本文選題：山羊 + 卵泡�。� 參考：《西南大學(xué)》2017年碩士論文

【摘要】：卵泡排卵是有著諸多因子參與調(diào)控的生殖生理過(guò)程,它決定著母畜的排卵數(shù),影響著母畜的繁殖性能。微小RNA(mircoRNA,或miRNA)是內(nèi)源性非編碼小分子RNA,研究發(fā)現(xiàn)miRNA對(duì)卵泡的生長(zhǎng)發(fā)育、閉鎖和黃體形成與消失等過(guò)程均有著重要作用,但在卵泡排卵上的研究報(bào)道還相對(duì)較少。本試驗(yàn)以大足黑山羊的排卵卵泡與從屬卵泡為實(shí)驗(yàn)材料,通過(guò)Illumina測(cè)序、生物信息學(xué)分析及qPCR表達(dá)驗(yàn)證等方法,來(lái)篩選與卵泡發(fā)育及排卵有關(guān)的miRNAs。主要實(shí)驗(yàn)結(jié)果如下:1、將大足黑山羊卵泡期卵泡按直徑大小分為六組(GF1~GF6),對(duì)其進(jìn)行sRNA高通量測(cè)序,平均得到了12,069,414拷貝的原始序列及11,876,770拷貝的干凈序列。其中長(zhǎng)度在22nt的sRNA序列平均占干凈序列的45%,通過(guò)將其在參考物種山羊基因組上的定位與miRBase數(shù)據(jù)庫(kù)(21.0版本)的比對(duì),共獲得418個(gè)已知miRNAs序列。同時(shí),利用miREvo及mirdeep等miRNA預(yù)測(cè)軟件預(yù)測(cè)到110個(gè)新的miRNAs序列。2、將GF1~GF6的六組卵泡分為排卵卵泡組(GF_d)和從屬卵泡組(GF_s)進(jìn)行表達(dá)差異分析,結(jié)果發(fā)現(xiàn)9個(gè)miRNAs存在差異(P0.01),其中4個(gè)miRNAs(chi-miR-582-5p、novel_130、chi-miR-214-3p及chi-miR-500-5p)在GF_d組中表達(dá)上調(diào),而5個(gè)miRNAs(chi-miR-383、chi-miR-130b-5p、chi-miR-92a-3p、chi-miR-125b-5p及novel-9)表達(dá)下調(diào)。為篩選更多差異表達(dá)miRNAs,本試驗(yàn)對(duì)六組卵泡進(jìn)行組內(nèi)差異分析,發(fā)現(xiàn)novel-344、chi-miR-10a-5p、chi-mi R-10a-3p及chi-miR-196b在GF1中表達(dá)上調(diào)(P0.01)。3、利用miRanda軟件及富集通路分析發(fā)現(xiàn),在上述13個(gè)顯著差異表達(dá)miRNAs中,其預(yù)測(cè)靶基因富集到卵巢激素合成通路(chx04913)的有chi-miR-130b-5p、chi-miR-214-3p、novel-344及chi-miR-10a-5p。其中,在排卵卵泡組與從屬卵泡組中差異表達(dá)的chi-miR-130b-5p及chi-miR-214-3p靶向于BMP15、GDF9、CYP19A1和LHR基因。同時(shí),novel-344和chi-miR-10a-5p與在顆粒細(xì)胞與膜細(xì)胞中表達(dá)的FSHR、GDF9、IGF1R、17β-HSD和LHR基因的mRNA 3’UTR序列有結(jié)合位點(diǎn)。4、對(duì)同批次GF_d和GF_s樣品進(jìn)行轉(zhuǎn)錄組測(cè)序,結(jié)果發(fā)現(xiàn)上述預(yù)測(cè)靶基因BMP15、CYP1A1、17β-HSD、IGF1R和LHR在GF_d中均表達(dá)上調(diào),而GDF9和FSHR表達(dá)下調(diào)。其中,BMP15與chi-miR-130b-5p、FSHR與novel-344以及GDF9與chi-miR-214-3p和chi-miR-10a-5p的上下調(diào)表達(dá)模式相反,符合miRNAs的一般調(diào)控特征。5、選取差異表達(dá)的6個(gè)miRNAs(chi-miR-200a、chi-miR-34c-5p、chi-miR-10a-3p、chi-miR-10a-5p、novel-344和chi-miR-196b)及3個(gè)mRNA(BMP15、GDF9和BMP6)進(jìn)行qPCR表達(dá)鑒定,實(shí)驗(yàn)結(jié)果與Illumina測(cè)序分析中得到的表達(dá)模式一致。本試驗(yàn)篩選出了山羊排卵卵泡與從屬卵泡中差異表達(dá)的miRNAs,這些miRNAs與其預(yù)測(cè)的靶基因可能參與卵泡中的激素合成過(guò)程,從而影響卵泡發(fā)育與排卵。
[Abstract]:Follicular ovulation is a reproductive physiological process controlled by many factors, which determines the ovulation number of female animals and affects the reproductive performance of female animals. MicroRNAs mirco RNAs or miRNAs are endogenous non-coding small RNAs. It has been found that miRNA plays an important role in follicular growth and development, atresia and luteal formation and disappearance, but there are few studies on follicular ovulation. The ovulatory follicles and subordinate follicles of Dazu black goat were used as experimental materials. Illumina sequencing, bioinformatics analysis and qPCR expression validation were used to screen miRNAs related to follicle development and ovulation. The main results were as follows: 1. The follicles of Dazu Black Goat were divided into six groups according to their diameters. The sRNA high-throughput sequencing showed that 12069414 copies of the original sequence and 11876770 copies of the clean sequence were obtained. Among them, the sRNA sequence with length in 22nt accounted for 45% of the clean sequence on average, and 418 known miRNAs sequences were obtained by comparing their location on the goat genome of reference species with the miRBase database (version 21. 