欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2011, Vol. 37 ›› Issue (01): 79-86.doi: 10.3724/SP.J.1006.2011.00079

• 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 γ-醇溶蛋白基因的克隆、原核表达与功能鉴定

王明霞1,高翔1,2,*,陈其皎1,2,*,董剑1,2,赵万春1,2,李艳亮1,李敏1   

  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 / 陕西省小麦新品种培育工程研究中心，陕西杨凌 712100
  • 收稿日期:2010-05-19 修回日期:2010-08-02 出版日期:2011-01-12 网络出版日期:2010-10-09
  • 基金资助:

    本研究由国家自然科学基金青年科学基金(30900896), 陕西省“13115”科技创新工程重大项目(2007ZDKG-01), 现代农业产业技术体系建设专项(NYCYTX-001)和西北农林科技大学基本科研业务费青年项目(QN2009007)资助。

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 Published:2011-01-12 Published online:2010-10-09

PDF

2050

被引次数

6

摘要： 利用设计合成的特异γ-醇溶蛋白基因引物，采用PCR方法从小麦品种陕253克隆获得一个γ-醇溶蛋白基因(GenBank登录号GQ857626)。序列分析表明，该基因编码产物的II区由于碱基转换产生一个额外的半胱氨酸残基。构建了GQ857626的原核表达载体并转入表达菌株E. coli Rosetta gami B(DE3)，IPTG诱导其成功表达。使用HisTrap HP组氨酸标记亲和层析柱纯化该基因的表达产物，并使用4 g粉质仪分析其功能。结果显示，将此纯化蛋白通过氧化还原反应整合到基础面粉后，面团的形成时间缩短，稳定时间减少，弱化度增加，导致主要的粉质指数明显下降，说明该亚基对面团流变学性质整体表现不利。

