山羊排卵卵泡与从属卵泡中差异表达miRNAs的筛选及其功能研究

发布时间：2018-05-01 21:37

  本文选题：山羊 + 卵泡　； 参考：《西南大学》2017年硕士论文

【摘要】：卵泡排卵是有着诸多因子参与调控的生殖生理过程,它决定着母畜的排卵数,影响着母畜的繁殖性能。微小RNA(mircoRNA,或miRNA)是内源性非编码小分子RNA,研究发现miRNA对卵泡的生长发育、闭锁和黄体形成与消失等过程均有着重要作用,但在卵泡排卵上的研究报道还相对较少。本试验以大足黑山羊的排卵卵泡与从属卵泡为实验材料,通过Illumina测序、生物信息学分析及qPCR表达验证等方法,来筛选与卵泡发育及排卵有关的miRNAs。主要实验结果如下:1、将大足黑山羊卵泡期卵泡按直径大小分为六组(GF1~GF6),对其进行sRNA高通量测序,平均得到了12,069,414拷贝的原始序列及11,876,770拷贝的干净序列。其中长度在22nt的sRNA序列平均占干净序列的45%,通过将其在参考物种山羊基因组上的定位与miRBase数据库(21.0版本)的比对,共获得418个已知miRNAs序列。同时,利用miREvo及mirdeep等miRNA预测软件预测到110个新的miRNAs序列。2、将GF1~GF6的六组卵泡分为排卵卵泡组(GF_d)和从属卵泡组(GF_s)进行表达差异分析,结果发现9个miRNAs存在差异(P0.01),其中4个miRNAs(chi-miR-582-5p、novel_130、chi-miR-214-3p及chi-miR-500-5p)在GF_d组中表达上调,而5个miRNAs(chi-miR-383、chi-miR-130b-5p、chi-miR-92a-3p、chi-miR-125b-5p及novel-9)表达下调。为筛选更多差异表达miRNAs,本试验对六组卵泡进行组内差异分析,发现novel-344、chi-miR-10a-5p、chi-mi R-10a-3p及chi-miR-196b在GF1中表达上调(P0.01)。3、利用miRanda软件及富集通路分析发现,在上述13个显著差异表达miRNAs中,其预测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,在排卵卵泡组与从属卵泡组中差异表达的chi-miR-130b-5p及chi-miR-214-3p靶向于BMP15、GDF9、CYP19A1和LHR基因。同时,novel-344和chi-miR-10a-5p与在颗粒细胞与膜细胞中表达的FSHR、GDF9、IGF1R、17β-HSD和LHR基因的mRNA 3’UTR序列有结合位点。4、对同批次GF_d和GF_s样品进行转录组测序,结果发现上述预测靶基因BMP15、CYP1A1、17β-HSD、IGF1R和LHR在GF_d中均表达上调,而GDF9和FSHR表达下调。其中,BMP15与chi-miR-130b-5p、FSHR与novel-344以及GDF9与chi-miR-214-3p和chi-miR-10a-5p的上下调表达模式相反,符合miRNAs的一般调控特征。5、选取差异表达的6个miRNAs(chi-miR-200a、chi-miR-34c-5p、chi-miR-10a-3p、chi-miR-10a-5p、novel-344和chi-miR-196b)及3个mRNA(BMP15、GDF9和BMP6)进行qPCR表达鉴定,实验结果与Illumina测序分析中得到的表达模式一致。本试验筛选出了山羊排卵卵泡与从属卵泡中差异表达的miRNAs,这些miRNAs与其预测的靶基因可能参与卵泡中的激素合成过程,从而影响卵泡发育与排卵。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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