二穗短柄草抗大麦条纹花叶病毒基因Bsr1的功能分析

发布时间：2018-01-27 06:37

本文关键词： 二穗短柄草大麦条纹花叶病毒 Bsr1 病毒抗性　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：病毒病是作物重要病害,造成严重的产量损失。发掘和克隆抗病毒基因是进行农作物抗病毒分子育种以及研究寄主与病毒互作的基础。目前已克隆的植物显性抗病毒基因主要来源于双子叶植物。大麦条纹花叶病毒(Barley stripe mosaic virus, BSMV)分布广泛,一般侵染大麦、小麦和燕麦等。BSMV有多个分离物和株系,被用于研究病毒致病性和运动机制。二穗短柄草是新兴的温带禾本科模式植物,广泛应用于大麦和小麦等禾本科植物的比较基因组学、功能基因组学以及寄主-病原物互作等研究。本实验室利用二穗短柄草自交系Bd3-1和Bd21对BSMV ND18株系的不同反应,从Bd3-1中图位克隆了一个抗BSMV的候选基因Bsr1,是典型的CC-NB-LRR抗病基因。同时,从Bd21中克隆到感病等位基因bsr1。对Bsrl基因功能的验证和研究有助于了解单子叶植物抗病毒基因的功能特点及单子叶植物尤其是禾本科植物和病毒互作机制。在此基础上,本研究将短柄草Bsrl基因转化对ND18感病的小麦和大麦品种,研究Bsrl基因在麦类作物是否具有抗BSMV的功能,探索Bsrl在麦类作物遗传改良中的应用可行性。此外,还利用本生烟瞬时表达体系分析了Bsrl和bsrl各结构域和片段对基因功能的影响。具体研究结果如下：1.构建短柄草抗BSMV基因Bsrl过表达载体pMBsr1和基因组互补载体pCBsr1,转化对BSMVND18株系感病的短柄草品系Bd21-3。转基因结果表明过表达Bsrl基因的短柄草获得了对ND18的抗性。这些结果证实Bsrl基因是控制短柄草Bd3-1对BSMV ND 18株系抗性的基因。2.将Bsrl基因的互补载体转化对BSMV ND18株系感病的大麦品种Golden Promise和小麦品种科农199,分子检测表明获得了转Bsrl基因的大麦和小麦植株。接种BSMV ND18株系的鉴定结果表明转Bsrl基因的大麦获得了对ND18的抗性,说明来源于短柄草的Bsrl基因在大麦中可以表达对ND18的抗性,为利用外源抗病基因进行大麦抗病毒育种提供了理论基础。3.利用抗BSMV ND18株系基因Bsrl和感BSMV ND18株系等位基因bsrl分别与BSMVND18株系共注射本生烟,结果表明Bsrl可引起本生烟叶片的过敏性坏死,而bsrl不能引起叶片坏死反应,证实Bsrl可诱导本生烟的细胞程序性死亡,抗病毒功能。根据Bsrl与bsrl氨基酸序列及结构差异,构建一系列结构域／片段互换的重组体。将重组体与ND18在本生烟叶片中瞬时表达,研究Bsrl各结构域对其功能的影响。结果发现仅重组体bNBLT和Bsrl一样能够诱导本生烟叶片的过敏性坏死,表明Bsrl的LRR结构域及C末端在Bsrl与bsrl抗病毒功能差异中具有重要的作用。4.对分别接种ND18(无毒株系)和ND18 mutant (ND18的TGB1双突变体TGB1R390K,T392K,有毒株系)的短柄草自交系Bd3-1进行RNA-seq,分析其中与水杨酸(Salicylic acid, SA)和茉莉酸(Jasmonic acid, JA)/乙烯(Ethylene, ET)信号途径相关基因的表达变化。结果发现：Bd3-1对ND18的防御反应中,SA通路的大多数基因未表达或者下调表达,仅AOX1B和2个WRKY转录因子在14dpi表达水平提高。而1 dpi时少数JA/ET信号途径的基因表达水平有所提高,14 dpi时主要是4个乙烯响应因子表达上调。Bd3-1与ND18 mutant的非亲和互作中前期SA和JA/ET信号途径的大多数基因未表达或者下调表达。后期大多数SA信号途径中的基因上调表达,而JA/ET信号途径中的基因下调表达。表明：在Bd3-1与BSMV ND18株系的亲和互作中SA信号途径可能被抑制,部分JA/ET信号通路的基因参与了Bsrl对ND18的防御反应。在Bd3-1与ND18mutant的非亲和互作中,SA信号途径被激活而JA/ET通路被抑制。
[Abstract]:Virus disease is an important disease of crops, causing serious yield loss. Explore and cloning of antiviral gene is the basis for crop molecular breeding and Research on antiviral host virus interaction. Plant antiviral gene has been cloned dominant mainly from dicotyledonous plants. Barley stripe mosaic virus (Barley stripe mosaic virus, BSMV) are widely distributed. The general infection of barley, wheat and oat.BSMV has multiple isolates and strains were used to study the virus pathogenicity and movement mechanism. Two distachyon is gramineous plants of temperate new model, widely used in comparative genomics of gramineous plants such as wheat and barley, functional genomics and host pathogen interactions. This laboratory using two distachyon inbred lines Bd3-1 and Bd21 different responses to BSMV strains of ND18, from the Bd3-1 map based cloning of an anti BSMV syndrome The gene Bsr1, CC-NB-LRR resistance gene is typical. At the same time, from Bd21 was cloned into the susceptible allele bsr1. of Bsrl gene function validation and research is helpful to understand the monocot plant antiviral gene features and monocotyledonous plants especially the interaction mechanism of gramineous plants and viruses. On this basis, the the research will be short of wheat and barley varieties distachyon transformation of Bsrl gene to ND18 infection, whether the study of Bsrl gene in Wheat with anti BSMV function, to explore the feasibility of the application of Bsrl in wheat genetic improvement. In addition, we