本文關(guān)鍵詞： 灰葡萄孢 甲基化 DNA甲基轉(zhuǎn)移酶 異染色質(zhì)蛋白1 H3K9甲基轉(zhuǎn)移酶　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：灰霉病是一種由葡萄孢屬真菌引起的重要病害,其中灰葡萄孢(Botrytis cinerea)的寄主范圍最為廣泛,可侵染200多種植物,每年造成嚴重的經(jīng)濟損失。表觀遺傳在動植物的生長發(fā)育過程中發(fā)揮重要的調(diào)控作用,我們通過氨基酸序列比對發(fā)現(xiàn)灰葡萄孢中存在三個分別編碼DNA甲基轉(zhuǎn)移酶、HP1(異染色蛋白1)和H3K9甲基轉(zhuǎn)移酶的基因BC1G_12419、BC1G_06432和BC1G_11188,分別命名為Bcdim-2,Bchp1和Bcdim-5,對三個基因的功能進行了初步研究,取得如下結(jié)果:1.Bcdim-2基因開放閱讀框4125bp,含有4個外顯子,3個內(nèi)含子,編碼1025個氨基酸,含有DNA甲基轉(zhuǎn)移酶保守結(jié)構(gòu)域;Bchp1基因開放閱讀框1118bp,含有6個外顯子,6個內(nèi)含子,編碼244個氨基酸,含有HP1典型的兩個保守結(jié)構(gòu)域:CD(Chromo Domain)和CSD(Chrome Shadow Domain);Bcdim-5基因開放閱讀框1752bp,含有4個外顯子,3個內(nèi)含子,編碼357個氨基酸,含有Pre-SET、SET和Post-SET三個保守結(jié)構(gòu)域。2.Real-time PCR結(jié)果表明這三個基因在灰葡萄孢的營養(yǎng)生長、菌核形成和致病階段均有表達,并且Bcdim-2基因在致病階段表達下調(diào),Bchp1基因在菌核形成和致病階段表達下調(diào),Bcdim-5基因在三個階段的表達水平并無顯著差異。3.根據(jù)同源重組的原理,分別對這三個基因進行敲除,獲得了基因的敲除轉(zhuǎn)化子。深入研究發(fā)現(xiàn),敲除Bcdim-2和Bchp1基因不會影響灰葡萄孢的菌絲生長速度、尖端形態(tài)、菌核產(chǎn)生、分生孢子形成及其致病力,但Bcdim-5基因敲除轉(zhuǎn)化子的生長速率減緩25%左右,菌絲尖端形態(tài)不變;菌落表面出現(xiàn)白色菌絲糾集,菌核變小變圓,每皿菌核干重下降;分生孢子產(chǎn)生時間推遲,分生孢子量變少;在番茄、擬南芥、大豆上的致病力均有所降低。對Bcdim-5基因進行互補可恢復(fù)敲除轉(zhuǎn)化子的菌絲生長速度、分生孢子和菌核形成能力,但其致病力不能完全恢復(fù)。4.選取灰葡萄孢中的致病相關(guān)基因,檢測其在Bcdim-5基因缺失突變體和B05.10菌株中的表達量變化,結(jié)果發(fā)現(xiàn)在突變體中大部分致病相關(guān)基因表達下調(diào),推測Bcdim-5基因敲除菌株致病力的下降與致病相關(guān)基因的下調(diào)表達有關(guān)。本研究初步證實Bcdim-5基因與灰葡萄孢的生長、產(chǎn)孢、菌核形成和致病過程相關(guān),Bcdim-5基因敲除后影響致病相關(guān)基因的表達,而Bcdim-2和Bchp1基因缺失后不會影響菌株的正常生長發(fā)育,進而推測H3K9的甲基化對灰葡萄孢的生長、產(chǎn)孢、菌核形成和致病均有重要作用,而DNA的甲基化對這些生物學(xué)過程調(diào)控作用不明顯。
[Abstract]:Botrytis cinerea is an important disease caused by Grapevine Fungi. Botrytis cinerea has the most extensive host range and can infect more than 200 species of plants. Causing serious economic losses every year. Epigenetics plays an important regulatory role in the growth and development of plants and animals, Three genes encoding DNA methyltransferase HP1 and H3K9 methyltransferase BC1G12419BC1G06432 and BC1G11188 named Bcdim-2Bchp1 and Bcdim-5 were found in grapevine by amino acid sequence alignment. The results are as follows: 1. Bcdim-2 open reading frame 4125bpcontaining 4 exons, 3 introns, encoding 1025 amino acids, containing DNA methyltransferase conserved domain Bchp1 open reading frame 1118bp, containing 6 exons and 6 introns, It encodes 244 amino acids and contains two typical conserved domains of HP1: CD1: Chromo domain) and CSD(Chrome Shadow domain Bcdim-5 gene. The open reading frame 1752 BP contains 4 exons and 3 introns, encoding 357 amino acids. The results of Real-time PCR with three conserved domains of Pre-set set and Post-SET showed that the three genes were expressed in the vegetative growth, sclerotia formation and pathogenicity of grapevine. Moreover, the down-regulation of Bcdim-2 gene expression in the sclerotia formation stage and the down-regulation of Bcdim-5 gene expression in the sclerotia stage showed no significant difference. 3. According to the principle of homologous recombination, the three genes were knockout respectively. Knockout transformants were obtained. It was found that knockout of Bcdim-2 and Bchp1 genes had no effect on hyphal growth rate, tip morphology, sclerotia production, conidial formation and pathogenicity of grapevine. However, the growth rate of Bcdim-5 knockout transformants slowed down about 25%, the morphology of hyphae tip remained unchanged, the white hyphae gathered on the surface of colony, the sclerotia became smaller and rounded, the dry weight of each dish sclerotia decreased, the time of conidial production was delayed and the amount of conidial spores decreased. The pathogenicity of tomato, Arabidopsis thaliana and soybean were all decreased. The ability of mycelium growth, conidium and sclerotia formation of knockout transformants could be recovered by complementing Bcdim-5 gene. However, its pathogenicity could not be recovered completely. 4. The pathogenicity related genes were selected from grapevine spores, and the expression levels of these genes were detected in Bcdim-5 mutant and B05.10 strain. The results showed that the expression of most pathogenicity related genes was down-regulated in the mutants. It is inferred that the decrease of pathogenicity of Bcdim-5 knockout strain is related to the down-regulation of pathogenicity related genes. The gene knockout of Bcdim-5 associated with sclerotia formation and pathogenicity affected the expression of pathogenicity related genes, but the deletion of Bcdim-2 and Bchp1 genes did not affect the normal growth and development of the strain, and the methylation of H3K9 could induce the growth and sporulation of grapevine. Sclerotia formation and pathogenicity are important, but methylation of DNA has little effect on these biological processes.
【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】：2015
【分類號】：S432.44

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本文編號：1537147