关键词: 普通小麦, γ-醇溶蛋白, 原核表达, 粉质参数

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

[1]Bushuk W. Wheat cultivar identification by gliadin electrophoregrams. Can J Plant Sci, 1978, 58: 505–515
[2]Harbred N P, Bartels D, Thompson R D. Analysis of the gliadin multigene loci in bread wheat using nulisomic tetrasomic lines. Mol Gen Genet, 1985, 198: 234–242
[3]Wrifley C W, Robinson P J, Williams W T. Association between electrophoretic patterns of gliadin proteins and quality characteristics of wheat cultivars. Aust Agric Res, 1982, 33: 409–418
[4]Redaelli R. Two-dimensional mapping of gliadins using biotypes and null mutants of common wheat cultivar Saratovskaya 29. Hereditas, 1994, 121: 131–137
[5]Peter R, Nigel G. Cereal seed storage proteins: structures properties and role in grain utilization. J Exp Pathol, 2002, 53: 947–958
[6]Müller S, Wieser H. The location of disulphide bonds in α-type gliadins. J Cereal Sci, 1995, 22: 21–27
[7]Anderson O D, Hsia C C, Torres V. The wheat γ-gliadin genes: characterization of ten new sequences and further understanding of γ-gliadin gene family structure. Theor Appl Genet, 2001, 103: 323–330
[8]Shewry P R, Tatham A S. Disulphide bonds in wheat gluten proteins. J Cereal Sci, 1997, 25: 207–227
[9]Branlard G, Dardevet M. Diversity of grain proteins and bread wheat quality: I. Correlation between gliadin bands and flour quality characteristics. Cereal Sci, 1985, 13: 329–343
[10]Metakovsky E V, Annicchiarico P, Boggini G, Pogna N E. Relationship between gliadin alleles and dough strength in Italian bread wheat cultivars. Cereal Sci, 1997, 25: 229–236
[11]Shi J-L(师俊玲), Wei Y-M(魏益民). Analysis of the relationship between protein and the quality of wheat. J Cereals and Oils (粮食与油脂), 1999, (4): 3–7 (in Chinese)
[12]Yan Y-M(晏月明), Ru Y-Y(茹岩岩), Yu J-Z(余建中), Liu G-T(刘广田), Prodanovic S. Analysis of gliadin allele composition at Gli-1 and Gli-2 loci in Chinese wheat cultivars. J Agric Biotechnol (农业生物技术学报), 2000, 6(1): 23–27 (in Chinese with English abstract)
[13]Pei Y-H(裴玉贺), Wang A-L(王爱丽), Yan Y-M(晏月明). Small-scale testing and application of wheat flour quality. J Triticeae Crops (麦类作物学报), 2005, 25(suppl): 104–108 (in Chinese with English abstract)
[14]Békés F, Gras P W, Gupta R B. Mixing properties as a measure of reversible reduction and oxidation of doughs. Cereal Chem, 1994, 71: 44–50
[15]Békés F, Gras P W. In vitro studies on gluten protein functionality. Cereal Food World, 1999, 44: 580–586
[16]Pei Y-H(裴玉贺), Sun H(孙辉), Song X-Y(宋希云), Yan Y-M(晏月明), Ji K-M(祭康敏), Li Q-Y(李巧云), He Z-H(何中虎), Liu L(刘丽), Huang X-F(黄兴峰). In vitro functional testing of high molecular glutenin subunits in wheat. Acta Agron Sin(作物学报), 2008, 34(11): 1910–1915 (in Chinese with English abstract)
[17]Uthayakumaran S, Stoddard F L, Gras P W, Bekes F. Effects of incorporated glutenins on functional properties of wheat dough. Cereal Chem, 2000, 77: 737–743
[18]Luo C, Griffin W B, Branlard G, McNeil D L. Comparison of low- and high-molecular-weight wheat glutenin allele effects on flour quality. Theor Appl Genet, 2001, 102: 1088–1098
[19]MacRitchie F. Evaluation of contributions from wheat protein fractions to dough mixing and bread making. Cereal Sci, 1987, 6: 259–269
