2. 北京市水产科学研究所，北京 100068;
3. 渔业生物技术北京市重点实验室，北京 100068
2. Beijing Fisheries Research Institute，Beijing 100068;
3. Beijing Key Laboratory of Fishery Biotechnology，Beijing Fishery Research Institute，Beijing 100068
鲟形目（Acipenseriformes）鱼类起源于泥盆纪（距今约4.5-3.5亿年），属于硬骨鱼纲（Osteicthyes）、辐鳍亚纲（Actinopterygii）、硬鳞总目（Chondrostei）、鲟形目，是目前世界上最古老的鱼类，现存鲟鱼类分为鲟科（Acipenseridae）和匙吻鲟科（Polyodontidae）2科共6属27种，有“活化石”之称[1, 2]。鲟鱼肉质鲜美，营养丰富，其鱼卵做成的鱼子酱富含人体必需氨基酸、多种不饱和脂肪酸、无机盐、维生素及铜、锌、锰等微量元素，价格昂贵，具有“黑色黄金”的美誉[3, 4, 5]，因此在养殖上，雌性鲟鱼比雄性具有更高的经济价值。鲟鱼个体大，性成熟晚，平均8龄以上达到性成熟，从外形上无法辨别雌雄，甚至在繁殖期也没有明显的第二性征[6, 7, 8]，很大程度上限制了鲟鱼性别鉴定的准确性，增加了养殖成本，因此鲟鱼性别鉴定及全雌化苗种培育和生产就成为鲟鱼产业化发展的研究重点[9]。目前鲟鱼性别鉴定的方法主要是活体手术法，还有需借助仪器的超声波、内窥镜等方法，但一般要到5龄左右才能保障鉴别的准确度[10, 11]。因此，采用现代分子生物学技术探究鲟鱼性别决定分子机制可以很大程度提高性别鉴定准确度、缩短性别鉴定年限，增加养殖经济效益，为实现鲟鱼全雌育种奠定基础。鲟鱼与高等鱼类不同，基于其特殊的进化地位，其性别决定类型及性别决定机制尚不明确，因此本文从性别分化相关候选基因入手，介绍鲟鱼性别分化相关基因的研究现状及研究进展，为鲟鱼性别分化及性别决定调控机制的深入研究奠定理论基础，以期实现鲟鱼全雌化苗种培育。
1 鲟鱼性别分化相关基因研究进展鱼类性别分化是指未分化性腺在个体发育过程中分化成为卵巢和精巢的过程[12]。在这个过程中，性别作为一种性状，主要受遗传物质的制约，但环境条件也起着极其重要的作用[13, 14, 15]。有研究表明，温度、pH、盐度、饲养密度、光照时间、摄食率及社会因素等环境因素均不同程度影响雌雄异体鱼类的性别比例，即影响鱼类的性别分化方向，其中最主要的影响因子是温度[15]。有研究发现，在性腺分化过程中，有些鱼类（如罗非鱼属）性腺形态学上的分化早于细胞学上的分化，而有些鱼类（如青鳉属）则反之[16, 17]。目前，对性别决定和性别分化分子机制的研究发现，在哺乳动物和鸟类上是以与性染色体上连锁的Sry（Sex determining region Y）和Dmrt1（Double-sex and mab-3 related transcription factor 1）基因为主导、多个基因参与的级联反应方式调控性别分化过程[18, 19, 20, 21]。在鱼类中，由于其性别决定的可塑性及性别分化机制的多样性，致使在物种间[如虹鳟（Oncorhynchus mykiss）、红鳍东方鲀（Takifugu rubripes）、银汉鱼（Odontesthes hatcheri）及青鳉（Oryzias latipes）等]性别决定与性别分化基因不尽相同，但均位于性染色体或性别决定基因座上，具有雄性特异性[22, 23, 24, 25]。
