灰葡萄孢细胞自噬相关基因BcATG26、BcATG17和BcATG14的功能研究

发布时间：2018-03-16 07:11

本文选题：灰葡萄孢　切入点：细胞自噬　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：灰葡萄孢(Botrytis cinerea)可危害一些重要经济作物,造成巨大的经济损失。目前细胞自噬在灰葡萄孢中的研究较少,在真核生物中,细胞自噬过程可维持细胞内的生理平衡,有助于细胞度过逆境,并影响一些丝状真菌细胞的分化及致病力。本研究在灰葡萄孢基因组数据库中下载3个自噬相关基因的序列,即BC1G_12424、BC1G_11270和BC1G_01868,对其进行了基因结构和同源性分析,采用Split-marker技术在野生型菌株B05.10中对其分别进行了敲除,最后对敲除突变体的菌丝生长、菌核产生、营养需求和致病力等方面进行了测定。取得的研究结果如下:(1)通过在NCBI上BLAST比对以及构建进化树,证实了BC1G_12424、BC1G_11270和BC1G_01868分别与多物种的自噬基因ATG26、ATG17和ATG14有同源性,因此命名为BcATG26、BcATG17和BcATG14。(2)通过Split-marker技术分别敲除基因BcATG26、BcATG17和BcATG14,经过PCR和Soutnern blot验证,获得了基因缺失突变体△BcATG26、△BcATG17和△BcATG14。(3)明确了自噬基因BcATG26、BcATG17和BcATG14在自噬过程中的重要作用。用MDC染色,B05.10的菌丝在激光共聚焦显微镜下出现大量的荧光颗粒,而△BcATG26-13、△BcATG17-6和△BcATG14-107的菌丝细胞中未见或者有极少数的荧光颗粒,自噬基因的敲除阻断了自噬过程,导致不能形成自噬体或者自噬小体。(4)细胞自噬影响灰葡萄孢菌丝生长和菌核发育。基因缺失突变体在菌丝生长和菌核形成方面与野生型菌株B05.10有差异。在PDA培养基上,△BcATG26-13相对于野生型菌株B05.10,菌核黑化延迟,15d仍为黄色;而△BcATG14-107只能形成很小的菌核。在1/8 PDA、DM、DM-C和DM-N培养基上,与野生型菌株B05.10相比,△BcATG26-13、△BcATG17-6和△BcATG14-107菌丝生长速度减慢,菌落发育畸形。(5)细胞自噬影响灰葡萄孢致病力。用菌丝块离体接种烟草叶片、活体接种烟草叶片和离体接种草莓叶片测定致病力,结果表明,△BcATG26-13、△BcATG17-6和△BcATG14-107的致病力与野生型菌株B05.10相比有极显著差异(P0.01)。(6)细胞自噬可能影响灰葡萄孢耐受过氧化氢(H_2O_2)和植保素(camalexin)的胁迫。在添加有H_2O_2或者植保素camalexin的PDA上,与野生型菌株B05.10相比,△BcATG26-13、△BcATG17-6和△BcATG14-107菌丝生长速度减慢,菌落发育畸形,即对H_2O_2和camalexin更为敏感。以上结果表明,细胞自噬在灰葡萄孢菌丝生长、菌核的形成和发育、对寄主植物的致病力以及病原菌耐受活性氧(ROS)和植保素camalexin的胁迫等方面都起着重要作用。解析灰葡萄孢细胞自噬相关基因的功能可为更好的防治灰霉病提供理论基础。
[Abstract]:Botrytis cinerea) can harm some important economic crops and cause huge economic losses. At present, there are few studies on autophagy in grapevine. In eukaryotes, the process of autophagy can maintain the physiological balance of cells. In this study, the sequence of three autophagy related genes were downloaded from the genome database of grapevine spore, and the differentiation and pathogenicity of some filamentous fungi cells were affected. That is, BC1G12424BC1GC11270 and BC1G1GS01868, the gene structure and homology of them were analyzed, and the wild strain B05.10 was knocked out by Split-marker technique. Finally, the mycelium growth and sclerotia production of the knockout mutants were studied. The nutritional requirements and pathogenicity were determined. The results are as follows: 1) by comparing BLAST on NCBI and constructing evolutionary tree, it is proved that BC1G12424BC1G11270 and BC1G_01868 have homology with ATG26, ATG17 and ATG14, respectively. So named BcATG26BcATG17 and BcATG14.2) the genes BcATG26, BcATG17 and BcATG14 were knockout by Split-marker technique, respectively, and verified by PCR and Soutnern blot. The gene deletion mutants BcATG26, BcATG17 and BcATG14.3) were obtained. The important roles of the autophagy genes BcATG26, BcATG17 and BcATG14 in autophagy were identified. The mycelium of B05.10 was stained with MDC, and a large number of fluorescent particles appeared under laser confocal microscope. However, in the mycelia of BcATG26-13, BcATG17-6 and BcATG14-107, no or very few fluorescent particles were found, and the knockout of autophagy gene blocked the autophagy process. The effect of autophagy on hypha growth and sclerotia development of grapevine was influenced by autophagy or autophagy. The gene deletion mutants were different from wild-type strain B05.10 in hyphal growth and sclerotia formation. Compared with wild-type strain B05.10, BcATG26-13 was still yellow for 15 days, but BcATG14-107 could only form very small sclerotia. Compared with wild strain B05.10, the growth rate of BcATG26-13, BcATG17-6 and BcATG14-107 mycelium was slower than that of wild strain B05.10 on 1/8 PDA-DMM-C and DM-N medium. The pathogenicity of grapevine was affected by autophagy. Tobacco leaves were inoculated with mycelium in vitro, tobacco leaves were inoculated in vivo and strawberry leaves were inoculated in vitro. The pathogenicity of BcATG26-13, BcATG17-6 and BcATG14-107 was significantly different from that of wild-type strain B05.10. The autophagy might affect the tolerance of Ash grape to the stress of H _ 2O _ 2 (H _ 2O _ 2) and C _ 2O _ 2 (P _ 2O _ 2 camalexin). Compared with wild-type strain B05.10, BcATG26-13, BcATG17-6 and BcATG14-107 hyphal growth rate was slower, and colony development malformation was more sensitive to H _ 2O _ 2 and camalexin. The results indicated that the cell autophagy was formed and developed in the sclerotia of grapevine. It plays an important role in the pathogenicity of host plants, the tolerance of pathogenic bacteria to reactive oxygen species (Ros) and the stress of phytatin camalexin, and the analysis of the function of autophagy related genes in the spores of grapevine can provide a theoretical basis for better prevention and control of grey mold.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44

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