本文關(guān)鍵詞： 鐮形扇頭蜱 MicroRNA 脂多糖 靶基因 先天性免疫　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：蜱是一類專門寄生于脊椎動(dòng)物的體表寄生蟲,是多種疾病的傳播媒介。先天性免疫反應(yīng)是蜱與病原互作的關(guān)鍵環(huán)節(jié),但迄今為止,對(duì)蜱的先天性免疫反應(yīng)機(jī)制了解甚少。Micro RNA,簡(jiǎn)稱mi RNA,是生物體內(nèi)18-25 nt的非編碼小RNA,作用于靶基因的3’端非翻譯區(qū)以抑制其表達(dá)或者降解m RNA,在后轉(zhuǎn)錄水平發(fā)揮著生物學(xué)調(diào)控作用,如生長(zhǎng)發(fā)育和先天性免疫等生理學(xué)功能。為了研究蜱的mi RNA在先天性免疫反應(yīng)中的作用,本論文在實(shí)驗(yàn)室已有的差異表達(dá)mi RNAs數(shù)據(jù)庫(kù)的基礎(chǔ)上,通過節(jié)肢動(dòng)物的先天性免疫刺激劑——脂多糖(LPS)注射鐮形扇頭蜱,分離鑒定若干蜱的先天性免疫相關(guān)mi RNAs分子,為闡明蜱的先天性免疫反應(yīng)機(jī)制提供基礎(chǔ)。本研究結(jié)果包括以下4個(gè)方面:一、通過實(shí)時(shí)熒光定量PCR驗(yàn)證了LPS注射后差異表達(dá)(下調(diào))的5個(gè)mi RNAs分子,即mi R-184、mi R-79-3p、mi R-71-5p、mi R-1-3p和mi R-133-3p,它們的下調(diào)倍數(shù)分別為4.2、3.6、3.4、3.3和5.0。應(yīng)用反轉(zhuǎn)錄PCR克隆獲得這些mi RNAs的前體序列,并且通過m Fold軟件預(yù)測(cè)了這些mi RNAs的前體發(fā)夾結(jié)構(gòu),進(jìn)一步驗(yàn)證了這些mi RNAs。二、通過實(shí)時(shí)熒光定量PCR發(fā)現(xiàn)這五種mi RNAs的表達(dá)具有階段特異性和器官特異性的特點(diǎn),相比較于其它發(fā)育階段和器官,這五種mi RNAs均在幼蜱和脂肪體中表達(dá)量是最高的。三、研究了mi R-133-3p在蜱吸血前后不同器官的表達(dá)特征,發(fā)現(xiàn)在吸血后蜱的脂肪體、唾液腺和卵巢的表達(dá)量相較于未吸血時(shí)明顯下調(diào),下調(diào)倍數(shù)分別為13.3、4.2和15.4;盡管實(shí)驗(yàn)組注射mi R-133-3p agomirna后,mi R-133-3p在半飽血蜱及其脂肪體表達(dá)量明顯增加,但吸血等生物學(xué)性狀并沒有顯著差異,表明mi R-133-3p在蜱吸血中無重要作用。但是,mi R-133-3p在感染有巴貝斯蟲的飽血幼蜱中表達(dá)量顯著下降。四、應(yīng)用雙熒光素酶報(bào)告基因檢測(cè)系統(tǒng)開展了mi R-133-3p和mi R-79-3p的預(yù)測(cè)靶基因驗(yàn)證。結(jié)果發(fā)現(xiàn)mi R-133-3p實(shí)驗(yàn)組產(chǎn)生的平均相對(duì)熒光活性為0.2,而對(duì)照組為2.5。使用t檢驗(yàn)法分析發(fā)現(xiàn)兩者的相對(duì)熒光活性差異極其顯著,說明預(yù)測(cè)的降血鈣素基因相關(guān)肽Ⅰ型受體(CGRPR)是mi R-133-3p的靶基因之一;而mi R-79-3p實(shí)驗(yàn)組產(chǎn)生的相對(duì)熒光活性為2.8,對(duì)照組為2.8,使用t檢驗(yàn)法分析其差異不顯著,說明富含脯氨酸和谷氨酰胺的剪接因子(SFPG)不是mi R-79-3p的靶基因。
[Abstract]:Ticks are a class of parasites that are specialized in vertebrates and are the transmission vectors of many diseases. Congenital immune response is a key link in the interaction between ticks and pathogens, but up to now, Very little is known about the innate immune response mechanism of ticks. Micro RNAs, referred to as mi RNAs, are non-coding small RNAs of 18-25 NT in organisms that act on the 3'untranslated region of the target gene to inhibit its expression or degrade m RNAs, and play a biological regulatory role at the post-transcriptional level. In order to study the role of mi RNA of ticks in congenital immune response, this paper is based on the database of differential expression of mi RNAs in laboratory. Lipopolysaccharide (LPS), a congenital immune stimulator of arthropods, was injected into ticks falciform fan head to isolate and identify some innate immune-associated mi RNAs molecules of several ticks. In order to elucidate the mechanism of innate immune response of ticks, the results of this study include the following four aspects: first, five differentially expressed (down-regulated) mi RNAs molecules after LPS injection were verified by real-time fluorescent quantitative PCR. That is, mi R-184t / mi R-79-3p / mi R-71-5p / mi R-133-3p, and their down-regulation times are 4.2 / 3.6/ 3.4/ 3.3and 5.0respectively. The precursor sequences of these mi RNAs were cloned by reverse transcription PCR, and the hairpin structure of these miRNAs was predicted by m Fold software to further verify these miRNAs. The expression of these five kinds of mi RNAs was found to be phase specific and organ specific by real-time fluorescence quantitative PCR. Compared with other developmental stages and organs, the expression of these five kinds of mi RNAs was the highest in juvenile ticks and fat bodies. The expression characteristics of mi R-133-3p in different organs of ticks before and after blood sucking were studied. It was found that the expression of miR-133-3p in fat body, salivary gland and ovary of ticks after blood sucking was significantly decreased compared with that of non-sucking ticks. The down-regulation times were 13.3% 4.2 and 15.4 respectively. Although the expression of miR-133-3p in the experimental group was significantly increased after the injection of mi R-133-3p agomirna, there was no significant difference in biological characters such as blood sucking. The results showed that mi R-133-3p had no important role in the blood sucking of ticks, but the expression of R-133-3p was significantly decreased in the infecting young ticks infected with Babes. The predictive target genes of mi R-133-3p and mi R-79-3p were verified by double luciferase reporter gene detection system. The results showed that the average relative fluorescence activity of the experimental group was 0.2, compared with 2.5 in the control group. The difference of relative fluorescence activity between them is extremely significant. The predicted CGRPRs were one of the target genes of mi R-133-3p, and the relative fluorescence activity of the experimental group was 2.8, and the control group was 2.8. There was no significant difference between the two groups by t-test. These results suggest that SFPG, a splicing factor rich in proline and glutamine, is not a target gene of mi R-79-3p.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7

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