大丽轮枝菌腺苷酸激酶和寡糖基转移酶STT3亚基毒力功能分析
发布时间：2018-09-09 18:57
【摘要】：棉花黄萎病(Verticillium Wilt of cotton)是一种土传、维管束系统性病害,被称为“棉花癌症”,给棉花的产量和棉纤维的质量都造成很大影响,其致病菌为大丽轮枝菌(Verticillium dahliae)。由于大丽轮枝菌极易产生新的生理小种,给研究带来相当大的难度和很多的不确定性,因此对大丽轮枝菌的研究一直以来都是热点。本实验室利用寄主诱导的基因沉默技术(Host-induced gene silencing),以高致病力的大丽轮枝菌株V991为实验材料,构建一系列针对大丽轮枝菌靶标基因的烟草脆裂病毒(tobacco rattle virus, TRV)干扰载体,通过植物体内dsRNA与靶标基因互作,并进行统计病情指数统计,从中筛选出20个与病原菌生长发育与致病性相关的基因。本文从中选择腺苷酸激酶(adenylate kinase, AK)和寡糖基转移酶STT3亚基(oligosaccharyl transferase STT3 subunit, OST STT3)进行深入研究,期望进一步研究病原菌基因生物学功能和拓宽植物抗逆基因数据库。本研究的主要结果如下：1、利用实验室构建的HIGS体系,扩大本氏烟草培养规模,用含有AK和STT-3靶标基因片段的农杆菌注射植株10天后,接种106个/mL大丽轮枝菌,统计植株病情指数,并利用qRT-PCR进行植株真菌生物量检测和靶标基因表达水平分析；2、利用融合PCR技术,将靶标基因上、下游片段以及潮霉素抗性表达盒整合成一个完整的片段,通过BP反应将其构建至缺失突变体载体中；通过酶切反应,将新霉素抗性表达盒以及靶标基因表达盒构建至pCAMBIA1302载体中,成为回补质粒；然后将回补质粒中的靶标基因开放阅读框替换为GFP基因开放阅读框,成为GFP表达质粒。最后,通过农杆菌介导的方法获得阳性转化子。3、将野生型、突变体以及回补体孢子接种本氏烟草,统计植株病情指数并进行生物量测定,分析不同菌株毒力水平。同时,用表达GFP的野生型以及缺失突变体孢子接种本氏烟草,然后通过共聚焦显微镜观察两者在植株根部侵染的区别,旨在进一步明确靶标基因在病原菌发病过程中所发挥的作用。4、将野生型、AK突变体以及回补体孢子滴加到察比克培养基上,分析在不同逆境情况下菌株生理指标(菌落直径和产孢量)的差异。同时,将野生型、STT3突变体以及回补体孢子滴加到含有不同碳源的培养基上,分析菌株生理指标(菌落直径和产孢量)的差异,从而进一步探索STT3基因在碳源利用率方面所发挥的作用。5、构建含有靶标基因稳定遗传的Gateway干扰载体,转化本氏烟草。对获得的阳性植株接种病原菌,并对其进行抗病性分析以及分子水平检测。本实验利用突变体和回补体的构建以及稳定遗传阳性植株的获得,进一步探索AK和STT3基因在病原菌生长发育以及毒力方面发挥的作用,为农作物病虫害的防治提供了一定的实验数据和理论基础。
[Abstract]:Cotton Verticillium Wilt (Verticillium Wilt of cotton) is a systemic disease of vascular bundle, which is called "cotton cancer". It has a great influence on cotton yield and cotton fiber quality. The pathogenic bacteria is (Verticillium dahliae)., the pathogen of Verticillium dahliae. Because it is easy to produce new physiological species, it brings a great deal of difficulty and uncertainty to the research, so the research of Dendrotus dahliensis has been a hot spot all the time. The host induced gene silencing technique (Host-induced gene silencing),) was used to construct a series of (tobacco rattle virus, TRV) interference vectors of tobacco embrittlement virus (TBV) targeting the target gene of Verticillium dahliae (V991). Through the interaction of dsRNA and target genes in plants and the statistics of disease index, 20 genes related to the growth and pathogenicity of pathogenic bacteria were screened out. In this paper, adenylate kinase (adenylate kinase, AK) and oligosyltransferase (STT3) subunit (oligosaccharyl transferase STT3 subunit, OST STT3) were selected for further study in order to further study the gene biological function of pathogenic bacteria and broaden the database of plant stress resistance genes. The main results of this study were as follows: 1. Using the HIGS system constructed in the laboratory, we expanded the scale of tobacco cultivation. After 10 days of inoculation with Agrobacterium tumefaciens containing AK and STT-3 target gene fragments, we inoculated 106 / mL Rhizoctonia dahliensis. Plant disease index was counted, and plant fungal biomass and target gene expression level were analyzed by qRT-PCR. Using fusion PCR technique, the upstream and downstream fragments of target gene and hygromycin resistant expression box were integrated into a complete fragment. It was constructed into the deletion mutant vector by BP reaction, and the neomycin resistant expression box and target gene expression box were constructed into the pCAMBIA1302 vector by enzyme digestion. Then the open reading frame of the target gene in the complement plasmid was replaced by the open reading frame of the GFP gene to become the GFP expression plasmid. Finally, the positive transformant. 3 was obtained by Agrobacterium-mediated method. Spores of wild type, mutant and compensator were inoculated into Bentner's tobacco. The disease index and biomass of different strains were measured and the virulence levels of different strains were analyzed. At the same time, the wild type expressing GFP and the spores of the mutant were inoculated with Bentner's tobacco, and then the difference of infection in the root of the two plants was observed by confocal microscope. The purpose of this study was to further clarify the role of target gene in the pathogenesis of pathogenic bacteria, and to add spores of wild type AK mutant and compensator to Chabbik medium. The differences of physiological indexes (colony diameter and spore yield) under different stress conditions were analyzed. At the same time, the spores of wild type STT3 mutant and compensator were added to the medium containing different carbon sources, and the differences of physiological indexes (colony diameter and sporulation) were analyzed. In order to further explore the role of STT3 gene in the utilization of carbon source. 5, construct the Gateway interference vector with stable inheritance of target gene, and transform the tobacco. The positive plants were inoculated with pathogenic bacteria, and the disease resistance and molecular level were analyzed. The purpose of this study was to explore the role of AK and STT3 genes in the growth and virulence of pathogenic bacteria by constructing mutants and complement and obtaining stable genetically positive plants. It provides some experimental data and theoretical basis for the control of crop diseases and insect pests.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S435.621.2
