欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2011, Vol. 37 ›› Issue (11): 2106-2110.doi: 10.3724/SP.J.1006.2011.02106

• 研究简报 • 上一篇    下一篇

利用BSMV-VIGS技术快速分析小麦TNBL1基因的抗黄矮病功能

赵丹1,2，赵继荣1，黄茜1,2，李宁1，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
  • 收稿日期:2011-02-18 修回日期:2011-06-25 出版日期:2011-11-12 网络出版日期:2011-09-06
  • 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发展计划(863计划)重点项目(2006AA10A104)资助。

Functional Analysis of TNBL1 Gene in Wheat Defense Response to Barley yellow dwarf virus Using BSMV-VIGS Technique

ZHAO Dan1,2,ZHAO Ji-Rong1,HUANG Xi1,2,LI Ning1,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
  • Received:2011-02-18 Revised:2011-06-25 Published:2011-11-12 Published online:2011-09-06
  • Contact: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781

PDF

2895

被引次数

8

摘要： 小麦黄矮病是由蚜虫介导的大麦黄矮病毒(Barley yellow dwarf virus, BYDV)侵染引起的小麦重要病害之一。利用cDNA-AFLP分析，筛选出在抗黄矮病小麦易位系YW642中特异表达的长度为292 bp的 cDNA片段，以此片段为启始序列，利用RACE和RT-PCR技术克隆出该基因的全长cDNA序列，推导该基因编码1个NBS-LRR蛋白，将其命名为TNBL1。本研究利用大麦条纹花叶病毒(BSMV)诱导的基因沉默(virus-inducing gene silencing, VIGS)技术，快速分析TNBL1是否参与小麦抗黄矮病反应。通过PCR添加酶切位点、定向酶切与连接，将TNBL1特异的292bp片段反向整合到BSMV-γ链的多克隆位点上，获得重组载体BSMV-γ: TNBL1as，体外转录BSMV-VIGS载体的3个组分(BSMV-TNBL1as、BSMV-α和BSMV-β)，等量混合、摩擦接种到抗黄矮病的小麦易位系YW642幼苗叶片上，使YW642中TNBL1基因沉默，然后接种BYDV病原进行黄矮病抗性鉴定。结果表明，TNBL1基因沉默后的YW642对BYDV敏感、显现感病症状，其体内BYDV含量较未发生基因沉默的YW642中的明显增加，证明TNBL1基因是正向调控小麦抗BYDV反应的1个重要基因。

关键词: 小麦, 中间偃麦草, 黄矮病, 病毒诱导的基因沉默, NBS-LRR

Abstract: Barley yellow dwarf virus (BYDV), transmitted by at least 25 species of aphids, causes one of the most serious virus diseases of wheat worldwide. Through cDNA-AFLP analysis, we identified a cDNA fragment with 292bp expressing in the BYDV-resistant wheat-Thinopyrun intermedium translocation line YW642, but not in susceptible wheat Zhong8601. The full-length cDNA sequence of the gene, namely TNBL1, was cloned by RACE and RT-PCR methods, which encodes a putative NBS-LRR protein. This study focused on the functional analysis of TNBL1 in wheat defense to BYDV infection using Barley stripe mosaic virus (BSMV)-based virus-inducing gene silencing method. After the specific fragment of TNBL1 was addedwith 2 restriction-enzyme sequences byPCR, and digested and ligased with the digested BSMV-γ, the recombinant BSMV-γ:TNBL1as construct was obtained. The three components of the BSMV-VIGS vectors, BSMV-TNBL1as、BSMV-α and BSMV-β were transcribed in vitro, and mixed with equal quantity and inoculated onto the first and second leaves of the resistant line YW642 seedlings at the two-leaf stage. As a result, the TNBL1 expression was obviously repressed (silenced) in YW642 treated by BSMV:TNBL1. These seedlings were further inoculated with BYDV aphids. The BYDV content was much higher in the TNBL1-silenced YW642 plants than that in the control YW642 plants without BSMV:TNBL1 treatment. Furthermore, the TNBL1-silenced YW642 plants were susceptible to BYDV infection with the viral symptom. These results indicated that the TNBL1 gene is an important gene positively involved in wheat defense response to BYDV infection.

