發(fā)布時間：2018-05-05 09:10

  本文選題：蛹蟲草 + 羅伯茨綠僵菌�。� 參考：《安徽農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：DNA甲基化作為真核生物中的一種重要表觀遺傳機(jī)制,在不同物種之間的分布模式和作用差異顯著。在真菌中,DNA甲基化在調(diào)控多種生物過程中起著重要作用,但是截止到目前真菌有性生長發(fā)育與DNA甲基化的關(guān)系尚未有研究。蛹蟲草以其易于獲得有性階段的特性,為我們研究真菌有性生長發(fā)育階段的表觀遺傳現(xiàn)象提供了寶貴資源。羅伯茨綠僵菌是研究真菌侵染昆蟲機(jī)制的一種模式系統(tǒng),同時也是化學(xué)農(nóng)藥殺蟲劑的替代品,但是目前對其DNA甲基化分子機(jī)制研究甚少。本論文首先通過克隆測序、酶活測定等方法在蛹蟲草中鑒定出兩個DNA甲基轉(zhuǎn)移酶(CmDMTA和CmDIM-2)。然后利用亞硫酸氫鹽高通量測序技術(shù)研究蛹蟲草有性階段和無性階段的DNA甲基化模式,并結(jié)合數(shù)字表達(dá)譜技術(shù)研究DNA甲基化在有性生長發(fā)育中的作用。最后,對羅伯茨綠僵菌中的兩個DNA甲基轉(zhuǎn)移酶(MrRID和MrDIM-2)進(jìn)行單敲除和雙敲除,以此來研究綠僵菌中DNA甲基轉(zhuǎn)移酶的功能。具體研究結(jié)果如下:(1)蛹蟲草DNA甲基轉(zhuǎn)移酶的研究通過同源檢索,發(fā)現(xiàn)在蛹蟲草中存在兩個發(fā)生DNA甲基化所必需的DNA甲基轉(zhuǎn)移酶(CmDMTA和CmDIM-2)。通過RACE及測序方法得到了CmDMTA和CmDIM-2基因的全長序列,并且進(jìn)行了系統(tǒng)發(fā)育關(guān)系分析。隨后,分別構(gòu)建原核表達(dá)載體并在大腸桿菌BL21中誘導(dǎo)表達(dá)。SDS-PAGE結(jié)果顯示,兩個重組蛋白都被成功的誘導(dǎo)表達(dá),而且Western blot結(jié)果顯示重組蛋白都能夠被抗HIS標(biāo)簽抗體特異性識別。然后采用鎳柱純化重組蛋白,獲得高純度重組蛋白。最后通過DNA甲基轉(zhuǎn)移酶活性檢測發(fā)現(xiàn)CmDMTA和CmDIM-2都具有DNA甲基轉(zhuǎn)移酶活性。(2)DNA甲基化在蛹蟲草有性生長發(fā)育中的作用(1)DNA甲基轉(zhuǎn)移酶在蛹蟲草不同階段的表達(dá):Real-time PCR結(jié)果顯示CmDMTA和CmDIM-2在蛹蟲草不同生長階段的表達(dá)變化趨勢相似。隨著分生孢子的不斷產(chǎn)生,CmDMTA和CmDIM-2基因的表達(dá)量越來越高,Cm DMTA和CmDIM-2基因的表達(dá)量在有性階段的初期達(dá)到最高。(2)蛹蟲草不同階段的總蛋白酶活性及總DNA甲基化水平我們選擇DNA甲基轉(zhuǎn)移酶表達(dá)差異最為明顯的兩個時期(3M和NF)作為進(jìn)一步研究對象。酶活測定結(jié)果顯示,3M的總蛋白DNA甲基轉(zhuǎn)移酶活性要低于有性階段NF總蛋白的酶活性。HPLC結(jié)果顯示蛹蟲草中確實存在DNA甲基化,且NF(0.42%)的DNA甲基化水平要高于3M(0.38%)時期。