本文選題：灰葡萄孢　切入點：細(xì)胞自噬　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：灰葡萄孢(Botrytis cinerea)可危害一些重要經(jīng)濟(jì)作物,造成巨大的經(jīng)濟(jì)損失。目前細(xì)胞自噬在灰葡萄孢中的研究較少,在真核生物中,細(xì)胞自噬過程可維持細(xì)胞內(nèi)的生理平衡,有助于細(xì)胞度過逆境,并影響一些絲狀真菌細(xì)胞的分化及致病力。本研究在灰葡萄孢基因組數(shù)據(jù)庫中下載3個自噬相關(guān)基因的序列,即BC1G_12424、BC1G_11270和BC1G_01868,對其進(jìn)行了基因結(jié)構(gòu)和同源性分析,采用Split-marker技術(shù)在野生型菌株B05.10中對其分別進(jìn)行了敲除,最后對敲除突變體的菌絲生長、菌核產(chǎn)生、營養(yǎng)需求和致病力等方面進(jìn)行了測定。取得的研究結(jié)果如下:(1)通過在NCBI上BLAST比對以及構(gòu)建進(jìn)化樹,證實了BC1G_12424、BC1G_11270和BC1G_01868分別與多物種的自噬基因ATG26、ATG17和ATG14有同源性,因此命名為BcATG26、BcATG17和BcATG14。(2)通過Split-marker技術(shù)分別敲除基因BcATG26、BcATG17和BcATG14,經(jīng)過PCR和Soutnern blot驗證,獲得了基因缺失突變體△BcATG26、△BcATG17和△BcATG14。(3)明確了自噬基因BcATG26、BcATG17和BcATG14在自噬過程中的重要作用。用MDC染色,B05.10的菌絲在激光共聚焦顯微鏡下出現(xiàn)大量的熒光顆粒,而△BcATG26-13、△BcATG17-6和△BcATG14-107的菌絲細(xì)胞中未見或者有極少數(shù)的熒光顆粒,自噬基因的敲除阻斷了自噬過程,導(dǎo)致不能形成自噬體或者自噬小體。(4)細(xì)胞自噬影響灰葡萄孢菌絲生長和菌核發(fā)育�；蛉笔蛔凅w在菌絲生長和菌核形成方面與野生型菌株B05.10有差異。在PDA培養(yǎng)基上,△BcATG26-13相對于野生型菌株B05.10,菌核黑化延遲,15d仍為黃色;而△BcATG14-107只能形成很小的菌核。在1/8 PDA、DM、DM-C和DM-N培養(yǎng)基上,與野生型菌株B05.10相比,△BcATG26-13、△BcATG17-6和△BcATG14-107菌絲生長速度減慢,菌落發(fā)育畸形。(5)細(xì)胞自噬影響灰葡萄孢致病力。用菌絲塊離體接種煙草葉片、活體接種煙草葉片和離體接種草莓葉片測定致病力,結(jié)果表明,△BcATG26-13、△BcATG17-6和△BcATG14-107的致病力與野生型菌株B05.10相比有極顯著差異(P0.01)。(6)細(xì)胞自噬可能影響灰葡萄孢耐受過氧化氫(H_2O_2)和植保素(camalexin)的脅迫。在添加有H_2O_2或者植保素camalexin的PDA上,與野生型菌株B05.10相比,△BcATG26-13、△BcATG17-6和△BcATG14-107菌絲生長速度減慢,菌落發(fā)育畸形,即對H_2O_2和camalexin更為敏感。以上結(jié)果表明,細(xì)胞自噬在灰葡萄孢菌絲生長、菌核的形成和發(fā)育、對寄主植物的致病力以及病原菌耐受活性氧(ROS)和植保素camalexin的脅迫等方面都起著重要作用。解析灰葡萄孢細(xì)胞自噬相關(guān)基因的功能可為更好的防治灰霉病提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S432.44

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