蛹虫草有性阶段DNA甲基化及绿僵菌DNA甲基转移酶功能研究

发布时间：2018-05-05 09:10

本文选题：蛹虫草 + 罗伯茨绿僵菌　；参考：《安徽农业大学》2016年博士论文

【摘要】：DNA甲基化作为真核生物中的一种重要表观遗传机制,在不同物种之间的分布模式和作用差异显著。在真菌中,DNA甲基化在调控多种生物过程中起着重要作用,但是截止到目前真菌有性生长发育与DNA甲基化的关系尚未有研究。蛹虫草以其易于获得有性阶段的特性,为我们研究真菌有性生长发育阶段的表观遗传现象提供了宝贵资源。罗伯茨绿僵菌是研究真菌侵染昆虫机制的一种模式系统,同时也是化学农药杀虫剂的替代品,但是目前对其DNA甲基化分子机制研究甚少。本论文首先通过克隆测序、酶活测定等方法在蛹虫草中鉴定出两个DNA甲基转移酶(CmDMTA和CmDIM-2)。然后利用亚硫酸氢盐高通量测序技术研究蛹虫草有性阶段和无性阶段的DNA甲基化模式,并结合数字表达谱技术研究DNA甲基化在有性生长发育中的作用。最后,对罗伯茨绿僵菌中的两个DNA甲基转移酶(MrRID和MrDIM-2)进行单敲除和双敲除,以此来研究绿僵菌中DNA甲基转移酶的功能。具体研究结果如下:(1)蛹虫草DNA甲基转移酶的研究通过同源检索,发现在蛹虫草中存在两个发生DNA甲基化所必需的DNA甲基转移酶(CmDMTA和CmDIM-2)。通过RACE及测序方法得到了CmDMTA和CmDIM-2基因的全长序列,并且进行了系统发育关系分析。随后,分别构建原核表达载体并在大肠杆菌BL21中诱导表达。SDS-PAGE结果显示,两个重组蛋白都被成功的诱导表达,而且Western blot结果显示重组蛋白都能够被抗HIS标签抗体特异性识别。然后采用镍柱纯化重组蛋白,获得高纯度重组蛋白。最后通过DNA甲基转移酶活性检测发现CmDMTA和CmDIM-2都具有DNA甲基转移酶活性。(2)DNA甲基化在蛹虫草有性生长发育中的作用(1)DNA甲基转移酶在蛹虫草不同阶段的表达:Real-time PCR结果显示CmDMTA和CmDIM-2在蛹虫草不同生长阶段的表达变化趋势相似。随着分生孢子的不断产生,CmDMTA和CmDIM-2基因的表达量越来越高,Cm DMTA和CmDIM-2基因的表达量在有性阶段的初期达到最高。(2)蛹虫草不同阶段的总蛋白酶活性及总DNA甲基化水平我们选择DNA甲基转移酶表达差异最为明显的两个时期(3M和NF)作为进一步研究对象。酶活测定结果显示,3M的总蛋白DNA甲基转移酶活性要低于有性阶段NF总蛋白的酶活性。HPLC结果显示蛹虫草中确实存在DNA甲基化,且NF(0.42%)的DNA甲基化水平要高于3M(0.38%)时期。(3)蛹虫草DNA甲基化特征BS-Seq结果表明,mC在蛹虫草基因组中分布较均匀,NF基因组DNA甲基化水平为0.41%(mCG:0.39%、mCHG:0.38%、mCHH:0.43%);3M基因组DNA甲基化水平为0.39%(mCG:0.37%、mCHG:0.39%、mCHH:0.4%)。进一步分析发现,虽然NF和3M整体甲基化水平相近,但是mC分布差异明显,说明DNA甲基化在蛹虫草生长发育过程中是一个动态变化过程。我们共鉴定出225个不同甲基化区域(DMRs),其中有141个分布在非基因区,84个位于基因区域。同时鉴定出136个DMR相关基因,GO和KEGG分析发现其中包括许多能量代谢、信号传导等与生长发育相关的基因。(4)蛹虫草DNA甲基化与有性生长发育的关系:将DMR相关基因与RNA-Seq结果结合起来分析发现,在NF中,48个高甲基化基因中有5个表达量是下调的;在61个低甲基化基因中有7个表达量是上调的。因此,符合高甲基化低表达这一规律的基因占总DMR相关基因的12/136(10%),这说明在蛹虫草中基因的甲基化和表达之间并没有高度的一致性。进一步分析这12个基因,并没有发现与蛹虫草生长发育相关的基因,因此我们没有找到DNA甲基化和有性生长发育直接相关的证据。但是将蛹虫草基因组信息与BS-Seq测序结果结合分析后,我们认为蛹虫草中确实存在RIP过程,且与DNA甲基化有关。(3)罗伯茨绿僵菌DNA甲基转移酶功能研究(1)生物信息学分析罗伯茨绿僵菌DNA甲基转移酶:通过同源检索,在罗伯茨绿僵菌中发现两个DNA甲基转移酶(MrRID和MrDIM-2),生物信息学分析发现MrRID和MrDIM-2分别与蛹虫草的CmDMTA和Cm DIM-2及脉孢菌中的RID和DIM-2同源关系较近。(2)MrRID和MrDIM-2在罗伯茨绿僵菌DNA甲基化中的功能分析:检测不同DNA甲基转移酶突变株(ΔRID、ΔDIM-2和ΔRID/ΔDIM-2)的基因组DNA甲基化情况,ΔRID、ΔDIM-2和ΔRID/ΔDIM-2菌株中mC位点数量分别占野生型菌株的~71%,~10%和~8%。深入分析发现,MrRID在DNA甲基化过程中起着识别DNA甲基化位点的功能,而且一些DNA位点的甲基化需要MrRID和MrDIM-2共同参与。(3)DNA甲基转移酶突变株的生物性状:ΔMr DIM-2和ΔRID/ΔDIM-2的生长速度和产孢能力明显弱于野生型和ΔMrRID,热激或紫外照射后突变株(尤其ΔMrDIM-2和ΔRID/ΔDIM-2)的孢子萌发率相对于野生型明显下降。但是所有突变株对化学物质耐受力与野生型菌株相比无明显差异。毒力测定实验显示,相对于ΔMrRID(LT50=4.6±0.8)和野生型(LT50=4.4±0.5)菌株,ΔMrDIM-2(LT50=6.5±0.9)和ΔRID/ΔDIM-2(LT50=7.3±1.2)的毒力明显下降,而且虫子尸体表面产孢能力也明显减弱。
[Abstract]:DNA methylation, as an important epigenetic mechanism in eukaryotes, has significant differences in distribution patterns and roles among different species. In fungi, DNA methylation plays an important role in regulating a variety of biological processes, but the relationship between sexual growth and DNA methylation of fungi has not yet been studied. Cordyceps militaris It is easy to obtain the characteristics of the sexual phase, which provides valuable resources for the study of epigenetic phenomena in the stage of sexual growth and development of fungi. Roberts Bacillus anisopliae is a model system for the study of the mechanism of fungal infection of insects, and is also a substitute for chemical pesticides. However, the molecular mechanism