本文编号：2233278
[Abstract]:Cotton Verticillium Wilt (Verticillium Wilt of cotton) is a systemic disease of vascular bundle, which is called "cotton cancer". It has a great influence on cotton yield and cotton fiber quality. The pathogenic bacteria is (Verticillium dahliae)., the pathogen of Verticillium dahliae. Because it is easy to produce new physiological species, it brings a great deal of difficulty and uncertainty to the research, so the research of Dendrotus dahliensis has been a hot spot all the time. The host induced gene silencing technique (Host-induced gene silencing),) was used to construct a series of (tobacco rattle virus, TRV) interference vectors of tobacco embrittlement virus (TBV) targeting the target gene of Verticillium dahliae (V991). Through the interaction of dsRNA and target genes in plants and the statistics of disease index, 20 genes related to the growth and pathogenicity of pathogenic bacteria were screened out. In this paper, adenylate kinase (adenylate kinase, AK) and oligosyltransferase (STT3) subunit (oligosaccharyl transferase STT3 subunit, OST STT3) were selected for further study in order to further study the gene biological function of pathogenic bacteria and broaden the database of plant stress resistance genes. The main results of this study were as follows: 1. Using the HIGS system constructed in the laboratory, we expanded the scale of tobacco cultivation. After 10 days of inoculation with Agrobacterium tumefaciens containing AK and STT-3 target gene fragments, we inoculated 106 / mL Rhizoctonia dahliensis. Plant disease index was counted, and plant fungal biomass and target gene expression level were analyzed by qRT-PCR. Using fusion PCR technique, the upstream and downstream fragments of target gene and hygromycin resistant expression box were integrated into a complete fragment. It was constructed into the deletion mutant vector by BP reaction, and the neomycin resistant expression box and target gene expression box were constructed into the pCAMBIA1302 vector by enzyme digestion. Then the open reading frame of the target gene in the complement plasmid was replaced by the open reading frame of the GFP gene to become the GFP expression plasmid. Finally, the positive transformant. 3 was obtained by Agrobacterium-mediated method. Spores of wild type, mutant and compensator were inoculated into Bentner's tobacco. The disease index and biomass of different strains were measured and the virulence levels of different strains were analyzed. At the same time, the wild type expressing GFP and the spores of the mutant were inoculated with Bentner's tobacco, and then the difference of infection in the root of the two plants was observed by confocal microscope. The purpose of this study was to further clarify the role of target gene in the pathogenesis of pathogenic bacteria, and to add spores of wild type AK mutant and compensator to Chabbik medium. The differences of physiological indexes (colony diameter and spore yield) under different stress conditions were analyzed. At the same time, the spores of wild type STT3 mutant and compensator were added to the medium containing different carbon sources, and the differences of physiological indexes (colony diameter and sporulation) were analyzed. In order to further explore the role of STT3 gene in the utilization of carbon source. 5, construct the Gateway interference vector with stable inheritance of target gene, and transform the tobacco. The positive plants were inoculated with pathogenic bacteria, and the disease resistance and molecular level were analyzed. The purpose of this study was to explore the role of AK and STT3 genes in the growth and virulence of pathogenic bacteria by constructing mutants and complement and obtaining stable genetically positive plants. It provides some experimental data and theoretical basis for the control of crop diseases and insect pests.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S435.621.2
【相似文献】
相关博士学位论文 前1条
1 苏晓峰;大丽轮枝菌腺苷酸激酶和寡糖基转移酶STT3亚基毒力功能分析[D];中国农业科学院;2016年
，本文编号：2233278
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2233278.html
