9-LOX家族脂氧合酶基因ZmLOX3和ZmLOX12调节玉米抗病机制研究
发布时间：2018-08-08 13:23
【摘要】：生物胁迫是限制作物生产的主要危害因子之一,它严重影响作物的产量和品质。因此研究作物抗病机制,解析作物与病原菌互作模式、挖掘抗病基因,是培育抗病品种的基础。本研究通过解析两个9-LOX家族脂氧合酶基因ZmLOX3和ZmLOX12的抗病机制,取得以下主要结果：(1)通过玉米LOX3基因突变体与黄曲霉脂氧合酶基因突变体互作系统研究宿主与病原真菌的脂氧合酶基因在病原真菌侵染宿主过程中的作用,进一步阐述宿主和病原真菌间脂氧合酶基因调节宿主抗病或感病分子机制以及研究宿主与病原真菌间的相互作用。研究结果发现：宿主ZmLOX3基因可以抑制黄曲霉菌的定植与产孢,同时病原真菌PpoA、PpoD、和PpoC氧化酶基因需要依靠宿主LOX3基因的作用来调节自身孢子的产生,而病原真菌脂氧合酶loxA基因不需要依靠宿主LOX3基因的作用即可调节自身的产孢,同时病原真菌PpoA控制自身黄曲霉毒素的合成,并且宿主中籽粒中ABA和IAA可以促进病原真菌黄曲霉毒素的积累。这一结果说明玉米LOX3基因可以抵抗黄曲霉菌的侵害,同时在黄曲霉菌体内的脂氧合酶基因参与其侵染宿主过程中,并且与宿主的脂氧合酶相互作用共同参与宿主对病原真菌的抗病与感病过程中。本研究证实脂氧合酶基因是宿主与病原真菌间相互作用的重要信号交流分子,为研究作物与病原真菌互作分子机制以及作物抗病育种提供优异的基因资源和理论基础。(2)ZmLOXS是禾生炭疽菌诱导植物产生系统诱导抗病反应(ISR)的负调控因子,但在lox3突变体中存在的ISR长距离抗性传导信号的调控机制尚不清楚。本研究通过发现在lox3突变体在根部接种木霉菌后ZmLOX12的表达水平增加,进一步对ZmLOX12基因突变体进行功能验证发现：在接种木霉菌后,相比野生型玉米材料,ZmLOX12缺陷型突变体lox12-1不仅缺失ISR信号,且更易感禾生炭疽菌。这说明LOX12基因的正常表达是产生ISR的必要条件,是宿主体内ISR信号正调控因子。对宿主内植物激素以及与茉莉酸合成有关的基因表达分析发现,根部接种木霉菌后lox12-1突变体内的OPDA含量(茉莉酸前体)以及JA-Ile含量显著低于未接种的突变体及野生型材料。茉莉酸是调节ISR的主要信号分子,这可以解释为什么经过木霉菌作用后的lox12-1突变体对禾生炭疽菌表现出系统诱导抗病性感病性。因此LOX12参与调解根部木霉菌诱导宿主产生系统诱导抗病性过程中的分子机制,通过调节茉莉酸合成相关基因的表达来间接调节宿主体内茉莉酸含量,进而参与木霉菌系统诱导抗病反应,是宿主系统诱导抗病机制的正调控因子。(3)为明确ZmWX12基因的诱导表达机制,我们分离了ZmLOX12基因的启动子序列。序列分析发现LOX12启动子含有许多重要的顺式作用元件,如：BIHD1OS、ASF1-motif、GCC-core、MYB-core和POLLEN1LELAT52。将启动子序列连接GUS基因转化拟南芥发现,木霉菌可以诱导ZmLOX12启动子激活GUS基因表达,外源施加MeJA有相同诱导效果,这从另一方向上证实玉米LOX12基因可以受到木霉菌和茉莉酸的诱导表达。本研究的结果表明两个玉米脂氧合酶基因均参与了宿主抗病性反应,增强宿主免疫抗病性,因此本研究结果可以为作物抗黄曲霉病与抗叶枯病分子育种以及作物与病原菌互作等方面提供了新的视角和理论依据,并具有指导作物抗病分子育种的应用价值。
[Abstract]:Biological stress is one of the main harmful factors that restrict crop production. It seriously affects crop yield and quality. Therefore, it is the basis of the study of crop disease resistance mechanism, the analysis of crop and pathogen interaction pattern, and the excavation of disease resistant genes. This study is based on the analysis of the resistance of two 9-LOX family lipoxygenase genes ZmLOX3 and ZmLOX12. The main results are as follows: (1) the role of lipoxygenase gene in the host and pathogenic fungi in the process of infecting the host by the gene mutants of the LOX3 gene and the lipoxygenase gene of Aspergillus flavus is studied, and the host and pathogenic fungi regulate the host resistance or sense of the host and pathogenic fungi. The molecular mechanism of the disease and the interaction between the host and the pathogenic fungi showed that the host ZmLOX3 gene could inhibit the colonization and sporulation of Aspergillus flavus, and the pathogenic fungi PpoA, PpoD, and PpoC oxidase genes need to rely on the host LOX3 gene to regulate the production of spores, and the pathogenic fungi lipoxygenase loxA The gene does not need to rely on the role of the host LOX3 gene to regulate its own sporulation, while the pathogenic fungus PpoA controls the synthesis of its own aflatoxin, and the ABA and IAA in the grain of the host can promote the accumulation of aflatoxin in the pathogenic fungi. This result shows that the maize LOX3 gene can resist the invasion of Aspergillus flavus and at the same time in the Yellow koji. The lipoxygenase gene in the mould participates in the process of infecting the host, and participates in the host's lipoxygenase interaction in the course of the host's disease resistance and susceptibility to pathogenic fungi. This study confirms that lipoxygenase gene is an important signaling molecule for the interaction between the host and the pathogenic fungi, in order to study the crop and the pathogenic fungi. Interplanting molecular mechanism and crop resistance breeding provide excellent genetic resources and theoretical basis. (2) ZmLOXS is a negative regulator of plant induced systemic induced resistance (ISR) induced by anthrax of grasses, but the regulation mechanism of ISR long distance resistance