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作物学报 ›› 2014, Vol. 40 ›› Issue (04): 600-610.doi: 10.3724/SP.J.1006.2014.00600

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定

杨华1,高翔1,2,*,陈其皎1,赵万春1,*,董剑1,2,李晓燕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研究中心, 陕西杨凌 712100
  • 收稿日期:2013-12-05 修回日期:2014-01-12 出版日期:2014-04-12 网络出版日期:2014-02-14
  • 通讯作者: 高翔, E-mail: gx@nwsuaf.edu.cn; 赵万春, E-mail: zhaowc2009@hotmail.com
  • 基金资助:

    本研究由国家自然科学基金项目(31171538, 30900896),国家现代农业产业技术体系建设专项(CARS-3-2-47)和西北农林科技大学唐仲英育种基金项目(A212020912)资助。

Isolation, Characterization and Farinograph Analysis of Novel HMW-GSs from Dasypyrum villosum

YANG Hua1,GAO Xiang1,2,*,CHEN Qi-Jiao1,ZHAO Wan-Chun1,*,DONG Jian1,2,LI Xiao-Yan1   

  1. 1 College of Agronomy, Northwest A&F University, Yangling 712100, China; 2 Wheat Engineering Research Center of Shaanxi Province/New Varieties Cultivation of Wheat Engineering Research Center of Shaanxi Province, Yangling 712100, China
  • Received:2013-12-05 Revised:2014-01-12 Published:2014-04-12 Published online:2014-02-14
  • Contact: 高翔, E-mail: gx@nwsuaf.edu.cn; 赵万春, E-mail: zhaowc2009@hotmail.com

摘要:

利用设计的HMW-GS特异引物,从簇毛麦TA10220中克隆到6HMW-GS基因序列(1.0~1.7 kb, GenBank登录号为KF887414~KF887419),比普通小麦HMW-GS基因序列小,其中KF887414~KF887417为能正常表达的基因,KF887418KF887419是编码区出现终止信号的假基因。综合推导氨基酸的序列分析以及基于编码蛋白全序列、N-端和C-端域的进化树分析,认为KF887414属于yHMW-GSKF887415~KF887419的氨基酸序列同时具有xy型的结构特征。将具有完整编码区的4个基因在宿主菌Escherichia coli Rosetta-gami B(DE3)中经IPTG诱导表达,通过SDS-PAGE分析表达的蛋白质和种子提取的HMW-GS,经Western blot进一步检测表达产物,证明蛋白质表达成功。通过His-Trap HP柱纯化表达蛋白,回收产物经SDS-PAGE分析后,低温冷冻干燥备用。将冷冻干燥的回收产物加入中国春基础面粉中,利用微量掺粉试验通过测定粉质参数来鉴定簇毛麦来源的HMW-GS对面粉品质的影响,结果表明,簇毛麦来源的4HMW-GS对于面粉加工品质具有显著的正向效应。

关键词: 簇毛麦, 高分子量谷蛋白亚基, 原核表达, 掺粉实验

Abstract:

Dasypyrum villosum carrying many novel HMW-GS alleles is an important genetic resource for wheat protein improvement. In this study, we isolated six HMW-GS genes from D. villosum TA10220 (1.0~1.7 kb, GenBank accession numbers: KF887414~KF887419), which were substantially smaller than those from common wheat, using a pair of specific primers. An in-frame stop codon was found in the coding sequences of KF887418 and KF887419 and thus these genes might be pseudogenes. The comprehensive analysis of deduced amino acid sequence, and phylogenetic and evolutionary analyses of full sequence, N- and C-terminal domains revealed that KF887414 was closely related to y-type HMW-GS, but KF887415~KF887416 had structural characteristics of both x- and y-types. DNA fragments of KF887414~KF887417 were subcloned into thepEASY-E2 expression vector and expressed in Escherichia coli Rosetta-gami B(DE3)cell under IPTG induction. The four genes were successfully expressed in E. coli system according to SDS-PAGE analysis (both the expressed protein and HMW-GS isolated from seed) and western-blotting assay. The fusion protein was purified and recovered by His-Trap affinity chromatography and low temperature cryodesiccation, and then integrated into the control flour by using a 4 g Micro-dough LAB Farinograph. Results showed that the four HMW-GSs originated from D. villosum had positive effects on dough quality property.

