本文選題：chi-miR-4110 + Smad2��； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：MicroRNA(miRNA)是一類重要的非編碼小RNA分子,在動物繁殖過程中發(fā)揮著重要的調(diào)控作用。miRNA通過調(diào)節(jié)靶基因的表達(dá)影響動物的繁殖機(jī)能。為進(jìn)一步揭示mi RNA對奶山羊生殖機(jī)能的調(diào)控作用,本研究在前期構(gòu)建的多羔(3-5羔/胎)和單羔(1羔/胎)奶山羊發(fā)情期卵巢差異表達(dá)miRNA文庫的基礎(chǔ)上,以關(guān)中奶山羊?yàn)檠芯繉ο?利用Real time-PCR檢測chi-miR-4110在多羔和單羔奶山羊發(fā)情期卵巢中的差異表達(dá)水平,并利用生物信息學(xué)、熒光素酶載體和Western blotting等技術(shù)驗(yàn)證chi-mi R-4110靶基因及其對靶基因的調(diào)控作用,為提高奶山羊產(chǎn)羔率提供試驗(yàn)依據(jù)和理論基礎(chǔ)。本研究獲得的主要結(jié)果如下:1、chi-miR-4110在多羔和單羔奶山羊卵巢組織中的表達(dá)水平利用生物信息學(xué),從卵巢差異表達(dá)miRNA文庫中篩選對繁殖性狀有重要調(diào)控作用且差異表達(dá)顯著的chi-miR-4110,采用Real time-PCR檢測chi-miR-4110在多羔和單羔奶山羊卵巢中的相對表達(dá)量顯示,chi-miR-4110在多羔奶山羊卵巢中的表達(dá)量極顯著(P0.01)高于單羔奶山羊卵巢中的表達(dá)量,表明chi-miR-4110可能對奶山羊產(chǎn)羔性狀有重要的調(diào)控作用。2、chi-miR-4110靶基因的預(yù)測及驗(yàn)證通過應(yīng)用生物信息學(xué)對chi-miR-4110的靶基因進(jìn)行綜合分析,篩選出與卵巢發(fā)育相關(guān)的靶基因Smad2。構(gòu)建含有Smad2-3'UTR區(qū)的雙熒光素酶載體,與chi-miR-4110共轉(zhuǎn)染到293T細(xì)胞中,利用熒光素酶檢測系統(tǒng)檢測熒光素酶的活性。結(jié)果顯示與chi-miR-4110共轉(zhuǎn)染的試驗(yàn)組的熒光素酶活性顯著低于陰性對照,表明chi-miR-4110通過與Smad2-3'UTR相互作用導(dǎo)致熒光素酶活性降低,初步鑒定Smad2為chi-miR-4110的靶基因。3、chi-miR-4110對靶基因的調(diào)控將chi-miR-4110和control分別轉(zhuǎn)染到奶山羊卵巢顆粒細(xì)胞中,利用Real time-PCR和Western blotting檢測Smad2 mRNA以及Smad2蛋白的表達(dá)量,結(jié)果表明,在奶山羊卵巢顆粒細(xì)胞中,過表達(dá)chi-miR-4110使得Smad2 mRNA以及Smad2蛋白的表達(dá)量均顯著降低,表明chi-miR-4110在轉(zhuǎn)錄后水平對Smad2起負(fù)調(diào)控的作用。以上研究結(jié)果顯示,Smad2是chi-mi R-4110的靶基因,在奶山羊卵巢顆粒細(xì)胞中,chi-miR-4110靶向調(diào)控Smad2的表達(dá)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S827

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相關(guān)期刊論文 前1條

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本文編號：1860826