性分化前后雌雄鸡胚中差异表达miRNA的鉴定与分析
发布时间：2019-03-27 13:32
【摘要】：鸡是一种重要的农业动物和模式生物。目前鸡的性别决定及分化机制尚未明确。近年来性别决定的体细胞自主性假说认为,个体性别早在性腺分化前已决定。基于此观点及mi RNA在动物发育中的重要调控作用,本研究采用二代高通量测序技术鉴定性分化前后(孵化2.5天和4.5天)鸡胚中表达的mi RNA,对测序结果进行了一系列的表达特性及靶基因功能分析,并采用定量PCR方法验证部分差异表达mi RNA,以期在mi RNA水平寻找鸡性别决定的早期作用因子,为进一步对重要候选mi RNA的深入研究提供基础资料。本研究获得的主要结果如下:(1)使用mi RDeep*将mi RNA比对到鸡基因组上,结果表明各样本中mi RNA种类在各染色体上的分布相似,但2.5天雄性鸡胚中Z染色体mi RNA种类比雌性多;通过多数据库序列比对,在2.5天雌、雄及4.5天雌、雄样本中分别鉴定出1687、1791、1288和1355个mi RNA,其中已知的鸡mi RNA分别为388(2.5d-f)、405(2.5d-m)、336(4.5d-f)和322(4.5d-m)个。(2)使用DESeq分别对2.5和4.5天同时期雌雄样本的已知mi RNA进行差异表达分析,共发现42个差异表达显著的mi RNA,其中有3个mi RNA在两个时期持续差异表达:gga-mi R-2954为雄性高表达,gga-mi R-6606-5p为雌性高表达,gga-mi R-6552-3p由雄性高表达转为雌性高表达。(3)对42个差异表达mi RNA进行靶基因预测和靶基因的GO基因功能富集分析和KEGG信号通路分析,发现wnt信号通路、泛素介导的蛋白水解,TGF-β信号途径,聚焦粘附,血管平滑肌收缩通路,MAPK信号通路等可能参与性别分化。在2个时期均富集显著的GO分类有20个,这些GO大致分为这几类:关于磷的代谢、基因表达调控、生物合成调控、发育、蛋白与酶的活性。(4)采用荧光定量PCR技术对16个mi RNA进行了表达验证并与测序结果进行比较,结果表明2.5天和4.5天分别有13和10个定量结果与测序结果的差异趋势基本一致。
[Abstract]:Chicken is an important agricultural animal and model creature. At present, the sex determination and differentiation mechanism of chicken are not clear. In recent years, the somatic autonomy hypothesis of sex determination suggests that individual sex has been determined before gonadal differentiation. Based on this point of view and the important role of mi RNA in animal development, the second generation high throughput sequencing technique was used to identify the expression of mi RNA, in chicken embryos before and after differentiation (2.5d and 4.5d). A series of expression characteristics and functional analysis of target genes were carried out by sequencing results, and partial differential expression of mi RNA, was verified by quantitative PCR in order to find out the early acting factors of sex determination in chicken at mi RNA level. It provides the basic data for the further study of important candidate mi RNA. The main results obtained in this study are as follows: (1) mi RNA was compared to chicken genome using mi RDeep*, and the results showed that the distribution of mi RNA species in each sample was similar on each chromosome. However, there were more mi RNA species on Z chromosome in male embryos than in females at 2. 5 days. Through multi-database sequence alignment, 1687, 1791, 1288 and 1355 mi RNA, were identified in the female, male and 4.5d female samples, respectively. The known chicken mi RNA was 388 (2.5d-f) and 405 (2.5d-m), respectively, in which the known chicken mi RNA was 388 (2.5d-f) and 405( 2.5d-m), respectively, and the male samples were 1687, 1791, 1288 and 1355 respectively. (4.5d-f) and 322 (4.5d-m). (2) DESeq was used to analyze the known mi RNA of the female and male samples at the same period of 2.5 and 4.5 days, respectively. A total of 42 mi RNA, with significant difference were found. Three of them continued to be differentially expressed at two stages: gga-mi Rx2954 was male and gga-mi RX6606p was highly expressed in the female, and the expression of mi RNA was significantly higher than that of the control group (P < 0. 05). (3) 42 differentially expressed mi RNA were detected by target gene prediction, functional enrichment analysis of GO gene and KEGG signaling pathway analysis of target gene. Wnt signaling pathway was found in 42 differentially expressed mi RNA. Ubiquitin-mediated proteolysis, TGF- 尾 signaling pathway, focusing adhesion, vascular smooth muscle contraction pathway and MAPK signaling pathway may be involved in sex differentiation. There are 20 GO classifications with significant enrichment in both stages. These GO are roughly classified into these categories: metabolism of phosphorus, regulation of gene expression, regulation of biosynthesis, regulation of development, regulation of metabolism of phosphorus, regulation of gene expression, regulation of biosynthesis, regulation of development, and so on. The activity of protein and enzyme. (4) the expression of 16 mi RNA was verified by fluorescence quantitative PCR (FQ-PCR) and compared with the result of sequencing. The results showed that there were 13 and 10 quantitative results on 2.5 days and 4.5 days, respectively, which were consistent with the results of sequencing.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
