chi-miRNA-4110对奶山羊Smad2基因靶向调控作用的研究

发布时间：2018-05-08 09:17

本文选题：chi-miR-4110 + Smad2　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：MicroRNA(miRNA)是一类重要的非编码小RNA分子,在动物繁殖过程中发挥着重要的调控作用。miRNA通过调节靶基因的表达影响动物的繁殖机能。为进一步揭示mi RNA对奶山羊生殖机能的调控作用,本研究在前期构建的多羔(3-5羔/胎)和单羔(1羔/胎)奶山羊发情期卵巢差异表达miRNA文库的基础上,以关中奶山羊为研究对象,利用Real time-PCR检测chi-miR-4110在多羔和单羔奶山羊发情期卵巢中的差异表达水平,并利用生物信息学、荧光素酶载体和Western blotting等技术验证chi-mi R-4110靶基因及其对靶基因的调控作用,为提高奶山羊产羔率提供试验依据和理论基础。本研究获得的主要结果如下:1、chi-miR-4110在多羔和单羔奶山羊卵巢组织中的表达水平利用生物信息学,从卵巢差异表达miRNA文库中筛选对繁殖性状有重要调控作用且差异表达显著的chi-miR-4110,采用Real time-PCR检测chi-miR-4110在多羔和单羔奶山羊卵巢中的相对表达量显示,chi-miR-4110在多羔奶山羊卵巢中的表达量极显著(P0.01)高于单羔奶山羊卵巢中的表达量,表明chi-miR-4110可能对奶山羊产羔性状有重要的调控作用。2、chi-miR-4110靶基因的预测及验证通过应用生物信息学对chi-miR-4110的靶基因进行综合分析,筛选出与卵巢发育相关的靶基因Smad2。构建含有Smad2-3'UTR区的双荧光素酶载体,与chi-miR-4110共转染到293T细胞中,利用荧光素酶检测系统检测荧光素酶的活性。结果显示与chi-miR-4110共转染的试验组的荧光素酶活性显著低于阴性对照,表明chi-miR-4110通过与Smad2-3'UTR相互作用导致荧光素酶活性降低,初步鉴定Smad2为chi-miR-4110的靶基因。3、chi-miR-4110对靶基因的调控将chi-miR-4110和control分别转染到奶山羊卵巢颗粒细胞中,利用Real time-PCR和Western blotting检测Smad2 mRNA以及Smad2蛋白的表达量,结果表明,在奶山羊卵巢颗粒细胞中,过表达chi-miR-4110使得Smad2 mRNA以及Smad2蛋白的表达量均显著降低,表明chi-miR-4110在转录后水平对Smad2起负调控的作用。以上研究结果显示,Smad2是chi-mi R-4110的靶基因,在奶山羊卵巢颗粒细胞中,chi-miR-4110靶向调控Smad2的表达。
[Abstract]:MicroRNAs miRNAs are a kind of important non-coding small RNA molecules, which play an important role in animal reproduction. MiRNAs affect the reproductive function of animals by regulating the expression of target genes. In order to further reveal the regulatory effect of mi RNA on reproductive function of dairy goats, the miRNA library of ovarian differential expression was constructed in the early stage of multilambs (3-5 lambs / fetus) and single lambs (1 lamb / fetus) of dairy goats during estrus. Real time-PCR was used to detect the differential expression of chi-miR-4110 in ovaries during estrus and bioinformatics in Guanzhong dairy goats. Luciferase vector and Western blotting were used to verify the target gene of chi-mi R-4110 and its regulation on target gene, which provided experimental and theoretical basis for improving lambing rate of dairy goats. The main results obtained in this study are as follows: 1) the expression level of 1: 1 chi-miR-4110 in ovaries of multi-lamb and single-lamb dairy goats using bioinformatics. Chi-miR-4110, which plays an important role in the regulation of reproductive traits and has significant differential expression, was screened from the miRNA library of ovarian differential expression. The relative expression of chi-miR-4110 in the ovaries of multiple lambs and single lamb dairy goats was detected by Real time-PCR. The results showed that chi-miR-4110 was expressed in the ovaries of multi-lamb dairy goats. The expression of P0.01 was significantly higher in the ovaries of the single lamb dairy goat than that in the single lamb dairy goat. It is suggested that chi-miR-4110 may play an important role in the regulation of lambing traits in dairy goats. The prediction and verification of target genes of chi-miR-4110 by bioinformatics were used to screen the target gene Smad2 related to ovarian development. A double luciferase vector containing Smad2-3'UTR region was constructed and co-transfected with chi-miR-4110 into 293T cells. Luciferase detection system was used to detect luciferase activity. The results showed that the luciferase activity in the co-transfected chi-miR-4110 group was significantly lower than that in the negative control group, indicating that the luciferase activity of chi-miR-4110 decreased through the interaction with Smad2-3'UTR. Smad2 was identified as the target gene of chi-miR-4110. The regulation of the target gene. Chi-miR-4110 and control were transfected into the ovarian granulosa cells of dairy goat respectively. The expression of Smad2 mRNA and Smad2 protein were detected by Real time-PCR and Western blotting. The results showed that the expression of Smad2 mRNA and Smad2 protein was detected in the granulosa cells of dairy goat ovary. Overexpression of chi-miR-4110 significantly reduced the expression of Smad2 mRNA and Smad2 protein, suggesting that chi-miR-4110 plays a negative role in Smad2 regulation at post-transcriptional level. These results suggest that Smad2 is a target gene of chi-mi R-4110, and it regulates the expression of Smad2 in dairy goat ovarian granulosa cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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