17β-雌二醇介导下大口黑鲈性别二态性表达基因Dmrt1的调控机制研究

Dmrt1 is one of the most conserved genes related to sex determination in fish. In previous studies, Dmrt1 was specifically overexpressed in the testis of large-mouthed bass, while the expression of Dmrt1 was upregulated during E2-induced feminization of largemouth bass (LMB). We speculate that E2 may affect the sex differentiation and testicular development of LMB by mediating the regulation of Dmrt1 expression. Further, this project intends to: study the changes of Dmrt1 expression induced by E2 and ER antagonists in vivo and in vitro during the whole reproductive cycle by intraperitoneal injection and incubation of primary germ cell of testes, and observe the growth and development of testis tissues and cells; construct luciferase reporter gene vector of contact promoter sequence of Dmrt1, and detect promoter activity under the treatment with different doses of E2 and ER antagonists; identify the core promoter region and potential estrogen response elements of Dmrt1 in LMB by serially truncated constructs, along with site-directed mutagenesis, EMSA and ChIP techniques; preliminarily explore other non-classical transcriptional regulation pathways. The purpose of this study was to improve the research on the molecular functions and regulatory networks of sex differentiation and testicular development of LMB, and lay the foundation for achieving sex control precisely and promoting industrial development of LMB ultimately.

Dmrt1是鱼类中已知的最为保守的一个与性别决定相关的基因。前期研究中，Dmrt1在大口黑鲈精巢中特异性高表达；E2诱导大口黑鲈雌性化过程中Dmrt1的表达受到抑制。我们推测E2可能通过介导Dmrt1的表达调控影响大口黑鲈性别分化和精巢发育。本课题拟进一步：利用腹腔注射以及精巢原代生殖细胞孵育，研究整个生殖周期中，E2、ER拮抗剂在体和离体诱导下Dmrt1的表达变化，并观测精巢组织、细胞的生长和发育情况；构建Dmrt1完整启动子序列荧光素酶报告基因载体，检测不同剂量E2、ER拮抗剂处理对大口黑鲈Dmrt1启动子活性的影响；通过启动子系列截短实验，结合定点突变、EMSA和ChIP等技术，鉴定大口黑鲈Dmrt1的核心启动子区域及潜在的雌激素反应元件；并初步探讨其它非经典转录调控途径。本研究旨在完善大口黑鲈性别分化、精巢发育的相关分子功能及调控网络，为实现性别精准控制最终促进产业发展提供依据。

鱼类的性别决定和分化是遗传因素和环境因子共同作用的结果。Dmrt1是目前已知的鱼类中最为保守的一个与性别决定相关的基因，在大口黑鲈中呈现精巢特异性表达。E2可诱导大口黑鲈雌性化，且该过程中Dmrt1的表达受到抑制，从而推测E2可能通过介导Dmrt1的表达调控影响大口黑鲈性别分化和精巢发育。E2是如何介导Dmrt1的表达调控过程？其具体的作用机制是什么？本项目对Dmrt1进行了基因组的定位、多基因组比对和共线性分析，为鱼类Dmrt基因家族的功能分析提供了分子基础；阐明了大口黑鲈生殖细胞的发生和性别分化时期，确立了性别分化诱导的关键时间点，为建立诱导大口黑鲈雌性化的方法提供了依据；在确立了大口黑鲈性别分化点的基础上，以丰年虫为E2载体，利用雌激素处理技术对其性别发育进行诱导，建立了大口黑鲈雌性化的方法；同时，利用E2、ER拮抗剂在体和离体诱导大口黑鲈精巢组织，确立E2、ER拮抗剂在体和离体诱导的最适浓度，证实E2处理通过ER介导抑制大口黑鲈Dmrt1的表达；构建了以pGL3-Basic为载体的3个系列截短的Dmrt1启动子荧光素酶报告基因载体 pGL3-1016bp、pGL3-792bp、pGL3-388bp，利用双荧光素酶系统检测证实大口黑鲈Dmrt1核心启动子区域及潜在的雌激素反应元件位于转录起始位点上游-792—-388bp处；完成3个正常卵巢与3个性反转卵巢共6个样品的转录组测序，其中正常卵巢与3个性反转卵巢差异表达基因200个，与正常卵巢相比，性反转卵巢中有42个基因上调，158个基因下调，为探讨其他非经典转录调控途径提供了转录组学证据。本研究对于完善大口黑鲈性别分化、精巢发育的相关分子功能及调控网络，还可为实现性别精准控制最终促进产业发展提供依据。