灰霉病菌Atg9基因的功能研究

发布时间：2018-03-14 07:50

本文选题：灰霉病菌　切入点：农杆菌介导的转化　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：植物灰霉病是由灰霉病菌（Botrytis cinerea）侵染引起的真菌性病害，在全球广泛分布，每年造成巨大的经济损失。灰霉病菌是典型的死体营养型植物病原真菌，在自然条件下多以分生孢子作为侵染寄主的初侵染和再侵染来源。灰霉病菌是气传植物病原菌，能侵染200多种寄主植物。自噬相关蛋白9（Atg9）是自噬体（autophagosome）膜形成所必需的一个泛素样蛋白质，由117个氨基酸残基组成，负责介导细胞中依赖于溶酶体/液泡等细胞器的生物大分子的周转。本研究主要阐述Atg9基因在灰霉病菌生长、发育和致病性中的作用，主要结果如下： 1.以田间灰霉病菌强致病力菌株Bc3-4为原始材料构建了含有3000多个T-DNA随机插入突变的转化子群体，采用番茄、草莓离体叶片分别对所获得的转化子群体独立进行至少3次的致病性筛选，共获得148个侵染表型明显下降的灰霉病菌T-DNA插入突变体。 2.采用热不对称交错PCR（Thermal Asymmetric Interlaced PCR, TAIL-PCR）技术对部分获得的侵染表型明显致弱的灰霉病菌突变体进行T-DNA插入灰霉病菌基因组的遗传座位分析，发现其中的一个突变体的T-DNA插入灰霉病菌Atg9基因的启动子区域，，Southern blot分析结果显示该突变是由单拷贝的T-DNA插入造成的。由于很少有关于自噬基因调控灰霉病菌生长发育及致病性的研究报导，因此，本研究对灰霉病菌Atg9基因进行了相关研究。 3.通过农杆菌介导的遗传转化(Agrobacterium tumefaciens mediatedtransformation, ATMT)法成功敲除灰霉病菌的Atg9基因，经过PCR等方法验证获得了灰霉病菌Atg9基因的敲除突变体△BcAtg9。 4.通过灰霉病菌野生型Bc05.10与其Atg9基因敲除突变体△BcAtg9菌株生长发育对比研究发现：在PDA培养基上，△BcAtg9与野生型菌株生长速率并无明显的加快或减慢现象，说明Atg9基因不影响菌丝体的生长；产孢量分析显示野生型每个平板的产孢量约为（3.1±0.4）×107个孢子/plate，而△BcAtg9突变株的产孢量约为（1.5±0.3）×107个孢子/plate，产量约下降一倍，显著低于野生型菌株分生孢子的产孢量；致病性测定结果表明，在接种3天后，野生型菌株侵染导致的病斑面积为（1.24±0.14）cm2，而突变株侵染导致的病斑面积为（0.34±0.06）cm2，显著小于野生型菌株侵染导致的病斑面积，说明灰霉病菌△BcAtg9突变株的致病力显著下降；上述结果与T-DNA插入灰霉病菌Atg9基因启动子导致突变体致病性下降的结果是一致的。上述研究结果表明：灰霉病菌Atg9基因不影响其菌丝体的正常生长和分子孢子的形态形成，但对产孢量和致病性有显著的影响，表明Atg9基因参与灰霉病菌的致病过程。
[Abstract]:Botrytis cinerea is a fungal disease caused by Botrytis cinerea. It is widely distributed in the world and causes huge economic losses every year. Under natural conditions, conidia are used as the primary and re-infective source of the host. Gray mold is the pathogen of airborne plants. Autophagosome 9 is a ubiquitin like protein necessary for autophagosome membrane formation and consists of 117 amino acid residues. In this study, the role of Atg9 gene in the growth, development and pathogenicity of Botrytis cinerea was studied. 1. A transformation population containing more than 3000 T-DNA insertion mutations was constructed by using the strong pathogenicity strain Bc3-4 in the field, and tomato was used. The isolated leaves of strawberry were screened for pathogenicity at least 3 times, and 148 T-DNA insertion mutants were obtained. 2. The T-DNA insertion genetic loci of the partially acquired mutants with obviously weakened infection phenotype were analyzed by PCR(Thermal Asymmetric Interlaced PCR (TAIL-PCR) technique, which were inserted into the genome of Botrytis cinerea. Southern blot analysis of the promoter region of the Atg9 gene of one of the mutants showed that the mutation was caused by a single copy of T-DNA insertion. There is little evidence that autophagy genes regulate the growth of cinerea cinerea. A study of development and pathogenicity, Therefore, the Atg9 gene of Botrytis cinerea was studied in this study. 3. Agrobacterium tumefaciens mediated transformation (ATMTT) method was used to successfully knockout the Atg9 gene of Botrytis cinerea. The Atg9 gene knockout mutant BcAtg9 was obtained by PCR and other methods. 4. By comparing the growth and development of wild type Bc05.10 and Atg9 knockout mutant BcAtg9, it was found that the growth rate of BcAtg9 and wild-type strain on PDA medium was not significantly accelerated or slowed down. The results showed that Atg9 gene had no effect on the growth of mycelia, the sporulation of wild type was about 3.1 卤0.4) 脳 107 spore / plateau, while that of BcAtg9 mutant was about 1.5 卤0.3) 脳 107 spore / plateau, and the yield of wild type was about double. The sporulation of conidia was significantly lower than that of wild-type strains, and the pathogenicity test showed that, after 3 days of inoculation, The spot area caused by wild-type strain infection was 1.24 卤0.14 cm ~ 2, while that of mutant strain was 0.34 卤0.06 cm ~ 2, which was significantly smaller than that caused by wild type strain infection, which indicated that the pathogenicity of BcAtg9 mutant was significantly decreased. The above results were consistent with the results of T-DNA insertion of the Atg9 gene promoter of gray mold bacteria resulting in the decrease of pathogenicity of the mutant. The results showed that the Atg9 gene did not affect the normal growth of mycelium and the formation of molecular spores, but had a significant effect on sporulation and pathogenicity, indicating that the Atg9 gene was involved in the pathogenicity of cinerella cinerea.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

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