本文選題：雞毒支原體　切入點(diǎn)：gga-miR-99a　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：雞毒支原體(Mycoplasma gallisepticum, MG)是禽類支原體感染中最重要的病源微生物之一,引起禽類的慢性呼吸道疾病(chronic respiratory disease, CRD),該病廣泛分布于世界上所有養(yǎng)禽的國家,對(duì)家禽業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。microRNAs是一類長(zhǎng)度18-25nt的非編碼微小分子RNAs,主要通過識(shí)別特殊序列進(jìn)行轉(zhuǎn)錄后基因表達(dá)的調(diào)控。當(dāng)外界病原微生物進(jìn)入宿主后,宿主體內(nèi)miRNAs呈現(xiàn)不同豐度的表達(dá),且在病原微生物感染不同時(shí)期以及由于病原微生物種類的不同,宿主體內(nèi)miRNAs表達(dá)量發(fā)生變化。因此,通過研究宿主細(xì)胞感染病原前后miRNAs的表達(dá)變化及其靶標(biāo)功能,不僅有助于揭示了該病的致病機(jī)制,還為實(shí)現(xiàn)基因治療提供理論依據(jù)。本實(shí)驗(yàn)組前期通過MG感染雞胚,深度測(cè)序發(fā)現(xiàn),處理組中g(shù)ga-miR-99a的表達(dá)量顯著下調(diào),gga-miR-146c的表達(dá)量顯著上調(diào),表明gga-miR-99a、gga-miR-146c可能在MG引起的疾病中發(fā)揮調(diào)控作用。本研究主要運(yùn)用Q-PCR研究不同時(shí)期MG感染雞胚肺組織中miR-99a和miR-146c的表達(dá)量；通過生物信息學(xué)方法預(yù)測(cè)靶基因,并進(jìn)行驗(yàn)證；利用超表達(dá)和RNAi技術(shù)研究靶基因的功能,初步闡明gga-miR-99a、gga-miR-146c在MG感染中的調(diào)控機(jī)制。本研究的主要成果如下：(1)采用Q-PCR檢測(cè)不同時(shí)期MG感染雞胚肺組織中miR-99a的表達(dá)量,13d、14d、16d、17d肺組織中g(shù)ga-miR-99a的相對(duì)表達(dá)量在感染MG后極顯著上調(diào)(p0.01),12d、15d、19d、20d肺組織中g(shù)ga-miR-99a的相對(duì)表達(dá)量在感染MG后極顯著下調(diào)(p0.01)。(2)通過TargetScan和miRDB預(yù)測(cè)gga-miR-99a的靶基因,初步選定SMARCA5作為gga-miR-99a的靶基因。進(jìn)一步運(yùn)用雙熒光素酶報(bào)告基因驗(yàn)證,結(jié)果顯示,與NC組、突變載體組、空載體組相比,gga-miR-99a能夠極顯著抑制SMARCA5 3'UTR的雙熒光素酶報(bào)告基因的表達(dá)(p0.01)。同時(shí),DF-1細(xì)胞中超表達(dá)gga-miR-99a后,與NC組相比,SMARCA5相對(duì)表達(dá)量極顯著下調(diào)(p0.01)；抑制gga-miR-99a后,與Inhibitor NC相比,SMARCA5相對(duì)表達(dá)量極顯著上調(diào)(p0.01)。此外,12d感染組肺組織中SMARCA5的相對(duì)表達(dá)量極顯著上調(diào)(p0.01)；16d感染組肺組織中SMARCA5的相對(duì)表達(dá)量極顯著下調(diào)(p0.01),該結(jié)果與同時(shí)期肺組織中g(shù)ga-miR-99a的表達(dá)趨勢(shì)相反。(3)DF-1細(xì)胞中轉(zhuǎn)染gga-miR-99a 24h、48h、72h,結(jié)果發(fā)現(xiàn),轉(zhuǎn)染72h后,DF-1細(xì)胞增殖率顯著下降(P0.05)；同時(shí),轉(zhuǎn)染gga-miR-99a后檢測(cè)DF-1細(xì)胞周期,與陰性對(duì)照相比,G1期水平明顯升高(P0.05), G2期基本保持不變(P0.05)。(4)采用Q-PCR檢測(cè)不同時(shí)期MG感染雞胚肺組織中miR-146c的表達(dá)量,13d、14d極顯著下調(diào)(p0.01),而18d、19d、20d極顯著上調(diào)(p0.01)；16d、17d顯著上調(diào)(p0.05)。(5)通過TargetScan和miRDB預(yù)測(cè)gga-miR-146c的靶基因,初步選定MMP16、 TRAF7作為gga-miR-146c的靶基因。進(jìn)一步運(yùn)用雙熒光素酶報(bào)告基因驗(yàn)證,結(jié)果顯示,與NC組、突變載體組、空載體組相比,gga-miR-146c能夠極顯著抑制MMP16、 TRAF7 3'UTR的雙熒光素酶報(bào)告基因的表達(dá)(p0.01)。同時(shí),DF-1細(xì)胞中超表達(dá)gga-miR-146c后,與NC組相比,MMP16、TRAF7的相對(duì)表達(dá)量極顯著下調(diào)(p0.01)；抑制gga-miR-146c后,與Inhibitor NC相比,MMP16、TRAF7相對(duì)表達(dá)量極顯著上調(diào)(p0.01)。(6)DF-1細(xì)胞中超表達(dá)gga-miR-146c后,與NC組相比,TLR6、TLR2、Myd88、 TNF-a相對(duì)表達(dá)量極顯著上調(diào)(p0.01), Apoal無顯著性差異；抑制gga-miR-146c后,與Inhibitor NC相比,Apoa I相對(duì)表達(dá)量極顯著上調(diào)(p0.01), TLR6、TLR2、 Myd88、TNF-α無顯著性差異。
[Abstract]:Mycoplasma gallisepticum (Mycoplasma gallisepticum MG) is one of the most important infection of pathogenic microorganisms of poultry mycoplasma, caused by avian chronic respiratory disease (chronic respiratory, disease, CRD), the disease is widely distributed in the world all countries of poultry, poultry industry caused serious economic losses in.MicroRNAs is non encoding small molecule RNAs the length of 18-25nt, mainly through the identification of special sequence of regulation of gene expression after transcription. When the external pathogenic microorganisms into the host, the host miRNAs showed different expression in abundance, and infection of pathogenic microorganisms in different periods and the different kinds of pathogenic microorganisms, the host miRNAs expression change. Therefore, the pathogen and expression the change of miRNAs and its target function of host cell infection, not only helps to reveal the pathogenic mechanism of the disease, but also for real We provide a theoretical basis for gene therapy. The experimental group by the early MG infected chicken embryo, deep sequencing found that the expression of gga-miR-99a in the treatment group were significantly reduced, the expression of gga-miR-146c was significantly up-regulated, indicating that gga-miR-99a, play to control the possible role of gga-miR-146c in the disease caused by MG. This research mainly uses the Q-PCR expression of