【作者】 刘杰；

【导师】 程汉华； 周荣家；

【作者基本信息】 武汉大学 ， 细胞生物学， 2014， 博士

【摘要】 鱼类作为最古老的脊椎动物,占据了脊椎动物物种中大约一半的数量。鱼类生殖方式的研究对脊椎动物物种的形成和演化研究具有重要理论意义；同时,鱼类性别决定和分化的人工控制对渔业养殖具有重要的实践意义。microRNA (miRNA)是一类由内源基因编码的长度约22个碱基的非编码单链小RNA分子,广泛存在于真核生物。成熟miRNA通过结合到mRNA 3’UTR靶位点,从而在转录后水平促进mRNA降解或者抑制蛋白翻译。研究显示miRNA参与了生殖细胞发生、配子形成、受精直至胚胎发育等生殖过程。黄鳝在进化中具有原始的地位,并且发育过程中天然地经历由雌性向雄性的性逆转过程,因此它是研究比较基因组学和性别分化的一种很好的模式物种。本研究通过高通量测序和miRNA芯片方法,分析了黄鳝性逆转过程中性腺差异表达的miRNA I口斑马鱼雌雄性腺差异表达的miRNA,并对这两种鱼类性腺差异的miRNA做了比较研究,取得了以下主要结果：1.通过高通量测序获得了1119个黄鳝性腺相关的miRNA分子,包括195个保守的miRNA和924个新的miRNA。分析显示,195个保守的miRNA对应133个miRNA基因,定位在234条前体序列上,其中有65个miRNA基因对应2条及以上的前体序列。保守的miRNA分子组成了84个miRNA家族,这些家族大多在脊椎动物中保守。新发现的924条miRNA由791个miRNA基因编码,定位在1372条前体序列上。2.黄鳝195个保守miRNA中,在雌性、雄性和间性性腺中均表达的miRNA有180个,其中42个miRNA具有间性偏好性(23.33%),12个miRNA具有雄性偏好性(6.67%),13个miRNA具有雌性偏好性(7.22%)。miRNA的差异表达分析显示黄鳝间性向雄性转变的过程中,miRNA表达活跃。3.通过miRNA芯片从斑马鱼成熟雌雄性腺中检测到了123个miRNA具有性别差异表达。黄鳝和斑马鱼性腺miRNA比较分析显示,77个miRNA分子在性腺中的表达具有保守性,其中10个miRNA分子表达十分丰富,57个miRNA分子在两种鱼类雌雄性腺中表达趋势一致,20个miRNA分子在两种鱼类雌雄性腺中表达趋势相反。原位杂交显示miR-125b, miR-205, miR-130c在斑马鱼和黄鳝性腺发育中的卵细胞和不同发育时期的精子细胞中表达,但其表达具有一定的差异性。4.miR-17-92基因簇在斑马鱼和黄鳝基因组上都具有2个连锁群。荧光定量PCR检测显示miR-17-92基因簇在两种鱼类成体组织中广谱表达,在卵巢中的表达高于精巢。在斑马鱼胚胎时期miR-17-92基因簇的表达呈逐渐上升的趋势。采用锁核苷酸探针(LNA)原位杂交验证了miR-17a-5p在性腺和胚胎期的表达。进一步采用生物信息学分析了miR-17-92基因簇的下游靶基因,荧光素酶报告基因分析证实了miR-17-92基因簇对性别分化相关的靶基因Wnt4a和Dmrtl具有负调控作用。更多还原

【Abstract】 Teleost fish as the most ancient vertebrate represents approximately half of the vertebrate species. Study of fish reproductive strategies may provide theoretical guidance for elucidating speciation and evolution of vertebrates, and practical implications to develop techniques for artificial control of fish sex. MicroRNAs (miRNAs) are an abundant family of small regulatory RNAs, which are endogenous non-coding molecules with about 22 nucleotides in length. The mature miRNAs exert down-regulation role at post-transcriptional level by degradation mRNA or by translation inhibition. The miRNA pathways are involved in regulations of reproductive process, expanding from germ cell biogenesis, gametogenesis to fertilization and embryo development.The swamp eel(Monopterus albus) is a good model for comparative genomic studies and sexual determination/differentiation, for the reason of its primitive evolutionary status and natural occurring sex reversal from female into male via intersex during its life cycle. In this study, the miRNome during the gonadal differentiation and the differential expressions of gonadal miRNAs were analyzed by high-throughput sequencing and miRNA array in swamp eel and zebrafish (Danio rerio). The main results are as follows:1. Firstly, we obtained 1119 mature miRNAs in gonads of the swamp eel by Illumina Solexa sequencing.195 miRNAs were conserved and 924 miRNAs were novel. The 195 miRNAs were classified as 133 conserved miRNA genes which located on 234 pre-miRNAs. Among them,65 miRNA genes located on more than one pre-miRNAs. The conserved miRNAs were clustered into 84 miRNA families, most of which were evolutionarily conserved in vertebtates.924 novel miRNAs were classified as 791 miRNA genes, which located on 1372 pre-miRNAs.2.180 from 195 conserved miRNAs were co-expressed among ovary,ovotestis and testis in swamp eel, while 42 miRNAs were intersex-biased (23.33%),12 miRNAs were male-biased (6.67%) and 13 miRNAs were female-biased (7.22%). Analysis of differential expression showed that the miRNAs become active during the gonadal-reversal, especially in the prosess of testis restructuring from the ovotestis.3. miRNA array analysis showed 123 differentially expressed miRNAs in mature ovary and testis of zebrafish. By contrast,77 miRNAs were co-expressed in gonads of both fishes.10 miRNAs were expressed abundantly, and 57 miRNAs of 77 co-expressed miRNAs exhibited coincident expression trends between the ovary and testis, while 20 miRNAs displayed opposite tendencies. In situ hybridization of miR-125b, miR-205 and miR-130c showed positive signals in developing ovary and different types of sperm cells during spermatogenesis with a certain difference in expression.4. Quantitative realtime PCR indicated that miR-17-92 cluster are ovary biased expressed in both two fishes. Expressions of six members in miR-17-92 cluster were upregulated during the embryogeny of zebrafish. Downstream target genes of miR-17-92 cluster were further analyzed through bioinformatic tools, and two direct target genes involved in the process of gonad differentiation were identified by luciferase reporter assay, in which Wnt4a was down-regulated by miR-17a/20a and Dmrtl was down-regulated by miR-19a/19b.更多还原