conduction signal in lox3 mutants is not clear. This study was found in lox3 process. The expression of ZmLOX12 increased after inoculation of Trichoderma. Further functional verification of ZmLOX12 gene mutants found that after inoculation of Trichoderma, compared with wild type corn, the ZmLOX12 deficient mutant lox12-1 was not only missing the ISR signal, but also more susceptible to the Bacillus grasses. This indicated that the normal expression of the LOX12 gene was produced. The necessary condition of ISR is a positive regulator of ISR signal in the host. The analysis of plant hormones in host and gene expression related to the synthesis of jasmonate found that the OPDA content (jasmonate precursor) and JA-Ile content in the lox12-1 mutation in the root inoculated with Trichoderma were significantly lower than that of the uninoculated mutant and the wild type material. The main signal molecule of ISR, which can explain why the lox12-1 mutant after Trichoderma action has a systemic induced resistance to the acanthoderma grasses. Therefore, LOX12 participates in the molecular mechanism of mediating root Trichoderma induced system induced disease resistance in the host, and by regulating jasmonic acid to synthesize related genes. Expression to indirectly regulate the content of jasmonic acid in the host, and then participate in the induction of disease resistance by Trichoderma system, is a positive regulator of the host system induced resistance mechanism. (3) we separate the promoter sequence of the ZmLOX12 gene in order to clarify the induction mechanism of ZmWX12 gene. The sequence analysis found that the LOX12 promoter contains many important CIS formulas. The functional components, such as BIHD1OS, ASF1-motif, GCC-core, MYB-core and POLLEN1LELAT52., connect the promoter sequence to the GUS gene transformation of Arabidopsis, and Trichoderma can induce ZmLOX12 promoter to activate GUS gene expression, and exogenous MeJA has the same induction effect, which confirms that the corn LOX12 gene can be subjected to Trichoderma and jasmine from the other side. The results of this study showed that two maize lipoxygenase genes were involved in host resistance response and enhanced host immune resistance. Therefore, the results of this study could provide a new perspective and theoretical basis for crop resistance to yellow aspergillosis and molecular breeding of resistance to leaf blight, as well as crop and pathogen interaction. To guide the application of crop resistance molecular breeding.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.13
本文编号：2171907
[Abstract]:Biological stress is one of the main harmful factors that restrict crop production. It seriously affects crop yield and quality. Therefore, it is the basis of the study of crop disease resistance mechanism, the analysis of crop and pathogen interaction pattern, and the excavation of disease resistant genes. This study is based on the analysis of the resistance of two 9-LOX family lipoxygenase genes ZmLOX3 and ZmLOX12. The main results are as follows: (1) the role of lipoxygenase gene in the host and pathogenic fungi in the process of infecting the host by the gene mutants of the LOX3 gene and the lipoxygenase gene of Aspergillus flavus is studied, and the host and pathogenic fungi regulate the host resistance or sense of the host and pathogenic fungi. The molecular mechanism of the disease and the interaction between the host and the pathogenic fungi showed that the host ZmLOX3 gene could inhibit the colonization and sporulation of Aspergillus flavus, and the pathogenic fungi PpoA, PpoD, and PpoC oxidase genes need to rely on the host LOX3 gene to regulate the production of spores, and the pathogenic fungi lipoxygenase loxA The gene does