(3)蛹蟲草DNA甲基化特征BS-Seq結(jié)果表明,mC在蛹蟲草基因組中分布較均勻,NF基因組DNA甲基化水平為0.41%(mCG:0.39%、mCHG:0.38%、mCHH:0.43%);3M基因組DNA甲基化水平為0.39%(mCG:0.37%、mCHG:0.39%、mCHH:0.4%)。進(jìn)一步分析發(fā)現(xiàn),雖然NF和3M整體甲基化水平相近,但是mC分布差異明顯,說明DNA甲基化在蛹蟲草生長發(fā)育過程中是一個動態(tài)變化過程。我們共鑒定出225個不同甲基化區(qū)域(DMRs),其中有141個分布在非基因區(qū),84個位于基因區(qū)域。同時鑒定出136個DMR相關(guān)基因,GO和KEGG分析發(fā)現(xiàn)其中包括許多能量代謝、信號傳導(dǎo)等與生長發(fā)育相關(guān)的基因。(4)蛹蟲草DNA甲基化與有性生長發(fā)育的關(guān)系:將DMR相關(guān)基因與RNA-Seq結(jié)果結(jié)合起來分析發(fā)現(xiàn),在NF中,48個高甲基化基因中有5個表達(dá)量是下調(diào)的;在61個低甲基化基因中有7個表達(dá)量是上調(diào)的。因此,符合高甲基化低表達(dá)這一規(guī)律的基因占總DMR相關(guān)基因的12/136(10%),這說明在蛹蟲草中基因的甲基化和表達(dá)之間并沒有高度的一致性。進(jìn)一步分析這12個基因,并沒有發(fā)現(xiàn)與蛹蟲草生長發(fā)育相關(guān)的基因,因此我們沒有找到DNA甲基化和有性生長發(fā)育直接相關(guān)的證據(jù)。但是將蛹蟲草基因組信息與BS-Seq測序結(jié)果結(jié)合分析后,我們認(rèn)為蛹蟲草中確實存在RIP過程,且與DNA甲基化有關(guān)。(3)羅伯茨綠僵菌DNA甲基轉(zhuǎn)移酶功能研究(1)生物信息學(xué)分析羅伯茨綠僵菌DNA甲基轉(zhuǎn)移酶:通過同源檢索,在羅伯茨綠僵菌中發(fā)現(xiàn)兩個DNA甲基轉(zhuǎn)移酶(MrRID和MrDIM-2),生物信息學(xué)分析發(fā)現(xiàn)MrRID和MrDIM-2分別與蛹蟲草的CmDMTA和Cm DIM-2及脈孢菌中的RID和DIM-2同源關(guān)系較近。(2)MrRID和MrDIM-2在羅伯茨綠僵菌DNA甲基化中的功能分析:檢測不同DNA甲基轉(zhuǎn)移酶突變株(ΔRID、ΔDIM-2和ΔRID/ΔDIM-2)的基因組DNA甲基化情況,ΔRID、ΔDIM-2和ΔRID/ΔDIM-2菌株中mC位點數(shù)量分別占野生型菌株的~71%,~10%和~8%。深入分析發(fā)現(xiàn),MrRID在DNA甲基化過程中起著識別DNA甲基化位點的功能,而且一些DNA位點的甲基化需要MrRID和MrDIM-2共同參與。(3)DNA甲基轉(zhuǎn)移酶突變株的生物性狀:ΔMr DIM-2和ΔRID/ΔDIM-2的生長速度和產(chǎn)孢能力明顯弱于野生型和ΔMrRID,熱激或紫外照射后突變株(尤其ΔMrDIM-2和ΔRID/ΔDIM-2)的孢子萌發(fā)率相對于野生型明顯下降。但是所有突變株對化學(xué)物質(zhì)耐受力與野生型菌株相比無明顯差異。毒力測定實驗顯示,相對于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)的毒力明顯下降,而且蟲子尸體表面產(chǎn)孢能力也明顯減弱。
[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.

