本文選題：辣椒疫霉 + PcF/SCR效應(yīng)分子��； 參考：《揚州大學(xué)》2017年碩士論文

【摘要】：辣椒疫霉(Phytophthora capsici Leonian)是一種寄主范圍很廣的重要植物病原卵菌,經(jīng)常危害茄科、葫蘆科、豆科等多種農(nóng)作物。在侵染過程中,疫霉菌通常分泌大量不同種類的效應(yīng)分子進入植物體內(nèi),干擾植物的生理功能,抑制植物的免疫反應(yīng),以利于自身的侵染。因此,明確疫霉菌效應(yīng)分子的功能,闡明疫霉菌致病的機理,對控制植物疫病具有重要意義。PcF/SCR型效應(yīng)分子是一類分泌到植物細胞間的卵菌效應(yīng)蛋白,但目前尚不清楚它們在侵染過程中的作用。本文以辣椒疫霉PcF/SCR型效應(yīng)分子SCR209為對象,分析其功能,獲得的主要結(jié)果是:(1)利用半定量RT-PCR技術(shù)分析究了 PcF/SCR型效應(yīng)分子SCR209編碼基因在辣椒疫霉生長發(fā)育階段(菌絲、游動孢子囊、游動孢子和萌發(fā)的休止孢)和侵染本氏煙階段(接種后1.5、3、6、12、24、36、72 h)的表達情況。結(jié)果發(fā)現(xiàn),scr209基因在辣椒疫霉生長發(fā)育階段和侵染本氏煙階段上調(diào)表達。(2)通過高保真擴增技術(shù)將該基因克隆到馬鈴薯X病毒(Potato virusX,PVX)表達載體中,利用農(nóng)桿菌介導(dǎo)的瞬時表達法分析該基因能否引起本氏煙和番茄的細胞死亡。結(jié)果發(fā)現(xiàn),SCR209能夠引起本氏煙和番茄產(chǎn)生細胞死亡。(3)通過高保真擴增技術(shù)將基因與紅色熒光蛋白融合,利用農(nóng)桿菌介導(dǎo)的瞬時表達法分析SCR209在侵染過程中在植物中的定位。結(jié)果發(fā)現(xiàn),在侵染過程中SCR209可能泌出并定位在植物細胞膜上。(4)為進一步了解該效應(yīng)分子的功能,本論文在辣椒疫霉中進行了基因沉默和CRISPR/Cas9基因敲除分析,測定基因沉默轉(zhuǎn)化子和敲除轉(zhuǎn)化子的致病力和其它生物學(xué)性狀是否發(fā)生了變化。結(jié)果顯示,scr209基因的沉默和敲除都能顯著削弱辣椒疫霉對寄主植物的致病力,而且基因沉默和基因敲除都導(dǎo)致辣椒疫霉喪失對不同類型的外源活性氧(過氧化氫和過氧化氫異丙苯)的抗性。結(jié)果說明PcF/SCR型效應(yīng)分子SCR209在辣椒疫霉侵染植物的過程中發(fā)揮重要作用,該蛋白可能是通過應(yīng)對植物中的活性氧防衛(wèi)而發(fā)揮致病作用。本論文分析了 PcF/SCR型效應(yīng)分子SCR209在辣椒疫霉侵染植物過程中的作用,研究結(jié)果為認識辣椒疫霉與植物的胞間互作機制提供了重要的實驗數(shù)據(jù),為進一步分析該類效應(yīng)分子的作用機理奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S432.4

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本文編號：1941920