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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (01): 79-86.doi: 10.3724/SP.J.1006.2011.00079

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

Cloning, Prokaryotic Expression, and Functional Testing of a γ-Gliadin Gene from Wheat Cultivar Shaan 253

WANG Ming-Xia1,GAO Xiang1,2,*,CHEN Qi-Jiao1,2,*,DONG Jian1,2,ZHAO Wan-Chun1,2,LI Yan-Liang1,LI Min1   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100; 2 Wheat Engineering Research Center of Shaanxi Province / New varieties cultivation of wheat Engineering Research Center of Shaanxi Province; Yangling 712100, China
  • Received:2010-05-19 Revised:2010-08-02 Online:2011-01-12 Published:2010-10-09

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Abstract: The γ-gliadin gene (GenBank accession number GQ857626) was isolated from a wheat (Triticum aestivum) cultivar with a hight quality Shaan 253, using inductuion a set of genome specific primers. The deduced amino acid sequence of GQ857626 exhibited an additional cysteine residue in the region II of mature γ-gliadin subunits compared to the previous report. A prokaryotic expression vector was constructed to express GQ857626 in the host bacterium Escherichia coli Rosetta-gami B (DE3). The SDS-PAGE and Western blot analysis confirmed that the fusion protein was expressed by induction of 0.1 mmol L-1 isopropyl-β-D-thiogalactoside (IPTG), and its actual molecular weight was about 50 kD nearly equal to the predicted. After separation and purification using HisTrap HP affinity chromatography, the function of fusion protein was tested by means of micro 4 g farinograph. When the subunit of GQ857626 was integrated into the control flour through oxidation-reduction reaction, many important farinograph parameters changed drastically, such as development time, dough stabilizing time, degree of softening. Although the gluten strength of the control flour could be improved in some degree, the subunit of GQ857626 showed a negative effect on the rheological properties of wheat flour.

Key words: Wheat, γ-gliadin, Prokaryotic expression, Farinograph parameter

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