生物钟调控缢蛏夜间产卵的生理和分子机制研究

Spawning is a key step in the reproductive process of oviparous animals，which is crucial for successful fertilization and offspring reproduction. The process of spawning is often affected by external environmental factors and endogenous neuro-hormones. Most of sexually mature mollusks would be stimulated to spawn by raising or lowering temperature, drying, running water and other suitable environment, while Sinonovacula constricta could only spawn in specific time at night. Circadian clock might be involved in the regulating nocturnal spawning time of Sinonovacula constricta. In previous studies, we preliminarily found that the diurnal changes of sex hormones in testicles and ovaries were consistent with the diurnal expression of some circadian clock genes(Per、Clock、Tim) . However, the key genes of the ovarian circadian clock and the molecular mechanisms of regulating nocturnal spawning are still unknown. We plan to carry out the following studies in the proposed project: analyze sex hormones and 17β-HSDs diurnal changes in testicles and ovaries of Sinonovacula constricta during sexual maturation; based on whole genome and comparative transcriptome, screen and identify key circadian clock genes; analyze expression changes of the circadian clock genes and proteins at mRNA and protein levels by qRT-PCR, Western blot and immunohistochemical techniques, and RNAi and overexpression techniques will be used to verify gene function. The main aim of this project is to reveal the molecular mechanisms of nocturnal spawning of Sinonovacula constricta, which will provide the theoretical basis for optimizing the factory seedling technology.

产卵是卵生动物繁殖过程的关键一步，对成功受精和后代繁衍至关重要，而产卵过程往往受到外界环境因子和内源神经-激素影响。对于绝大多数性成熟贝类，受到升降温、阴干、流水等适宜环境刺激即会产卵，而缢蛏则只能在夜间特定时间段产卵，推测其受生物钟控制。申请人前期研究发现，缢蛏卵/精巢中性激素分泌量呈昼夜节律变化，且与Per、Clock、Tim等生物钟基因昼夜表达量变化趋势一致，然而卵巢生物钟关键基因及其调控夜间产卵的分子机制至今尚不清楚。本项目拟开展：分析成熟性腺中性激素及关键酶17β-HSDs的昼夜变化规律；基于全基因组和比较转录组，初筛卵巢生物钟基因；用qRT-PCR、Western blot和免疫组化技术，检测关键基因mRNA和表达蛋白的变化，用RNAi和过表达技术验证基因功能；研究RNA甲基化对生物钟基因的调控。本项目旨在揭示生物钟调控缢蛏夜间产卵的分子机制，为优化工厂化育苗技术提供理论依据。

产卵是卵生动物繁殖过程的关键一步，对成功受精和后代繁衍至关重要，而产卵过程往往受到外界环境因子和内源神经-激素影响。大多数性成熟贝类受到升降温、阴干、流水等环境刺激即会产卵，而缢蛏则只在夜间特定时间段产卵，推测其受卵巢生物钟控制，然而卵巢生物钟关键基因及其调控夜间产卵的分子机制尚不清楚。本项目分析了缢蛏生理生化指标的昼夜变化规律；基于卵巢昼/夜比较转录组分析，筛查和鉴定了卵巢生物钟关键基因；基于缢蛏性腺多组学联合分析，鉴定了调控性腺发育、产卵的关键基因/蛋白/代谢物及代谢通路；用qRT-PCR和免疫荧光技术，检测关键基因mRNA和蛋白的表达变化，并用RNA干扰和过表达技术验证基因/蛋白的功能。项目自实施以来，取得了一系列重要成果：①发现缢蛏摄食率、消化酶活/基因表达、心率、性激素含量均存在昼低夜高的变化规律，表明缢蛏是夜行性动物，性激素含量的昼夜变化可能是导致夜间产卵的关键因素；②初次开展了缢蛏卵巢昼/夜比较转录组分析，鉴定了Bmal1、Period、Timeless等产卵相关生物钟基因及KEGG通路，表明卵巢生物钟参与调控缢蛏的夜间产卵；③系统开展了生物钟基因Bmal1、Period、Timeless的克隆、表达、细胞定位及与性激素分泌的关联性分析，并用RNA干扰技术初步探究了其对夜间产卵的调控作用；④率先基于缢蛏雌、雄性腺多组学（转录组+蛋白组+代谢组）联合分析，鉴定了调控性腺发育、产卵的关键基因/蛋白/代谢物及代谢通路等；⑤系统研究了性别相关基因ScSox9、ScFem-1b、ScFem-1c、ScDmrt4/5-1和ScDmrt4/5-2及蛋白在性腺发育中的表达变化，并用RNA干扰和过表达技术探究了其在性腺发育、产卵中的功能；对缢蛏17β-HSDs基因家族成员进行了鉴定和表达分析，表明其可能通过介导性激素代谢调控性腺发育和产卵，为深入解析缢蛏夜间产卵的分子机制提供了有价值参考。本项目已发表SCI论文3篇，申请发明专利1项。该项目系统研究了生物钟调控缢蛏夜间产卵的生理和分子机制，将对缢蛏及其他贝类产卵分子机制研究和工厂化育苗育种实践具有重要应用价值和借鉴意义。