黄颡鱼性腺分化的转录组学证据

Sex determination is very complex reproductive and evolutionary process that involves the interaction among sex determination genes and a series of sex-related genes which are initiated by sex-determining genes in genetic pathway during critical periods of gonadal development. Thus, understanding the genetic regulation of gonadal sex differentiation is of great importance in studies of fish sex determination. However, those sex determination genes cannot be totally screened by traditional candidate gene approach, in turn, little is known about the interaction between those sex determination genes. Whereas, transcriptome sequencing technique can obtain gene expression mapping at whole genome scale and proceed the quantitative study of the gene expression differences at transcriptomic level，it may elucidate polygenetic sex determination pattern of fish. Significant differences of size and growth rate exist in yellow catfish (Pelteobagrus fulvidraco) male and female siblings at same age under the same condition. More energy should be devoted to clarify the genetic mechanism of sex determination in order to improve the method of yellow catfish breeding and reproduction. Up to date, genetic regulation mechanism of gonadal sex differentiation in yellow fish at transcriptomic level is still unknown. Here, using yellow catfish from full-sib families, we monitor and analyze histological structure and the pattern of dynamic characteristics of the sex steroid hormone levels at different stages of gonadal development. Gene expression profiling in different periods of gonadal development could then be obtained for male and female yellow catfish at transcriptomic level. Dominant genes controlling the gonadal sex differentiation will be screened and testified. This study will provide evidence of genetic mechanism for gonadal development and differentiation processes at transcriptomic level for fish.

鱼类性别决定是一个复杂的生物发育和进化过程，是由性别决定基因启动一系列性别相关基因共同参与的级联过程。性腺性别分化的基因调控机制对研究鱼类性别决定至关重要。传统候选基因法无法筛选出所有与性别分化相关的基因，更无法阐述性别决定这一级联过程。转录组测序技术可以实现全基因组表达图谱绘制和基因转录水平差异性定量研究，为阐明性别多基因决定模式提供可能。同龄黄颡鱼(Pelteobagrus fulvidraco)生长速度存在显著性别差异性，研究性别决定遗传机制将有助于黄颡鱼人工繁育的进行。目前，国内外在转录组水平上对黄颡鱼性腺发育及分化的分子调控机制研究未见报道。本研究选择全同胞家系黄颡鱼为研究对象，通过对性腺发育不同时期组织结构和性类固醇激素水平动态特征的研究，从转录组水平上获得雌雄黄颡鱼性腺发育的基因差异表达图谱，筛选并验证出控制性腺分化的主效基因，为性腺发育及分化的分子调控机制提供转录组学证据。

本项目开展了黄颡鱼受精卵的孵化和实验鱼的养殖，收集研究标本，开展了性腺不同发育时期的组织学观察研究；确定早期性腺分化发育的关键期，从而确定黄颡鱼性别决定、性别差异及重叠区的各个临界时期的取样点，并完成几个重要分化期转录组测序样本的采集；完成基于Illumina Hiseq2000的转录组测序，并利用转录组分析软件对短片段序列进行de novo 序列组装，获得性腺不同发育时间的基因差异表达谱，并进行GO和PATHWAY 等基因功能分析；利用比较基因组方法对转录组水平上得到的雌雄黄颡鱼的基因和雌雄沟鲶的全基因组序列分别进行比对，最终确定雌雄黄颡鱼的基因列表；综合转录组和比较基因组所得到的结果，筛选出调控黄颡鱼性别分化的主效基因；验证筛选出调控性别分化主效基因的工作，包括Real-time PCR 的验证和代谢蛋白差异验证，同时进一步确定调控黄颡鱼性别分化的主效基因。本课题利用现有的最先进的高通量测序技术结合生物信息学的数据装配和分析鉴定了黄颡鱼的性腺及脑转录组中性别分化相关功能基因，为黄颡鱼基因资源的开发奠定了坚实的基础。课题利用de novo序列组装技术进行高效准确地转录本拼接。结合已有的序列装配软件，开创了更适合鱼类基因组全基因组序列的技术路线和流程。课题获得了许多与性别相关的差异表达基因及特异表达基因，为黄颡鱼性别决定机制的理解提供了重要信息。本课题对分子标记的挖掘分析及表达谱的构建为进一步的研究黄颡鱼性别决定基因提供了重要的基因资源，同时对其它鱼类基因资源的开发具有参考价值。本课题发表论文7篇，其中SCI论文5篇，中文核心期刊2篇，授权实用新型专利3项，共培养硕士研究生2名并成功取得硕士学位，研究内容与本项目直接密切相关。