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作物学报 ›› 2009, Vol. 35 ›› Issue (4): 672-678.doi: 10.3724/SP.J.1006.2009.00672

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦品种“陕253”低分子量谷蛋白亚基基因的克隆及原核表达

吴丹1，高翔12*，于旭1，董剑12，赵万春12，陈其皎12，庞红喜12，李哲清12   

  1. 1西北农林科技大学农学院，陕西杨凌712100；2陕西省小麦工程技术研究中心，陕西杨凌712100
  • 收稿日期:2008-09-08 修回日期:2008-12-12 出版日期:2009-04-12 网络出版日期:2009-02-16
  • 通讯作者: 高翔
  • 基金资助:

    本研究由陕西省“13115”科技创新工程重大项目（2007ZDKG-01）；农业部小麦现代产业技术体系建设陕西小麦综合试验重大项目（NYCYTX-001）资助。

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 Published:2009-04-12 Published online:2009-02-16
  • Contact: GAO Xiang

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2153

被引次数

15

摘要：

利用LMW-GS特异引物, 从强筋小麦品种“陕253”中克隆了1个1 498 bp的片段(GenBank登录号为FJ172533)，该片段包含全长为912 bp的低分子量谷蛋白亚基的完整编码序列。经比较推导氨基酸序列的同源性，发现该基因属于Glu-D3位点编码低分子量谷蛋白亚基的基因，编码产物N-端具有LMW-m型低分子量谷蛋白亚基的典型特征，系统演化分析也支持这一结果。构建了该基因的表达载体pET32a-GluD3-S253，在宿主菌E. coli Rosetta-gami B (DE3)中经IPTG诱导表达融合蛋白。SDS-PAGE和Western blot检测表达产物，证实融合蛋白表达成功。

