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Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (4): 672-678.doi: 10.3724/SP.J.1006.2009.00672

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Prokaryotic Expression of a Low Molecular Weight Glutenin Gene from Wheat Variety"Shaan 253"

WU Dan1,GAO Xiang12*,YU Xu1,DONG Jian12,ZHAO Wan-Chun12,CHEN Qi-Jiao12,PANG Hong-Xi12,LI Zhe-Qing12   

  1. 1College of Agronomy,Northwest A&F University,Yangling 712100,China;2Wheat Engineering Research Center in Shaanxi Province,Yangling 712100,China
  • Received:2008-09-08 Revised:2008-12-12 Online:2009-04-12 Published:2009-02-16
  • Contact: GAO Xiang E-mail:gx@nwsuaf.edu.cn

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Abstract:

Low-molecular-weight glutenin subunits (LMW-GS) play an important role in the determination of flour viscoelastic properties in wheat (Triticum aestivum L.). LMW-GS has large polymorphism and variation in molecular size, thus, it is difficult to be isolated using one-dimensional electrophoresis. Shaan 253 is a wheat variety with characteristics of high yield and early maturity, especially, with elite high-molecular-weight glutenin subunits, such as 5+10 on 1D, 14+15 and 20 on 1B, and 1 on 1A. The purpose of this study was to understand the contribution of LMW-GS to the processing quality in Shaan 253. Using a pair of specific primers of LMW-GS and pMD19-T vector, one DNA fragment of 1 498 bp (GenBank accession No. FJ172533) was obtained from Shaan 253. The fragment contained the complete coding sequence of 912 bp and encoded 304 amino acid residues. According to sequence analysis, this gene was involved in Glu-D3 loci, and had high similarities to other known LMW-GS genes with the highest identity of 99.34%. Deduced amino acid sequence showed there were typical features of LMW-m type in N-terminal region, which was confirmed by the phylogenetic analysis. The expression vector of pET32a-GluD3-S253 was constructed and transformed into the host bacteria Escherichia coli Rosetta-gami B (DE3). The expression product was testified using SDS-PAGE and Western-blot, indicating that the fusion protein was successfully expressed.

Key words: Wheat, Low-molecular-weight glutenin subunits(LMW-GS), Gene cloning, Fusion protein, Prokaryotic expression

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