镰形扇头蜱的几种先天性免疫相关miRNAs分子研究

发布时间：2018-02-11 05:17

本文关键词： 镰形扇头蜱 MicroRNA 脂多糖靶基因先天性免疫　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：蜱是一类专门寄生于脊椎动物的体表寄生虫,是多种疾病的传播媒介。先天性免疫反应是蜱与病原互作的关键环节,但迄今为止,对蜱的先天性免疫反应机制了解甚少。Micro RNA,简称mi RNA,是生物体内18-25 nt的非编码小RNA,作用于靶基因的3’端非翻译区以抑制其表达或者降解m RNA,在后转录水平发挥着生物学调控作用,如生长发育和先天性免疫等生理学功能。为了研究蜱的mi RNA在先天性免疫反应中的作用,本论文在实验室已有的差异表达mi RNAs数据库的基础上,通过节肢动物的先天性免疫刺激剂——脂多糖(LPS)注射镰形扇头蜱,分离鉴定若干蜱的先天性免疫相关mi RNAs分子,为阐明蜱的先天性免疫反应机制提供基础。本研究结果包括以下4个方面:一、通过实时荧光定量PCR验证了LPS注射后差异表达(下调)的5个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,它们的下调倍数分别为4.2、3.6、3.4、3.3和5.0。应用反转录PCR克隆获得这些mi RNAs的前体序列,并且通过m Fold软件预测了这些mi RNAs的前体发夹结构,进一步验证了这些mi RNAs。二、通过实时荧光定量PCR发现这五种mi RNAs的表达具有阶段特异性和器官特异性的特点,相比较于其它发育阶段和器官,这五种mi RNAs均在幼蜱和脂肪体中表达量是最高的。三、研究了mi R-133-3p在蜱吸血前后不同器官的表达特征,发现在吸血后蜱的脂肪体、唾液腺和卵巢的表达量相较于未吸血时明显下调,下调倍数分别为13.3、4.2和15.4;尽管实验组注射mi R-133-3p agomirna后,mi R-133-3p在半饱血蜱及其脂肪体表达量明显增加,但吸血等生物学性状并没有显著差异,表明mi R-133-3p在蜱吸血中无重要作用。但是,mi R-133-3p在感染有巴贝斯虫的饱血幼蜱中表达量显著下降。四、应用双荧光素酶报告基因检测系统开展了mi R-133-3p和mi R-79-3p的预测靶基因验证。结果发现mi R-133-3p实验组产生的平均相对荧光活性为0.2,而对照组为2.5。使用t检验法分析发现两者的相对荧光活性差异极其显著,说明预测的降血钙素基因相关肽Ⅰ型受体(CGRPR)是mi R-133-3p的靶基因之一;而mi R-79-3p实验组产生的相对荧光活性为2.8,对照组为2.8,使用t检验法分析其差异不显著,说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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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