与高等硬骨鱼类不同，鲟鱼在其进化过程中经历了多次基因组加倍过程[26]，其染色体数目及多倍性情况仍处于争论之中[27]，加大了鲟鱼基因组测序的难度，也限制了对其性染色体筛选及性别决定和性别分化相关基因的研究。Chapman等[28]检测了鲟鱼成鱼群体的性别比例呈1：1；通过在高首鲟（Acipenser transmontanus）、短吻鲟（A. brevirostrum）和bester（Huso huso×A. ruthenus）上雌核发育研究推断鲟鱼是雌性个体为异形配子的性别决定遗传方式[29, 30, 31]，这些研究结果提示鲟鱼的性别决定及分化不是由环境因素决定，可能存在与性别决定和分化相关基因座。近些年，随着分子生物学技术的不断发展，研究者们通过ISSR（Inter-sample sequence repeats）、AFLP（Amplified fragment length polymorphism）、RAPD（Random amplified polymorphic DNA）等技术检测了西伯利亚鲟（A. baerii）、俄罗斯鲟（A. gueldenstaedtii）、小体鲟（A. ruthenus）、意大利鲟（A. naccarii）、湖鲟（A. fulvescens）、波斯鲟（A. percicus）及欧洲鳇等品种雌雄个体基因组差异，但目前尚未检测到鲟鱼上的性别特异性标记[32, 33, 34, 35]。随着性别决定及性别分化分子机制及相关候选基因在鱼类上的深入研究，为鲟鱼积累了大量的研究素材，为进一步探究奠定基础。
1.1 cyp19a1基因在类固醇激素的代谢过程中，需要多种酶参与催化，有一种酶在其中起着关键的作用，称为芳香化酶（P450arom），隶属细胞色素P450蛋白（CYPs）家族，能够催化雄激素（睾酮和雄烯二酮）转化为雌激素（雌酮和雌二醇），是雌激素生物合成的关键酶和限速酶，广泛存在于脊椎动物的脑和性腺中[36]。编码P450arom的基因为cyp19a1（cytochrome P450，family 19，subfamily A，polypeptide 1）基因，在大多数哺乳动物中，芳香化酶由cyp19a1单基因编码[37]，但在鱼类却发现了两种不同基因编码的P450arom，即性腺型芳香化酶（cyp19a1a）和脑型芳香化酶（cyp19a1b）[38, 39]，它们以明显不同的形式分别存在于性腺和脑中，且鱼类脑中芳香化酶表达量是哺乳动物的100-1 000倍[40]。鱼类中cyp19a1a主要存在于卵泡的颗粒细胞中，可将扩散入颗粒细胞的雄烯二酮芳香化为雌激素，促进卵泡的发育，同时可促进肝脏合成卵黄蛋白原，保证了卵母细胞发育前期物质能量的积累[41, 42, 43, 44, 45]，但过高的活性在卵母细胞发育后期则呈现出抑制作用[46]。cyp19a1b是中枢雌二醇生物合成的决定物，主要参与中枢神经系统发育过程中脑的分化[40]和性行为的调节[47]，也有研究认为其参与性腺分化的调节，但作用机制尚不明确[48]。
近年来，cyp19a1基因已成功在斑马鱼、虹鳟、罗非鱼、牙鲆、欧洲鲈鱼及日本鳗鲡等[41, 49, 50, 51, 52, 53]鱼类中克隆，但在鲟鱼方面几乎没有报道，仅在施氏鲟上成功克隆出其部分cDNA序列，但未标明该序列为性腺型（cyp19a1a）还是脑型（cyp19a1b）。本课题组前期在小体鲟上通过设计简并引物，成功在性腺中克隆出cyp19a1基因部分cDNA序列，组织分布结果（图 1）表明该基因在雌雄性腺及脑中均表达，且在雌性性腺和脑中表达量远远高于雄性性腺，初步推测鲟鱼cyp19a1基因的性别分化调控机制更接近于哺乳类动物，无脑型和性腺型的分化，但仍需进一步验证。
