Expression, Purification And In Vitro Functional Identification Of The Low-Molecular-Weight Glutenin With Different Repetitive Domain

Low molecular weight glutenin subunits, occupied 40% of wheat seed storage protein, is an important component of flour. LMW-GSs interact with high-molecular-weight glutenin thought disulfide affecting the processing-quality which endows it with extensibility.Lack of effective method to separate LMW-GSs, the contribution of LMW-GSs to the quality of flour is known less. Many researches about the relation between LMW-GSs and processing quality based on the traditional statistical analysis exists big differences,because this method only studies on the alleles, but not single protein subunit, and also neglect the interactions between the environments and genotypes, the genes and genes.To study the different repeats units of low molecular weight glutenin subunits on flour quality, this experiment cloned three low-molecular-weight glutenin subunit genes from strong gluten wheat variety"Shaan 253"using the primer pairs, which were designed according to the conservative sequences of 5'and 3'terminal domains, then constructs expression vector expressed in Escherichia coli. The fusion protein is purified byHis-Ni2+ affinity chromatography, and then mixed into flour individually. Their effects on flour mixing properties were investiged by 4g farinograph. The results are followed:1. Three full-length coding regions(open reading frame, ORF)of low-molecular-weight glutenin subunit(LMW-GS)gene were amplified from cultivar"Shaan253"(GenBank accession number GQ892581,GQ892582,GQ892589). The length of three nucleotide sequences were 1053bp,966bp,900bp. Three genes had higher similarity with other known LMW-GS genes at Glu-D3 locus (with the highest similarity of 99%, respectively).2. Sequence analysis suggested that three translated amino acid sequence have the typical LMW-GS structures: with a 20 amino-acids N-terminal signal peptide; 13 amino-acid conservative N-terminal; the repeat region, as well as conservative C-terminal sequence. Moreover they have eight typical cysteine residues. The comparison of the three novel genes demonstrated that repetitive domain variation resulted from deletions and insertions of some repeat units.3. These three novel genes were sub-cloned into the prokaryotic expression vector pET32a for fusion expression in the host bacteria E.coli Rosetta-gami B (DE3). Induced by IPTG and the production was examined by SDS-PAGE and western blot. The result shows that fusion protein was successful expressed.4. The fusion protein is purified by His-Ni2+ affinity chromatography and good purification result is obtained.5. The purified protein mixed into flour individually. There effects on flour mixing properties were investiged by 4g farinograph. The result shows these proteins have great influence on dough properties. The farinographic performance of GQ892582 missing a set of PPFS(Q)n (n = 4, 9) PVLPQQ significantly outperformed both GQ892581 with complete repeat unit and GQ892589 missing two sets of PPFS(Q)n (n = 4, 9) PVLPQQ. It suggested that the spatial structure of LMW Glutenin was significantly effected by repeat unit, not simply by the change of secondary structure element.This paper studied on the LMW-GS genes of strong gluten wheat variety"Shaan 253", and also successfully expressed in E.coli. The results not only provide a reference for the resource of good quality trait and potential influencing factors, but also lay the foundation for the study of a single LMW-GS in vitro.