V型肌球蛋白在大丽轮枝菌与棉花互作过程中的机制研究

发布时间：2018-10-20 06:54

【摘要】：目的:棉花(Gossypium hirsutum)是世界上重要的经济作物,也是天然纤维的主要来源。黄萎病(Verticillium Wilt)是棉花的主要病害,严重影响了棉花的产量和品质,制约了棉花的生产。黄萎病是一种土壤传播的维管束病害,可以危害两百多种双子叶植物,包括重要的经济作物棉花,番茄和生菜等。在我国,黄萎病是棉花的主要病害;其主要致病菌大丽轮枝菌是一种土壤传播的、半活体营养的丝状真菌,主要定殖在植物的木质部,由于其休眠形式的微菌核可以在土壤中存活数十年,因此人们一直没有找到防治黄萎病的有效措施。近年来,研究人员通过基因组、转录组、蛋白质组、T-DNA突变体库和基因敲除等手段对大丽轮枝菌致病相关基因进行了广泛的研究,取得了阶段性进展,特别是在大丽轮枝菌侵染结构的研究方面取得了重要进展。大丽轮枝菌基因组编码七百多种分泌蛋白,但人们对分泌蛋白的研究还不是很深入,分泌蛋白的分泌过程也不清楚。目前仅少数几个被鉴定与病原菌的致病性相关。由于分泌蛋白的多样性,导致研究较为困难,但是分泌途径相关的组分相对保守,通过对分泌途径相关组分的研究,将为棉花黄萎病的防治提供一个新的靶点。胞吐作用是真核生物细胞内基于囊泡运输的传统分泌途径,此外还存在一些非传统的分泌途径。真菌生长所需的营养物质和致病相关的因子等通过囊泡运输的方式被转运到细胞质膜的特定位置行使功能。囊泡依赖于马达分子肌球蛋白Myosin V(Myo V)提供动力和能量沿微丝提供的细胞轨道进行运输。Myosin V在丝状真菌中相对保守,在模式真菌酵母和构巢曲霉中已经得到了很好的研究,但是在大丽轮枝菌中还没有被研究。本研究对大丽轮枝菌中V型Myosin在病原菌致病过程中的功能进行了研究,以此希望能为棉花黄萎病的防治提供一个新的理论依据。方法:(1)根据已报导的模式丝状真菌构巢曲霉中的V型Myosin蛋白序列在大丽轮枝菌的数据库中进行同源序列比对,找到了大丽轮枝菌中同源的V型肌球蛋白Myosin2(Myo2),并构建了丝状真菌中V型肌球蛋白的系统进化树。(2)利用农杆菌介导的同源重组方法获得了大丽轮枝菌Myo2的单一敲除突变体菌株△Vdmyo2,并对其菌落形态、萌发速率及菌丝生长习性等进行观察。(3)构建Myo2-GFP融合表达载体,转化△Vdmyo2菌株获得了回复菌株△Vdmyo2/Vd Myo2-GFP。通过转盘式激光共聚焦显微镜观察了大丽轮枝菌菌丝生长过程中Myo2的时空动态模式。(4)利用扫描电子显微镜观察了大丽轮枝菌的入侵过程。(5)利用注射法和蘸根法侵染了大丽轮枝菌的宿主,棉花和拟南芥,比较分析了突变体菌株和对照野生型菌株的致病力差异。(6)利用超速离心浓缩的方法收集了大丽轮枝菌(突变体和野生型对照)的分泌蛋白,进行蛋白i TARQ质谱实验,利用Scaffold 4.0软件进行差异蛋白筛选,利用Blast2GO软件进行差异蛋白功能注释,差异蛋白GO分析,差异蛋白功能分类以及差异蛋白的表达分析,阐述了肌球蛋白Myo2动力系统介导的大丽轮枝菌的致病机理。结果与结论:(1)大丽轮枝菌中V型肌球蛋白Myo2与其他丝状真菌的同源性相对较高,在丝状真菌中功能相对保守。敲除突变体菌株表现出严重的生长缺陷,白色菌丝明显减少,黑色素增多,微菌核增加,菌落生长速率减慢。同时也严重影响了分生孢子的形态和菌丝的极性。分生孢子呈圆形,菌丝失去了极性呈三维立体式生长,菌丝变粗,分支和分隔明显增加,上述结果表明Myo2参与了分生孢子的发育和菌丝的极性生长过程。(2)大丽轮枝菌Myo2-GFP在胞质中呈点状体,在菌丝顶端的顶体处呈圆形体,且与顶体的Marker FM4-64共定位,该结果进一步显示大丽轮枝菌的Myo2在囊泡运输过程发挥重要作用。(3)宿主棉花和拟南芥接种实验显示突变体菌株的致病力显著下降。接种突变体菌株的棉花茎的维管束感病程度较轻,并且在茎切断中也不能分离出病原菌,表明Myo2对大丽轮枝菌的致病力是必需的。通过拟南芥的入侵实验说明突变体菌株仍然能侵入宿主细胞,推测致病下降的原因可能不是由侵染过程受阻导致的,主要的原因可能是Myo2缺失后在分泌蛋白外泌过程中囊泡运输上发生了功能缺陷。(4)通过分泌蛋白的蛋白质组数据我们发现Myo2缺失后大丽轮枝菌分泌蛋白的能力在同等条件下降低了。参与碳水化合物的转运和修饰以及细胞内转运、分泌、囊泡转运过程蛋白均存在显著差异。因此认为Myo2作为蛋白分泌途径中的重要组分,通过调节大丽轮枝菌致病相关的分泌蛋白的分泌来影响大丽轮枝菌的致病性,在大丽轮枝菌与宿主植物互作的过程中发挥重要的作用。
[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.
【学位授予单位】：石河子大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S435.621.2

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