Key words: Wheat, Barley yellow dwarf virus, Thinopyrum intermedium, Virus-induced gene silencing, NBS-LRR

[1]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
[2]Zhang Z Y, Xu J S, Xu Q J, Larkin P , Xin Z Y. Development of novel PCR markers linked to the BYDV resistance gene Bdv2 useful in wheat for marker-assisted selection. Theor Appl Genet, 2004, 109: 433–439
[3]Dangl J L, Jones J D G. Plant pathogens and integrated defence responses to infection. Nature, 2001, 411: 826–833
[4]Yi T-Y(易图永), Xie B-Y(谢丙炎), Zhang B-X(张宝玺), Gao B-D(高必达). Application of plant resistance gene analogs in cloning and mapping resistance genes .Biotechnol Bull (生物技术通报), 2002, (2): 16–20 (in Chinese with English abstract)
[5]Qin G-J(秦跟基), Li W-L(李万隆), Chen P-D(陈佩度). Update of resistance genes and resistance gene analogs in plants. J Nanjing Agric Univ (南京农业大学学报), 1999, 22(3): 102–107 (in Chinese with English abstract)
[6]Jones D A, Jones J D G. The roles of leucine-rich repeats in plants defences. Adv Bot Res, 1997, 24: 89–167
[7]Kajava A V. Structural diversity of leucine-rich repeat proteins. J Mol Biol, 1998, 227: 519–527
[8]Whitham S, McCormick S, Baker B. The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc Natl Acad Sci USA, 1996, 93: 8776–8781
[9]Spassova M I, Prins T W, Folkertsma R T, Klein-Lankhorst R M, Hille J, Goldbach R W, Prins M. The tomato gene Sw-5 is a member of the coiled coil, nucleotide binding, leucine-rich repeat class of plant resistance genes and confers resistance to TSWV in tobacco. Mol.Breed, 2001, 7: 151–161
[10]Ratcliff F, Harrison B D, Baulcombe D C. A similarity between viral defense and gene silencing in plants. Science, 1997, 276: 1558–1560
[11]Wang H-Z(王宏芝), Li R-F(李瑞芬), Wang G-Y(王国英), Ma R-C(马荣才), Wei J-H(魏建华). Virus induced gene silence and its application in plant gene functional genomics. Prog Nat Sci (自然科学进展), 2005, 15(1): 8–14 (in Chinese)
[12]Scofield S R, Huang L, Brandt A S, Bikram S G. Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol, 2005, 138: 2165–2173
[13]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)
[14]Lu R, Martin-Hernandez A M, Peart J R, Malcuit I, Baulcombe D C. Virus-induced gene silencing in plants. Methods, 2003, 30: 296–303
[15]Holzberg S, Brosio P, Gross C, Pogue G P. Barley stripe mosaic virus-induced gene silencing in a monocot plant .Plant J, 2002, 30: 315–327
[16]Lu 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)
[17]Zhou H B, Li S F, Deng Z Y, Wang X P, Chen T, Zhang J S, Chen S Y, Ling H Q, Zhang A M, Wang D W. Zhang X Q. Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection. Plant J, 2007, 52: 420–434
