Cloning, Chromosomal Location And In Vitro Functional Analysis Of Three Novel LMW-GS From Dasypyrum Villosum

Low molecular weight glutenin subunits, occupied 40% of wheat seed storage protein, is an important component of wheat protein. LMW-GSs interact with Low molecular weight glutenin subunits and high-molecular-weight glutenin thought inter-intramolecular disulfide affecting the processing-quality which endows it with extensibility. Compared to HMW-GS, The study of LMW-GS Gene family has been isolated and characterized in only a few cases in common wheat, and the intensive selection and breeding over past decades resulted in less diversity of LMW-GS in cultivars ,Therefore, it is necessary to mine novel superior-quality alleles in wild species.In order to study and find the new mutant LMW-GS and its quality effect,In this work, three the low-molecular-weight glutenin subunit (LMW-GS) genes were isolated from Dasypyrum villosa (TA2127) by PCR technique. CS-Dasypyrum villosa chromosome addition lines and translocation lines were used to locate of the LMW-GS genes based on single nucleotide polymorphisms (SNPs) markers. The DNA fragment of LMW-GS was subcloned into the pET32a expression vector and expressed in E. coli BL21 (DE3) under IPTG induction. Small-scale dough-testing was conducted by incorporating the purified subunit into the basic flour by oxidation-reduction reactions, and the farinograph parameters were determined using a 10 g Farinograph.The results are as follows :1. Three novel LMW-GS genes from TA2127 were obtained, which were named as LMW-DV1, LMW-DV12 and LMW-DV15 (Gene Bank no. HQ293219, HQ293220, HQ293221, respectively). Their lengths of the open reading frame (ORF) were 1,020bp, 849bp and 831bp, respectively, which encode the proteins containing 340, 283 and 277 amino acid residues. In addition ,they had the intact promoter sequences, which included the endosperm box with 30bp. respectively.2. Comparative analysis showed that the three LMW-GS had similar structures: signal peptide domain, N-terminal repetitive domain, and C-terminal domain, and all that lacked a conserved sequence of N-terminal and Particularly, the LMW-DV1 was a pseudo gene and had a Cys in the signal peptide. In addition, we identified the SNP and Indels of this three LMW-GS genes.3. The SNP analysis specific primer was designed to anchored the locations of LMW-DV12 and LMW-DV15 on chromosome 1VS, and the LMW-DV1 was not located which need to investigate further.4. The expression product of LMW-DV12 and LMW-DV15 was testified using SDS-PAGE and Western-blot, indicating that the fusion protein was successfully expressed.5. Using affinity chromatography to purify the fusion protein, obtained a single band, showed that the effect of the purification was better.6.The farinograph results of LMW-DV12 and LMW-DV15 showed a positive effect on improving the dough quality.This work, we used the Dasypyrum villosum(TA2127) as the materials, studied the structure characteristic of the LMW-GS gene of Dasypyrum villosum, and also successfully expressed in E.coli, and test the function of these LMW-GSs. The result lay the foundation for the study of a single LMW-GS in vitro, and provide a reference for the resource of good quality trait and potential influencing factors, and enrich the genetic pool to improve the processing quality and afford index for screening.