Heterologous Expression, Separation And Function Analysis Of Low-Molecular-Weight Glutenin Subunit

Gluten is a giant proteins complex consisting of high molecular weight gluteninsubunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS). Theindispensable role of HMW-GS in quality of wheat was well demonstrated. However, a largenumber of gene copies with frequent mutation as well as the close linkage with gene encodingglidins lead to difficulty in separating single LMW-GS, which limited the research progress ofLMW-GS.In our study, LMW-GS genes were cloned from Triticum aestivum variety Cheyenne.Expression vectors were constructed and high-level expression of recombinant proteins werecarried out by optimizing expression conditions. Then, recombinant proteins were purifiedand dialyzed, followed by dough experiments to determine the quality of LMW-GS. Thecontent of research and the main results were as follows:(1) Cloning and characterization of LMW-GS genes. Three genes, named lmw-cnd1,lmw-cnd2, lmw-cnd3with ORFs of1053bp,903bp,969bp, respectively, were cloned fromCheyenne. The genes show high similarity with LMW-GS genes from other Triticum aestivumand wheat relatives, with similarity about90%~93%. The three LMW-GS differ only inrepetitive domain of sequences due to insertion or deletion of repeats in this domain. Besides,secondary structure prediction of proteins indicated that, in the three LMW-GS, random loopaccounts for about59%~65%, α-helix amounts to27%~33%, average, and only8%~8.7%isβ-sheet.(2) Prokaryotic expression of LMW-GS. Expression vectors of target genes wereconstructed and transferred into E.coli to produce proteins. High level expression of the threeLMW-GS were detected after optimizing the expression conditions as follows: the targetgenes were inserted into pET-30b as expression vectors, and then transferred intoE.coli C43(DE3) and cultivated in LB with0.3mmol/L IPTG, induced4hours at37℃.(3) Purification and function determination of LMW-GS. Highly purified recombinantproteins were obtained by metal-chelated affinity chromatography. Character parameters ofdough with addition of the three LMW-GS were determined. The results show that theaddition of each LMW-GS led to the increase on elasticity of dough. LMW-CND2and LMW-CND3promoted the strength of dough. The elasticity and strength of dough went up asthe increase on the addition of LMW-CND3. Texture analysis shows that the addition of50mg of each LMW-GS caused the decrease on handness and increase on springiness andcohesiveness of dough after mixing in farinograph.