[20]Weegels P L, Marseille J P, Bosveld P, Hamer R J. Large-scale separation of gliadins and their bread making quality. Cereal Sci, 1994, 20: 253–264
[21]Murray H G, Thompson W F. Rapid isolation of high molecular weight DNA. Nucl Acids Res, 1980, 8: 4321–4325
[22]Wu D(吴丹), Gao X(高翔), Yu X(于旭), Dong J(董剑), Zhao W-C(赵万春), Chen Q-J(陈其皎), Pang H-X(庞红喜), Li Z-Q(李哲清). Cloning and prokaryotic expression of a low molecular weight glutenin gene from wheat variety Shaan 253. Acta Agron Sin (作物学报), 2009, 35(4): 672–678 (in Chinese with English abstract)
[23]Scheets K, Hedgcoth C. Nucleotide sequence of a gamma gliadin gene: comparisons with other gamma gliadin sequences show the structure of gamma gliadin genes and the general primary structure of gamma gliadins. J Plant Sci, 1988, 57: 141–150
[24]Qi P F, Wei Y M, Ouellet T, Chen Q, Tan X, Zheng Y L. The gamma-gliadin multigene family in common wheat (Triticum aestivum) and its closely related species. BMC Genomics, 2009, 10: 168
[25]Izard J W, Doughty M B, Kendall D A. Physical and conformational properties of synthetic idealized signal sequences parallel their biological function. Biochemistry, 1995, 34: 9904–9912
[26]Crooks G E, Hon G, Chandonia J M, Brenner S E. WebLogo: A sequence logo generator. Gen Res, 2004, 14: 1188–1190
[27]Schneider T D, Stephens R M. Sequence Logos: A new way to display consensus sequences. Nucl Acids Res, 1990, 18: 6097–6100
[28]Poreeddu E, Turehetta T, Masci S, Ovidio R D, Lafiandra D, Kasarda D D, Impiglia A, Nachit M M. Variation in endosperm protein composition and technological quality properties in durum wheat. Euphytica, 1998, 100: 197–205
[1] 余国武, 青芸, 何珊, 黄玉碧. 玉米SSIIb蛋白多克隆抗体的制备及其应用[J]. 作物学报, 2022, 48(1): 259-264.
[2] 靳义荣, 刘金栋, 刘彩云, 贾德新, 刘鹏, 王雅美. 普通小麦氮素利用效率相关性状全基因组关联分析[J]. 作物学报, 2021, 47(3): 394-404.
[3] 王珍, 姚梦楠, 张晓莉, 曲存民, 卢坤, 李加纳, 梁颖. 甘蓝型油菜BnMAPK1的原核表达、亚细胞定位及酵母双杂交文库筛选[J]. 作物学报, 2020, 46(9): 1312-1321.
[4] 张平平,姚金保,王化敦,宋桂成,姜朋,张鹏,马鸿翔. 江苏省优质软麦品种品质特性与饼干加工品质的关系[J]. 作物学报, 2020, 46(4): 491-502.
[5] 杨芳萍,刘金栋,郭莹,贾奥琳,闻伟鄂,巢凯翔,伍玲,岳维云,董亚超,夏先春. 普通小麦‘Holdfast’条锈病成株抗性QTL定位[J]. 作物学报, 2019, 45(12): 1832-1840.
[6] 王林生,张雅莉,南广慧. 普通小麦-大赖草易位系T5AS-7LrL·7LrS分子细胞遗传学鉴定[J]. 作物学报, 2018, 44(10): 1442-1447.
[7] 赵德辉, 张勇, 王德森, 黄玲, 陈新民, 肖永贵, 阎俊, 张艳, 何中虎. 北方冬麦区新育成优质品种的面包和馒头品质性状[J]. 作物学报, 2018, 44(05): 697-705.
[8] 苗永杰, 阎俊, 赵德辉, 田宇兵, 闫俊良, 夏先春, 张勇, 何中虎. 黄淮麦区小麦主栽品种粒重与籽粒灌浆特性的关系[J]. 作物学报, 2018, 44(02): 260-267.
[9] 肖永贵,Susanne DREISIGACKER,Claudia NU?EZ-RíOS,胡卫国,夏先春,何中虎. 基于FLUOstar平台的小麦dsDNA荧光定量与基因型分析[J]. 作物学报, 2017, 43(07): 947-953.
[10] 董雪,刘梦,赵献林,冯玉梅,杨燕. 普通小麦近缘种低分子量麦谷蛋白亚基Glu-A3基因的分离和鉴定[J]. 作物学报, 2017, 43(06): 829-838.
[11] 刘凯,邓志英,张莹,王芳芳,刘佟佟,李青芳,邵文,赵宾,田纪春*,陈建省*. 小麦茎秆断裂强度相关性状QTL的连锁和关联分析[J]. 作物学报, 2017, 43(04): 483-495.
[12] 宫希,蒋云峰,徐彬杰,乔媛媛,华诗雨,吴旺,马建,周小鸿,祁鹏飞,兰秀锦. 利用普通六倍体小麦和西藏半野生小麦杂交衍生的重组自交系定位小麦芒长QTL[J]. 作物学报, 2017, 43(04): 496-500.
[13] 苏亚春,王竹青,李竹,刘峰,许莉萍,阙友雄,戴明剑,陈允浩. 甘蔗过氧化物酶基因ScPOD02的克隆与功能鉴定[J]. 作物学报, 2017, 43(04): 510-521.
[14] 喻时周,杨成龙,郭建春,段瑞军. 海马齿甜菜碱醛脱氢酶基因克隆、高效表达及酶学特性分析[J]. 作物学报, 2016, 42(10): 1569-1574.
[15] 王鑫,马莹雪,杨阳,王丹峰,殷慧娟,王洪刚. 小麦矮秆种质SN224的鉴定及农艺性状QTL分析[J]. 作物学报, 2016, 42(08): 1134-1142.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2