本文编号：2448228
[Abstract]:Chicken is an important agricultural animal and model creature. At present, the sex determination and differentiation mechanism of chicken are not clear. In recent years, the somatic autonomy hypothesis of sex determination suggests that individual sex has been determined before gonadal differentiation. Based on this point of view and the important role of mi RNA in animal development, the second generation high throughput sequencing technique was used to identify the expression of mi RNA, in chicken embryos before and after differentiation (2.5d and 4.5d). A series of expression characteristics and functional analysis of target genes were carried out by sequencing results, and partial differential expression of mi RNA, was verified by quantitative PCR in order to find out the early acting factors of sex determination in chicken at mi RNA level. It provides the basic data for the further study of important candidate mi RNA. The main results obtained in this study are as follows: (1) mi RNA was compared to chicken genome using mi RDeep*, and the results showed that the distribution of mi RNA species in each sample was similar on each chromosome. However, there were more mi RNA species on Z chromosome in male embryos than in females at 2. 5 days. Through multi-database sequence alignment, 1687, 1791, 1288 and 1355 mi RNA, were identified in the female, male and 4.5d female samples, respectively. The known chicken mi RNA was 388 (2.5d-f) and 405 (2.5d-m), respectively, in which the known chicken mi RNA was 388 (2.5d-f) and 405( 2.5d-m), respectively, and the male samples were 1687, 1791, 1288 and 1355 respectively. (4.5d-f) and 322 (4.5d-m). (2) DESeq was used to analyze the known mi RNA of the female and male samples at the same period of 2.5 and 4.5 days, respectively. A total of 42 mi RNA, with significant difference were found. Three of them continued to be differentially expressed at two stages: gga-mi Rx2954 was male and gga-mi RX6606p was highly expressed in the female, and the expression of mi RNA was significantly higher than that of the control group (P < 0. 05). (3) 42 differentially expressed mi RNA were detected by target gene prediction, functional enrichment analysis of GO gene and KEGG signaling pathway analysis of target gene. Wnt signaling pathway was found in 42 differentially expressed mi RNA. Ubiquitin-mediated proteolysis, TGF- 尾 signaling pathway, focusing adhesion, vascular smooth muscle contraction pathway and MAPK signaling pathway may be involved in sex differentiation. There are 20 GO classifications with significant enrichment in both stages. These GO are roughly classified into these categories: metabolism of phosphorus, regulation of gene expression, regulation of biosynthesis, regulation of development, regulation of metabolism of phosphorus, regulation of gene expression, regulation of biosynthesis, regulation of development, and so on. The activity of protein and enzyme. (4) the expression of 16 mi RNA was verified by fluorescence quantitative PCR (FQ-PCR) and compared with the result of sequencing. The results showed that there were 13 and 10 quantitative results on 2.5 days and 4.5 days, respectively, which were consistent with the results of sequencing.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
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1 刘超;哺乳动物性别控制研究进展[J];黄牛杂志;1997年01期
2 杨秀荣;蒋和生;杨宁;;鸟类性别决定与性别分化机制[J];遗传;2012年04期
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