缢蛏生物钟基因Tim的鉴定、昼夜表达模式及对性激素分泌的影响  

Identification and circadian expression patterns of circadian clock gene Tim and its effects on sex hormone secretion in Sinonovacula constricta

作　　者：郦琪钧 郑向阳 柳燕子 严玉箫 陈妍蕾 姚韩韩 董迎辉 Li Qijun;Zheng Xiangyang;Liu Yanzi;Yan Yuxiao;Chen Yanlei;Yao Hanhan;Dong Yinghui(Zhejiang Key Laboratory of Aquatic Germplasm Resources,College of Biological&Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,China;Ninghai Marine Biological Seed Industry Research Institute,Ninghai 315604,China)

机构地区：[1]浙江万里学院生物与环境学院浙江省水产种质资源高效利用技术研究重点实验室,浙江宁波315100 [2]宁海海洋生物种业研究院,浙江宁海315604

出　　处：《海洋学报》2023年第12期101-111,共11页

基　　金：国家自然科学青年基金项目(31902393);宁波市重大科技攻关暨“揭榜挂帅”项目(2021Z114);宁波市公益性科技计划项目(2022S168);国家海洋水产种质资源库项目(2023);浙江省一流学科“生物工程”学生创新项目(CX2023007)。

摘　　要：Tim(Timeless)是继Per基因发现后的第2个核心生物钟基因,在动物繁殖调控中发挥重要作用。为探讨生物钟基因Tim对缢蛏夜间产卵的调控作用,本研究克隆了缢蛏Tim基因的cDNA全长序列并进行序列分析,利用qRT-PCR和免疫荧光技术研究了Tim基因/蛋白在不同组织中的昼夜表达模式,并用siRNA干扰技术初步研究了Tim基因对性激素分泌的影响。结果显示,缢蛏Tim基因开放阅读框序列为3 090 bp,编码1 029个氨基酸,具备3个保守结构域(TIMELESS superfamily、TIMELESS_C和FIil);Tim基因在缢蛏各个组织中均有表达,其中在肝胰腺、水管、卵巢、精巢中的表达水平较高;Tim基因在卵巢和精巢中呈现昼夜规律性表达,均在白天12:00–18:00较高,夜间00:00–06:00较低;Tim蛋白主要定位在肝胰腺的肝细胞、水管末端的上皮细胞、卵巢的成熟卵母细胞及精巢的精子细胞中,且在卵巢和精巢中的阳性信号均是白天12:00稍强于夜间00:00;siRNA干扰后,Tim基因在精巢、卵巢中的表达量均在干扰后第3-7天显著下降(P <0.05),而精巢中睾酮和卵巢中雌二醇含量均在干扰后第1-9天极显著下降(P <0.01)。由此可见,Tim基因/蛋白在精巢和卵巢中的表达均呈昼夜规律性变化,并且其被干扰后性激素含量显著降低,推测Tim基因可能通过介导性激素的分泌进而调控缢蛏夜间产卵过程。本研究初步探讨了缢蛏生物钟基因Tim的昼夜表达模式及其对性激素分泌的影响,将对深入解析贝类产卵机制和优化工厂化育苗技术具有重要应用价值。Tim(Timeless)is the second core circadian clock gene identified after the discovery of Per,which plays an important role in the regulation of animal reproduction.In order to explore the molecular mechanisms of Tim to regulate spawning in Sinonovacula constricta,the cDNA sequence of Tim was cloned and analyzed,the circadian expression patterns of Tim gene/protein in different tissues were analyzed by qRT-PCR and immunofluorescence technology,and the regulatory effects of Tim on sex hormone secretion were investigated by siRNA technology.The ORF sequence of Tim was 3090 bp,encoding 1029 amino acids and containing three conserved domains(TIMELESS superfamily、TIMELESS_C and FIil).Tim was expressed in test tissues,and showed higher expression levels in the hepatopancreas,siphon,ovary and testis.The expression levels of Tim were higher at 12:00–18:00 in the daytime and lower at 00:00–06:00 in the night time,which exhibited circadian rhythmic expression patterns in the ovary and testis.Tim protein was mainly expressed in hepatocytes of the hepatopancreas,epithelial cells of siphons,mature oocytes of the ovary,and spermatids of the testis.And protein fluorescence signals of Tim were slightly stronger at 12:00 during the day than at 00:00 during the night in the ovary and testis.After siRNA injection,the expression levels of Tim significantly decreased from the 3rd to 7th day in the siRNA treatment groups of the ovary and testis(P<0.05).Meanwhile,the contents of testosterone in the testis and estradiol in the ovary decreased significantly from the 1st to 9th day after siRNA injection(P<0.01).Consequently,it could be concluded that Tim gene/protein exhibited circadian rhythmic expression patterns in the ovary and testis,and the contents of sex hormones significantly decreased after Tim interference,which suggested that Tim might regulate the nocturnal spawning by mediating the secretion of sex hormones in S.constricta.These findings preliminarily explored the regulatory effects of circadian clock gene Tim on nocturna

关 键 词：缢蛏 生物钟 TIM 基因表达 SIRNA 性激素 

分 类 号：P735[天文地球—海洋生物学]