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Gene Cloning, Prokaryotic Expression and Functional Analysis of γ-Gliadin Genes from Wheat Cultivar Shann 253

Author: WangMingXia
Tutor: GaoXiang
School: Northwest University of Science and Technology
Course: Seed project
Keywords: Common wheat gamma - gliadin gene The prokaryotic expression Functional identification Farinograms
CLC: S512.1
Type: Master's thesis
Year: 2010
Downloads: 23
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Abstract

Gliadins total protein content of 40% to 50%, determine the ductility of the gluten, wherein the γ-gliadin accounted for 30% of the total. Currently, most people think of the γ-gliadin that have a positive role in the quality of wheat flour have a negative effect, there is no more consistent assertion. In addition, for the study of gamma-gliadin function is mainly based on statistical inference of protein subunits frequency, the less accurate method for determining the quality effect of a single gamma-gliadin subunits. Final, Australia and other countries in the 1980s began to use in vitro trace with powder method to detect the wheat storage protein function, they will be integrated into the control wheat dough exogenous protein subunits, by measuring the stability of the dough to form a time, peak, etc. rub mix and changes in the parameters of the tensile properties to quickly determine the effect of the quality of the protein subunits, a single storage protein subunits in vitro identification, the technology is widely used in determining the function of high and low molecular weight glutenin subunits. The present study attempts farinograph method of subunits in vitro functional identification, through the novel γ-gliadin gene cloned in E. coli expression experiments, the expression product was purified, to add it to the base powder observed farinograms changes, in order to determine the individual subunits of wheat quality characteristics. The results are as follows: (1) clone 10 γ-gliadin protein gene, GenBank No.: GQ857626 GQ871769-GQ871777. Gene fragment length of 777-945bp, encoding 259-302 amino acid residues. Sequence analysis showed that: clone 10 sequence in the repeat region, the polyglutamine region showed a significant difference. 5 sequence (GQ857626, GQ871770, GQ871772, GQ871776, GQ87177) a nucleotide encoding the first 14 amino acids occurs in zone II, a C → G conversion, resulting in a Ser → Cys, so that the encoded product contains additional cysteine residues. Further, the four gene containing the regulatory sequences of the initiation codon upstream -300 zone (GQ871770, GQ871772, GQ871774, GQ871776) analysis, and construct a component schematic diagram of the endosperm box sequence, this results in \\(2) GQ857626 subcloned into the expression vector pET-32a pre-built on the positive plasmid by IPTG induced expression in host strain Rosetta gami B (DE3) plysS. By Western blot and SDS-PAGE analysis, the product showed a 55kD a fusion protein with the predicted molecular weight and DNAMAN, confirmed that the target protein expression. (3) using HisTrap HP histidine tag affinity chromatography to purify the target protein, by oxidation - reduction reaction integrated into the basic flour, and take advantage of the 4g farinograph the rheological properties measurement results show that: the addition of 50 the mgγ-prolamin subunits after 10 major farinograms changes have taken place, and lift the trend vary. By oxidation - reduction reaction is the incorporation of exogenous protein dough process, the spatial structure of the dough has undergone some drastic change. Stable time significantly shorten the formation time significantly advance; dough weakening increased overall decline in the ability to withstand stirring. Within five minutes after the peak of the dough viscosity, by adding the protein to effectively increase the strength of the gluten, but this increase in the strength of the effect is not lasting, but makes offline time and rupture time in advance. This indicates that Judging from the overall effect of the the GQ857626 expression of γ-gliadin subunit Dough quality caused by the significant negative effects, this may be due to the presence of additional cysteine ??residues in the protein, unexpectedly terminated dough Valley The spatial extension of the protein aggregates, thus impeding the growth of the skeleton underlying flour.

Gene Cloning, Prokaryotic Expression and Functional Analysis of γ-Gliadin Genes from Wheat Cultivar Shann 253

Abstract

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