灰葡萄孢中甲基化相关基因的功能研究

发布时间：2018-02-26 07:42

本文关键词： 灰葡萄孢甲基化 DNA甲基转移酶异染色质蛋白1 H3K9甲基转移酶　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：灰霉病是一种由葡萄孢属真菌引起的重要病害,其中灰葡萄孢(Botrytis cinerea)的寄主范围最为广泛,可侵染200多种植物,每年造成严重的经济损失。表观遗传在动植物的生长发育过程中发挥重要的调控作用,我们通过氨基酸序列比对发现灰葡萄孢中存在三个分别编码DNA甲基转移酶、HP1(异染色蛋白1)和H3K9甲基转移酶的基因BC1G_12419、BC1G_06432和BC1G_11188,分别命名为Bcdim-2,Bchp1和Bcdim-5,对三个基因的功能进行了初步研究,取得如下结果:1.Bcdim-2基因开放阅读框4125bp,含有4个外显子,3个内含子,编码1025个氨基酸,含有DNA甲基转移酶保守结构域;Bchp1基因开放阅读框1118bp,含有6个外显子,6个内含子,编码244个氨基酸,含有HP1典型的两个保守结构域:CD(Chromo Domain)和CSD(Chrome Shadow Domain);Bcdim-5基因开放阅读框1752bp,含有4个外显子,3个内含子,编码357个氨基酸,含有Pre-SET、SET和Post-SET三个保守结构域。2.Real-time PCR结果表明这三个基因在灰葡萄孢的营养生长、菌核形成和致病阶段均有表达,并且Bcdim-2基因在致病阶段表达下调,Bchp1基因在菌核形成和致病阶段表达下调,Bcdim-5基因在三个阶段的表达水平并无显著差异。3.根据同源重组的原理,分别对这三个基因进行敲除,获得了基因的敲除转化子。深入研究发现,敲除Bcdim-2和Bchp1基因不会影响灰葡萄孢的菌丝生长速度、尖端形态、菌核产生、分生孢子形成及其致病力,但Bcdim-5基因敲除转化子的生长速率减缓25%左右,菌丝尖端形态不变;菌落表面出现白色菌丝纠集,菌核变小变圆,每皿菌核干重下降;分生孢子产生时间推迟,分生孢子量变少;在番茄、拟南芥、大豆上的致病力均有所降低。对Bcdim-5基因进行互补可恢复敲除转化子的菌丝生长速度、分生孢子和菌核形成能力,但其致病力不能完全恢复。4.选取灰葡萄孢中的致病相关基因,检测其在Bcdim-5基因缺失突变体和B05.10菌株中的表达量变化,结果发现在突变体中大部分致病相关基因表达下调,推测Bcdim-5基因敲除菌株致病力的下降与致病相关基因的下调表达有关。本研究初步证实Bcdim-5基因与灰葡萄孢的生长、产孢、菌核形成和致病过程相关,Bcdim-5基因敲除后影响致病相关基因的表达,而Bcdim-2和Bchp1基因缺失后不会影响菌株的正常生长发育,进而推测H3K9的甲基化对灰葡萄孢的生长、产孢、菌核形成和致病均有重要作用,而DNA的甲基化对这些生物学过程调控作用不明显。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44

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