of DNA methylation is rarely studied. Two DNA methyltransferases (CmDMTA and CmDIM-2) were identified in Cordyceps militaris by cloning sequencing, enzyme activity assay and other methods. Then, the DNA methylation pattern of Cordyceps militaris at sexual and vegetative stages was studied by high flux sequencing technology of hydrogen sulphite, and DNA methylation in sexual growth was studied with the combination of digital expression spectroscopy. In the end, two DNA methyltransferases (MrRID and MrDIM-2) in Bacillus anisopliae (MrRID and MrDIM-2) were knocked out by single knockout and double knockout to study the function of DNA methyltransferase in Bacillus anisopliae. The results were as follows: (1) the study of DNA methyltransferase of Cordyceps militaris was found by homologous retrieval, and two of the existence of DNA in Cordyceps militaris was found. DNA methyltransferase (CmDMTA and CmDIM-2) necessary for methylation. A full-length sequence of CmDMTA and CmDIM-2 genes was obtained by RACE and sequencing, and the phylogenetic relationship was analyzed. Subsequently, the prokaryotic expression vector was constructed and the expression of.SDS-PAGE in Escherichia coli BL21 was induced to show that the two recombinant proteins were successful. Induced expression, and Western blot results showed that the recombinant protein could be identified by anti HIS labeling antibody. Then the recombinant protein was purified with nickel column to obtain high purity recombinant protein. Finally, the activity of CmDMTA and CmDIM-2 was found to have DNA methyl transferase activity by DNA methyltransferase activity. (2) DNA methylation was in the presence of Cordyceps militaris. The role of the growth and development (1) the expression of DNA methyltransferase at different stages of Cordyceps militaris: Real-time PCR results showed that the expression of CmDMTA and CmDIM-2 in different stages of the growth of Cordyceps Cordyceps was similar. With the continuous generation of conidium, the expression of CmDMTA and CmDIM-2 genes increased and the expression of Cm DMTA and CmDIM-2 genes was in the same period. The initial stage of the sexual phase reached the highest. (2) the total protease activity and total DNA methylation level in different stages of Cordyceps militaris we chose the two period (3M and NF) as the further study. The results of enzyme activity assay showed that the activity of total egg white DNA methyltransferase in 3M was lower than that of the sexual phase NF total. The results of enzyme activity.HPLC showed that DNA methylation did exist in Cordyceps militaris, and the DNA methylation level of NF (0.42%) was higher than that of 3M (0.38%). (3) the DNA methylation characteristic of Cordyceps militaris BS-Seq results showed that mC was distributed more evenly in the genome of Cordyceps militaris, and the NF genome DNA methylation level was 0.41% (mCG:0.39%, mCHG:0.38%, etc.); The DNA methylation level of the group was 0.39% (mCG:0.37%, mCHG:0.39%, mCHH:0.4%). Further analysis showed that although the overall methylation level of NF and 3M was similar, the distribution of mC was