Key words: Dasypyrum villosum, High-molecular-weight glutenin subunits (HMW-GS), Prokaryotic expression, Farinograph

[1]Wrigley C W. Biopolymers-Giant proteins with flour power. Nature, 1996, 381: 738–739



[2]Payne P I. Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Annu Rev Plant Phys, 1987, 38: 141–153



[3]Shewry P R, Halford N G, Tatham A S. High molecular weight subunits of wheat glutenin. J Cereal Sci, 1992, 15: 105–120



[4]Lawrence G J, Shepherd K W. Variation in glutenin protein subunits of wheat. Aust J Biol Sci, 1980, 33: 221–233



[5]Lawrence G J, Shepherd K W. Inheritance of glutenin protein subunits of wheat. Theor Appl Genet, 1981, 60: 333–337



[6]Payne P I, Law C N, Mudd E E. Control by homoeologous group 1 chromosomes of the high-molecular-weight subunits of glutenin, a major protein of wheat endosperm. Theor Appl Genet, 1980, 58: 113–120



[7]Payne P I, Holt L M, Law C N. Structural and genetical studies on the high- molecular-weight subunits of wheat glutenin. Theor Appl Genet, 1982, 63: 129–138



[8]Payne P I, Nightingale M A, Krattiger A F, Holt L M. The relationship between HMW glutenin subunit composition and the bread-making quality of British-grown wheat varieties. J Sci Food Agric, 1987, 40: 51–65



[9]Mackie A M, Lagudah E S, Sharp P J, Lafiandra D. Molecular and biochemical characterization of HMW glutenin subunits from T. tauschii and the D genome of hexaploid wheat. J Cereal Sci, 1996, 23: 213–225



[10]Harberd N P, Bartels D, Thompson R D. DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat. Biochem Genet, 1986, 24: 579–596



[11]Shewry P R, Halford N G, Tatham A S. The high molecular weight subunits of wheat, barley and rye: genetics, molecular biology, chemistry and role in wheat gluten structure and functionality. Oxford Surv Plant Mol Cell Biol, 1989, 6: 163–219



[12]Dovidio R, Masci S, Porceddu E. Development of a set of oligonucleotide primers specific for genes at the Glu-1 complex loci of wheat. Theor Appl Genet, 1995, 91: 189–194



[13]Gradzielewska A. The genus Dasypyrum: 1. The taxonomy and relationships within Dasypyrum and with Triticeae species. Euphytica, 2006, 152: 429–440



[14]Gradzielewska A. The genus Dasypyprum: 2. Dasypyrum villosum: a wild species used in wheat improvement. Euphytica, 2006, 152: 441–454



[15]De Pace C, Snidaro D, Ciaffi M, Vittori D, Ciofo A, Cenci A, Tanzarella O A, Qualset C O, Scarascia Mugnozza G T. Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphytica, 2001, 117: 67–75



[16]Zhao W C, Qi L L, Gao X, Zhang G S, Dong J, Chen Q J, Bernd F, Bikram S G. Development and characterization of two new Triticum aestivum?Dasypyrum villosum Robertsonian translocation lines T1DS•1V#3L and T1DL•1V#3S and their effect on grain quality. Euphytica, 2010, 175: 343–350



[17]董剑, 杨华, 赵万春, 李晓燕, 陈其皎, 高翔. 普通小麦中国春-簇毛麦异位系T1DL•1VS和T1DS•1VL的农艺和品质特性. 作物学报, 2013, 39: 1386–1390



Dong J, Yang H, Zhao W C, Li X Y, Chen Q J, Gao X. Agronomic traits and grain quality of Chinese Spring?Dasypyrum villosum translocation lines T1DL•1VS and T1DS•1VL. Acta Agron Sin, 2013, 39: 1386–1390 (in Chinese with English abstract)



[18]Montebove L, De Pace C, Jan C C, Scarascia Mugnozza G T, Qualse C O. Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L.) Candargy. Theor Appl Genet, 1987, 73: 836–845



[19]Shewry P R, Parmar C, Pappin D C. Characterization and genetic control of the prolamins of Haynaldia villosa: relationship to cultivated species of the Triticeae (rye, wheat, and barley). Biochem Genet, 1987, 25: 309–325



[20]张瑞奇. 簇毛麦籽粒硬度基因和贮藏蛋白基因的染色体定位及易位系选育. 南京农业大学博士学位论文, 2010



Zhang R Q. Chromosome Location of Genes for Hardness Locus and Storage Proteins in H. villosa and Creation the Translocation Lines. PhD Dissertation of Nanjing Agricultural University, Nanjing, China, 2010 (in Chinese with English abstract)



[21]庞玉辉, 陈新宏, 赵继新, 武军, 程雪妮, 刘淑会, 杨群慧, 杜万里, 陈林刚. 簇毛麦HMW-GS及其启动子基因的克隆与序列分析. 西北植物学报, 2009, 29: 859–866



Pang Y H, Chen X H, Zhao J X, Wu J, Cheng X N, Liu S H, Yang Q H, Du W L, Chen L G. Cloning and sequence analysis of the HMW-GS gene and its promoter from Dasypyrum villosum. Acta Bot Boreali-Occident Sin, 2009, 29: 859–866 (in Chinese with English abstract)



[22]陈凡国, 朱翔宇, 夏光敏. 簇毛麦中一种新型高分子量麦谷蛋白亚基基因序列的研究. 西北植物学报, 2005, 25: 1410–1414



Chen F G, Zhu X Y, Xia G M. Sequence of a new HMW glutenin subunit gene in Haynaldia villosa. Acta Bot Boreali-Occident Sin, 2005, 25: 1410–1414 (in Chinese with English abstract)