1.2 Dmrt1与Sox9基因Dmrt（Doublesex and mab-3 related transcription factor）基因家族是目前发现的对性别决定和性别分化起重要作用的基因家族。该家族成员编码一个具有DNA结合能力的保守序列，即DM（Doublesex和Mab-3）结构域，并以锌指结构与特异DNA序列相结合的一类转录因子。dmrt1是DMRT家族中的一员，是目前已知从无脊椎动物到人最保守的性别分化相关基因，在性别分化期的雄性性腺及雄性成体的性腺中表达，而在雌性成鱼卵巢中表达较弱，与雄性性腺的分化和形成有关，被认为是性别决定与性腺发育的主效基因之一[54]。鱼类dmrt1基因相关研究工作开展较多，已在青鳉、虹鳟、罗非鱼、河豚、斑马鱼、黑鲷、半滑舌鳎和石斑鱼等多种鱼类中发现并克隆[55, 56, 57, 58, 59]，对于发育时期组织表达的雌雄差异性也在很多鱼类中有报道[60, 61, 62, 63, 64, 65]。目前有研究发现在青鳉Y染色体上存在一个Dmrt1基因的拷贝基因——dmy（Double sex/ mab-3domain of Y chromosome）基因，在青鳉胚胎发育、幼鱼及成鱼阶段均表现为雄性特异性表达，是青鳉雄鱼精巢发生、发育和分化直接相关的性别决定主效基因[24]。但目前未在其他的鱼类中发现该基因的存在[22, 66, 67]。有研究发现dmy可直接或通过结合因子SF1（Splicing factor 1）间接结合到cyp19a基因的启动子区，下调该基因的表达[68]。
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| 图 1 小体鲟不同组织中cyp19a1 基因的表达 |
Sox9（Sex determining region Y-box 9）基因是目前发现的决定大多数脊椎动物睾丸发育的主要基因之一，隶属于Sox基因家族，被认为与性逆转、性别分化胚胎期的细胞分化以及精原细胞的形成有关。Takamatsu 等[69]研究发现Sox9在虹鳟精巢中显著表达；Zhou等[70, 71]通过Northern杂交的方法同样发现了该基因在大鳞副泥鳅精巢中高表达，显示该基因可能与精巢分化和发育相关。但有研究者发现Sox9基因在青鳉中的表达有所不同，其在卵巢中高表达而在精巢中几乎不表达，其功能仍需深入研究[72]。Dong 等[73]成功克隆出半滑舌鳎Sox9基因，其组织表达结果显示，其在雄鱼成鱼的脑、垂体和性腺中表达量较高，且在幼鱼性腺中的表达量要高于雄性成鱼，在性腺分化前，其表达量有一个先上升再下降的过程。
在鲟鱼方面，目前已成功克隆中华鲟（A. sinensis）、高首鲟、小体鲟、西伯利亚鲟和密西西比铲鲟（Scaphirhynchus platorynchus）Dmrt1基因cDNA序列；成功获得西伯利亚鲟、施氏鲟（A.schrenckii）和大西洋鲟上Sox9基因cDNA序列。Amberg等[74]对61尾密西西比铲鲟性腺中Dmrt1基因的表达进行荧光定量PCR研究发现，其在雌雄性腺中均有表达，雄性性腺的表达量要高于雌性，但未达到显著水平。Berbejillo等[75, 76]研究发现未成熟的西伯利亚鲟性腺中Dmrt1在精巢中的表达量高于脑、肝脏、肌肉、肾脏等其他组织；在幼鱼已分化性腺中Dmrt1呈明显的性别二态性表达，在雄性中的表达量极显著高于雌性（P ＜ 0.01），而Sox9基因雌雄间性别二态性不明显。Hett等[77]对大西洋鲟Sox基因家族展开研究，该家族表达产物具有高度保守的HMG-box区段，克隆得到该家族8个Sox基因序列（Sox2、Sox3、Sox4、Sox9、Sox11、Sox17、Sox19和Sox21），对Sox9基因深入研究发现大西洋鲟Sox9基因由3个外显子和两个内含子组成，且该基因在鲟鱼上并不像其他硬骨鱼类一样存在基因复制现象，在性腺中的表达也不存在性别特异性[78]。在史氏鲟中克隆获得了4个与青鳉、虹鳟和人相应Sox基因的高度同源性片段，命名为ASSox9、ASSox2、ASSox4和ASSox11，且在性腺中的表达与大西洋鲟相似，无性别二态性[79]。
