【摘要】：DNA甲基化是一種重要的表觀遺傳現(xiàn)象,與基因表達調(diào)控、基因印跡等密切相關(guān)。馬氏珠母貝(Pinctada fucata martensii)作為我國重要的海水珍珠培育貝類之一,其礦化機制、免疫防御系統(tǒng)一直受到研究者的關(guān)注。對馬氏珠母貝外套膜DNA甲基化的研究有助于了解DNA甲基化與貝類的礦化、免疫機制的關(guān)系。本研究對馬氏珠母貝外套膜三個區(qū)域開展DNA甲基化基礎(chǔ)研究,篩選甲基化修飾差異基因并進行功能驗證。主要研究結(jié)果如下:(1)通過甲基化敏感多態(tài)性擴增技術(shù),檢測馬氏珠母貝外套膜的邊緣膜區(qū)(Mantle edge,ME),套膜區(qū)(Mantle pallial,MP)和中央膜區(qū)(Mantle central,MC)基因組DNA甲基化修飾水平。結(jié)果顯示ME、MP、MC的基因組甲基化水平分別為(17.02±1.98)%、(14.46±1.94)%、(19.04±2.55)%,具有顯著性差異(P0.05);基因組的基因組甲基化水平由高到低排列依次是MCMEMP;對特異性片段進行回收測序獲得iHog(interference hedgehog)并進行定量分析,iHog在ME,MP和MC中均有表達,以ME表達量最高,MC表達量最低,差異顯著,其與甲基化修飾呈現(xiàn)負相關(guān)(P0.05),推測iHog的DNA序列的甲基化修飾抑制了該基因在Mc組織中的表達。(2)使用DNA甲基化免疫共沉淀測序法(MeDIP-Seq)篩選ME和MC之間的DNA甲基化修飾差異基因。測序結(jié)果顯示ME、MC都得到122 M條原始Reads,以及分別獲得58702和55721條Peaks。通過生物信息學分析得到ME和MC具甲基化修飾差異基因約311個;挑選具甲基化修飾差異基因TRAF2、TRAF3以及MyD88基因進行熒光定量驗證,結(jié)果表明三個基因在ME、MC上的表達具有差異(P0.05)。(3)通過RACE技術(shù)成功獲得Pm-My D88的序列全長1591 bp,共編碼358個氨基酸;實時熒光定量PCR表明Pm-MyD88基因mRNA在鰓的表達量最高;LPS刺激后,其表達水平在12 h時達到最高水平(P0.05);原位雜交試驗則表明Pm-MyD88存在于血淋巴細胞中,并伴隨LPS刺激而表現(xiàn)出較強的陽性信號;共轉(zhuǎn)染pcDNA3.1-Pm-MyD88重組質(zhì)粒能使p NFκB-Luc的相對雙熒光素酶活性提高1.9372倍;軟件分析獲得miR-4047為Pm-MyD88的調(diào)控miRNA,體內(nèi)過表達miR-4047后,Pm-MyD88基因的表達量下調(diào)90.16%(P0.05),下游基因NF-kB和IL-17的表達量分別下調(diào)97.09%(P0.05)和99.99%(P0.01);雙熒光素酶共轉(zhuǎn)染實驗表明Pm-My D88-3'UTR重組質(zhì)粒共轉(zhuǎn)染miR-4047后,其相對熒光素酶活性下降36.95%;以上實驗結(jié)果表明Pm-My D88能夠參與并激活NFkB信號通路,且miR-4047和Pm-MyD88具有靶向關(guān)系。(4)利用RACE技術(shù)獲得甲基轉(zhuǎn)移酶:Pm-Dnmt1、Pm-Dnmt1b、Pm-Dnmt3a、Pm-Dnmt3b的cDNA序列全長,分別為4848 bp、2661 bp、2405 bp、2775 bp、其各自編碼1550、797、705和803個氨基酸。實時熒光定量PCR分析顯示四個基因在全組織中均有表達,其中除Pm-Dnmt1在中央膜中的表達量最高(P0.05),余下3個基因mRNA均在性腺中的表達量最高(P0.05),推測Pm-Dnmt1可通過DNA甲基化協(xié)同參與貝殼的形成。DNA甲基轉(zhuǎn)移酶抑制劑(Decitabine,DAC)去甲基化實驗結(jié)果則表明:DAC處理后甲基轉(zhuǎn)移酶在ME、MC上的表達均明顯下降,TRAF2、TRAF3以及MyD88基因的表達量則顯著上調(diào)(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).
【學位授予單位】：廣東海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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