0). At the same time, 110 new miRNAs sequences .2were predicted by miRNA prediction software such as miREvo and mirdeep. The six groups of GF1~GF6 follicles were divided into ovulatory follicles group (P < 0.01) and subordinate follicles group (P < 0.05). The results showed that there was a significant difference in 9 miRNAs, including 4 miRNAschi-miR-582-5pNovelsThe expression of miRNAschi-miR-214-3p and chi-miR-500-5p) was up-regulated in GF_d group, while the expression of 5 miRNAs-Aschi-miR-383chi-miR-130b-5pchi-miR-92a-92a-3pchi-125b-5p and Novel-9) was down-regulated. In order to screen more differentially expressed miRNAss, we analyzed the differences in the six groups of follicles. The results showed that Novel-344nchi-miR-10a-5pnchi-mi R-10a-3p and chi-miR-196b were up-regulated in GF1. The results of miRanda software and enrichment pathway analysis showed that, among the 13 significantly differentially expressed miRNAs, Novel-344nchi-miR-10a-5pnchi-mi and chi-miR-196b were up-regulated in GF1. The predicted target genes were chi-miR-130b-5pnchi-miR-214-3pNovel-344 and chi-miR-10a-5pNovel-344 and chi-miR-10a-5p. The differential expression of chi-miR-130b-5p and chi-miR-214-3p in ovulatory follicles and subordinate follicles was targeted at CYP19A1 and LHR genes of BMP15 GDF9. At the same time, Novel-344 and chi-miR-10a-5p had binding sites .4 to the mRNA 3'UTR sequence of the IGF1Rn17 尾 -HSD and LHR gene expressed in granulosa cells and membrane cells. The transcriptome sequencing of the same batch of GF_d and GF_s samples showed that the expression of BMP15A117 尾 -HSDIGF1R and LHR were up-regulated in GF_d. The expression of GDF9 and FSHR was down-regulated. The down-regulated expression patterns of BMP15 and chi-miR-130b-5pFSHR and novel-344, GDF9 and chi-miR-214-3p and chi-miR-10a-5p were opposite, which were consistent with the general regulatory characteristics of miRNAs. The qPCR expression patterns of 6 miRNAschi-miR-200achi-miR-34c-5pchi-miR-10a-3pchi-miR-10a-5pNovel-344 and chi-miR-196b) and three mRNABMP15GDF9 and BMP6 were identified. The results were consistent with the expression patterns obtained by Illumina sequencing. In this study, the differential expression of miRNAsin in ovulation follicles and subordinate follicles in goats was screened. These miRNAs and their predicted target genes may be involved in hormone synthesis in follicles, thus affecting follicle development and ovulation.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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