DNA甲基化介导的低温诱导红鳍东方鲀雄性化分子机制研究

DNA methylation plays many crucial roles during sex determination and differentiation in fish. Because of the high quality of meat, delicious spermary and their high price of the male Tiger Pufferfish (Takifugu rubripes), it has important academic significance and application value to study on all male seed breeding technology of Tiger Pufferfish. In recent years, 75% of male Tiger Pufferfish was produced using low-temperature induction technology by our project team, and also the SNP (single nucleotide polymorphism) genotyping system was verified by using SNP sex-specific markers which established by Kamiya et al.. But the regulation and molecular mechanism of DNA methylation on Tiger Pufferfish (Takifugu rubripes) with masculinization due to low-temperature induction is not clear. In this study, the Tiger Pufferfish that treated by low-temperature as the samples, the rapid identification technology of pseudo male is established by genetic sex and phenotypic sex identification; the single base-resolution DNA methylation profiles of pseudo-male, female and normal male are depicted by the whole genome bisulfite sequencing (WGBS); and we use BSP (bisulfite sequencing PCR) to make accurate identification of methylation patterns in promoter region and regulation mechanism which related to the sex determination and differentiation. This study will lay a foundation for further study about the regulation mechanism of methylation in sex determination and differentiation of fish, and then provide a theoretical basis for guiding seed rearing on masculinization of Tiger Pufferfish.

DNA甲基化在鱼类性别决定和分化过程中起着重要作用。红鳍东方鲀性成熟雄鱼肉质好、精巢味美、价格高。因此，开展红鳍东方鲀全雄制种技术研究具有重要的学术意义和应用价值。近年来，本项目组采用低温诱导雄性化技术，获得75%雄性苗种，并参考Kamiya等建立的SNP性别特异标记进行SNP基因分型体系的建立与验证，但关于DNA甲基化对低温诱导红鳍东方鲀雄性化的调控作用及机制尚不清楚。本项目以前期低温处理的红鳍东方鲀为研究对象，利用已建立的性别特异SNP标记进行低温处理组的遗传性别鉴定，结合生理性别鉴定，建立快速鉴定伪雄鱼技术；通过全基因组甲基化测序，构建单碱基分辨率的全基因组甲基化图谱；利用BS-PCR法验证基因表达和甲基化水平的关联性，解析DNA甲基化介导的低温诱导红鳍东方鲀雄性化分子表观机制,为阐明鱼类性别决定和分化过程中甲基化调控机制奠定基础，进而为指导红鳍东方鲀全雄苗种培育提供理论依据。

DNA甲基化在鱼类性别决定和分化过程中起着重要作用。红鳍东方鲀性成熟雄鱼肉质好、精巢味美、价格高。因此，开展红鳍东方鲀全雄制种技术研究具有重要的学术意义和应用价值。本研究选用L9(34)设计，进行三因素三水平的正交试验，建立了低温诱导红鳍东方鲀雄性化的技术体系，研究发现虽然低温处理期间对生长速度有影响，但低温解除后各处理组的生长速度逐渐恢复正常。该研究结果为低温诱导红鳍东方鲀雄性化技术在生产实践上的应用具有一定的指导意义。在低温诱导基础上，通过组织切片进行生理性别鉴别及SNP技术进行遗传性别鉴别，获得5尾伪雄鱼，建立了快速、准确的伪雄鱼鉴定技术。同时，为探究红鳍东方鲀性别分化中的DNA甲基化模式，我们对红鳍东方鲀的正常雄鱼（M），正常雌鱼(F)和伪雄鱼(P)的性腺进行了全基因组甲基化测序（WGBS）和分析，绘制了单碱基分辨率甲基化图谱，并进行了全基因组DNA甲基化差异分析，通过对全基因组甲基化水平的两两比较发现，雌鱼和雄鱼有3173个差异甲基化区域（DMRs），雄鱼和伪雄鱼有3037个DMRs，分别对应基因组长度的0.232％和0.223％。雌鱼和伪雄鱼仅有1635个DMRs，对应基因组长度的0.127％。许多与性别决定和分化相关的差异甲基化基因（DMGs）被筛选出来，例如amhr2和pfcyp19a。通过BSP验证后，amhr2基因在正常雄鱼和伪雄鱼中表现出低甲基化水平，在正常雌鱼中甲基化水平较高，pfcyp19a基因在正常雌鱼中甲基化水平较低，在正常雄鱼和伪雄鱼中甲基化水平较高。为验证此结论，对amhr2和pfcyp19a基因进行了qPCR检测。结果发现在amhr2中，M的相对表达高于F和P，阴性接近假阳性。对于pfcyp19a，F高于M和P，M接近P。以上结果为低温诱导红鳍东方鲀雄性化的分子遗传机制提供了深刻见解，提高了我们对此种重要水产养殖鱼类的性别决定和分化机制的认识。