鸡miR-1704靶基因的初步鉴定及生物学功能研究

发布时间：2018-06-07 10:03

本文选题：miR-1704 + 组织表达　；参考：《河南农业大学》2015年硕士论文

【摘要】：MicroRNAs是内源性的非编码小RNA分子,通过结合到靶基因mRNA的3’UTR,从而在转录后水平负调控基因的表达,参与机体内许多重要的生物过程。前人在鸡胚中发现mi R-1704,但有关其功能的研究尚未见报道。本研究利用荧光定量PCR技术检测了mi R-1704在固始鸡1日龄、6周龄和16周龄的9种不同组织中的表达情况;利用TargetScan在线软件预测了miR-1704的靶基因,对预测的靶基因在基因功能注释数据库DAVID和GO Term Finder进行了GO富集和KEGG通路分析,并通过双荧光报告系统初步鉴定了miR-1704的靶基因;以实验室固始鸡-安卡鸡F2代资源群体为试验素材,分析了miR-1704单核苷酸多态性与资源群体经济性状间的关联性,并通过构建miR-1704多态位点不同等位基因的真核表达载体对其功能进行了探索。主要结果如下:1.鸡mi R-1704在所检测的组织中呈广泛性表达,且在不同时期及不同组织间差异性表达。MiR-1704在1日龄和6周龄时低丰度表达,在16周龄时高丰度表达。1日龄时,miR-1704在腿肌、下丘脑和肝脏中的表达量较高,在肾脏和大脑中的表达量较低;6周龄时,miR-1704在各组织中的表达量均较低;16周龄时,miR-1704在脾脏、腿肌和下丘脑中的表达量较高,在小肠中的表达量最低。2.TargetScan预测得到miR-1704的靶基因173个。GO富集和KEGG通路分析结果表明,miR-1704靶基因显著富集于基因表达调控、生物合成、物质代谢、生物过程调节等生物学过程,以及细胞周期、钙信号通路和Hedgehog信号通路。利用双荧光素酶报告系统验证miR-1704与预测靶基因BMP2和IRS2的靶标关系,初步证实BMP2和IRS2是miR-1704的靶基因。3.对mi R-1704多态位点rs14668705 CG的关联分析结果表明,该突变与鸡出生重、6周体重、8周体重、10周体重、12周体重、4周胫围、8周胫围、12周胫围、屠体重、半净膛重、腿肌失水率、胸肌p H和谷草转氨酶显著相关(P0.05),且除胸肌pH值外,各性状CC型和GC型个体表型均值大于GG型。CG位点不同等位基因的表达研究表明,该突变显著影响成熟miR-1704的表达(P0.05)。由以上试验结果,可得出如下结论:1.miR-1704在鸡不同时期不同组织中广泛性、差异性表达。2.初步证实BMP2、IRS2是miR-1704的靶基因。3.miR-1704前体区rs14668705 CG与鸡的体重、胫围、屠体重等性状显著相关(P0.05),CC型个体表型均值大于GG型,且CG显著影响成熟miR-1704的生成(P0.05)。
[Abstract]:MicroRNAs is an endogenous non-coding small RNA molecule, which is involved in many important biological processes in the organism by binding to the target gene mRNA and negatively regulating the expression of genes at the post-transcriptional level. Mi R-1704 was found in chicken embryo, but the study on its function has not been reported. In this study, the expression of miR-1704 was detected by fluorescence quantitative PCR in 9 tissues of Gushi chicken at the age of 6 weeks and 16 weeks, and the target gene of miR-1704 was predicted by TargetScan online software. The predicted target genes were enriched by go and analyzed by KEGG pathway in DAVID and GO Term Finder, and the target genes of miR-1704 were preliminarily identified by double fluorescence reporting system. The relationship between miR-1704 single nucleotide polymorphisms and resource population economic traits was analyzed by using F _ 2 resource population of Gushi Chicken and Anka Chicken in laboratory as experimental materials. The function of Eukaryotic expression vector with different alleles of miR-1704 polymorphic loci was explored. The main results are as follows: 1. The expression of miR-1704 was widespread in the tissues examined, and the expression of MiR-1704 was low at 1 day and 6 weeks of age, and 1 day old at 16 weeks old, and miR-1704 in leg muscle at 16 weeks of age. The expression of miR-1704 was higher in the spleen, leg muscle and hypothalamus than in the spleen, leg muscle and hypothalamus at the age of 16 weeks. TargetScan predicted the enrichment of 173 target genes of miR-1704 and the analysis of KEGG pathway. The results showed that the target genes were significantly enriched in biological processes such as gene expression regulation, biosynthesis, substance metabolism, biological process regulation, and so on. And cell cycle, calcium signaling pathway and Hedgehog signaling pathway. Double luciferase reporting system was used to verify the target relationship between miR-1704 and predicted target gene BMP2 and IRS2. It was preliminarily confirmed that BMP2 and IRS2 were the target genes of miR-1704. The results of rs14668705 CG association analysis showed that the mutation was associated with chicken birth weight at 6 weeks and the body weight at 8 weeks and 10 weeks, and the body weight at 12 weeks and 4 weeks, tibia around 8 weeks and tibia around 8 weeks, slaughtering weight, half bore weight, and water loss rate of leg muscle. There was a significant correlation between pH and glutamic oxaloacetic transaminase in pectoralis muscle, and the phenotypic mean values of individuals with CC and GC were higher than those of GG type. CG alleles showed that the mutation significantly affected the expression of mature miR-1704. From the above results, we can draw the following conclusions: 1. The different expression of miR-1704 in different tissues of chicken at different stages. It was preliminarily confirmed that BMP2 / IRS2 was the target gene of miR-1704. 3.miR-1704 rs14668705 CG was significantly correlated with chicken body weight, tibia circumference and slaughter weight. The phenotypic mean of CC type was significantly higher than that of GG type, and CG significantly affected the production of mature miR-1704.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

【相似文献】