欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2011, Vol. 37 ›› Issue (06): 998-1004.doi: 10.3724/SP.J.1006.2011.00998

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦抗病基因类似序列BRG1的分离与功能分析

李宁1,黄茜1,2,刘燕1,赵丹1,2,刘艳3,黄占景1,2,张增艳1,*   

  1. 1中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程 / 农业部作物遗传育种重点开放实验室, 北京100081; 2河北师范大学生命科学院, 河北石家庄 050016; 3中国农业科学院植物保护研究所, 北京 100193
  • 收稿日期:2010-10-25 修回日期:2011-03-28 出版日期:2011-06-12 网络出版日期:2011-04-12
  • 通讯作者: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)重点项目(2006AA10A104)和国家转基因生物新品种培育科技重大科技专项(2008ZX08002-001)资助。

Identification and Functional Analysis of a Wheat Resistance Analogous Gene BRG1

LI Ning1,HUANG Xi1,2,LIU Yan1,ZHAO Dan1,2,LIU Yan3,HUANG Zhan-Jing2,ZHANG Zeng-Yan1,*   

  1. 1 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences / Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 College of Life Science, Hebei Normal University, Shijiazhuang 050016, China; 3 Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • Received:2010-10-25 Revised:2011-03-28 Published:2011-06-12 Published online:2011-04-12
  • Contact: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781

PDF

1804

被引次数

5

摘要： 利用cDNA-AFLP技术筛选到1个在抗黄矮病的小麦-中间偃麦草易位系YW642中特异表达的小麦抗病基因类似序列(BYDV resistance-related gene1, BRG1)的基因片段。利用cDNA末端快速扩增技术(RACE)和RT-PCR技术，从YW642中分离出BRG1基因全长cDNA序列，获得了一个通读的抗病同源基因cDNA序列，编码由645个氨基酸组成的蛋白质，包含1个NB-ARC保守结构域和3个LRR结构域，该蛋白属于nucleotide-site binding, leucine-rich repeats (NBS-LRR)家族。荧光定量或半定量RT-PCR表达分析表明，BRG1在抗病小麦易位系YW642叶片中优势表达，受BYDV的诱导，BYDV接种后48 h表达量最高，BRG1在感病小麦亲本中8601中表达量始终较低，随BYDV接种时间延长呈轻微的下调趋势；而且外源激素水杨酸(SA)与茉莉酸(JA)处理可上调该基因在YW642中的表达。利用病毒介导的基因沉默技术分析了BRG1基因的功能，结果表明该基因沉默的抗病小麦易位系YW642中BYDV相对含量增加，但未造成抗病性表型显著改变，说明该基因参与抗黄矮病反应，但不是小麦抗黄矮病重要基因。

关键词: 小麦-中间偃麦草易位系, 小麦黄矮病抗性, cDNA-AFLP, 抗病基因类似序列, 病毒介导的基因沉默

Abstract: Barley yellow dwarf virus (BYDV) can cause wheat yellow dwarf. In this study, we isolated a fragment of a wheat resistance analogous gene, tentatively named BYDV response gene1 (BRG1),which was expressed in the BYDV resistant wheat-Thinopyrum intermedium translocation line YW642 but was not expressed in the BYDV susceptible wheat Zhong 8601 by using cDNA-AFLP technique.The full-length cDNA sequence of the gene BRG1 was obtained by rapid amplification of cDNA end (RACE) and RT-PCR methods. The gene BRG1 encodes a NBS-LRR protein consisting of 645 amino acid residues，which possesses one typical NB-ARC domain and three leucine-rich domains. The result of expression analysis by Q-RT-PCR method indicated that the expression of BRG1 gene was predominant in the BYDV resistant translocation line YW642 and induced by BYDV infection, reached the peak at 48 hour post inoculation with BYDV, whereas the express level of the gene in the susceptible wheat parent Zhong8601 was lower than that in the resistant wheat YW642, and showed a decline tendency with BYDV infection time. The mRNA expressionof BRG1 gene in YW642 wasup-regulated by salicylic acid (SA) and jasmonate (JA). Virus induced gene silencing technique was used to conduct functional analysis on the gene BRG1. The results showed that after BYDV infection, BYDV relative content in BRG1 knocked-down YW642 was higher than that in YW642 expressing BRG1 gene, whereas the silenced BRG1 gene did not obviously alter the plant phenotype to BYDV infection, suggesting that the gene BRG1 may be involved in the host response to BYDV infection but not be an important gene.

