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Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (08): 1340-1349.doi: 10.3724/SP.J.1006.2014.01340


Cloning, Prokaryotic Expression and in vitro Functional Analysis of α-Gliadin Genes from Dasypyrum villosum

YANG Fan1,CHEN Qi-Jiao1,2,GAO Xiang1,2,*,ZHAO Wan-Chun1,2,*,JIANG Qin-Qin1,WU Dan1,MENG Min1   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100, China; 2 New Varieties Cultivation of Wheat Engineering Research Centre of Shaanxi Province, Yangling 712100, China
  • Received:2014-01-10 Revised:2014-06-04 Online:2014-08-12 Published:2014-06-13
  • Contact: 高翔, E-mail: gx@nwsuaf.edu.cn, Tel: 13709124775; 赵万春, E-mail: zhaowc2009@hotmail.com, Tel: 13110439969


Gliadin, which has a great effect on wheat quality, is one of main components in gluten. According to the full lengths of α-gliadin genes deposited in NCBI database, a conserved primer pair was designed to clone α-gliadin genes in five Dasypyrum villosum lines. A total 52 sequences (816 to 873 bp in length) were isolated (GenBank accession numbers: KJ004676 to KJ004727) including eight pseudogenes and another sequence KJ004680 without stop codon. Deduced amino acid sequence anaylsis showed that KJ004677, KJ004686, and KJ004714 contain an extra Cys from the Tyr → Cys mutation, whereas, the extra Cys in KJ004696 resulted from the Ser → Cys mutation. Amino acid variation mainly occurred in N-terminal repetitive region and polyglutamine domain I. Variation in N-terminal repetitive region formed five groups in the 43 α-gliadins. To study the effects of an extra Cys on dough quality, we constructed the prokaryotic expression vectors forKJ004708 (with the typical six Cys residues) and KJ004714 (with an extra Cys) and obtained proteins of ~30 kD from Escherichia coli BL21(DE3) under the induction of isopropyl-β-D-thiogalactoside (IPTG) with the predicted molecular weight. These expressed proteins were verified by matrix-assisted laser desorption-ionization time-of-flight MALDI-TOF/TOF tandem mass spectrometry analysis. The result showed that these α-gliadins were expressed correctly in E. coli. After purification, renaturation, and freeze-drying process, the functions of the expressed proteins were tested with 4 g Farinograph. Both KJ004708 and KJ004714 had positive effects on flour quality, especially KJ004714 with an extra Cys.

Key words: Dasypyrum villosum, Alfa-gliadin, Prokaryotic expression, Functional analysis, MALDI-TOF/TOF tandem mass spectrometer

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