基于高通量测序的尼罗罗非鱼（Oreochomis niloticus）性别二态性研究

发布时间：2018-01-07 05:28

本文关键词：基于高通量测序的尼罗罗非鱼（Oreochomis niloticus）性别二态性研究　出处：《山西农业大学》2016年博士论文　论文类型：学位论文

【摘要】：性别二态性是有性繁殖动物的普遍现象。虽然雌性和雄性在形态、生理、行为等方面有明显的差别,但是它们分享着几乎一致的基因组,性别二态性的形成与不同基因的表达有关。miRNA作为转录后水平调控基因表达的小RNA,参与性别二态性的形成。性腺是有性繁殖动物产生配子和类固醇激素的重要的繁殖器官,由于不同的发育通路,精巢和卵巢呈现出明显的形态上的二态性,它们经常作为研究mRNA和miRNA表达的靶器官。因此,从转录组水平探索精巢和卵巢差异表达的基因和miRNA,对于解析雌雄间的差异及揭示该差异形成的分子机理具有重要作用。尼罗罗非鱼是世界性养殖鱼类,其雄鱼比雌鱼生长快30%,全雄鱼的养殖具有更好的经济价值。在研究性别二态性方面,尼罗罗非鱼具有成熟时间短,产卵周期短的优点,且罗非鱼全基因组测序已经完成并全面公开,具有良好的基因组资源。因而,以罗非鱼为材料开展有关性别二态性的研究,可以兼顾基础与应用两个方面。本研究以尼罗罗非鱼为实验材料,采用新一代高通量测序技术在转录组水平上对雌雄性腺miRNA表达谱和mRNA数字化表达谱进行分析。通过miRNA-seq寻找罗非鱼性别差异显著的miRNA,对其靶基因进行预测。通过标签的数字基因表达系统(DGE)研究雌雄性腺转录水平的变化,以期得到大量的差异表达的基因和Pathway。通过二者的关联分析,可以在转录水平和转录后水平寻找性别决定的新的通路。探讨雌雄激素对性腺miRNA的影响,解析：miRNAs介导的调控网络在鱼类性别决定中的作用机制。本研究主要结果如下：(1)尼罗罗非鱼性腺组织mRNA表达谱的性别二态性研究取60日龄尼罗罗非鱼雌雄鱼各6尾,分别构建精巢和卵巢组织RNA文库,利用新一代高通量测序技术对其精巢和卵巢组织差异表达基因进行分析,结果表明,在精巢和卵巢中分别有16909和13495个基因表达。在精巢中表达量最高的基因有：1个40S核糖体蛋白和4个60S核糖体蛋白,精浆糖蛋白和热休克蛋白。在卵巢中表达量最高的基因有：泛素偶联酶2C、卵母细胞特异性连接组蛋白、两种脂肪酸结合蛋白和配子体特异性因子。这些高表达的基因呈现出精巢和卵巢完全不同的发育途径。在精巢和卵巢之间共发现有5559个差异表达基因,与精巢相比,2229个基因在卵巢中上调,3260个基因在卵巢中下调。对差异表达基因进行功能注释,发现这些差异表达基因主要被富集到代谢相关通路外,还有部分基因参与繁殖生物学相关的通路。(2)尼罗罗非鱼性腺组织miRNA组的性别二态性研究利用RNA-seq技术对以上尼罗罗非鱼的精巢和卵巢的小RNA文库进行了测序,分析miRNA在精巢和卵巢中的表达模式。结果表明,在精巢和卵巢组织的小RNA文库中分别获得了26675871和13030780个clean reads 。精巢和卵巢差异表达miRNAs的数量为152个,差异显著的为76个,在卵巢中显著高表达的miRNA为67个,在精巢中显著高表达的为9个。在表达的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,这些差异表达的miRNA以及特有的SNP有助于性别二态性的形成。(3)尼罗罗非鱼性腺组织miRNA组与mRNA组的联合分析利用Allen和Schwab的方法预测差异表达的miRNA的靶基因,利用Venny软件对差异表达基因与靶基因取交集,得到5555个交集基因。根据miRNA与mRNA间的负调控关系,获得miRNA与靶基因的数据集,降低每个差异表达miRNA的靶基因范围。对交集基因进行KEGG pathway分析,对类固醇激素生成通路的差异表达基因与相关miRNA进行荧光定量和相关性分析。结果得到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对在序列上具有互补同时表达量也显著负相关的靶标关系。这些差异表达的miRNA抑制性别相关的靶基因的表达进而促进尼罗罗非鱼性别二态性的形成。(4)雌二醇和甲基睾酮对尼罗罗非鱼性腺组织miRNA表达的影响用雌二醇和甲基睾酮处理尼罗罗非鱼的雌雄鱼,研究这两种激素对精巢和卵巢中差异表达的8个miRNA (miR-122、miR-214、miR-202、let-7b、let-7h、miR-17-5p、miR-200和miR-143)的影响,结果表明,总体水平仍然呈现在卵巢中的表达量显著高于精巢。雌二醇处理均引起精巢和卵巢这8个miRNA的显著降低。甲基睾酮处理后,在雌鱼miRNA表达的总体趋势是先显著降低,随着时间的推移再缓慢升高；在雄鱼,引起miRNA表达大幅度降低。同时对这些miRNA的表达量与性别特异的基因(Cypl9ala、 Foxl2和Dmrt1)表达量进行相关性分析,结果为：Cypl9ala和Foxl2的表达量与8个性别特异性的miRNA的表达量显著正相关,而Dmrtl的表达量与这8个miRNA的表达量显著负相关,且这些miRNA之间彼此也显著相关。性类固醇激素可以影响性别相关的]miRNA的表达变化,从而影响尼罗罗非鱼的性别二态性。
[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.

【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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