Key words: Wheat-Thinopyrum intermedium translocation line, Resistance to barley yellow dwarf virus (BYDV), cDNA-AFLP, Virus-induced gene silencing

[1]Larkin P, Kleven S, Banks P. Utilizing Bdv2, the Thinopyrum intermedium source of BYDV resistance, to develop wheat cultivars. Recent Advances and Future Strategies, M. Henry and A. McNab eds. (CIMMYT Texcoco, Mexico), 2002. pp 60–63
[2]Friebe B, Mukai Y, Gill B S, Canderon Y. C-banding and in situ hybridization analyses of Agropyron intermedum, a partial wheat × Ag. intermedium amploid, and six derived chromosome addition lions. Theor Appl Genet, 1992, 84: 899–905
[3]Sharma H, Ohm H, Goulart L, Lister R, Appels R., Benlhabib O. Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat. Genome, 1995, 38: 406–413
[4]Larkin P, Baeva K, Banks P, Lagudah E, Appels R, Chen X, Xin Z, Ohm H, McIntosh R. Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances. Genome, 1995, 38: 385–394
[5]Zhang Z Y, Lin Z S, Xin Z Y. Research progress in BYDV resistance genes derived from wheat and its wild relatives. J Genet Genom, 2009, 36: 567–573
[6]Cauderon Y, Saigne B, Dauge M. The resistance to wheat rusts of Agropyron intermedium and its use in wheat improvement. In: Sears E R, Sears L M S, eds. Proc Intl Wheat Genet Symp: Wheat Improvement, Vol. 4. University of Missouri, Columbia, MD, USA, 1973. pp 401–407
[7]Xin Z, Zhang Z, Chen X., Lin Z, Ma Y, Xu H, Banks P, Larkin P. Development and characterization of common wheat–Thinopyrum intermedium translocation lines with resistance to barley yellow dwarf virus. Euphytica. 2001, 119: 161–165
[8]Zhang Z-Y(张增艳), Xin Z-Y(辛志勇), Chen X(陈孝), Qian Y-T(钱幼亭), Lin Z-S(林志珊). Molecular cytogenetic characterization of a new wheat line Yw443 with resistance to barley yellow dwarf virus. Acta Genet Sin (遗传学报), 2000, 27(7): 614–620 (in Chinese with English abstract)
[9]Xie H(谢皓), Chen X(陈孝), Zhang Z-Y(张增艳), Xin Z-Y(辛志勇), Lin Z-S(林志珊), Du L-P(杜丽璞), Ma Y-Z(马有志), Xu H-J(徐惠君). Development and molecular cytogenetic characterization of a wheat line YW243 resistant to BYDV. Acta Agron Sin (作物学报), 2000, 26(6): 687–691 (in Chinese with English abstract)
[10]Banks P, Larkin P, Bariana H, Lagudah E, Appels R, Waterhouse P, Brettell R, Chen X, Xu H, Xin Z, Qian Y, Zhou M, Cheng Z, Zhou G. The use of cell culture for sub-chromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat. Genome, 1995, 38: 395–405
[11]Liu X D, Zhang Z Y, Xin Z Y. Molecular evidence of barley yellow dwarf virus replication/movement suppressed by the resistance gene Bdv2 derived from Th. intermedium. J Genet Genomics, 2005, 932: 942–947
[12]Ayala L, Bariana H, Singh R, Gibson J, Gibson A, Mechanicos P. Trigenomic chromosomes by recombination of Thinopyrum intermedium and Th. ponticum translocations in wheat. Theor Appl Genet, 2007, 116: 63–75
[13]Jones J D G, Dangl J L. The plant immune system. Nature, 2006, 444: 323–329
[14]Maule A, Caranta C, Boulton M. Sources of natural resistance to plant viruses: Status and prospects. Mol Plant Pathol, 2007, 8: 223–231
[15]Wang X, Liu W, Chen X, Tang C, Dong Y, Ma J, Huang X, Wei G, Han Q, Huang L, Kang Z. Differential gene expression in incompatible interaction between wheat and stripe rust fungus revealed by cDNA-AFLP and comparison to compatible interaction. BMC Plant Biol, 2010, 10: 9