关键词: 小麦, 低分子量谷蛋白亚基, 基因克隆, 融合蛋白, 原核表达

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

[1]Singh N K, Shepherd K W. Linkage mapping of genes controlling endosperm storage proteins in wheat: 1. Genes on the short arms of group 1 chromosomes. Theor Appl Genet, 1988, 75: 628–641
[2]Gupta R B, Shepherd K W. Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutenin: 1. Variation and genetic control of the subunits in hexaploid wheats. Theor Appl Genet, 1990, 80: 65–74
[3]Gupta R B, MacRitchie F. Allelic variation at glutenin subunit and gliadin loci, Glu-1, Glu-3 and Gli-1 of common wheats: II. Biochemical basis of the allelic effects on dough properties. J Cereal Sci, 1994, 19: 19–29
[4]Flaete N E S, Uhlen A K. Association between allelic variation at the combined Gli-1, Glu-3 Loci and protein quality in common wheat (Triticum aestivum L.). J Cereal Sci, 2003, 37: 129–137
[5]Cherdouh A, Khelifi D, Carrillo J M, Nieto-Taladriz M T. The high and low molecular weight glutenin subunit polymorphism of Algerian durum wheat landraces and old cultivars. Plant Breed, 2008, 124: 338–342
[6]Sreeramulu G, Singh N K. Genetic and biochemical characterization of novel low molecular weight glutenin subunits in wheat (Triticum aestivum L.). Genome, 1997, 40: 41–48
[7]Cassidy B G, Dvorak J, Anderson O D. The wheat low-molecular-weight glutenin gene: characterization of six new gene and process in understanding gene family structure. Theor Appl Genet, 1998, 96: 743–750
[8]Wei Y-Y(魏燕燕), Zhao H-X(赵惠贤), Li Y-C(李勇超), Wang Y-P(王银萍), Guo A-G(郭蔼光). Cloning and sequencing of LMW-GS gene at Glu-B3 in wheat. Acta Bot Boreali-Occident Sin (西北植物学报), 2006, 26(9): 1864–1869 (in Chinese with English abstract)
[9]D’Ovidio R. The low-molecular-weight glutenin subunits of wheat gluten. J Cereal Sci, 2004, 39: 321–339
[10]Liu L(刘丽), He Z-H(何中虎), Yu Y-X(于亚雄), Yang J-H(杨金华), Cheng G(程耿), Hu Y-X(胡银星). Research progress in wheat glutenin. Southwest China J Agric Sci (西南农业学报), 2004, 37(1): 8–14 (in Chinese with English abstract)
[11]Feilet P, Autoran J. The low molecular glutenin proteins in the determina tion of cooking guality. Cereal Chem, 1989, 66: 26–30
[12]Yang W-Q(杨文强). Low molecular weight glutenin subunits (LMW-GS) and their relationship with quality of wheat. J Northeast Agric Univ (东北农业大学学报), 2003, 34(4): 482–485 (in Chinese with English abstract)
[13]Lei D-F(雷东锋), Ma J-G(马建岗), Zhao W-M(赵文明). The progress of study on wheat seed storage proteins. Acta Bot Boreali-Occident Sin (西北植物学报), 2001, 21(3): 584–593 (in Chinese with English abstract)
[14]D’Ovidio R, Masci S. The low-molecular-weight glutenin subunits of wheat gluten. J Cereal Sci, 2004, 39: 321–339
[15]D’Ovidio R, Tanzarella O A, Porceddu E. Nucleotide sequence of a low-molecular-weight glutenin from Triticum durum. Plant Mol Biol, 1992, 18: 781–784
[16]Lee Y K, Bekes F, Ciaffi M. The low-molecular-weight glutenin subunit protenins of primitive wheats: Ⅳ. Functional properties of products from individual genes. Theor Appl Genet, 1999, 98: 149–155
[17]Cloutier S, Rampitsch C, Penner G A, Lukow O M. Cloning and expression of a LMW-i glutenin gene. J Cereal Sci, 2001, 33: 143–154
[18]Xu Z-F(徐兆飞), Zhang H-Y(张惠叶), Zhang D-Y(张定一). Wheat quality and its improvement. Beijing: China Meteorological Press, 2000. pp 47–51 (in Chinese)
[19]Murray H G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucl Acids Res, 1980, 8: 4321–4325
[20]An X, Zhang Q, Yan Y, Li Q, Zhang Y, Wang A, Pei Y, Tian J, Wang H, Hsam S L K, Zeller F J. Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.). Theor Appl Genet, 2006, 113: 383–395
[21]Bietz J A, Wall J S. Isolation and characterization of gliadinlike subunits from glutenins. Cereal Chem, 1973, 50: 537–547
[22]Cassidy B G, Dvorak J, Anderson O D. The wheat low-molecular weight glutenin genes: Characterization of six new genes and progress in understanding gene family structure. Theor Appl Genet, 1998, 96: 743–750
[23]Gupta R B, Shepherd K W. Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutelin. Theor Appl Genet, 1990, 80: 65–74
[24]Jackson E A, Holt L M, Payne P L. Characterization of high-molecular-weight gliadins low-molecular-weight glutenin subunit of wheat endosperm by two-dimensional electrophoresis and the chromosomal localization of their controlling gene. Theor Appl Genet, 1983, 66: 29–37
[25]Lew E J L, Kuzmicky D D, Kasarda D D. Characterization of low molecular weight glutenin subunits by reversed-phase high-performance liquid chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing. Cereal Chem, 1992, 69: 508–515
[26]Sissons M J, Bekes F, Skerritt J H. Isolation and functionality testing of low molecular weight glutenin subunits. Cereal Chem, 1998, 75: 30–36
[27]Cloutier S, Rampitsch C, Penner G A, Lukow O M. Cloning and expression of a LMW-i glutenin gene. J Cereal Sci, 2001, 33: 143–154
[28]Masci S, D’Ovidio R, Lafiandra D, Kasarda D D. Characterization of a Low-molecular-weight glutenin subunit gene from bread wheat and the corresponding protein that represents a major subunit of the glutenin polymer. Plant Physiol, 1998, 118: 1147–1158
[29]Tabiki T, Ikeguchi S, Ikeda T M. Effects of high-molecular-weight and low-molecular-weight glutenin subunit alleles on common wheat flour quality. Breed Sci, 2006, 56: 131–136
[30]Ikeda T M, Nagamine T, Fukuoka H, Yano H. Characterization of new low-molecular-weight glutenin subunit genes in wheat. Theor Appl Genet, 2002, 104: 680–687
[31]Han B(韩彬), Shepherd K W. The correlations between low glutenin subunits and gliadins and their effects on bread-making quality in the progeny of two wheats. Sci Agric Sin (中国农业科学), 1991, 24(4): 19–25 (in Chinese with English abstract)
[32]Gianibelli M C, Larroque O R, Macritchie F, Wrigley C W. Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chem, 2001, 78: 635–646
[33]Kolster P, Krechting C F, Van Gelder W M J. Quantification of individual high molecular weight glutenin subunits using SDS-PAGE and scanning densitometry. J Cereal Sci, 1992, 15: 49–61
[34]Zhang P-P(张平平), Zhang Y(张勇), Xia X-C(夏先春), He Z-H(何中虎). Protocol establishment of reversed-phase high-performance liquid chromatography (RP-HPLC) for analyzing wheat gluten protein. Sci Agric Sin (中国农业科学), 2007, 40(5): 1002–1009 (in Chinese with English abstract)
[35]Benmoussa M, Vezina L P, Page M, Gelinas P, Yelle S, Laberge S. Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene. Biotechnol Bioen, 2004, 87: 495–500
[36]Wang X Y, Liu K F, Guo W Z. Cloning and expression of low molecular weight glutenin genes from the Chinese elite wheat cultivar “Xiaoyan 54”. J Integr Plant Biol, 2006, 48: 212–218
[37]Patacchini C, Masci S, D’Ovidio R, Lafiandra D. Heterologous expression and purification of native and mutated LMW-GS from durum wheat. J Chromatogr B, 2003, 786: 215–220
[38]Tamás L, Shewry P R. Heterologous expression and protein engineering of wheat gluten proteins. J Cereal Sci, 2006, 43: 259–274
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