發(fā)布時間：2018-01-27 06:37

  本文關鍵詞： 二穗短柄草 大麥條紋花葉病毒 Bsr1 病毒抗性　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：病毒病是作物重要病害,造成嚴重的產量損失。發(fā)掘和克隆抗病毒基因是進行農作物抗病毒分子育種以及研究寄主與病毒互作的基礎。目前已克隆的植物顯性抗病毒基因主要來源于雙子葉植物。大麥條紋花葉病毒(Barley stripe mosaic virus, BSMV)分布廣泛,一般侵染大麥、小麥和燕麥等。BSMV有多個分離物和株系,被用于研究病毒致病性和運動機制。二穗短柄草是新興的溫帶禾本科模式植物,廣泛應用于大麥和小麥等禾本科植物的比較基因組學、功能基因組學以及寄主-病原物互作等研究。本實驗室利用二穗短柄草自交系Bd3-1和Bd21對BSMV ND18株系的不同反應,從Bd3-1中圖位克隆了一個抗BSMV的候選基因Bsr1,是典型的CC-NB-LRR抗病基因。同時,從Bd21中克隆到感病等位基因bsr1。對Bsrl基因功能的驗證和研究有助于了解單子葉植物抗病毒基因的功能特點及單子葉植物尤其是禾本科植物和病毒互作機制。在此基礎上,本研究將短柄草Bsrl基因轉化對ND18感病的小麥和大麥品種,研究Bsrl基因在麥類作物是否具有抗BSMV的功能,探索Bsrl在麥類作物遺傳改良中的應用可行性。此外,還利用本生煙瞬時表達體系分析了Bsrl和bsrl各結構域和片段對基因功能的影響。具體研究結果如下：1.構建短柄草抗BSMV基因Bsrl過表達載體pMBsr1和基因組互補載體pCBsr1,轉化對BSMVND18株系感病的短柄草品系Bd21-3。轉基因結果表明過表達Bsrl基因的短柄草獲得了對ND18的抗性。這些結果證實Bsrl基因是控制短柄草Bd3-1對BSMV ND 18株系抗性的基因。2.將Bsrl基因的互補載體轉化對BSMV ND18株系感病的大麥品種Golden Promise和小麥品種科農199,分子檢測表明獲得了轉Bsrl基因的大麥和小麥植株。接種BSMV ND18株系的鑒定結果表明轉Bsrl基因的大麥獲得了對ND18的抗性,說明來源于短柄草的Bsrl基因在大麥中可以表達對ND18的抗性,為利用外源抗病基因進行大麥抗病毒育種提供了理論基礎。3.利用抗BSMV ND18株系基因Bsrl和感BSMV ND18株系等位基因bsrl分別與BSMVND18株系共注射本生煙,結果表明Bsrl可引起本生煙葉片的過敏性壞死,而bsrl不能引起葉片壞死反應,證實Bsrl可誘導本生煙的細胞程序性死亡,抗病毒功能。根據Bsrl與bsrl氨基酸序列及結構差異,構建一系列結構域／片段互換的重組體。將重組體與ND18在本生煙葉片中瞬時表達,研究Bsrl各結構域對其功能的影響。結果發(fā)現僅重組體bNBLT和Bsrl一樣能夠誘導本生煙葉片的過敏性壞死,表明Bsrl的LRR結構域及C末端在Bsrl與bsrl抗病毒功能差異中具有重要的作用。4.對分別接種ND18(無毒株系)和ND18 mutant (ND18的TGB1雙突變體TGB1R390K,T392K,有毒株系)的短柄草自交系Bd3-1進行RNA-seq,分析其中與水楊酸(Salicylic acid, SA)和茉莉酸(Jasmonic acid, JA)/乙烯(Ethylene, ET)信號途徑相關基因的表達變化。結果發(fā)現：Bd3-1對ND18的防御反應中,SA通路的大多數基因未表達或者下調表達,僅AOX1B和2個WRKY轉錄因子在14dpi表達水平提高。而1 dpi時少數JA/ET信號途徑的基因表達水平有所提高,14 dpi時主要是4個乙烯響應因子表達上調。Bd3-1與ND18 mutant的非親和互作中前期SA和JA/ET信號途徑的大多數基因未表達或者下調表達。后期大多數SA信號途徑中的基因上調表達,而JA/ET信號途徑中的基因下調表達。表明：在Bd3-1與BSMV ND18株系的親和互作中SA信號途徑可能被抑制,部分JA/ET信號通路的基因參與了Bsrl對ND18的防御反應。在Bd3-1與ND18mutant的非親和互作中,SA信號途徑被激活而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.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S432.41

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1 王國鑫;二穗短柄草抗大麥條紋花葉病毒基因Bsr1的功能分析[D];中國農業(yè)大學;2016年

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本文編號：1467844