【作者】 王怡；

【导师】 骆剑； 王永波；

【作者基本信息】 海南大学 ， 渔业， 2020， 硕士

【摘要】 社会性雌雄同体的鱼类在其生命周期中具有性别可塑性,并表现出受群体社会结构影响的性逆转。波纹唇鱼是雌雄同体的社会性性逆转礁栖鱼类,其性逆转机制尚不清楚。P450芳香化酶是波纹唇鱼雌雄激素合成的转换开关,研究其编码基因启动子的转录调节机制是揭示波纹唇鱼性别反转规律的重要环节。本文通过RT-qPCR技术检测类固醇生成因子(SF-1)和叉头转录因子(Foxl2)在波纹唇鱼中的组织分布情况,利用质谱法对启动子DNA甲基化位点进行分析,采用双荧光素酶报告基因测定DNA甲基化对cyp19a1a和cyp19a1b启动子转录活性的影响,探索凝胶迁移实验(EMSA)以期鉴定Foxl2与cyp19a1b启动子转录结合位点。为揭示波纹唇鱼性反转分子调控机制奠定基础。1.通过序列分析波纹唇鱼cyp19a1a和cyp19a1b启动子CpG位点,采用Sequenom质谱法检测该启动子在未分化、雌鱼和雄鱼个体中不同CpG位点DNA甲基化情况,结果显示,cyp19a1a和cyp19a1b启动子中的16个CpG位点在不同个体中同一CpG位点甲基化率存在差异。基于前期数据中SF-1和Foxl2在cyp19a1a和cyp19a1b启动子上关键结合位点的结果,选取cyp19a1a(+114bp至-462bp)和cyp19a1b(-633 bp至-1144 bp)启动子部分序列诱导甲基化,并与SF-1和Foxl2的真核表达载体共转染至HEK293T细胞系,以检测甲基化对启动子转录活性的影响。结果显示波纹唇鱼cyp19a1a和cyp19a1b启动子诱导甲基化显著抑制Foxl2和SF-1体外启动子转录刺激。2.组织分布显示,SF-1和Foxl2在波纹唇鱼的鳃、肾脏、脾脏、大脑、性腺、垂体、肝脏、小脑、中脑、下丘脑组织均有表达。SF-1在肝脏、垂体、小脑和肾脏中高表达。Foxl2在垂体和肝脏中高表达,其次是鳃和下丘脑。SF-1和Foxl2在波纹唇鱼中的表达具有一定物种特异性。3.采用双酶切的方法构建SF-1和Foxl2原核表达载体,以不同IPTG(终浓度为0.1 mmol/L、0.5 mmol/L、1 mmol/L)在16℃、30℃、37℃诱导蛋白原核表达,其表达产物仅出现在包涵体中。为获得有活性的蛋白,采用诱导真核表达方式,将SF-1和Foxl2的真核表达载体转染进HEK293T细胞中表达,提取核蛋白通过Western Blot技术检测到Foxl2蛋白,未检测到SF-1蛋白。通过优化转模条件,聚丙烯酰胺凝胶配制等,摸索凝胶迁移实验条件,为后续研究建立凝胶迁移实验平台。更多还原

【Abstract】 Hermaphroditic fish shows a plastic sex and exhibited natural sex reversal during their lifespans.However,the underlying mechanisms for the sex reversal remaine unclear.Cheilinus undulates,a hermaphrodite fish,whose aromatase is a synthesis switch from androgen to estrogen.Accordingly,it is important work to figure out the regulatory mechanism of the Cheilinus undulates cyp19a1a and cyp19a1b during sex reverse.In the present thesis,tissue distribution levels of steroidogenic factor 1(SF-1)and Forkhead box L2(Foxl2)in Cheilinus undulates were evaluated by RT-q PCR.Possible methylation patterns of the promoter were analyzed.A luciferase reporter analysis revealed that effect of DNA methylation on the transcriptional activity of cyp19a1a and cyp19a1b promoter.The electrophoretic mobility shift assay(EMSA)was established to identify the exact bingding site of SF-1 and Foxl2 on the cyp19a1a and cyp19a1b promoter.The current study provides important basis for revealing the molecular mechanism of sex reversal in Cheilinus undulates.1.Methylation profile of cyp19a1a and cyp19a1b promoter at different CpG sites in immature,female,and male individuals were determined by Sequenom Mass Array.The results showed that the methylation rate of the same CpG site in different individuals was different.According to the key binding sites of SF-1 and Foxl2 on cyp19a1a and cyp19a1b obtained in our previous study,cyp19a1a(+114bp to-462bp)and cyp19a1b(-633bp to-1144bp)were selected for the expression vector construction.The methylated promoter constructed into luciferase reporter was co-transfected with eukaryotic expression vectors of SF-1 or Foxl2 into HEK293T cells to examine the effect of methylation on promoter transcriptional activity.The results showed that induced hypermethylation of the Cheilinus undulates cyp19a1a and cyp19a1b promoter completely suppressed promoter transcription stimulation in vitro by Foxl2 and SF-1 in combination.2.The transcripts of SF-1 genes were detected in Gil,Kidney,Spleen,Brain,Gonad,Pituitary,Liver,Cerebellum,Mesencephalon and Hypothalamu.SF-1 transcript was mainly expressed in Liver,Pituitary,Cerebellum and Head-kidney.The transcripts of Foxl2genes were detected in Gil,Kidney,Spleen,Brain,Gonad,Pituitary,Liver,Cerebellum,Mesencephalon and Hypothalamu.Foxl2 transcript was highly expressed in Pituitary and Liver.3.Prokaryotic expression vectors coding SF-1 and Foxl2 were constructed by double digestion.The expression of both proteins wereinduced at 16℃,30℃,37℃ with different concentration of IPTG(final concentrations of 0.1mmol/L,0.5mmol/L,1mmol/L).However,the products were only expressed in inclusion bodies.Instead,eukaryotic expression was employed to obtain both proteins.The eukaryotic expression vectors coding SF-1 and Foxl2 were transfected into HEK293T cells,while,only Foxl2 was detected by Western Blot,but SF-1.The conditions for EMSA were explored,and an experimental platform was established for EMSA.更多还原