also used smoke transient expression system to analyze the effects of Bsrl and BSRL structure domain and fragment of gene function. The results are as follows: 1. construction of Brachypodium BSMV resistant gene Bsrl expression vector pMBsr1 and genome complementary vector pCBsr1 transformed to BSMVND18 strain susceptible strains of Brachypodium Bd21-3. The results show that the resistance gene on the expression of ND18 Bsrl genes in Brachypodium. These results confirmed that the Bsrl gene is the control of Brachypodium Bd3-1 gene.2. of BSMV ND 18 strains transformed the complementary vector of Bsrl gene of BSMV ND18 strain susceptible barley varieties Golden and Promise wheat cultivar Kenong 199. Molecular detection indicated that the obtained transgenic barley and wheat Bsrl genes. The identification results were inoculated with BSMV ND18 strain showed that Bsrl transgenic barley was resistant to ND18, indicating that Bsrl gene from Brachypodium could express resistance to ND18 in barley, provides a theoretical basis for the use of anti.3. BSMV ND18 strains Bsrl and BSMV genes of ND18 strains were BSRL alleles and BSMVND18 strains were injected nicotianabenthamiana for the use of exogenous genes for antiviral breeding of barley, the results show that Bsrl can cause the leaf allergy Necrosis, while BSRL can not cause leaf necrosis reaction, confirmed that Bsrl can induce programmed cell death nicotianabenthamiana, antiviral function. According to the difference between Bsrl and BSRL amino acid sequence and structure, construct a series of domain / recombinant fragment exchange. Recombinant ND18 and transient expression in the raw tobacco in effect study on the Bsrl domain of the protein. The results showed that recombinant bNBLT and Bsrl can induce hypersensitive of the tobacco leaf, showed that the terminal LRR domain and C Bsrl plays an important role in.4. were inoculated with ND18 difference between Bsrl and BSRL in antiviral function (nontoxic strains) and ND18 mutant (ND18 TGB1 double mutant TGB1R390K, T392K, toxic strains) Brachypodium inbred lines Bd3-1, RNA-seq, and the analysis of salicylic acid (Salicylic acid, SA) and jasmonic acid (Jasmonic acid, JA) / ethylene (Ethylene, ET) signal pathway The expression changes of related genes. The results showed that the defense reaction of Bd3-1 on ND18, most of the genes of the SA pathway is not expressed or down regulated expression, only the expression of AOX1B and 2 WRKY transcription factors at the level of 14dpi increased. And 1 DPI a JA/ET signaling pathway gene expression level increase, 14 DPI is 4 a non affinity ethylene response factor.Bd3-1 and ND18 expression mutant in the interaction between the pre SA and JA/ET signaling pathway gene expression or down-regulation most did not upregulate gene expression. The majority of SA signaling pathway, JA/ET signaling pathway in gene expression. Bd3-1 and BSMV showed that the ND18 strain of mutual affinity in the SA signaling pathway may be inhibited, part of the JA/ET signaling pathway genes involved in defense response of Bsrl to ND18. Bd3-1 and ND18mutant in the incompatible interaction, SA signaling pathway is activated by JA/ET The road is suppressed.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.41

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