脊椎动物促泌乳素（prolactin）适应性进化的分子基础研究

Prolactin (PRL) is an anterior pituitary synthesized and secreted hormone in vertebrates, which has been reported to influence over 300 physiological actions, such as stimulating production, lactogenesis, growth, development, osmoregulation, metabolism, etc. In teleosts, PRL was known as "freshwater-adapting hormone" to regulate the ion conservation and water secretion processes. We have knock-out the zebrafish PRL via transcription activator-like effector nucleases (TALEN) and two independent mutant lines were obtained. We found that the process of Na+, K+, and Cl- ion uptake was severely affected in PRL-deficient embryos and all these embryos died at 6-16 days post fertilization. However, in brackish water (5175 mg/L ocean salt), PRL-deficient embryos can survive, thrive through adulthood without obvious physiological defects. In this project, in situ hybridization, qPCR, western-blot and ELISA will be employed to examine PRL target genes expression, PRL downstream JAK-STAT signaling pathway and PRL level in response to ion concentrations. Then we will answer if PRL still has a conserved role in regulating ion uptake in different species in vertebrates, such as human, mouse, xenopus, common carp, black porgy and so on. Ultilizing analysis of bioinformations, we also want to discover if PRL evolve adaptively in freshwater fishes to answer if selection drives PRL evolve faster. Finally, we focused on the role of PRL in sea fishes. Thus, we will generate black porgy (Acanthopagrus schlegelii) PRL knock-out lines to study its function in sea fish via TALEN strategy. This study should provide invaluable insights into the relationship between PRL stimulated ion-uptake in different species in vertebrates and freshwater adaptive process in different species.

促泌乳素(Prolactin, PRL)是脊椎动物脑垂体前叶腺体合成与分泌的一种重要的多肽类激素，已被报道了生殖和泌乳、生长和发育、内分泌和代谢等300多种功能；在硬骨鱼类中被认为是“淡水适应激素”，在调节离子吸收方面具有重要功能。我们已获得PRL敲除的斑马鱼品系，发现其表现了典型的Na+、K+、Cl-的离子吸收缺陷；在常规培养液中培养，会在6-16天内全部死亡，但若在5175mg/L的海盐中则可以存活、生长至性成熟，并且与正常斑马鱼无显著差异。本研究拟利用生物信息学分析和我们已经获得的斑马鱼品系，回答PRL在淡水环境的选择压力下是否显示了某些分子进化特征，并获得其他物种PRL在调节离子吸收方面的功能认识；探讨进化过程中，不同物种的PRL及其靶基因和JAK-STAT信号通路对盐离子浓度变化的响应差异，拟回答淡水适应性是否加速了PRL序列的正向进化；并期望挖掘海水鱼中PRL执行的功能。

鱼类的垂体分泌的促泌乳素Prolactin(PRL)在维持体内离子吸收方面不可或缺。在前期的工作中，我们学科组发现了prl的敲除斑马鱼的鳃部slc12a10.2和肾脏的slc12a3等离子通道表达量显著降低，相应的，其体内的Na+、K+、Cl-的离子浓度显著降低，造成prl纯合子斑马鱼在6-16天时死亡。为了验证哺乳动物的PRL是否具有促进离子吸收的功能，我们用人的PRL重组蛋白和人的prl mRNA对prl纯合子斑马鱼进行注射和浸泡，但未能检测到具有挽救效果，说明人的PRL重组蛋白不能挽救prl纯合子斑马鱼的离子吸收。.但鱼类对外界离子的吸收和调控机制的探索一直是本项目所关注的。因此，我们也对PRL信号通路的离子通道和其相关的信号通路和分子的敲除品系斑马鱼进行了分析，从而解析PRL信号通路及其以外调节离子吸收的内分泌途径及调控机制。我们对PRL的靶基因，鳃部表达的slc12a10.2和肾脏表达的slc12a3，分别进行了敲除（二者均属于slc12a家族，且都被报道负责Na+和Cl-的吸收，因此其二者的敲除若存在差异，则可说明鱼类机体对离子吸收的调控存在多样性），并对纯合子进行了生理学分析和比较。我们首先在slc12a10.2和slc12a3的纯合子中，均发现垂体prl的表达均显著上升，提示对二者的敲除是有效的。鳃部表达的slc12a10.2和肾脏表达的slc12a3虽然被报道具有相同的功能（负责Na+和Cl-的吸收），但二者的纯合子的Na+含量虽然与对照组并无显著变化，但二者仍表现了众多不同的表型：slc12a10.2的纯合子在9-17天时由于Cl-的缺乏导致死亡，但slc12a3的纯合子并无缺Cl-的表型，且可正常生长，并至性成熟繁殖。通过对机制的解析，发现slc12a3的纯合子中，PRL的下游靶基因slc9a3.2、slc12a10.2和atp1a1a.5在肾脏中均发生补偿性上调；而PRL下游靶基因在肾脏中的补偿性上调在slc12a10.2的纯合子中却没有出现。总之，我们揭示了鱼类在应对不同的渗透压吸收压力时存在多样化的响应策略，并首次提出了PRL的下游离子通道的表达和功能具有对上游的促泌乳素Prolactin的响应选择性。.本项目资助经费为24.00万元，实际支出为23.0979万元；发表相关SCI论文4篇；培养在读博士研究生1名（舒婷婷博士）。