【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S567.35;S476.12

【相似文獻(xiàn)】

相關(guān)會議論文 前6條

1 李文學(xué);陳雯;;DNA甲基轉(zhuǎn)移酶[A];第五屆廣東省環(huán)境誘變劑學(xué)會暨第三屆廣東省預(yù)防醫(yī)學(xué)會衛(wèi)生毒理專業(yè)委員會學(xué)術(shù)交流會論文集[C];2006年

2 韓麗;范高超;沈清明;姜立萍;朱俊杰;;基于能量轉(zhuǎn)移策略構(gòu)建的高靈敏DNA甲基轉(zhuǎn)移酶光電化學(xué)傳感[A];中國化學(xué)會第29屆學(xué)術(shù)年會摘要集——第04分會：納米生物傳感新方法[C];2014年

3 劉文斌;劉晉yN;敖琳;周紫垣;周燕虹;崔志鴻;曹佳;;大鼠肺鱗癌癌變過程中DNA甲基轉(zhuǎn)移酶表達(dá)水平的動態(tài)研究[A];中國環(huán)境誘變劑學(xué)會第14屆學(xué)術(shù)交流會議論文集[C];2009年

4 王上上;施偉民;;DNA甲基轉(zhuǎn)移酶對系統(tǒng)性紅斑狼瘡發(fā)病的影響[A];中華醫(yī)學(xué)會第14次全國皮膚性病學(xué)術(shù)年會論文匯編[C];2008年

5 李淵;武淑蘭;卜定方;朱燕;朱強(qiáng);曹香紅;;急性白血病/骨髓增生異常綜合征患者DNA甲基轉(zhuǎn)移酶亞型的表達(dá)[A];第九屆全國實驗血液學(xué)會議論文摘要匯編[C];2003年

6 顧松;;DNA甲基轉(zhuǎn)移酶異常剪接體DNMT3B7對神經(jīng)母細(xì)胞瘤相關(guān)基因表達(dá)及細(xì)胞增殖影響的研究[A];中華醫(yī)學(xué)會第八次全國小兒外科學(xué)術(shù)會論文集[C];2010年

相關(guān)博士學(xué)位論文 前1條

1 王玉龍;蛹蟲草有性階段DNA甲基化及綠僵菌DNA甲基轉(zhuǎn)移酶功能研究[D];安徽農(nóng)業(yè)大學(xué);2016年

相關(guān)碩士學(xué)位論文 前10條

1 楊娟;辛伐他汀對急性髓系白血病NB4細(xì)胞株DNA甲基轉(zhuǎn)移酶的影響[D];山東大學(xué);2015年

2 崔競紅;孕鼠脂多糖暴露致子代腎臟組織DNA甲基轉(zhuǎn)移酶表達(dá)變化的初步研究[D];第三軍醫(yī)大學(xué);2015年

3 郭為偉;胚胎停育絨毛組織中DNA甲基轉(zhuǎn)移酶的表達(dá)及臨床意義[D];青島大學(xué);2016年

4 李亞林;沉默DNA甲基轉(zhuǎn)移酶上調(diào)NDRG1表達(dá)對前列腺癌DU145細(xì)胞生物學(xué)行為的影響[D];天津醫(yī)科大學(xué);2016年

5 劉培;基于發(fā)卡DNA及G四聯(lián)體DNA甲基轉(zhuǎn)移酶傳感器的構(gòu)建及應(yīng)用研究[D];山東農(nóng)業(yè)大學(xué);2016年

6 王超;DNA甲基轉(zhuǎn)移酶在TGF-β誘導(dǎo)EMT中的功能研究[D];浙江大學(xué);2015年

7 周將;外周血PBMC中DNA甲基轉(zhuǎn)移酶及CD11a mRNA表達(dá)在ANCA相關(guān)性小血管炎發(fā)病作用的研究[D];廣西醫(yī)科大學(xué);2014年

8 張勇燕;鄰苯二甲酸二丁酯宮內(nèi)暴露下的多代雄性生殖毒性及睪丸DNA甲基轉(zhuǎn)移酶的改變[D];第三軍醫(yī)大學(xué);2009年

9 葉振;DNA甲基轉(zhuǎn)移酶在胃癌及癌前病變中的表達(dá)及意義[D];天津醫(yī)科大學(xué);2009年

10 張春嬌;可卡因誘導(dǎo)腦內(nèi)成癮相關(guān)核團(tuán)DNA甲基轉(zhuǎn)移酶表達(dá)改變的研究[D];吉林大學(xué);2012年

，

本文編號：1847095