has been paying more attention and investigating from different aspects because it plays an important role in wh'eat end-product-processing quality. Analysis of HMW glutenin subunits in hexaploid wheat showed that there were limited type HMW subunits in bread wheat to meet the need demand of flour with good quality. People focus on the polymorphism of high molecular weight subunits (HMW-GS) composition of the wild relatives closed to wheat, especially the genus Aegilops L., and looking for new types of good quality HMW-GS become a very important reach field in wheat quality improvement.Species Ae. speltoides, Ae. bicornis, Ae. longissima, Ae. searsii and Ae. sharonesis in Aegilops Sitopsis were used in the study for isolated and characterized the HMW-GS composition and polymorphism by SDS-PAGE method. Ae. searsii and Ae. bicornis were use to amplify the complete ORFs and 5'upstream region of HMW gfutenin genes of in genomic PCR reactions with the degenerate primers based on the published nucleotide acid sequences of HMW glutenin subunit genes. Using the cloning and the nested deletion method, we have obtained eight full-length nucleotide sequences. The eight mature proteins of HMW glutenin subunit from Ae. searsii and Ae. bicornis were expressed and isolated in vitro successfully in coli form BL21, and were identified with Western-blot.The results showed that all accessions of Aegilops Sitpsis have the gene loci encoding x-and y-type HMW-GS respectively and the composition of HMW glutenin subunits showed polymorphism.Two DNA fragments encoding x- and y-type HMW glutenin were amplified from two Ae. searsii accessions (IG49077 and IG48586) and two Ae. bicornis accessions (Y52 and Ae4) based on the SDS-PAGE electrophoretic mobilities. Analysis of the nucleotide acid sequences and amino acid sequences derived from the cloned ORFs showed HMW glutenin subunits from Ae. searsii and Ae. bicornis were similar to that of previous published HMW-GSs from hexaploid wheat, no intron structure, but full-length ORF contained the conserved structure consisting of signal peptide, N- and C-terminal region and central repetitive domain were found. The difference of molecular weight was determined by the length of the central repetitive domain. The cloning HMW-GS genes accord with the typical x-type and y-type characteristics of cysteine residue, i.e. x-type N-terminal containing 3 cysteine residues andC-terminal containing one cysteine residue, and y-type N-terminal containing 5 cysteine residues, C-terminal and central repetitive domain containing one cysteine residue each. Ninety-eight percent homology sequence of nucleotide acid was found in HMW-GS 5'upstream region of the eight cloned HMW-GSs from Ae.searsii and Ae. bicornis when compare with hexaploid wheat. The main difference came from the change of single nucleotide base and short nucleotide sequence deletion or insertion. All promoter regions contained the structure of "TATA-box" and "cereal-box".49077-x, cloning from an Ae. searsii accession IG49077, is a new type HMW-GS and has an extra cysteine residue in the C-terminal region of the repetitive domain, and is provided with the essential characteristics of good quality structure of HMW-GS. Amino acid sequence and motif of C-terminal region in central repetitive domain of 49077-x is the same as that of y-type HMW-GS. The identification of 159 amino acid sequences of 49077-x and 49077-y showed that probably caused by the deletion/replacement of partly nucleotide acid sequence during the evolution. However, the contribution of 49077-x to wheat flour quality would be confirmed by microscale dough -mixing test and gene transfer further. The molecular weight of Y52-y, cloning from Ae. bicornis accession Y52, is higher than that of y-type subunits from hexaploid wheat and should be a new type HMW-GS. Y52-y has a constitution with 18 consecutive hexapeptide and nonapeptide, and this structure did not reported in the other y-type HMW-GS.Homology analysis of nucleotide and amino acid sequences of the conservative region showed that the high homology occurred between the HMW-GSs of diploid Aegilops Sitopsis and T. aestivum, there was only involved the change of individual nucleotide base deletion, insertion or replacement. Analysis of the homology tree based on signal peptide, N- and C-terminal amino acid sequences and 5' flanking sequences indicated that the HMW glutenin subunits coding by Ae. searsii, Ae. bicornis and those coding by Glu-Dl of T. aestivum were clustered together with the highest boot strap values, and the conclusion could be drawn that Ae. searsii andAe. bicornis might be not the donor of B genome in hexaploid wheat.