| Low-molecular-weight glutenin (LMW-GS) is abundant in wheat grain that can not be ignored in wheat processing. However, LMW-GS of wheat is a complex family, so it is difficult to study. So far, the research of LMW-GS has been lagging far behind high-molecular-weight glutenin (HMW-GS) and gliadin. In our country, the research on genetic variation of LMW-GS and its impact on the quality are still in its infancy. This study was designed on the characterization of LMW-GS in wheat grain. And then the coding gene of LMW-GS are cloned and analyzed in order to enrich the LMW-GS gene family and provide a theoretical reference for the improvement of the quality of wheat.Six common wheat varieties (lines):R146, H6, American hard red, American soft red, R111, R291 were selected in this experiment. R146, R111 and R291 were provided by Sichuan Agricultural University, Key Laboratory of Crop Genetics and Breeding. H-6, American hard red and American soft red were provided by International Wheat and Maize Improvement Center. In this study, using SDS-PAGE to analyze their storage protiens, and then 5 pairs of specific primers (LMW-1, LMW-2, LMW-3, LMW-4,LMW-5) were used for PCR amplification. DNA sequence aligment analysis, protein secondary prediction and phlogenetic analysis were carried out to the cloning. The results were as follows:1. LMW-GS of these six common wheat varieties were initially separated by SDS-PAGE. The results showed that according to their different molecular weight, LMW-GS were mainly divided into two major regions:B subunits and C subunits. D subunits existed in some species. The LMW-GS encoded by new LMW-H6-5-2 (GeneBank no. JF736507) which cloned form this experiment was in C region.2. By PCR cloning,18 LMW-GS genes were got (4 of them from R146,4 from H6,2 from American hard red,4 from American soft red,2 from R111 and 2 from R291). There were 14 sequences had completed Open Reading Frames,4 sequences were silencers because of terminator codon in their coding region. In the upstream of LMW-GS genes, there contained some special squence structures such as TATA boxes, CACA boxes, Endosperm box. The amino acid sequences encoded by these cloning genes had the typical structure of LMW-GS:Composed of 20 amino acids signal region (Sig), Composed of 13 amino acids N-terminal conserved region (I), Composed of 70-186 amino acids that are glutamine and proline-rich (Ⅱ), contained 5 cysteine amino acids C-terminal region (Ⅲ), contained 1 cysteine amino acid glutamine-rich region (IV) and contained the last cysteine amino acid C-terminal conserved region (V). They all had 8 cysteine amino acids. The LMW-GS number of the first cysteine amino acid in I region is 8, and the first cysteine amino acid inⅡregion of other 10 LMW-GS.8 cysteine amino acids all in C-terminal region can't be found. Since the distribution of C residues, these proteins are also different between functions, and its mechanism is not clearly.3. By sequence aligment analysis, these 18 sequences had high consistency with the LMW-GS genes of wheat. It demonstrated that these 18 sequences were LMW-GS genes. In these sequences, the amino acids sequence encoded by gene LMW-H6-5-2 was a new LMW-GS, and compared with the amino acid sequence published on NCBI, the highest similarity was just 74% with ABM66824. There were big differences on the repeat units in the central region, the number of the repeat units and the content and distribution of Q between these two sequences.4. Made a protein structure prediction with SPIPRED v3.0 secondary structure prediction method for the new LMW-GS encoded by LMW-H6-5-2 and high quality subunit AY263369. The results showed that the content ofβ-fold of new LMW-GS encoded by LMW-H6-5-2 was more than AY263369, so it could be a high quality subuint. But according to its molecular weight, it was a C subunit and some research indicated that C subunit was closely related to the poor quality. Therefore we should consider the influence of both the amount and distribution of a-helix and the content ofβ-fold on the quality of wheat. The real influence of new LMW-GS encoded by LMW-H6-5-2 on wheat quality required further experimental study.5. Made a phylogenetic analysis between these 18 genes with DANMAN software. The results showed that they had high homology and closer relationship with each other because they were all LMW-m enconding genes. Despite the use of different primers, some squences cloned from different varieties also showed higher similarity. This indicated that different varieties also had similar gene type and these genes may have origenated in the same ancestor site. |
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