本文關鍵詞：基于高通量測序的尼羅羅非魚（Oreochomis niloticus）性別二態(tài)性研究　出處：《山西農業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 尼羅羅非魚 性別二態(tài)性 性腺 microRNA mRNA RNA-seq 雌二醇 甲基睪酮

【摘要】：性別二態(tài)性是有性繁殖動物的普遍現(xiàn)象。雖然雌性和雄性在形態(tài)、生理、行為等方面有明顯的差別,但是它們分享著幾乎一致的基因組,性別二態(tài)性的形成與不同基因的表達有關。miRNA作為轉錄后水平調控基因表達的小RNA,參與性別二態(tài)性的形成。性腺是有性繁殖動物產生配子和類固醇激素的重要的繁殖器官,由于不同的發(fā)育通路,精巢和卵巢呈現(xiàn)出明顯的形態(tài)上的二態(tài)性,它們經常作為研究mRNA和miRNA表達的靶器官。因此,從轉錄組水平探索精巢和卵巢差異表達的基因和miRNA,對于解析雌雄間的差異及揭示該差異形成的分子機理具有重要作用。尼羅羅非魚是世界性養(yǎng)殖魚類,其雄魚比雌魚生長快30%,全雄魚的養(yǎng)殖具有更好的經濟價值。在研究性別二態(tài)性方面,尼羅羅非魚具有成熟時間短,產卵周期短的優(yōu)點,且羅非魚全基因組測序已經完成并全面公開,具有良好的基因組資源。因而,以羅非魚為材料開展有關性別二態(tài)性的研究,可以兼顧基礎與應用兩個方面。本研究以尼羅羅非魚為實驗材料,采用新一代高通量測序技術在轉錄組水平上對雌雄性腺miRNA表達譜和mRNA數(shù)字化表達譜進行分析。通過miRNA-seq尋找羅非魚性別差異顯著的miRNA,對其靶基因進行預測。通過標簽的數(shù)字基因表達系統(tǒng)(DGE)研究雌雄性腺轉錄水平的變化,以期得到大量的差異表達的基因和Pathway。通過二者的關聯(lián)分析,可以在轉錄水平和轉錄后水平尋找性別決定的新的通路。探討雌雄激素對性腺miRNA的影響,解析：miRNAs介導的調控網絡在魚類性別決定中的作用機制。本研究主要結果如下：(1)尼羅羅非魚性腺組織mRNA表達譜的性別二態(tài)性研究取60日齡尼羅羅非魚雌雄魚各6尾,分別構建精巢和卵巢組織RNA文庫,利用新一代高通量測序技術對其精巢和卵巢組織差異表達基因進行分析,結果表明,在精巢和卵巢中分別有16909和13495個基因表達。在精巢中表達量最高的基因有：1個40S核糖體蛋白和4個60S核糖體蛋白,精漿糖蛋白和熱休克蛋白。在卵巢中表達量最高的基因有：泛素偶聯(lián)酶2C、卵母細胞特異性連接組蛋白、兩種脂肪酸結合蛋白和配子體特異性因子。這些高表達的基因呈現(xiàn)出精巢和卵巢完全不同的發(fā)育途徑。在精巢和卵巢之間共發(fā)現(xiàn)有5559個差異表達基因,與精巢相比,2229個基因在卵巢中上調,3260個基因在卵巢中下調。對差異表達基因進行功能注釋,發(fā)現(xiàn)這些差異表達基因主要被富集到代謝相關通路外,還有部分基因參與繁殖生物學相關的通路。(2)尼羅羅非魚性腺組織miRNA組的性別二態(tài)性研究利用RNA-seq技術對以上尼羅羅非魚的精巢和卵巢的小RNA文庫進行了測序,分析miRNA在精巢和卵巢中的表達模式。結果表明,在精巢和卵巢組織的小RNA文庫中分別獲得了26675871和13030780個clean reads 。精巢和卵巢差異表達miRNAs的數(shù)量為152個,差異顯著的為76個,在卵巢中顯著高表達的miRNA為67個,在精巢中顯著高表達的為9個。在表達的miRNA中有大量的SNP存在,有些是精巢和卵巢共有的,有些是精巢或卵巢特有的,這些差異表達的miRNA以及特有的SNP有助于性別二態(tài)性的形成。(3)尼羅羅非魚性腺組織miRNA組與mRNA組的聯(lián)合分析利用Allen和Schwab的方法預測差異表達的miRNA的靶基因,利用Venny軟件對差異表達基因與靶基因取交集,得到5555個交集基因。根據(jù)miRNA與mRNA間的負調控關系,獲得miRNA與靶基因的數(shù)據(jù)集,降低每個差異表達miRNA的靶基因范圍。對交集基因進行KEGG pathway分析,對類固醇激素生成通路的差異表達基因與相關miRNA進行熒光定量和相關性分析。結果得到miR-456/Amh、miR-138/Amh、 miR-17-5p/Dmrt1、miR-20a/Dmrtl、miR-138/Cypl7a2、miR-338/Cyp17a2和miR-200a/ Cyp17a2共7對在序列上具有互補同時表達量也顯著負相關的靶標關系。這些差異表達的miRNA抑制性別相關的靶基因的表達進而促進尼羅羅非魚性別二態(tài)性的形成。(4)雌二醇和甲基睪酮對尼羅羅非魚性腺組織miRNA表達的影響用雌二醇和甲基睪酮處理尼羅羅非魚的雌雄魚,研究這兩種激素對精巢和卵巢中差異表達的8個miRNA (miR-122、miR-214、miR-202、let-7b、let-7h、miR-17-5p、miR-200和miR-143)的影響,結果表明,總體水平仍然呈現(xiàn)在卵巢中的表達量顯著高于精巢。雌二醇處理均引起精巢和卵巢這8個miRNA的顯著降低。甲基睪酮處理后,在雌魚miRNA表達的總體趨勢是先顯著降低,隨著時間的推移再緩慢升高；在雄魚,引起miRNA表達大幅度降低。同時對這些miRNA的表達量與性別特異的基因(Cypl9ala、 Foxl2和Dmrt1)表達量進行相關性分析,結果為：Cypl9ala和Foxl2的表達量與8個性別特異性的miRNA的表達量顯著正相關,而Dmrtl的表達量與這8個miRNA的表達量顯著負相關,且這些miRNA之間彼此也顯著相關。性類固醇激素可以影響性別相關的]miRNA的表達變化,從而影響尼羅羅非魚的性別二態(tài)性。
[Abstract]:Two state of sex is a common phenomenon in sexual reproduction animal. Although the male and female in morphology, physiology, behavior and other aspects have obvious difference, but they share almost the same as the.MiRNA genome, regulation of gene expression in post transcriptional expression of small RNA form the gender two states with different genes involved in the formation. Two states. Sex gonad is an important reproductive organ of the sexual reproduction of animal produce gametes and steroid hormones, due to the development of different pathways, testis and ovary showed two distinct