[16]Chen G P, Ma W S, Huang Z J, Xu T, XueY B, Shen Y Z. Isolation and characterization of TaGSKl involved in wheat salt tolerance. Plant Sci, 2003, 165: 1369–1375
[17]Zhang Z-Y(张增艳), Yao W-L(姚乌兰), Xin Z-Y(辛志勇). Advance in virus-induced gene silencing, a novel powerfully tools for functional analysis of plant genes. J Plant Genet Resour (植物遗传资源学报), 2006, 7(1): 100–105 (in Chinese with English abstract)
[18]Gabriëls S, Takken F, Jong C, Liu Q, Turk S, Wachowski L, Peters J, Witsenboer H, Pierre J, Wit D, Joosten1 M. cDNA-AFLP combined with functional analysis reveals novel genes involved in the hypersensitive response. Mol Plant-Microbe Interact, 2006, 19: 567–576
[19]Zhang Z Y, Yao W L, Dong N, Liang H X, Liu H X, Huang R F. A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments. J Exp Bot, 2007, 58: 2993–3003
[20]Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M. AFLP: a new technique for DNA-fingerprinting. Nucl Acids Res, 1995, 23: 4407–4414
[21]Zhao J-R(赵继荣), Li N(李宁), Liu H-X(刘红霞), Bi Y(毕阳), Zhang Z-Y(张增艳). Confirmation of cDNA-AFLP differential expressed fragments of related defense genes against BYDV in wheat. J Plant Genet Resour (植物遗传资源学报), 2009, 10(1): 16–20 (in Chinese with English abstract)
[22]Liu X-D(刘晓东), Zhang Z-Y(张增艳), Yao W-L(姚乌兰), Xin Z-Y(辛志勇). Implement of barley stripe mosaic virus-based induced gene silencing in wheat. Acta Agron Sin (作物学报), 2005, 31(11): 1518–1520 (in Chinese with English abstract)
[23]Zhang Z-Y(张增艳), Yao W-L(姚乌兰), Li H-T(李宏涛), Xin Z-Y(辛志勇). Progress in molecular genetics of barley and wheat. J Triticeae Crops (麦类作物学报), 2005, 25(6): 139–143 (in Chinese with English abstract)
[24]Ayliffe M A, Lagudah E S. Molecular genetics of disease resistance in cereals. Ann Bot, 2004, 94: 765–773
[25]Collins N, Drake J, Ayliffe M, Sun Q, Ellis J, Hulbert S, Pryor T. Molecular characterization of the maize Rp1-D rust resistance haplotype and its mutants. Plant Cell, 1999, 11: 1365–1376
[26]Bruggeman R, Rosoks N, Kudrna N, Kilian A, Han F, Chen J, Druka A, Steffenson B, Kleinhofs A. The barley stem rust resistance gene Rpg1 is a novel disease resistance gene with homology to receptor kinase. Proc Natl Acad Sci USA, 2002, 99: 9328–9333
[27]Han F, Kilian A, Chen J P, Kudrna D, Steffenson B, Yamamoto K, Matsumoto T, Sasaki T, Kleinhofs A. Sequence analysis of a rice BAC covering the syntenous barley Rpg1 region. Genome, 1999, 42: 1071–1076
[1] 陈坤梅,李宏伟,林凡云,陈耀锋,李滨,郑琪,李振声. 利用病毒介导基因沉默方法研究小麦抗光氧化相关基因[J]. 作物学报, 2014, 40(11): 1905-1913.
[2] 陈荣平, 刘磊, 万秀清, 邱恩建, 王春军, 宋宝刚, 颜培强, 杨铁钊. TMV侵染烟草基因差异表达的cDNA-AFLP分析[J]. 作物学报, 2012, 38(01): 62-70.
[3] 张岗，董艳玲，夏宁，张毅，王晓杰，屈志鹏，李依民，黄丽丽，康振生. 利用cDNA-AFLP技术分析小麦成株抗条锈性差异基因表达特征[J]. 作物学报, 2010, 36(3): 401-409.
[4] 韩利涛,姜伟,杨守萍,喻德跃,盖钧镒. 大豆细胞质雄性不育系MADS-box基因的分离分析[J]. 作物学报, 2010, 36(06): 905-910.
[5] 谷俊涛,鲍金香,王效颖,郭程瑾,李小娟,路文静,肖凯. 利用cDNA-AFLP技术分析小麦应答低磷胁迫的特异表达基因[J]. 作物学报, 2009, 35(9): 1597-1605.
[6] 程立宝，李淑艳，景新明，何光源. 表达大豆GMCHI基因能够提高原核生物对耐低温性[J]. 作物学报, 2009, 35(6): 1156-1160.
[7] 张一;倪中福;姚颖垠;孙其信. 小麦杂交种与亲本之间穗下节间基因差异表达分析[J]. 作物学报, 2008, 34(05): 770-776.
[8] 李浩;张平平;查向东;夏先春;何中虎. 优质面包小麦品种济南17和豫麦34灌浆期高温胁迫差异表达基因的分离[J]. 作物学报, 2007, 33(10): 1644-1653.
[9] 吴敏生;高志环;戴景瑞. 利用cDNA—AFLP技术研究玉米基因的差异表达[J]. 作物学报, 2001, 27(03): 339-342.
Viewed
Full text


Abstract

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