马氏珠母贝外套膜不同区域的DNA甲基化差异及功能研究

发布时间：2019-05-25 03:40

【摘要】：DNA甲基化是一种重要的表观遗传现象,与基因表达调控、基因印迹等密切相关。马氏珠母贝(Pinctada fucata martensii)作为我国重要的海水珍珠培育贝类之一,其矿化机制、免疫防御系统一直受到研究者的关注。对马氏珠母贝外套膜DNA甲基化的研究有助于了解DNA甲基化与贝类的矿化、免疫机制的关系。本研究对马氏珠母贝外套膜三个区域开展DNA甲基化基础研究,筛选甲基化修饰差异基因并进行功能验证。主要研究结果如下:(1)通过甲基化敏感多态性扩增技术,检测马氏珠母贝外套膜的边缘膜区(Mantle edge,ME),套膜区(Mantle pallial,MP)和中央膜区(Mantle central,MC)基因组DNA甲基化修饰水平。结果显示ME、MP、MC的基因组甲基化水平分别为(17.02±1.98)%、(14.46±1.94)%、(19.04±2.55)%,具有显著性差异(P0.05);基因组的基因组甲基化水平由高到低排列依次是MCMEMP;对特异性片段进行回收测序获得iHog(interference hedgehog)并进行定量分析,iHog在ME,MP和MC中均有表达,以ME表达量最高,MC表达量最低,差异显著,其与甲基化修饰呈现负相关(P0.05),推测iHog的DNA序列的甲基化修饰抑制了该基因在Mc组织中的表达。(2)使用DNA甲基化免疫共沉淀测序法(MeDIP-Seq)筛选ME和MC之间的DNA甲基化修饰差异基因。测序结果显示ME、MC都得到122 M条原始Reads,以及分别获得58702和55721条Peaks。通过生物信息学分析得到ME和MC具甲基化修饰差异基因约311个;挑选具甲基化修饰差异基因TRAF2、TRAF3以及MyD88基因进行荧光定量验证,结果表明三个基因在ME、MC上的表达具有差异(P0.05)。(3)通过RACE技术成功获得Pm-My D88的序列全长1591 bp,共编码358个氨基酸;实时荧光定量PCR表明Pm-MyD88基因mRNA在鳃的表达量最高;LPS刺激后,其表达水平在12 h时达到最高水平(P0.05);原位杂交试验则表明Pm-MyD88存在于血淋巴细胞中,并伴随LPS刺激而表现出较强的阳性信号;共转染pcDNA3.1-Pm-MyD88重组质粒能使p NFκB-Luc的相对双荧光素酶活性提高1.9372倍;软件分析获得miR-4047为Pm-MyD88的调控miRNA,体内过表达miR-4047后,Pm-MyD88基因的表达量下调90.16%(P0.05),下游基因NF-kB和IL-17的表达量分别下调97.09%(P0.05)和99.99%(P0.01);双荧光素酶共转染实验表明Pm-My D88-3'UTR重组质粒共转染miR-4047后,其相对荧光素酶活性下降36.95%;以上实验结果表明Pm-My D88能够参与并激活NFkB信号通路,且miR-4047和Pm-MyD88具有靶向关系。(4)利用RACE技术获得甲基转移酶:Pm-Dnmt1、Pm-Dnmt1b、Pm-Dnmt3a、Pm-Dnmt3b的cDNA序列全长,分别为4848 bp、2661 bp、2405 bp、2775 bp、其各自编码1550、797、705和803个氨基酸。实时荧光定量PCR分析显示四个基因在全组织中均有表达,其中除Pm-Dnmt1在中央膜中的表达量最高(P0.05),余下3个基因mRNA均在性腺中的表达量最高(P0.05),推测Pm-Dnmt1可通过DNA甲基化协同参与贝壳的形成。DNA甲基转移酶抑制剂(Decitabine,DAC)去甲基化实验结果则表明:DAC处理后甲基转移酶在ME、MC上的表达均明显下降,TRAF2、TRAF3以及MyD88基因的表达量则显著上调(P0.05)。
[Abstract]:DNA methylation is an important epigenetic phenomenon, which is closely related to gene expression regulation, gene blot and so on. Pinctada fucata barrensis is one of the most important pearl-breeding shellfish in China, and its mineralization mechanism and immune defense system have been concerned by the researchers all the time. The study of the DNA methylation of the outer mantle of the mother-of-pearl of the Martensii is helpful to understand the relationship between the DNA methylation and the mineralization and the immune mechanism of the shellfish. In this study, a study was conducted to study the DNA methylation of the three regions of the outer mantle of the mother's pearl, and to screen the differentially expressed genes and to perform the function verification. The main results of the study were as follows: (1) The methylation level of the genomic DNA of the mantle, the mantle region (MP) and the central membrane region (MC) of the outer mantle of the mother of the horse's pearl was detected by the methylation sensitive polymorphism. The results showed that the genomic methylation levels of ME, MP and MC were (17.02-1.98)%, (14.46-1.94)% and (19.04-2.55)%, respectively. The specific fragment was recovered and sequenced to obtain iHog (reference hedgehog) and