patterns of the state, they often make the target organ for the study of mRNA expression and miRNA. Therefore, exploring gene and expression of miRNA in testis and the difference from the ovarian transcriptome, plays an important role in analysing the difference between male and female and reveal the molecular differences in the formation of the mechanism. The Nile tilapia is the world of fish, the male than female The fish grow nearly 30%, all male breeding has better economic value. In the study of gender on two states, the Nile tilapia has mature in short time, spawning cycle shorter and tilapia genome sequencing has been completed and fully open, with good genomic resources. Therefore, the research on gender for tilapia two states the development of materials, both basic and application two aspects. In this study, Nile tilapia as experimental materials, the transcriptional level of sexual expression of miRNA spectrum analysis and mRNA digital expression profiling using a new generation of high-throughput sequencing technology. Through the miRNA-seq miRNA looking for tilapia significant gender differences, the prediction of the the expression of target genes. By digital gene tag (DGE) changes of gonad at transcriptional level. In order to get a large number of differentially expressed genes and Pathway. by two Analysis of the association, looking for sex determination in transcriptional and post transcriptional analysis of a new pathway. To investigate the effects of estrogen and androgen on gonadal miRNA: mechanism of miRNAs mediated regulation network in fish sex determination. The main results of this study are as follows: (1) tilapia gonad tissue mRNA expression study on gender two state spectra of 60 day old male Nile tilapia fish tail 6, testis and ovarian tissue RNA library were constructed using a new generation of high-throughput sequencing gene expression analysis, the difference of the testis and ovary tissues. The results showed that there were 16909 and 13495 genes expressed in testis and ovary the highest expression level in testis. The gene is 1 40S ribosomal protein and 4 60S ribosomal protein, seminal plasma glycoprotein and heat shock protein in the ovary. The highest expression level of genes: the ubiquitin conjugating enzyme 2C, oocyte Cell specific linker histone, two kinds of fatty acid binding protein and gametophyte specific factor. These highly expressed genes showing developmental pathways of testis and ovary. Completely different between testis and ovary were found 5559 differentially expressed genes, and the testis compared up-regulated and 2229 genes in ovary, down 3260 a gene in the ovary. The expression of genes for functional annotation of differences, found that these differentially expressed genes were mainly enriched to the metabolism pathway, and some genes involved in pathways related to reproductive biology. (2) the Nile tilapia gland tissue in group miRNA the gender two states using the RNA-seq technology of Nile tilapia and testis ovary small RNA library by sequencing, expression pattern analysis of miRNA in testes and