distinct, indicating that DNA methylation was a dynamic process during the growth and development of Cordyceps militaris. We identified 225 different methylation regions (DMRs), of which 141 136 DMR related genes were identified in the non gene region and 84 in the gene region. GO and KEGG analysis found that many energy metabolism, signal transduction and other genes related to growth and development. (4) the relationship between DNA methylation and sexual growth of Cordyceps militaris: the analysis of DMR related genes and RNA-Seq results was found. In NF, 5 of the 48 hypermethylation genes were down regulated, and 7 of the 61 low methylation genes were up-regulated. Therefore, the gene that conforms to the low expression of hypermethylation accounts for the 12/136 (10%) of the total DMR related genes, which indicates that there is no high consistency between the methylation and expression of the gene in the Cordyceps militaris. Further analysis of these 12 genes did not find genes related to the growth and development of Cordyceps militaris, so we did not find evidence of direct correlation between DNA methylation and sexual growth. But after the analysis of the genomic information of Cordyceps militaris and BS-Seq sequencing, we believe that the RIP process does exist in the Cordyceps militaris and DNA methylation. (3) study on the function of Roberts DNA methytransferase (1) bioinformatics analysis of DNA methytransferase of Bacillus anisopliae: through homologous retrieval, two DNA methyltransferases (MrRID and MrDIM-2) were found in Roberts Bacillus anisopliae. Bioinformatics analysis found that MrRID and MrDIM-2 were respectively with CmDMTA and Cm DIM-2 and spore of Cordyceps militaris, respectively. The homology of RID and DIM-2 in the bacteria was close. (2) functional analysis of MrRID and MrDIM-2 in DNA methylation of Bacillus anisopliae: detection of genomic DNA methylation of different DNA methyltransferase mutant strains (delta RID, Delta DIM-2 and delta RID/ Delta DIM-2). And ~8%. in-depth analysis found that MrRID plays a role in identifying DNA methylation sites in the process of DNA methylation, and the methylation of some DNA sites requires the joint participation of MrRID and MrDIM-2. (3) the biological traits of the DNA methyltransferase mutant strain: the growth rate and sporulation capacity of delta Mr DIM-2 and delta RID/ DIM-2 are significantly weaker than those of the wild type and the delta. The spore germination rate of the mutant (especially Delta MrDIM-2 and delta RID/ Delta DIM-2) was significantly lower than that of the wild type, but the tolerance of all the mutants to the wild type was not significantly different. The test of virulence test showed that, compared to the delta MrRID (LT50=4.6 + 0.8) and the wild type (LT50=4.4 + 0.5), Delta MrDIM-2 (LT) (LT) The toxicity of 50=6.5 + 0.9 and delta RID/ DIM-2 (LT50=7.3 + 1.2) decreased significantly, and the ability of sporulation on the surface of the dead body was also significantly reduced.

【学位授予单位】：安徽农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.35;S476.12

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