not need to rely on the role of the host LOX3 gene to regulate its own sporulation, while the pathogenic fungus PpoA controls the synthesis of its own aflatoxin, and the ABA and IAA in the grain of the host can promote the accumulation of aflatoxin in the pathogenic fungi. This result shows that the maize LOX3 gene can resist the invasion of Aspergillus flavus and at the same time in the Yellow koji. The lipoxygenase gene in the mould participates in the process of infecting the host, and participates in the host's lipoxygenase interaction in the course of the host's disease resistance and susceptibility to pathogenic fungi. This study confirms that lipoxygenase gene is an important signaling molecule for the interaction between the host and the pathogenic fungi, in order to study the crop and the pathogenic fungi. Interplanting molecular mechanism and crop resistance breeding provide excellent genetic resources and theoretical basis. (2) ZmLOXS is a negative regulator of plant induced systemic induced resistance (ISR) induced by anthrax of grasses, but the regulation mechanism of ISR long distance resistance conduction signal in lox3 mutants is not clear. This study was found in lox3 process. The expression of ZmLOX12 increased after inoculation of Trichoderma. Further functional verification of ZmLOX12 gene mutants found that after inoculation of Trichoderma, compared with wild type corn, the ZmLOX12 deficient mutant lox12-1 was not only missing the ISR signal, but also more susceptible to the Bacillus grasses. This indicated that the normal expression of the LOX12 gene was produced. The necessary condition of ISR is a positive regulator of ISR signal in the host. The analysis of plant hormones in host and gene expression related to the synthesis of jasmonate found that the OPDA content (jasmonate precursor) and JA-Ile content in the lox12-1 mutation in the root inoculated with Trichoderma were significantly lower than that of the uninoculated mutant and the wild type material. The main signal molecule of ISR, which can explain why the lox12-1 mutant after Trichoderma action has a systemic induced resistance to the acanthoderma grasses. Therefore, LOX12 participates in the molecular mechanism of mediating root Trichoderma induced system induced disease resistance in the host, and by regulating jasmonic acid to synthesize related genes. Expression to indirectly regulate the content of jasmonic acid in the host, and then participate in the induction of disease resistance by Trichoderma system, is a positive regulator of the host system induced resistance mechanism. (3) we separate the promoter sequence of the ZmLOX12 gene in order to clarify the induction mechanism of ZmWX12 gene. The sequence analysis found that the LOX12 promoter contains many important CIS formulas. The functional components, such as BIHD1OS, ASF1-motif, GCC-core, MYB-core and POLLEN1LELAT52., connect the promoter sequence to the GUS gene transformation of Arabidopsis, and Trichoderma can induce ZmLOX12 promoter to activate GUS gene expression, and exogenous MeJA has the same induction effect, which confirms that the corn LOX12 gene can be subjected to Trichoderma and jasmine from the other side. The results of this study showed that two maize lipoxygenase genes were involved in host resistance response and enhanced host immune resistance. Therefore, the results of this study could provide a new perspective and theoretical basis for crop resistance to yellow aspergillosis and molecular breeding of resistance to leaf blight, as well as crop and pathogen interaction. To guide the application of crop resistance molecular breeding.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.13
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