辣椒疫霉质外体效应分子SCR209的功能分析

发布时间：2018-05-27 11:39

本文选题：辣椒疫霉 + PcF/SCR效应分子　；参考：《扬州大学》2017年硕士论文

【摘要】：辣椒疫霉(Phytophthora capsici Leonian)是一种寄主范围很广的重要植物病原卵菌,经常危害茄科、葫芦科、豆科等多种农作物。在侵染过程中,疫霉菌通常分泌大量不同种类的效应分子进入植物体内,干扰植物的生理功能,抑制植物的免疫反应,以利于自身的侵染。因此,明确疫霉菌效应分子的功能,阐明疫霉菌致病的机理,对控制植物疫病具有重要意义。PcF/SCR型效应分子是一类分泌到植物细胞间的卵菌效应蛋白,但目前尚不清楚它们在侵染过程中的作用。本文以辣椒疫霉PcF/SCR型效应分子SCR209为对象,分析其功能,获得的主要结果是:(1)利用半定量RT-PCR技术分析究了 PcF/SCR型效应分子SCR209编码基因在辣椒疫霉生长发育阶段(菌丝、游动孢子囊、游动孢子和萌发的休止孢)和侵染本氏烟阶段(接种后1.5、3、6、12、24、36、72 h)的表达情况。结果发现,scr209基因在辣椒疫霉生长发育阶段和侵染本氏烟阶段上调表达。(2)通过高保真扩增技术将该基因克隆到马铃薯X病毒(Potato virusX,PVX)表达载体中,利用农杆菌介导的瞬时表达法分析该基因能否引起本氏烟和番茄的细胞死亡。结果发现,SCR209能够引起本氏烟和番茄产生细胞死亡。(3)通过高保真扩增技术将基因与红色荧光蛋白融合,利用农杆菌介导的瞬时表达法分析SCR209在侵染过程中在植物中的定位。结果发现,在侵染过程中SCR209可能泌出并定位在植物细胞膜上。(4)为进一步了解该效应分子的功能,本论文在辣椒疫霉中进行了基因沉默和CRISPR/Cas9基因敲除分析,测定基因沉默转化子和敲除转化子的致病力和其它生物学性状是否发生了变化。结果显示,scr209基因的沉默和敲除都能显著削弱辣椒疫霉对寄主植物的致病力,而且基因沉默和基因敲除都导致辣椒疫霉丧失对不同类型的外源活性氧(过氧化氢和过氧化氢异丙苯)的抗性。结果说明PcF/SCR型效应分子SCR209在辣椒疫霉侵染植物的过程中发挥重要作用,该蛋白可能是通过应对植物中的活性氧防卫而发挥致病作用。本论文分析了 PcF/SCR型效应分子SCR209在辣椒疫霉侵染植物过程中的作用,研究结果为认识辣椒疫霉与植物的胞间互作机制提供了重要的实验数据,为进一步分析该类效应分子的作用机理奠定了基础。
[Abstract]:Phytophthora capsici Leonian (Phytophthora capsici) is an important plant pathogen with a wide range of hosts, which often harms many crops such as Solanaceae, Cucurbitaceae, Leguminosae and so on. In the process of infection, Phytophthora infestans usually secrete a large number of different effector molecules into the plant, which interferes with the physiological function of the plant and inhibits the immune response of the plant in order to facilitate its infection. Therefore, clarifying the function of the effector molecules of Phytophthora and elucidating the pathogenesis of Phytophthora infestans is of great significance to the control of plant blight. PcF- SCR effector molecules are a kind of oocyte effector proteins secreted into plant cells. However, their role in the process of infection is unclear. In this paper, SCR209, an effector molecule of Phytophthora capsici, was used to analyze its function. The main result was: (1) A semi-quantitative RT-PCR technique was used to analyze the SCR209 coding gene of PcF/SCR type effector molecule in the growth and development stage of Phytophthora capsici (hyphae). The expression of zoosporosporus, zoospore and germinating endospores, and the stage of infecting Bensi's tobacco (1. 5%) and the stage of infection (1. 5%) after inoculation (1. 5%, 2. 12, 24, 36, 36, 72 h after inoculation). The results showed that the expression of scr209 gene was up-regulated in Phytophthora capsici at the stage of growth and development and at the stage of infecting Phytophthora capsici. The gene was cloned into the expression vector of Potato virus XNV PVX by high fidelity amplification. The transient expression mediated by Agrobacterium tumefaciens was used to analyze whether the gene could cause cell death in tobacco and tomato. The results showed that SCR209 could cause cell death in tobacco and tomato. The gene was fused with red fluorescent protein by high fidelity amplification, and the localization of SCR209 in plants was analyzed by the transient expression mediated by Agrobacterium tumefaciens. The results showed that SCR209 might be secreted and located on the cell membrane of plants during infection. In order to further understand the function of the effector molecule, gene silencing and CRISPR/Cas9 knockout analysis were carried out in Phytophthora capsici. The pathogenicity and other biological characters of transformants and knockout transformants were determined. The results showed that the silencing and knockout of scr209 gene significantly weakened the pathogenicity of Phytophthora capsici to host plants. Gene silencing and gene knockout both lead to the loss of resistance of Phytophthora capsici to different types of exogenous active oxygen species (hydrogen peroxide and cumene peroxide). The results showed that PcF/SCR type effector molecule SCR209 played an important role in the process of infecting plants by Phytophthora capsici, and the protein may play a pathogenic role by responding to the defense of reactive oxygen species in plants. The role of PcF/SCR type effector molecule SCR209 in the process of infecting plants by Phytophthora capsici was analyzed in this paper. The results provided important experimental data for understanding the mechanism of intercellular interaction between Phytophthora capsici and Phytophthora capsici. It lays a foundation for further analysis of the mechanism of this kind of effector molecules.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.4

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