9-LOX家族脂氧合酶基因ZmLOX3和ZmLOX12調(diào)節(jié)玉米抗病機制研究
發(fā)布時間：2018-08-08 13:23
【摘要】：生物脅迫是限制作物生產(chǎn)的主要危害因子之一,它嚴重影響作物的產(chǎn)量和品質(zhì)。因此研究作物抗病機制,解析作物與病原菌互作模式、挖掘抗病基因,是培育抗病品種的基礎(chǔ)。本研究通過解析兩個9-LOX家族脂氧合酶基因ZmLOX3和ZmLOX12的抗病機制,取得以下主要結(jié)果：(1)通過玉米LOX3基因突變體與黃曲霉脂氧合酶基因突變體互作系統(tǒng)研究宿主與病原真菌的脂氧合酶基因在病原真菌侵染宿主過程中的作用,進一步闡述宿主和病原真菌間脂氧合酶基因調(diào)節(jié)宿主抗病或感病分子機制以及研究宿主與病原真菌間的相互作用。研究結(jié)果發(fā)現(xiàn)：宿主ZmLOX3基因可以抑制黃曲霉菌的定植與產(chǎn)孢,同時病原真菌PpoA、PpoD、和PpoC氧化酶基因需要依靠宿主LOX3基因的作用來調(diào)節(jié)自身孢子的產(chǎn)生,而病原真菌脂氧合酶loxA基因不需要依靠宿主LOX3基因的作用即可調(diào)節(jié)自身的產(chǎn)孢,同時病原真菌PpoA控制自身黃曲霉毒素的合成,并且宿主中籽粒中ABA和IAA可以促進病原真菌黃曲霉毒素的積累。這一結(jié)果說明玉米LOX3基因可以抵抗黃曲霉菌的侵害,同時在黃曲霉菌體內(nèi)的脂氧合酶基因參與其侵染宿主過程中,并且與宿主的脂氧合酶相互作用共同參與宿主對病原真菌的抗病與感病過程中。本研究證實脂氧合酶基因是宿主與病原真菌間相互作用的重要信號交流分子,為研究作物與病原真菌互作分子機制以及作物抗病育種提供優(yōu)異的基因資源和理論基礎(chǔ)。(2)ZmLOXS是禾生炭疽菌誘導植物產(chǎn)生系統(tǒng)誘導抗病反應(ISR)的負調(diào)控因子,但在lox3突變體中存在的ISR長距離抗性傳導信號的調(diào)控機制尚不清楚。本研究通過發(fā)現(xiàn)在lox3突變體在根部接種木霉菌后ZmLOX12的表達水平增加,進一步對ZmLOX12基因突變體進行功能驗證發(fā)現(xiàn)：在接種木霉菌后,相比野生型玉米材料,ZmLOX12缺陷型突變體lox12-1不僅缺失ISR信號,且更易感禾生炭疽菌。這說明LOX12基因的正常表達是產(chǎn)生ISR的必要條件,是宿主體內(nèi)ISR信號正調(diào)控因子。對宿主內(nèi)植物激素以及與茉莉酸合成有關(guān)的基因表達分析發(fā)現(xiàn),根部接種木霉菌后lox12-1突變體內(nèi)的OPDA含量(茉莉酸前體)以及JA-Ile含量顯著低于未接種的突變體及野生型材料。茉莉酸是調(diào)節(jié)ISR的主要信號分子,這可以解釋為什么經(jīng)過木霉菌作用后的lox12-1突變體對禾生炭疽菌表現(xiàn)出系統(tǒng)誘導抗病性感病性。因此LOX12參與調(diào)解根部木霉菌誘導宿主產(chǎn)生系統(tǒng)誘導抗病性過程中的分子機制,通過調(diào)節(jié)茉莉酸合成相關(guān)基因的表達來間接調(diào)節(jié)宿主體內(nèi)茉莉酸含量,進而參與木霉菌系統(tǒng)誘導抗病反應,是宿主系統(tǒng)誘導抗病機制的正調(diào)控因子。(3)為明確ZmWX12基因的誘導表達機制,我們分離了ZmLOX12基因的啟動子序列。序列分析發(fā)現(xiàn)LOX12啟動子含有許多重要的順式作用元件,如：BIHD1OS、ASF1-motif、GCC-core、MYB-core和POLLEN1LELAT52。將啟動子序列連接GUS基因轉(zhuǎn)化擬南芥發(fā)現(xiàn),木霉菌可以誘導ZmLOX12啟動子激活GUS基因表達,外源施加MeJA有相同誘導效果,這從另一方向上證實玉米LOX12基因可以受到木霉菌和茉莉酸的誘導表達。本研究的結(jié)果表明兩個玉米脂氧合酶基因均參與了宿主抗病性反應,增強宿主免疫抗病性,因此本研究結(jié)果可以為作物抗黃曲霉病與抗葉枯病分子育種以及作物與病原菌互作等方面提供了新的視角和理論依據(jù),并具有指導作物抗病分子育種的應用價值。
[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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.13
本文編號：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.
【學位授予單位】：四川農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.13
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