绵羊不同发情状态下松果体若干miRNAs表达差异及其与AANAT靶关系鉴定

发布时间：2018-02-09 19:08

本文关键词： miRNA AANAT 表达量松果体靶关系双荧光素酶报告基因检测　出处：《扬州大学》2015年硕士论文　论文类型：学位论文

【摘要】：miRNAs是一类长度为21-24nt的非编码小RNA,通过与靶mRNAs的3'-UTR结合在转录后水平上沉默基因表达,参与多种生理、生化、病理过程。松果体是动物季节性发情主要生理途径的重要组成部分,通过分泌褪黑激素,调节下丘脑促性腺激素释放激素的脉冲式释放,进一步通过下丘脑-垂体-性腺轴调控季节性繁殖活动。芳基烷基胺-氮-乙酰转移酶基因(AANAT)是合成褪黑激素的关键酶,参与调控动物季节性繁殖。本研究旨在探究miRNA在绵羊不同发情状态松果体组织中的表达差异及其对AANAT的表观遗传调控作用。根据前期获得的绵羊转录组测序结果,验证绵羊松果体中部分miRNAs及AANAT在不同时期的表达差异,运用生物信息学方法预测候选miRNAs和AANAT的靶关系,最后利用双荧光素酶报告基因检测系统对其进行验证。主要研究内容和结果如下：1、基于前期通过转录组分析确定的绵羊不同发情状态松果体组织差异表达的AANAT基因和5个候选miRNA(miR-118,-247,-364,-406,-892),进行荧光定量分析,结果表明绵羊AANAT基因的表达乏情期高于发情间期,发情间期和发情前期保持上调趋势至发情期达到峰值,miR-364、miR-892、miR-406在四个时期间的表达趋势与AANAT基因相反,miR-118、miR-247的表达量在乏情期至发情间期升高,发情间期至发情前期降低,发情前期至发情期升高。经过分析发现AANAT与miR-364、miR-406呈显著负相关(R2=0.572；P0.05),与miR-892呈显著负相关(R2=0.669；P0.05)。2、利用3'RACE法扩增出AANAT的3'-UTR并进行测序,然后运用miRanda和RNAhybrid等生物信息学软件预测靶AANAT 3'-UTR区的miRNAs,确定miR-118、miR-247与AANAT存在靶结合位置。3、miR-118、miR-247与AANAT的靶关系验证。将合成的AANAT 3'-UTR野生型(WT)和突变型(MUT)序列构建到pmirGLO载体中,将miR-118、miR-247的成熟序列和miRNA阴性对照(NE)构建到pcDNA6.2TM-GW/EmGFP-miR载体中。实验分别分为4个实验组(WT+miRNA组,MUT+miRNA组,WT+NE组,MUT+NE组)和1个空白对照组,每个组设3个重复,然后转染到293FT细胞中,双荧光素酶报告检测表明miR-118对野生型AANAT 3'-UTR重组质粒表达的荧光素酶活性有显著地抑制效果(P0.01),说明miR-118可以与AANAT的3'-UTR区序列相互作用并且明确了具体作用的靶位点,证实miR-118和AANAT存在靶关系。然而,miR-247对野生型AANAT 3'-UTR重组质粒表达的荧光素酶活性没有显著地抑制效果,表明miR-247与AANAT基因不存在靶关系。
[Abstract]:MiRNAs is a kind of non-coding small RNAs 21-24nt in length. It is involved in many physiological, biochemical and pathological processes by silencing gene expression at the post-transcriptional level by binding to the target mRNAs 3H-UTR. Pineal gland is an important part of the main physiological pathway of seasonal estrus in animals. By secreting melatonin, regulating the pulse release of hypothalamic gonadotropin releasing hormone, Further regulation of seasonal reproductive activity through hypothalamus-pituitary-gonad axis. Aryl alkylamine-azo-acetyltransferase gene (AANATA) is the key enzyme for melatonin synthesis. The aim of this study was to investigate the difference of miRNA expression in the pineal tissues of sheep in different estrous states and its effect on the epigenetic regulation of AANAT. The expression differences of miRNAs and AANAT in the pineal gland of sheep at different stages were verified. The target relationship between candidate miRNAs and AANAT was predicted by bioinformatics. At last, the double luciferase reporter gene detection system was used to verify it. The main contents and results were as follows: 1, based on the differentially expressed AANAT base of pineal tissues in different estrous states of sheep identified by transcriptome analysis. Fluorescence quantitative analysis was carried out for miR-118N -247C ~ (-364N) -406N ~ (-892N), and 5 candidate miRNAs (miRNAs) and 5 candidate miRNAs (miRNAs). The results showed that the expression of AANAT gene in sheep was higher in estrus than in estrus. During estrus and early estrus, the expression trend of miR-364miR-892miR-406 increased from estrus to estrus, and decreased from estrus to preestrus, contrary to that of AANAT gene, and the expression of miR-247 increased during estrus to estrus, and decreased from estrus to preestrus. From early estrus to estrus, it was found that there was a significant negative correlation between AANAT and miR-364miR-406 and miR-892, and a significant negative correlation between AANAT and miR-892. The AANAT 3G -UTR was amplified by 3G race method and sequenced. Then using bioinformatics software, such as miRanda and RNAhybrid, to predict the miRNAss in the target AANAT 3O -UTR region, and to determine the target binding position between miR-118 miR-247 and AANAT. The target relationship between miR-247 and AANAT was verified. The synthesized AANAT 3H-UTR wild type WTand mutants were constructed into pmirGLO vector. The mature sequence of miR-118mmiR-247 and miRNA negative control neon) were constructed into pcDNA6.2TM-GW/EmGFP-miR vector. The experiment was divided into four experimental groups: WT miRNA group, mut miRNA group (WT NE group) and a blank control group. Each group was transfected into 293FT cells with 3 repeats. The detection of double luciferase report showed that miR-118 could inhibit the luciferase activity of wild-type AANAT _ 3 ~ (-UTR) recombinant plasmid significantly, indicating that miR-118 could interact with the 3 ~ (-) -UTR region of AANAT and identify the specific target sites. It was confirmed that there was a target relationship between miR-118 and AANAT, however, the activity of luciferase expressed in wild type AANAT 3N-UTR recombinant plasmid was not significantly inhibited by mmiR-247, indicating that there was no target relationship between miR-247 and AANAT gene.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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