[23]刘守斌. 簇毛麦染色体分子标记的筛选及其高分子量谷蛋白亚基基因的克隆. 中国农业大学博士论文, 2002



Liu S B. The Characterization of V-Genome Specific Molecular Marker and Cloning of High-Molecular-Weight Glutenin Subunit Genes in Haynaldia villosa. PhD Dissertation of China Agricultural University, Beijing, China, 2002 (in Chinese with English abstract)



[24]陈晓燕. 簇毛麦储藏蛋白基因的克隆及小麦的抗白粉病育种. 山东大学硕士论文, 2009



Chen X Y. Glutenin and Gliadin Genes Cloning from Dasypyrum villosum and Powdery Mildew Resistance Breeding in Wheat. MS Thesis of Shandong University, Tai’an, Shandong, China, 2009 (in Chinese with English abstract)



[25]段淑娥, 赵文明. 小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析. 陕西师范大学学报(自然科学版), 2004, 32(1): 77–79



Duan S E, Zhao W M. Rapid separation and SDS-PAGE analysis of wheat glutenin subunits. J Shaanxi Normal Univ (Nat Sci Edn), 2004, 32(1): 77–79 (in Chinese with English abstract)



[26]Verbruggen I M, Veraverbeke W S, Vandamme A, Delcour A. Simultaneous isolation of wheat high molecular weight and low molecular weight glutenin subunits. J Cereal Sci, 1998, 28: 25–32



[27]Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321–4325



[28]Xu H, Wang R J, Shen X, Zhao Y L, Sun G L, Zhao H X, Guo A G. Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar. Theor Appl Genet, 2006, 113: 1295–1303



[29]Chen F G, Zhao F, Liu R K, Xia G M. Functional properties of two low-molecular-weight glutenin subunits carrying additional cysteine residues from hybrid introgression line II-12 derived from Triticum aestivum and Agropyron elongatum. Food Chem, 2011, 127: 1773–1776



[30]Forde J, Malpica J M, Halford N G, Shewry P R, Anderson O D, Greene F C, Miflin B J. The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L.). Nucl Acids Res, 1985, 13: 6817–6832



[31]De Bustos A, Rubio P, Jouve N. Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat. Theor Appl Genet, 2000, 100: 1085–1094



[32]Wan Y, Wang D, Shewry P, Halford N. Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica. Theor Appl Genet, 2002, 104: 828–839



[33]Anderson O D, Greene F C. The characterization and comparative analysis of high-molecular-weight glutenin genes from genomes A and B of a hexaploid bread wheat. Theor Appl Genet, 1989, 77: 689–700



[34]Zhong G Y, Qualset C O. Allelic diversity of high-molecular-weight glutenin protein subunits in natural populations of Dasypyrum villosum (L.) Candargy. Theor Appl Genet, 1993, 86: 851–858



[35]Feng D S, Chen F G, Zhao S Y, Xia G M. High-molecular-weight glutenin subunit genes in decaploid Agropyron elongatum. Acta Bot Sin, 2004, 46: 489–496



[36]封德顺, 陈凡国, 赵双宜, 夏光敏. 高冰草中一种新型高分子量谷蛋白亚基编码序列的研究. 西北植物学报, 2004, 24: 237–242



Feng D S, Chen F G, Zhao S Y, Xia G M. Study on a novel HMW glutenin subunit coding region from Agropyron elongatum. Acta Bot Boreali-Occident Sin, 2004, 24: 237–242 (in Chinese with English abstract)



[37]Wang J R, Yan Z H, Wei Y M, Zheng Y L. A novel high-molecular-weight glutenin subunit gene Ee1.5 from Elytrigia elongate (Host) Nevski. J Cereal Sci, 2004, 40: 289–294



[38]Wang J R, Yan Z H, Wei Y M, Zheng Y L. Characterization of high-molecular-weight glutenin subunit genes from Elytrigia elongata. Plant Breed, 2006, 125: 89–95



[39]Guo Z F, Yan Z H, Wang J R, Wei Y M, Zheng Y L. Characterizationof HMW prolamines and their coding sequences from Crithopsis delileana. Hereditas, 2005, 142: 56–64



[40]Liu S W, Zhao S Y, Chen F G, Xia G M. Generation of novel high quality HMW-GS genes in two introgression lines of Triticum aestivum/Agropyron elongatum. Evol Biol, 2007, 7: 76



[41]Liu S W, Gao X, Xia G M. Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding. Theor Appl Genet, 2008, 116: 325–334



[42]Jiang Q T, Wei Y M, Lu Z X, Pu Z E, Lan X J, Zheng Y L. Structural variation and evolutionary relationship of novel HMW glutenin subunits from Elymus glaucus. Hereditas, 2010, 147: 136–14

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