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Cloning, Chromosomal Location and in Vitro Functional Analysis of Three Novel LMW-GS from Dasypyrum Villosum

Author: WangYanPeng
Tutor: GaoXiang
School: Northwest University of Science and Technology
Course: Crop Genetics and Breeding
Keywords: H.villosa Low molecular weight glutenin Chromosomal localization Protein Purification The flour experiment
CLC: S512.1
Type: Master's thesis
Year: 2011
Downloads: 14
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Abstract


The low molecular weight glutenin subunits (LMW-GS) is the WHEAT GLUTENIN important part, accounted for 40% of the grain storage proteins content. They form large aggregates through intramolecular and intermolecular disulfide bonds with high molecular weight glutenin subunits (HMW-GS), together determine the quality of wheat flour processing, giving gluten extensibility. Compared with the high molecular weight glutenin, low molecular weight glutenin subunits (LMW-GS) focused on better quality common wheat, breeding to accelerate the pace in recent years, the scope of LMW-GS gene mutations smaller , and many of the results is the study of the relationship between the allele and processing quality, and few studies on the function of a single low molecular weight glutenin subunits. In order to study the effect of the variation of new types of LMW-GS genes in closely related species of wheat, as well as its subunits quality, this experiment LMW-GS-specific primers, from leaves of H.villosa genomic clone three new types of low molecular weight glutenin gene complete coding sequence, and were constructed prokaryotic expression vector, expressed in E. coli, these two genes, affinity purification method to purify the target protein, by oxidation - reduction reaction subunits integrated into the basic flour do The flour-experimental study its function. The results are as follows:. Clone the H. villosa three low-molecular-weight glutenin subunit gene (GenBank accession number points HQ293219, HQ293220 HQ29322) named LMW-DV1 LMW-DV12 in LME-DV15, all genes with complete coding area, a length of between 831-846, 277 and 282 amino acid residues may be encoded to provide the low molecular weight glutenin gene with GenBank sequence having some homology to identify the genes for the low molecular weight glutenin subunit gene, but LMW-DV1 appear in the reading frame of the termination codon, and it is presumed to be a pseudogene. In addition, these three genes has a complete promoter structure and contains the wheat crops protein specific endosperm box. The sequence analysis showed that the translated amino acid sequence are typical of the LMW-GS structure: N-terminal conserved region, the N-terminal repeat region, C-terminal repeats containing a N-terminal signal peptide composed of 20 amino acids; zone. Institute cloned the protein sequences of the three genes encoding the missing section of the N-terminal conserved region, and the N-terminal of the direct repeat region (QQQLPQQP); sequence containing 8 cysteine ??residues typical. After comparison with wheat planted 15 LMW-GS gene sequences identified three LMW-GS SNP loci. By specific SNP analysis, specific primers were designed H.villosa LMW-GS, the use of the Chinese Spring - H.villosa additional and translocation lines, the the H. villosa LMW-DV12 and LMW-DV15 positioning in 1VS, the LMW-DV1 positioning controversial, further research is needed. Build of LMW-DV12 and LMW-DV15, two gene fragments of Trx fusion expression vector, respectively, in the host bacteria E. coli Rosetta-gami B (DE3) by IPTG induction of fusion protein expression, SDS-PAGE and Western-blot detecting the expression product, and confirmed that the fusion protein was successfully expressed. 5. Using affinity chromatography and isolated and purified fusion protein, single strip proved better purification effect. With purified samples do flour-experiment, two proteins added to make the dough properties had a great impact, the results show that the characteristics of the LMW-GS H.villosa Dough has a positive effect. The experiment H.villosa TA2127 research material to study the structural characteristics of LMW-GS gene H.villosa build a low-molecular-weight glutenin subunit gene expression vector, successfully achieved the integration of these two genes in prokaryotic expression system expression and the function of a single subunit. The experimental results enriched wheat LMW-GS gene type, as well as provide the potential for improved quality of wheat reference value, and to lay the foundation for a single LMW-GS in vitro studies.

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CLC: > Agricultural Sciences > Crop > Cereal crops > Wheat > Wheat
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