炭疽菌侵染后茶树cDNA-AFLP体系构建和基因差异表达研究

发布时间：2018-04-25 06:05

本文选题：茶树 + cDNA-AFLP　；参考：《福建农林大学》2016年硕士论文

【摘要】：茶叶是世界上最流行的健康饮料之一,具有抗氧化、利尿、抗突变强心解痉等功效,茶叶需求量也节节攀升。为保证茶叶的品质和产量,茶树病害的有效防治显得越发重要。茶树炭疽病遍及各大主要茶区,严重影响茶叶的品质和产量,为此,有必要探究炭疽菌-茶树的互作机理,为有效防治茶炭疽病提供一些理论依据。本研究通过对不同茶树品种叶片分别接种不同炭疽病病原菌菌株,茶炭疽病病斑多为圆形、半圆形,有的会被叶脉隔断,形状不规则,有的病斑则串联到一起,病害严重的叶片蜷缩成卷形；颜色以褐色、暗褐色为主,有的为暗绿色,从病斑中心位置至病斑边缘渐渐转化为灰白色,病斑中心密生小黑点并呈现隆起状.。不同茶树品种对炭疽病病原菌的抗病能力强弱也有差异,铁观音、肉桂、大叶乌龙属于抗性较弱品种,毛蟹、福鼎大白茶抗性较强,本研究结合茶树形态学为基础对炭疽病病原菌的致病能力分化进行了探索,筛选获得强致病菌株ZRG,较弱致病力菌株ATG、FSX,从形态上分析强弱菌株的附着胞、分生孢子等结构差异特性。针对强致病力菌株ZRG侵染茶树叶片后部分生理生化反应进行了探究,以期为揭开炭疽菌-茶树互作机制提供理论依据,并通过qRT-PCR对茶树受病原菌侵染主要几种抗氧化酶类表达进行分析,旨在为茶树生理生化研究提供一种新的模式和茶树分子手段选育抗病品种的开展提供借鉴。利用炭疽病病原菌强致病菌株ZRG侵染茶树品种毛蟹后,对茶树叶片基因差异表达谱,即cDNA-AFLP体系进行了构建。对146条差异条带功能进行分析归类,其中参与碳水化合物和能量代谢11.6%、脂质代谢2.7%、核酸代谢6.8%、蛋白质代谢15.1%、应激反应21.9%、生物调控与信号转导17.8%、运输4.8%、细胞壁与细胞骨架代谢3.4%和其他代谢过程12.3%。对其中部分差异条带进行进一步的qRT-PCR验证cDNA-AFLP图谱,验证设立三次重复实验,一致率为92.6%,证明了cDNA-AFLP体系的可靠性。在其中发现了WEKY转录因子、乙烯转录因子和过氧化物酶等与植物抗逆境密切相关基因表达上调。总之,本研究筛选出茶树被炭疽菌侵入后差异相关基因,并对这些基因进行分析,但未对其在茶树抗炭疽病中的功能进行研究,这些工作有待在还需在茶树中进行进一步转基因验证。
[Abstract]:Tea is one of the most popular health drinks in the world, with antioxidant, diuretic, anti-mutation, strong spasmolysis and other functions, tea demand is also rising. In order to guarantee the quality and yield of tea, the effective control of tea plant disease becomes more and more important. Anthracnose of tea plants is widespread in major tea areas, which seriously affects the quality and yield of tea leaves. Therefore, it is necessary to explore the interaction mechanism of anthrax and tea plants in order to provide some theoretical basis for effective control of tea anthracnose. In this study, different strains of anthracnose were inoculated into different leaves of different tea varieties. Most of the anthracnose spots were round and semicircular, some of them were separated by leaf veins, some of them were irregular in shape, and some of them were connected together. The leaves with severe disease were curled up into coils. The color was brown, dark brown, and some of them were dark green. From the center of the spot to the edge of the spot, the leaf gradually changed into gray and white, and the center of the disease was dense with small black spots and appeared as a bulge. The resistance of different tea varieties to anthracnose pathogens was also different. Tieguanyin, cinnamon and Oolong were among the less resistant varieties, hairy crab and Fuding Dabai tea had stronger resistance to anthracnose. Based on the morphology of tea plant, the pathogenicity differentiation of anthracnose pathogen was studied, and the strong pathogenic strain ZRG, the weak pathogenic strain ATGG FSX were obtained, and the attachment cells of the strong and weak strains were analyzed from morphological point of view. Conidia and other structural differences. The physiological and biochemical responses of the strongly pathogenic strain ZRG to tea leaves were studied in order to provide theoretical basis for uncovering the interaction mechanism of anthrax and tea plants. The expression of several antioxidant enzymes in tea plants infected by pathogens was analyzed by qRT-PCR in order to provide a new model for the physiological and biochemical studies of tea plants and to provide reference for the selection of resistant varieties by molecular means of tea plants. The gene differential expression profile of tea leaves, or cDNA-AFLP system, was constructed after infecting the tea variety hairy crab with ZRG, a strong pathogen of anthracnose. The function of 146 different bands was analyzed and classified. Among them, 11.6 involved in carbohydrate and energy metabolism, 2.7 in lipid metabolism, 6.8 in nucleic acid metabolism, 15.1in protein metabolism, 21.9in stress reaction, 17.8in biological regulation and signal transduction, 4.8in transport, 3.4% in cell wall and cytoskeleton metabolism and 12.3% in other metabolic processes. Some of the differential bands were further verified by qRT-PCR to verify the cDNA-AFLP map, and three repeated experiments were established, and the consistency rate was 92.6, which proved the reliability of the cDNA-AFLP system. The expression of WEKY transcription factors, ethylene transcription factors and peroxidase were up-regulated. In conclusion, the differentially related genes of tea plants after invasion by anthrax were screened and analyzed, but their functions in anthracnose resistance of tea plants were not studied. These efforts need to be further verified in tea plants.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.711

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