The Structural Characters Of The Coding Gene Of Y-type HMW-GS In Triticum Araraticum Jakubz.

The high-molecular-weight glutenin of Triticeae species is one of the important storage proteins. The contents and compositions of HMW-GS play an important role in determining the processing quality of wheat flours. Triticum araraticum Jakubz. (2n=4x=28, AAGG) is the secondary gene pool of common wheat. There is no literature reported the isolation and characteriation of y-type HMW-GS in T. araraticum. In this study, their coding genes cloning, sequencing, structural analysis, prokaryotic expression, and sequence alignment were carried out. The main results are as follows:(1) Genomic PCR reaction was carried out by using degenerate primers specific for y-type HMW-GS and DNA fragments that represented the complete coding region sequences were obtained from PI427305. The sizes of the amplified fragments were 2.2 and 1.8 kb, respectively. The DNA fragments amplified were separately cloned into the pMD18-T vector. Then, DNA sequencing was carried out by using overlapping sub-clones. Their sizes were 2202 and 1866 bp, respectively. GenBank accession numbers were HM131806 and HM131807, respectively. Comparison analysis by using BLAST of NCBI, it was found that the DNA sequnces showed the highest homology with that of previously published 1Gy7* of CWI17006 in Triticum timopheevii Zhuk., and 1Ay of PI428281 in Triticum urartu Tum.. Therefore, it was concluded that the HMW-GS genes were 1Gy and 1Ay.(2) The complete coding region of IGy gene was 2202bp. It encoded 732 amino acid residues. The coding sequence began with start codon ATG and ended with double stop codon TGATAG. The deduced amino acid sequence had the typical structure of y-type HMW-GS including a signal peptide with 21 amino acid residues, a non-repetitive N-terminal domain with 104 amino acid residues (5 Cys), a central repetitive domain with 565 amino acid residues (1 Cys) and a non-repetitive C-terminal domain with 42 amino acid residues (1 Cys). The repetitive domain was rich in polypeptide with the total of 85, including the types like 12-merpeptide, undecapeptide, nonapeptide, hexapeptide, tripep-tide. Of which, hexapeptide was the most, up to 68.24%.(3) The secondary structure of the amino acid sequence was predicted by using PSIPRED. The result showed that a-helix and coil were in N-terminal domain, C-terminal domain and the repetitive domain.(4) A pair of primers without the sequence encoding signal peptide was designed to amplify 1Gy gene from PMD18-T 1Gy. The amplified fragment was inserted into the pET-30a, then transformed into E.coli strain BL21(DE)plySs, which induced by isopropyl-B-D-thiogalactoside (IPTG). And the encoding gene was expressed highly in E.coli. The mobility of 1Gy expressed was slower than that of seed by using SDS-PAGE. And therefore the 1Gy gene was normally expressed in E.coli.(5) Using D-hordein as an outgroup, both N-+C-terminal amino acid sequences and the whole amino acid sequences were used to construct a dendrogram of IGy and other 21 kinds of HMW glutenin subunits including eight x-types and thirteen y-types from Triticum, Aegilops and Secale. It was obtained that all the 21 subunits were firstly divided into two categories like x- and y-type. The 1Gy was classified together with 1Gy7* at the reliability of more than 96%. These results suggested that the 1Gy subunit from T. araraticum had an obvious genetic similarity with the 1Gy7* subunit from T. timopheevii.(6) There was a gap of 36 bp in the repetitive domain from 1486 to 1521 bp between the expressed 1Ay of PI428281 in T. urartu and the silent 1Ay of PI427305. The primary structure of the expressed 1Ay contained a signal peptide (21 amino acid residues), an N-terminal domain (104 amino acid residues), a C-terminal domain (42 amino acid residues) and a central repetitive domain (441 amino acid residues). If the gap was ignored, their acid sequences would resemble highly. Therefore, the 1Ay was silent because of the presence of a premature stop codon via base substitution of C→T.