the quantitative analysis was carried out. The iHog was expressed in the ME, MP and MC, with the highest ME expression, the lowest expression of the MC and the significant difference, and it was negatively correlated with the methylation modification (P0.05). It is assumed that the methylation modification of the DNA sequence of iHog inhibits the expression of the gene in the Mc tissue. (2) the DNA methylation-modified differential gene between ME and MC was screened using a DNA methylation-immunoprecipitation sequencing method (MeDIP-Seq). The sequencing results showed that the ME and MC both obtained 122M original Reads and 58702 and 55721 Peaks, respectively. The results showed that the expression of three genes in ME and MC was different (P0.05). (3) The total length of the Pm-MyD88 was 1591 bp by the RACE technique, and 358 amino acids were encoded. The real-time fluorescence quantitative PCR showed that the expression of Pm-MyD88 gene was the highest, and the expression level reached the highest level at 12 h after LPS stimulation (P0.05). The in situ hybridization assay showed that Pm-MyD88 was present in the blood lymphocytes and showed a strong positive signal with the stimulation of LPS; the co-transfection of the pcDNA3.1-Pm-MyD88 recombinant plasmid can increase the relative double-luciferase activity of the p-NF-B-Luc by 1.9372 times; the software analysis obtains the regulated miRNA of the miR-4047 as Pm-MyD88, the in vivo expression of the miR-4047, The expression of Pm-MyD88 gene was reduced by 90.16% (P0.05). The expression of the downstream gene NF-kB and IL-17 was reduced by 97.09% (P0.05) and 99.99% (P0.01). The co-transfection of the double-luciferase showed that the relative luciferase activity decreased by 36.95% after the transfection of the miR-4047 with the recombinant plasmid of Pm-My D88-3 'UTR. The above experimental results show that Pm-My D88 can participate in and activate the NFkB signaling pathway, and miR-4047 and Pm-MyD88 have targeted relationships. (4) The full length of the cDNA sequence of the methyltransferase: Pm-Dnmt1, Pm-Dnmt1b, Pm-Dnmt3a, Pm-Dnmt3b was 4848 bp,2661 bp,2405 bp, and 2775 bp, respectively, using the RACE technique. The real-time fluorescence quantitative PCR analysis showed that the expression of the four genes in the whole tissue was the highest (P0.05). The expression of the remaining three gene mRNA in the gonad was the highest (P0.05), and it was estimated that Pm-Dnmt1 could be co-involved in the formation of the shell by the DNA methylation. The results of the demethylation of the DNA methyltransferase inhibitor (DDAC) showed that the expression of the methyltransferase in the ME and the MC decreased significantly after the DAC treatment, and the expression of TRAF2, TRAF3 and MyD88 was significantly increased (P0.05).
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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