【摘要】：目的:棉花(Gossypium hirsutum)是世界上重要的經(jīng)濟作物,也是天然纖維的主要來源。黃萎病(Verticillium Wilt)是棉花的主要病害,嚴重影響了棉花的產(chǎn)量和品質(zhì),制約了棉花的生產(chǎn)。黃萎病是一種土壤傳播的維管束病害,可以危害兩百多種雙子葉植物,包括重要的經(jīng)濟作物棉花,番茄和生菜等。在我國,黃萎病是棉花的主要病害;其主要致病菌大麗輪枝菌是一種土壤傳播的、半活體營養(yǎng)的絲狀真菌,主要定殖在植物的木質(zhì)部,由于其休眠形式的微菌核可以在土壤中存活數(shù)十年,因此人們一直沒有找到防治黃萎病的有效措施。近年來,研究人員通過基因組、轉(zhuǎn)錄組、蛋白質(zhì)組、T-DNA突變體庫和基因敲除等手段對大麗輪枝菌致病相關(guān)基因進行了廣泛的研究,取得了階段性進展,特別是在大麗輪枝菌侵染結(jié)構(gòu)的研究方面取得了重要進展。大麗輪枝菌基因組編碼七百多種分泌蛋白,但人們對分泌蛋白的研究還不是很深入,分泌蛋白的分泌過程也不清楚。目前僅少數(shù)幾個被鑒定與病原菌的致病性相關(guān)。由于分泌蛋白的多樣性,導(dǎo)致研究較為困難,但是分泌途徑相關(guān)的組分相對保守,通過對分泌途徑相關(guān)組分的研究,將為棉花黃萎病的防治提供一個新的靶點。胞吐作用是真核生物細胞內(nèi)基于囊泡運輸?shù)膫鹘y(tǒng)分泌途徑,此外還存在一些非傳統(tǒng)的分泌途徑。真菌生長所需的營養(yǎng)物質(zhì)和致病相關(guān)的因子等通過囊泡運輸?shù)姆绞奖晦D(zhuǎn)運到細胞質(zhì)膜的特定位置行使功能。囊泡依賴于馬達分子肌球蛋白Myosin V(Myo V)提供動力和能量沿微絲提供的細胞軌道進行運輸。Myosin V在絲狀真菌中相對保守,在模式真菌酵母和構(gòu)巢曲霉中已經(jīng)得到了很好的研究,但是在大麗輪枝菌中還沒有被研究。本研究對大麗輪枝菌中V型Myosin在病原菌致病過程中的功能進行了研究,以此希望能為棉花黃萎病的防治提供一個新的理論依據(jù)。方法:(1)根據(jù)已報導(dǎo)的模式絲狀真菌構(gòu)巢曲霉中的V型Myosin蛋白序列在大麗輪枝菌的數(shù)據(jù)庫中進行同源序列比對,找到了大麗輪枝菌中同源的V型肌球蛋白Myosin2(Myo2),并構(gòu)建了絲狀真菌中V型肌球蛋白的系統(tǒng)進化樹。(2)利用農(nóng)桿菌介導(dǎo)的同源重組方法獲得了大麗輪枝菌Myo2的單一敲除突變體菌株△Vdmyo2,并對其菌落形態(tài)、萌發(fā)速率及菌絲生長習(xí)性等進行觀察。(3)構(gòu)建Myo2-GFP融合表達載體,轉(zhuǎn)化△Vdmyo2菌株獲得了回復(fù)菌株△Vdmyo2/Vd Myo2-GFP。通過轉(zhuǎn)盤式激光共聚焦顯微鏡觀察了大麗輪枝菌菌絲生長過程中Myo2的時空動態(tài)模式。(4)利用掃描電子顯微鏡觀察了大麗輪枝菌的入侵過程。(5)利用注射法和蘸根法侵染了大麗輪枝菌的宿主,棉花和擬南芥,比較分析了突變體菌株和對照野生型菌株的致病力差異。(6)利用超速離心濃縮的方法收集了大麗輪枝菌(突變體和野生型對照)的分泌蛋白,進行蛋白i TARQ質(zhì)譜實驗,利用Scaffold 4.0軟件進行差異蛋白篩選,利用Blast2GO軟件進行差異蛋白功能注釋,差異蛋白GO分析,差異蛋白功能分類以及差異蛋白的表達分析,闡述了肌球蛋白Myo2動力系統(tǒng)介導(dǎo)的大麗輪枝菌的致病機理。結(jié)果與結(jié)論:(1)大麗輪枝菌中V型肌球蛋白Myo2與其他絲狀真菌的同源性相對較高,在絲狀真菌中功能相對保守。敲除突變體菌株表現(xiàn)出嚴重的生長缺陷,白色菌絲明顯減少,黑色素增多,微菌核增加,菌落生長速率減慢。同時也嚴重影響了分生孢子的形態(tài)和菌絲的極性。分生孢子呈圓形,菌絲失去了極性呈三維立體式生長,菌絲變粗,分支和分隔明顯增加,上述結(jié)果表明Myo2參與了分生孢子的發(fā)育和菌絲的極性生長過程。(2)大麗輪枝菌Myo2-GFP在胞質(zhì)中呈點狀體,在菌絲頂端的頂體處呈圓形體,且與頂體的Marker FM4-64共定位,該結(jié)果進一步顯示大麗輪枝菌的Myo2在囊泡運輸過程發(fā)揮重要作用。(3)宿主棉花和擬南芥接種實驗顯示突變體菌株的致病力顯著下降。接種突變體菌株的棉花莖的維管束感病程度較輕,并且在莖切斷中也不能分離出病原菌,表明Myo2對大麗輪枝菌的致病力是必需的。通過擬南芥的入侵實驗說明突變體菌株仍然能侵入宿主細胞,推測致病下降的原因可能不是由侵染過程受阻導(dǎo)致的,主要的原因可能是Myo2缺失后在分泌蛋白外泌過程中囊泡運輸上發(fā)生了功能缺陷。(4)通過分泌蛋白的蛋白質(zhì)組數(shù)據(jù)我們發(fā)現(xiàn)Myo2缺失后大麗輪枝菌分泌蛋白的能力在同等條件下降低了。參與碳水化合物的轉(zhuǎn)運和修飾以及細胞內(nèi)轉(zhuǎn)運、分泌、囊泡轉(zhuǎn)運過程蛋白均存在顯著差異。因此認為Myo2作為蛋白分泌途徑中的重要組分,通過調(diào)節(jié)大麗輪枝菌致病相關(guān)的分泌蛋白的分泌來影響大麗輪枝菌的致病性,在大麗輪枝菌與宿主植物互作的過程中發(fā)揮重要的作用。
[Abstract]:Objective: Cotton (Gyppium hirsutum) is an important economic crop in the world, and is also the main source of natural fiber. Verticillium Wilt is the main disease of cotton, which seriously affects the yield and quality of cotton and restricts the production of cotton. Verticillium wilt is a kind of soil-borne vascular disease, which can harm more than two hundred kinds of plants, including important cash crop cotton, tomato, lettuce and so on. Verticillium dahliae is the main disease of cotton in China, and its main pathogenic bacteria are the filamentous fungi of soil-borne and semi-living nutrition, mainly