池蝶蚌性别分化相关环境因子及Fem-1和Tra-2基因家族的表达调控研究

Diversity of sex differentiation exhibited in the different animals, especially in the inferior animals. The sex differentiation was easily influenced by environmental factors (such as temperature). Fresh water shellfish occupy an important place in evolution and ecological system, and to be known its the sex differentiation was easily influenced by natural environment. However, the key environmental factors which can affect sex differentiation was unclear. There is no systematic study about sex differentiation mechanisms in fresh water shellfish. Accordingly, we selected the freshwater pearl mussel Hyriopsis schlegelii as experiment object to research. Based on the sequence information from the spermary and ovary tissue transcriptomes and the previous study that special hermaphroditic phenomenon could be observed in certain stage of sexual gland development, we would investigate the sex differentiation mechanisms of the Hyriopsis schlegelii. The content of this research includes there parts: First, some factors about the influence of different ages, temperature, and pH on the sex differentiation of Hyriopsis schlegelii will be investigated. Second, the expression characteristic of two gene families (Fem-1a，Fem-1b，Fem-1c, Tra-2α，and Tra-2β) related to hermaphrodite will be determined. Third, the cascade relationship between Tra-2 and Fem-1 family will be investigated, and the related signal pathways will be analyzed. The important regulatory mechanism of sex determination genes on the Hyriopsis schlegelii could be understand by mastering the environmental factor influencing on the sex differentiation for fresh water shell.

性别分化在不同动物中表现出多样性，尤其是低等生物的性别分化极易受到环境因素比如温度等的影响。淡水贝类在进化地位以及生态系统中占有重要地位，已知其性别分化易受环境影响，而影响其性别分化的关键环境因子以及相关联基因的表达调控机制仍无系统研究。因此，本研究拟以优质淡水珍珠蚌池蝶蚌为对象，以我们前期获得其雌雄两性性腺转录组高通量测序信息、以及所发现在特定阶段出现特殊的雌雄同体现象为基础，开展性别分化调控机制研究。研究内容包括：1、观察不同发育年龄、温度、pH等重要环境因子对其性别分化的影响；2、该因子对与性别相关重要基因家族Fem-1和Tra-2（Fem-1a，Fem-1b，Fem-1c和Tra-2α，Tra-2β）的表达特征进行研究；3、开展Fem-1和Tra-2互作与级联反应研究，分析其可能的信号通路。通过研究掌握影响池蝶蚌性别分化的关键环境因子，进一步认识相关的重要性别决定基因的调控机制。

本研究以优质淡水珍珠蚌池蝶蚌为对象，以前期获得其雌雄两性性腺转录组高通量测序信息、以及所发现在特定阶段出现特殊的雌雄同体现象为基础，开展性别分化调控机制研究，取得了以下结果：.1）对池蝶蚌成熟配子进行了形态学研究，在池蝶蚌性腺组织切片观察到池蝶蚌从26月龄到32月龄，性腺中出现雌、雄性滤泡同时存在的雌雄同体现象。.2）首次通过使用Iso-seq和RNA-seq相结合，生成了不同性别池蝶蚌H.schlegelii的全长转录组；.3）通过一系温度刺激，研究了温度胁迫的雌雄间性腺的差异表达基因（DEGs）。这些数据不仅清楚地反映了性腺的转录组动力学响应温度胁迫，而且还将进一步阐明性腺发育的可能适应机制；.4）获得了4个性别相关基因，feminization-1基因的b、c两个亚型（Hsfem-1b、Hsfem-1c）、Transformer两个亚型（Transformer2α、Transformer 2β），初步分析了其结构特征；检测了这4个性别相关基因在多个组织中及不同发育年龄阶段性腺中的表达；开展了不同环境因子（温度、17β-雌二醇和曲洛斯坦）胁迫下，池蝶蚌性腺中fem-1b和Tra2α基因的表达，合成敲降fem-1b和Tra2α基因表达的siRNA，探究siRNA干扰技术在池蝶蚌性别决定研究中的应用；.5）首次克隆了两个凋亡抑制基因IAP（定义为HsIAP-1及HsIAP-2）基因并进行了系统分析；对IAP基因进行了组织表达分析，进行了重组HsIAP-1的功能研究；.6）研究了与雄性生殖细胞发育相关的Dmrt1基因。初步探究了池蝶蚌性腺发育过程中温度与Dmrt1的因果关系；研究了性别相关基因Dmrt1在性腺早期阶段及雌雄同体阶段的时空表达，表明池蝶蚌可能发生性逆转或雌雄同体现象的阶段是4、5月龄-36、37月龄。.本研究到目前已基本清楚了控制性别的相关基因和因素，并已开始进行全人工性别调控技术研究，为获得更多优质珍珠提供理念和技术依据。