Prokaryotic Expressing And Function Identification Of XYGluD3-LMWGS1 Gene In Commom Wheat

Triticum aestivum low-molecular-weight glutenins subunits (LMW-GS) is a kind of important storage protein and account for 40% of wheat gluten protein content. These proteins play a significant role on flour-processing properties. LMW and High-molecular-weight glutenins subunits (HMW-GS) are considered to significantly affect dough-quality characteristics. They incorporate in glutenin macropolymer (GMP) through intermolecular disulphide bonds. It was reported that the content,component and size distribution significantly affect the quality characteristics of wheat flour. The composition and quanity of glutenins affect the size distribution of glutenin macropolymer (GMP). So, wheat flour processing properties can be improved by enhancing the quanity of GMP and altering GMP component through introducing genes related to good quality into wheat cultivar. XYGluD3-LMWGS1(access no. AY263369) reported previously is the firstgene discovered so far that encoden LMW-GS with 9 Cys residues , In order to confirm its quality function, Cloning the coding region of XYGluD3-LMWGS1, expressing it in E.coli, and testing functional properties were carried out in this study. The main results are as follows : 1.The prokaryotic expression vector pET32-XYGluD3-LMWGS of the gene XYGluD3-LMWGS1 was constructed, and the gene was expressed on large scale in E.coli Rosetta(DE3)-plysS after induction by IPTG. The condition of the induction was as follows:37℃,1.0mM IPTG, 6hours. And the N-terminal amino-acid sequencing showed that the expressed protein was the targeted protein. 2.The prokaryotic expressed protein could be effectively purified by using Reactive Red 120-Agarose resin ,and the lactic acid–sodium lactate (pH 4.5) contained low concentration SDS system should be used for elution 3.The purified protein was supplemented into a base wheat flour Yuzhan2000,and mixing test showed the protein caused an significant increase in mixing time. It had a positive effect on dough strength. 4.The results of the total glutenin polymer content (TGP%), glutenin macropolymer content (GMP%), and the relative content of glutenin macropolymer (GMP/TGP) of the resulted dough showed that GMP, GMP/TGP of the dought had significately increased. This indicated the targeted protein had incorporated into glutenin macropolymer and played an important role in dough properties.