Isolation, Characterization And Farinograph Analysis Of Novel Hmw-gss From Dasypyrum Villosum

High molecular weight glutenin subunits (HMW-GSs), occupied10%of wheat seedprotein, is an important component of wheat (Triricum aestivum L.) glutenins. HMW-GSsinteract with HMW-GSs and LMW-GSs (low molecular weight glutenin subunits) thoughtinter-intramolecular disulfide affecting the wheat processing quality. Wheat related specieshave a rich variation for storage protein genes, excavation and identification of new genesfrom them is a method to expand the genetic basis of the wheat quality improvement.Dasypyrum villosum carrying many novel HMW-GS alleles is an important genetic resourcefor wheat protein improvement.This study focuses on studying the seed storage protein genetic resources for wheatquality improvement. In this work, six HMW-GSs were isolated from Dasypyrum villosumTA10220using a pair of specific primers. DNA fragments of HMW-Dv1~HMW-Dv4weresubcloned into the pEASY-E2expression vector and expressed in Escherichia coliRosetta-gami B (DE3) cell under IPTG induction. The fusion protein was purified byHis-Trap affinity chromatography. Small-scale dough-testing was used to test the function ofHMW-GSs of Dasypyrum villosum. The results are as follows:1. Six HMW-GSs were isolated from Dasypyrum villosum TA10220(1.0~1.7kb,GenBank accession numbers: KF887414~KF887419), which were named as HMW-Dv1~HMW-Dv6and substantially smaller than those from common wheat, using a pair of specificprimers. An in-frame stop codon was found in the coding sequences of HMW-Dv5andHMW-Dv6and thus these genes might be pseudogenes.2. The analysis of deduced amino acid sequence showed that the six HMW-GS had thetypical structures of wheat: signal peptide domain, N-terminal domain, C-terminal domain,and repetitive domain. The comprehensive analysis of deduced amino acid sequence, andphylogenetic and evolutionary analyses of full sequence, N-and C-terminal domains revealedthat HMW-Dv1was closely related to y-type HMW-GS, but HMW-Dv2~HMW-Dv6hadstructural characteristics of both x-and y-types.3. DNA fragments of HMW-Dv1~HMW-Dv4were subcloned into the pEASY-E2 expression vector and expressed in Escherichia coli Rosetta-gami B (DE3) cell under IPTGinduction. The four genes were successfully expressed in E. coli system according toSDS-PAGE analysis (both the expressed protein and HMW-GS isolated from seed) andwestern blotting assay.4. The fusion protein was purified by His-Trap affinity chromatography. The proteinpurification was better according to SDS-PAGE analysis with a single band.5. The purified protein was recovered by low temperature cryodesiccation, and thenintegrated into the control flour by using a4g Micro-dough LAB Farinograph. Resultsshowed that the four HMW-GSs originated from Dasypyrum villosum had positive effects ondough quality property.In this work, we used the Dasypyrum villosum (TA10220) as the material, studied thestructure characteristic of HMW-GS of Dasypyrum villosum, successfully expressed fourgenes (HMW-Dv1~HMW-Dv4) in E. coli system, and also tested the function of HMW-GSsof Dasypyrum villosum. The results enrich the HMW-GS genetic pool, and provide a referenceto improve the processing quality.