colonizing the wood of the plant, and the microsclerotium in its dormant form can survive in the soil for several decades. Therefore, people have not found effective measures to prevent verticillium wilt. In recent years, researchers have made extensive research on the virulence-related genes of the large-li-wheelset by genome, transcriptome, proteome, T-DNA mutant library and gene knockout. in particular, important progress has been made in that research of the infection structure of the large-li wheel branch fungus. There are more than 700 secreted proteins encoded by the genome of the L. rivulus L., but the research on the secreted protein is not very deep, nor the secretion process of the secretory protein is unclear. Only a few have been identified as being related to the pathogenicity of pathogenic bacteria. Due to the diversity of secretory proteins, the research is difficult, but the components related to the secretory pathway are relatively conservative, and the research on the relevant components of the secretory pathway will provide a new target for the prevention and treatment of verticillium wilt of cotton. The exocytosis is a traditional secretory pathway based on vesicle transport in a true nuclear biological cell, and there are some non-traditional secretory pathways. The nutrients and virulence related factors required for the growth of the fungus are transported to a specific location of the cytoplasmic membrane by way of vesicle transport. Vesicles are dependent on motor molecular omyosin Myosin V (Myo V) to provide power and energy for transport along the cell tracks provided by the microfilaments. Myosin V is relatively conserved in filamentous fungi, and has been well studied in the mode fungal yeasts and Aspergillus nidulans, but has not yet been studied in the large-leaf fungus. This study studied the function of V-type Myosin in the pathogenic process of pathogenic bacteria, hoping to provide a new theoretical basis for the prevention and treatment of verticillium wilt of cotton. Methods: (1) The V-type Myosin2 (Myo2) was found to be homologous to the L-type Myosin (Myto2), in which the V-type Myosin protein sequence in Aspergillus nidulatum was sequenced according to the reported model. The phylogenetic tree of V-type Myosin in filamentous fungi was constructed and constructed. (2) The single knockout mutant strain Vddmyo2 was obtained by Agrobacterium-mediated homologous recombination, and the colony morphology, germination rate and growth habit of mycelium were observed. (3) The expression vector of Myo2-GFP was constructed, and the mutant Vddmyo2/ Vd Myo2-GFP was obtained by transforming Vddmyo 2 strain. The space-time dynamic model of Myo2 was observed by rotating disc laser confocal microscope. (4) The process of invasion was observed by scanning electron microscope (SEM). (5) The host, cotton and Arabidopsis were infected by injection method and dip root method, and