Cloning,Rokaryotic Expression And In Vitro Functional Analysis Of Three Storage Protein Genes From Wheat(Triticum Aestivum L.)

Wheat storage protein is generally divided into glutenin and gliadin,which together determine the processing quality of wheat flour,accounting for more than 80% of the total wheat protein content.Glutenin is known as HMW-GS and LMW-GS.In this study,we designed respectively a pair of specific primers for HMW-GS,LMW-GS and ?-gliadin according to the known complete gene sequences in the NCBI.Then the sequences of three kind of proteins were obtained by PCR amplification,cloning and sequencing from wheat varieties "Xinong 538" and "Xinong 558".After sequence alignment and phylogenetic tree analysis,we induced the expression of the three genes that were obtained from "Xinong 538" in Escherichia coli expression system.And then,we analyzed the effect of the three expressive proteins on wheat flour by protein purification and Farinograph.The experimental results were as follows:1.A total of 6 gene sequences that all contained single and complete open reading frame(ORFs)without introns were obtained using the designed primers.The GenBank accession numbers(GenBank acc.No.)of the gene sequences of HMW-GS,LMW-GS and ?-gliadin cloned from "Xinong 538" were KX452083,KX452081 and KX452085,and the lengths of coding region were 1812 bp,915bp and 879 bp,respectively.The GenBank acc.No.of these sequences cloned from "Xinong 558" were KX452084,KX452082 and KX452086 with the lengths of 1812 bp,915bp and 897 bp,respectively.2.After alignment to the deduced amino acid sequences,we found that KX452083 and KX452084 were all mainly composed of signal peptide,N-terminal region,repeated and C-terminal region.They all contained seven cysteine(Cys)residues,the intermediate repeated region contained typical repeating units of hexapeptide,nonapeptide and their variants,but there was no repeating tripeptide which just exists in x-type subunits.So we suggested that KX452083 and KX452084 belonged to y-type subunits.In addition,according to BLASTp and phylogenetic analysis,respectively,the two sequences were highly similar to1Dy10 and 1Dy12 subunit.KX452081 and KX452082 were mainly made up of signal peptide,N-terminus,repeated region with abundant glutamine(Q)and proline(P),C-terminalconserved region I with 5 Cys,C-terminal region II containing a Cys and being rich in Q and C-terminal region III containing a Cys.Then we speculated that KX452081 and KX452082 belonged to Glu-D3,Type V(Group 10),m-type,C-group LMW-GS by analysis of sequence alignment,the composition of N-/C-terminal amino acids(METRCIPG······VGTGVGAY),the location of Cys,and phylogenetic tree.KX452085 and KX452086 consisted of six parts:signal peptide,N-terminus,polyglutamine(Poly Q)region I,unique domain I,Poly Q region II and unique domain II.KX452085 contained 6 Cys that could form three pairs of intra-molecular disulfide bonds.KX452086 contained 7 Cys that not only could form three pairs of intra-molecular disulfide bonds,but also the rest might form an intermolecular disulfide bond.3.According to the main purpose of this experiment and the previous researches,we used the three sequences that KX452083(HMW-GS),KX452081(LMW-GS)and KX452085(?-gliadin)cloned from "Xinong 538" as the researchful objects.We induced their recombinants using IPTG to in vitro express in Escherichia coli Trans B(DE3),and the results of SDS-PAGE and Western-blot analysis indicated that the three genes were successfully expressed.4.The three genes were in vitro expressed in E.coli,and then a series of related parameters were obtained by protein purification,cryopreservation and mixing test in which Chinese spring was taken as the control flour.The results showed that KX452083(HMW-GS)was positively related to flour gluten strength,but had negative effects on dough elasticity and extension.KX452081(LMW-GS)caused the increase of dough flexibility,while resulted in the decrease of dough extension,and its effects on flour gluten were too comeplex to draw a conclusion.KX452085(?-gliadin)had a negative effect on flour gluten strength,but made the dough be more susceptible to rheological changes and had significant improvements in dough elongation and elasticity.Farinograph quality number(FQN)is usually used as an index to evaluate the flour quality,so FQN is used as the standard,we found that the order of the degree to which three storage proteins isolated from "Xinong 538" in this experiment affected flour processing quality was KX452083(HMW-GS)> KX452085(?-gliadin)> KX452081(LMW-GS).