different periods of MG infection miR-99a and miR-146c in lung tissues of chicken embryo in quantity; through the method of bioinformatics prediction of target genes, and verified; using overexpression and RNAi technology to study the target gene function, elucidate the regulatory mechanism of gga-miR-146c gga-miR-99a in MG infection. The main results of this study are as follows: (1) the expression of Q-PCR detection in different stages of MG infection of miR-99a in lung tissue of chick embryos in 13D, 14d, 16d, relative expression of gga-miR-99a 17D in lung tissue infected with MG significantly up-regulated (P0.01), 12D, 15d The relative expression of gga-miR-99a, 19d, 20d in lung tissue was significantly reduced in MG after infection (P0.01). (2) the target gene TargetScan and miRDB prediction gga-miR-99a, selected SMARCA5 as the target gene of gga-miR-99a. Further results show that using the dual luciferase reporter verified, with the NC group, the mutant vector group. Compared with the empty vector group, gga-miR-99a can significantly inhibit the expression of luciferase reporter gene SMARCA5 of 3'UTR (P0.01). At the same time, the expression of gga-miR-99a in DF-1 cells, compared with the NC group, the relative expression of SMARCA5 was significantly reduced (P0.01); the inhibition of gga-miR-99a, compared with Inhibitor NC, the relative expression of SMARCA5 was significantly up-regulated (P0.01). In addition, the relative expression of SMARCA5 12D in the lung tissues of the infected group was significantly increased (P0.01); the relative expression of SMARCA5 16d in the lung tissues of the infected group were significantly reduced (P0.01), and the results The trend of the expression of gga-miR-99a in lung tissue at the same time the opposite. (3) DF-1 cell transfer 24h 48h, 72h gga-miR-99a staining, results showed that, after 72h transfection significantly decreased DF-1 cell proliferation rate (P0.05); at the same time, detection of cell cycle of DF-1 cells after transfection of gga-miR-99a, compared with the negative control, G1 level increased significantly (P0.05), G2 (P0.05) remained unchanged. (4) the expression of Q-PCR, MG in the different stages of miR-146c infection detection of lung tissue in chicken 13D, 14d was significantly reduced (P0.01), 18D, 19d, 20d was significantly increased (P0.01); 16d, 17D were significantly increased (5 (P0.05). The target gene TargetScan and miRDB) of gga-miR-146c, selected MMP16, TRAF7 as a target gene of gga-miR-146c. Further results show that using the dual luciferase reporter verified, with the NC group, compared with the mutant vector group, vector group, gga-miR-146c significantly inhibited MMP16, TRAF7 3'UTR The expression of the dual luciferase reporter gene (P0.01). At the same time, the expression of gga-miR-146c in DF-1 cells, compared with group NC, MMP16, TRAF7 expression was significantly reduced (P0.01); the inhibition of gga-miR-146c, compared with Inhibitor, NC MMP16, the relative expression was significantly up-regulated the expression of TRAF7 (P0.01) (6). The expression of gga-miR-146c in DF-1 cells, compared with group NC, TLR6, TLR2, Myd88, relative expression was significantly up-regulated the expression of TNF-a (P0.01), no significant difference in Apoal; the inhibition of gga-miR-146c, compared with Inhibitor NC, Apoa I relative expression was very significantly increased (P0.01), TLR6, TLR2, Myd88, no significant differences of TNF- alpha.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855

【二級(jí)參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 高俊偉;畢丁仁;胡思順;彭秀麗;;雞毒霉形體黏附素截短蛋白表達(dá)條件的優(yōu)化[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào);2007年04期

2 董靜;施毅;宋勇;印潔;劉遠(yuǎn)韜;呂鏜鋒;朱美英;;小鼠感染肺炎衣原體后肺組織TLR2、TLR4基因表達(dá)的變化及意義[J];中國感染與化療雜志;2006年02期

3 畢丁仁,冀敄霖;雞源霉形體的分離和鑒定[J];畜牧獸醫(yī)學(xué)報(bào);1988年S1期

4 ;Polymorphisms of some cytokines and chronic hepatitis B and C virus infection[J];World Journal of Gastroenterology;2009年44期

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本文編號(hào)：1614059