the difference of pathogenicity between mutant strain and control wild-type strain was analyzed. (6) By using the method of ultracentrengation and concentration, the secretion protein of the large-sized verticillium (mutant and wild-type control) was collected, the protein i TARQ mass spectrum experiment was carried out, the difference protein was screened by using the Saffold 4.0 software, and the difference protein GO analysis was performed by using the Blast2GO software. The functional classification of differential protein and the expression of differential protein were analyzed. Results and Conclusion: (1) The homology of V-type Myo2 with other filamentous fungi was relatively high, and the function was relatively conservative in filamentous fungi. The knockout mutant strain showed serious growth defect, the white mycelium was obviously reduced, the melanin increased, the microsclerotium increased, and the colony growth rate was slowed down. At the same time, the morphology of the conidiospore and the polarity of the mycelium are also seriously affected. The results showed that Myo2 was involved in the development of conidiospore and the process of polarity growth of mycelium. (2) Myo 2-GFP was a punctate in the cytoplasm, and was located at the top of the top of the hypha and co-located with Marker FM4-64 of the top body. The result further showed that Myo2 played an important role in the vesicle transport. (3) The host cotton and Arabidopsis inoculation experiment showed that the pathogenicity of mutant strain decreased significantly. The cotton stalk of the inoculated mutant strain has a relatively light vascular bundle sense, and the pathogenic bacteria can not be isolated in the stem cut-off, which indicates that Myo2 is necessary for the pathogenic force of the large-leaf fungus. The invasion experiment of Arabidopsis proves that the mutant strain can still invade the host cell, and it is estimated that the cause of the disease decrease may not be caused by the obstruction of the infection process, and the main reason may be that the functional defect of the vesicle transport during the secretion of the secreted protein after the loss of Myo2 may be the main reason. (4) Through the protein group data of the secretory protein, we found that the ability of Myo2 to secrete protein was reduced under the same conditions. There were significant differences in the transport and modification of carbohydrate and intracellular transport, secretion and vesicle transport. Therefore, Myo2 is regarded as an important component in the pathway of protein secretion, and it plays an important role in the process of interaction between the host plants and the host plants by modulating the secretion of secreted proteins related to the pathogenesis of the large-li-wheel-branched bacteria.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S435.621.2

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