ovaries. The results show that the small RNA Library in testis and ovary tissue were obtained for 26675871 And 13030780 clean reads. The expression of testis and ovary difference of miRNAs number is 152, significant differences between the 76, in the ovary was significantly higher expression of miRNA was 67, significantly higher expression in the testis was 9. The expression of SNP in the presence of a large number of miRNA, some testis and ovary in common, some testis or ovary specific, these differentially expressed miRNA and unique SNP contribute to the formation of gender in two states. (3) the joint analysis of Nile tilapia gonad tissue of miRNA group and mRNA group by using the method of Allen and Schwab prediction difference expression of the target gene miRNA and target gene expression. Take the intersection of different genes by using Venny software, get the 5555 intersection. According to the miRNA gene between mRNA and negative regulation between miRNA and target gene data set, reduce the difference of each target gene expression miRNA gene by KEGG pathway of the intersection. Analysis of differences on the steroidogenic pathway and related gene expression were analyzed by fluorescence quantitative miRNA and correlation. Results miR-456/Amh, miR-138/Amh, miR-17-5p/Dmrt1, miR-20a/Dmrtl, miR-138/Cypl7a2, miR-338/Cyp17a2 and miR-200a/ Cyp17a2 in a total of 7 sequences with complementary expression and a significant negative relationship. The target expression of these differentially expressed miRNA inhibiting target gene related to sex and gender and promote the formation of Nile Tilapia on two states. (4) estradiol and testosterone on the expression of Nile tilapia gonad tissue with miRNA estradiol and testosterone treatment of Nile tilapia and fish, to study the two kinds of hormones on 8 miRNA differentially expressed in testis and ovary (miR-122 miR-214, miR-202, let-7b, let-7h, miR-17-5p, miR-200, and miR-143) effects, results show that the overall level is still in the egg The expression of the nest was significantly higher than that in testis. Estradiol treatment caused significant reduction in testis and ovary. The 8 miRNA methyl testosterone treatment, the overall trend in the female miRNA expression is significantly reduced, with the passage of time and then slowly increased; in males, induced the expression of miRNA is greatly reduced. At the same time, the expression of gender specific of these miRNA genes (Cypl9ala, Foxl2 and Dmrt1) expression by correlation analysis. The results are as follows: significant positive correlation expression of Cypl9ala and Foxl2 with 8 sex-specific miRNA, while the expression of Dmrtl and the 8 miRNA had a significant negative correlation, and the miRNA between each other are significantly related. The expression of sex steroid hormones can influence]miRNA sex related, thus affecting the Nile tilapia sex two state.

【學位授予單位】：山西農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S917.4

【參考文獻】

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1 謝兆輝;李學貴;許y斏，

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