Gene Cloning, Prokaryotic Expression And Functional Analysis Of γ-Gliadin Genes From Wheat Cultivar Shann 253

The gliadin content was 40%-50% in the total wheat protein, determining the extensibility of gluten.γ-gliadin made up 30% of the total gliadin. At present, most people thinkγ-gliadin acted both positive and negative role on wheat quality, lacking a united conclusion.In addition, theγ-gliadin function researches mainly based on the single subunits frequency statistics. This method is not very accorate. In the late 1980s, Australia and other countries began to test wheat protein function by micro mixed dough detection. They measured the variation of dough stabilizing time,dough time,peak time index of common difference et.al after adding the protein subunit, had achieving the goal of fast determining the subunit quality. This technique was widely applied in the HWM-GS subunit function testing.This study attempts to analysis theγ-gliadin subunit functional by micro 4g Farinograph. Fusion protein cloning by us was induced in the host strain E. coli Rosetta gami B (DE3) by IPTG. The expression product was purified by affinity chromatography, and then rheological properties were tested. The chief results are showed as fo11ows:(1) Primers were designed according to conserved ends of the coding region ofγ-gliadin genes, 10 sequences was cloned from Shaan 253(GenBank No.GQ857626,GQ871769-GQ871777). Sequence analysis showed that the fragment contained the full length of 777-945bp coding sequence, encoded peptides of 259-302 amino acids. Sequence alignment showed that these squences have remarkable difference in both repeated domain and Polyglutamine region. Four sequences(GQ871770,GQ871772,GQ871774,GQ871776) contained control regions upstream 300bp of the ATG codon was analysis, and construc the pattern of Endosperm Box. This study was published in Theor Appl Genet.(2) GQ857626 was cloned into the pMD19-T vector. Whereafter, one of these positive clones was subcloned into the expression system pET32. Subsequently, the recombinant plasmid was identified by sequencing and digestion with restriction enzymes, and then was transformed into the host bacteria E. coli Rosetta-gami B (DE3). SDS-PAGE and Western-blot were used to detect the expression products, of which was existed mainly as inclusion body. It could provide a foundation for quality improvement of wheat as this novel gliadin genes were cloned and the fusion protein was successfully expressed.(3) The expression product was purified by affinity chromatography, and then rheological properties were tested by micro 4g Farinograph. It is found that the change of dough stabilizing time reduced and the measure of weakness increased after addingγ-gliadin subunits. Although the mechanical endurance around the peak increased, the total index of dough decreased remarkably. It shows the subunit have some influence on dough stability before the maximum of consistency, but the wheat flour quality resulting from weaken of the gluten strength are declining.