[18]Ding X S, Schneider W L, Chaluvadi S R, Mian M A R, Nelson R S. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Mol Plant Microbe Interactions, 2006, 19, 1229–1239
[19]Vander-Linde K, Kastner C, Kumlehn J, Kahmann R, Doehlemann G. Systemic virus-induced gene silencing allows functional characterization of maize genes during biotrophic interaction with Ustilago maydis. New Phytol, 2011, 189: 471–483
[20]Zhang Z-Y(张增艳), Ma Y-Z(马有志), Xin Z-Y(辛志勇), Chen X( 陈孝), Wu D-L(武东亮), Lin Z-S(林志珊). Analysis of the chromosome constitution of wheat lines resistant to Barley yellow dwarf virus by genomic in situ hybridization. Sci Agric Sin (中国农业科学), 1998, 31(3): 1–4 (in Chinese with English abstract)
[21]Meyers B C, Dickeman A W, Michelmore R W, Sivaramakrishnan S, Sobral B W, Young N D. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding super family. Plant J, 1999, 20: 317–332
[1] 翟胜男, 曹新有, 李豪圣, 李吉虎, 李法计, 刘金栋, 夏先春, 吕莹莹, 马瑞峰, 王颖, 耿洪伟, 刘建军. 小麦Pod-A1、Pod-D1和Pod-2D位点等位变异对籽粒过氧化物酶活性的遗传效应分析[J]. 作物学报, 2026, 52(6): 1593-1603.
[2] 习千辉, 徐梓瑗, 刘梦梦, 王宏艺, 郎凯琳, 井震海, 陈锋, 赵磊. 小麦籽粒铜含量的全基因组关联分析及候选基因预测[J]. 作物学报, 2026, 52(6): 1604-1617.
[3] 毛嘉琦, 黄朋雨, 赵佳佳, 郑兴卫, 武棒棒, 郝宇琼, 屈非, 刘成, 马朋涛, 郑军. 山西小麦品种白粉病抗性评价及抗病基因分子检测[J]. 作物学报, 2026, 52(6): 1669-1681.
[4] 胡川, 赵凯男, 黄修利, 吴金芝, 任开明, 王贺正, 付国占, 黄明, 李友军. 一次灌溉下耕作方式和氮肥用量对旱地小麦产量和品质的影响[J]. 作物学报, 2026, 52(6): 1830-1846.
[5] 陈雪燕, 何华川, 李政嘉, 董新盼, 李藕琪, 刘小云, 李丹萍, 陈志伟, 刘国霞, 吕胜源, 吴印莹, 赵振东, 曹新有, 万何平. 水培盐碱复合胁迫下‘济麦60’苗期根系有机酸分泌动态变化及其转录调控机制[J]. 作物学报, 2026, 52(6): 1859-1875.
[6] 高沛阳, 李瑾璇, 董宇奎, 石玉, 张振, 张永丽. 测墒补灌下小麦分蘖发生和成穗对施氮量的响应[J]. 作物学报, 2026, 52(6): 1847-1858.
[7] 张献丰, 郭利建, 李康春, 孔斌雪, 刘玉芳, 车卓, 杨德龙. 小麦ABHD6基因家族鉴定与粒重功能标记开发[J]. 作物学报, 2026, 52(6): 1711-1727.
[8] 王壮壮, 武紫君, 张永新, 张芯源, 袁丽雪, 陈如雪, 刘世举, 段剑钊, 冯伟, 王同朝, 王永华. 豫东南黏壤潮土区水氮优化协同提高冬小麦产量和氮素利用效率[J]. 作物学报, 2026, 52(5): 1501-1521.
[9] 何万龙, 耿洪伟, 张飞飞, 米克热阿依·阿巴白克热, 罗紫洋, 李鹏程, 周钊宇, 程宇坤. 基于深度学习的小麦重要病害图像识别系统的研究[J]. 作物学报, 2026, 52(5): 1401-1417.
[10] 张振, 冯连杰, 石玉, 于振文, 张永丽. 节水补灌下不同穗型小麦产量形成差异研究[J]. 作物学报, 2026, 52(5): 1522-1535.
[11] 侯思宇, 王国璀, 韦金贵, 谢玮欣, 殷文, 樊志龙, 柴强, 胡发龙. 绿肥配施化学氮肥对西北干旱灌区小麦干物质积累及产量形成的影响[J]. 作物学报, 2026, 52(4): 1208-1219.
[12] 尚云秋, 赵竹, 陈欢, 丁永刚, 乔玉强, 李玮, 张向前, 曹承富, 杜世州. 长期定位耕作方式对雨养小麦籽粒灌浆和产量形成的影响[J]. 作物学报, 2026, 52(4): 1236-1250.
[13] 乔宇馨, 李程越, 康晓玉, 张鑫琪, 贾绍辉, 刘倩, 曹亚丽, 史鑫蕊, 郝兴宇, 李萍. 基于APSIM模型的长期免耕秸秆覆盖对旱地小麦增产效应研究[J]. 作物学报, 2026, 52(4): 1181-1192.
[14] 李灿, 张喜伟, 朱博涛, 张沛沛. 小麦GSK激酶TaSK41的功能分析及互作蛋白的筛选[J]. 作物学报, 2026, 52(3): 677-687.
[15] 侯洁, 付朵朵, 武海峰, 郝宇琼, 郑兴卫, 武棒棒, 周凯, 李晓华, 郑军, 赵佳佳. 山西省小麦地方品种的染色体多样性及遗传效应分析[J]. 作物学报, 2026, 52(3): 746-763.
Viewed
Full text


Abstract

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