性分化前后雌雄雞胚中差異表達(dá)miRNA的鑒定與分析
發(fā)布時(shí)間：2019-03-27 13:32
【摘要】：雞是一種重要的農(nóng)業(yè)動物和模式生物。目前雞的性別決定及分化機(jī)制尚未明確。近年來性別決定的體細(xì)胞自主性假說認(rèn)為,個體性別早在性腺分化前已決定�；诖擞^點(diǎn)及mi RNA在動物發(fā)育中的重要調(diào)控作用,本研究采用二代高通量測序技術(shù)鑒定性分化前后(孵化2.5天和4.5天)雞胚中表達(dá)的mi RNA,對測序結(jié)果進(jìn)行了一系列的表達(dá)特性及靶基因功能分析,并采用定量PCR方法驗(yàn)證部分差異表達(dá)mi RNA,以期在mi RNA水平尋找雞性別決定的早期作用因子,為進(jìn)一步對重要候選mi RNA的深入研究提供基礎(chǔ)資料。本研究獲得的主要結(jié)果如下:(1)使用mi RDeep*將mi RNA比對到雞基因組上,結(jié)果表明各樣本中mi RNA種類在各染色體上的分布相似,但2.5天雄性雞胚中Z染色體mi RNA種類比雌性多;通過多數(shù)據(jù)庫序列比對,在2.5天雌、雄及4.5天雌、雄樣本中分別鑒定出1687、1791、1288和1355個mi RNA,其中已知的雞mi RNA分別為388(2.5d-f)、405(2.5d-m)、336(4.5d-f)和322(4.5d-m)個。(2)使用DESeq分別對2.5和4.5天同時(shí)期雌雄樣本的已知mi RNA進(jìn)行差異表達(dá)分析,共發(fā)現(xiàn)42個差異表達(dá)顯著的mi RNA,其中有3個mi RNA在兩個時(shí)期持續(xù)差異表達(dá):gga-mi R-2954為雄性高表達(dá),gga-mi R-6606-5p為雌性高表達(dá),gga-mi R-6552-3p由雄性高表達(dá)轉(zhuǎn)為雌性高表達(dá)。(3)對42個差異表達(dá)mi RNA進(jìn)行靶基因預(yù)測和靶基因的GO基因功能富集分析和KEGG信號通路分析,發(fā)現(xiàn)wnt信號通路、泛素介導(dǎo)的蛋白水解,TGF-β信號途徑,聚焦粘附,血管平滑肌收縮通路,MAPK信號通路等可能參與性別分化。在2個時(shí)期均富集顯著的GO分類有20個,這些GO大致分為這幾類:關(guān)于磷的代謝、基因表達(dá)調(diào)控、生物合成調(diào)控、發(fā)育、蛋白與酶的活性。(4)采用熒光定量PCR技術(shù)對16個mi RNA進(jìn)行了表達(dá)驗(yàn)證并與測序結(jié)果進(jìn)行比較,結(jié)果表明2.5天和4.5天分別有13和10個定量結(jié)果與測序結(jié)果的差異趨勢基本一致。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S831
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S831
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 劉超;哺乳動物性別控制研究進(jìn)展[J];黃牛雜志;1997年01期
2 楊秀榮;蔣和生;楊寧;;鳥類性別決定與性別分化機(jī)制[J];遺傳;2012年04期
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