1.3 Foxl2基因Foxl2基因（Forkhead transcription factor gene 2）属翼状螺旋/叉头转录因子超家族成员，参与脊椎动物雌性性腺早期发育与分化，在生殖嵴时期开始表达，通过调控靶基因的转录影响靶基因蛋白表达。该基因主要在垂体和卵巢颗粒细胞中表达，参与颗粒细胞增殖分化。研究发现Foxl2可直接结合到靶基因cyp19a启动子区，上调cyp19a基因的表达，也可通过与sf1蛋白结合，增强sf1对cyp19a基因转录的激活作用[80, 81]。在对半滑舌鳎的研究中发现，Foxl2基因在雌性性腺中表达量较高，孵化过程中在原肠胚时期表达量要高于其他时期，孵化后第37天表达量达到峰值，与半滑舌鳎性别分化时间相符，提示Foxl2与半滑舌鳎性别分化和胚胎期的细胞分化过程有关[73]。Nakamoto等[82]成功克隆了青鳉的Foxl2基因cDNA序列发现，该基因最先在XX型卵巢分化起始时的生殖细胞中表达，在成鱼卵巢的前卵黄囊和卵黄囊中表达，在后卵黄囊中不表达，且在成鱼精巢中不表达，表明该基因与青鳉颗粒细胞分化有关。在牙鲆上通过瞬时转染技术发现Foxl2可以在体外激活cyp19a1基因的转录，提示在牙鲆性腺分化过程中Foxl2可以调控cyp19a1基因的转录[83]。在对cyp19a1基因的转录调控过程中，wt1（Wilms tumor transcription factor 1）和dax1（Orphan nuclear receptor Dax-1）基因也参与其中。有研究表明，wt1可直接或通过不同类型、不同剪切方式分别调节雌雄性腺中Foxl2和Dmrt1的表达，从而间接调节cyp19a基因的表达和雌激素的生成[12]。在罗非鱼中已发现其cyp19a1基因5'区存在SF1、wt1和sry的结合位点[84]。青鳉Dax1仅在雌性成鱼卵巢的后卵黄囊中表达，在雄性成鱼精巢中不表达，通过共转染试验发现，dax1抑制Ad4BP/Sf1和Foxl2介导的cyp19a1基因表达[85]。
在鲟鱼方面，目前已成功克隆获得密西西比铲鲟、俄罗斯鲟、小体鲟和西伯利亚鲟的cDNA序列。Foxl2基因的研究在鲟鱼上报道较少，仅在密西西比铲鲟上报道了Foxl2基因在性别分化成熟的雌鱼性腺中表达量是雄鱼表达量的9倍，达到了极显著的水平（P ＜ 0.01），认为Foxl2可以作为鲟鱼性别标记基因[74]。
2 第三代测序技术在鲟鱼性别分化相关基因研究中的应用随着第三代高通量测序技术的开发与应用，对鲟鱼转录组测序及构建cDNA文库成为当前的研究热潮。Hale等[86]通过454焦磷酸测序技术对两尾性成熟湖鲟（13-14龄）性腺转录组测序，获得了共125 MB的序列信息及473577高质量的测序读长，构建了雌雄两个性腺cDNA文库，筛选出73个湖鲟候选性别决定标记，对Sox3、Sox4、Sox5、Sox9、Sox18、cyp19a1、Dmrt1、Foxl2、Rspo1、Wt1、Wnt1、Tra-1及Sry性别决定候选基因雌雄间表达情况进行统计分析，发现Dmrt1在雄鱼上的表达量极显著高于雌鱼（P ＜0.001）；而Tra-1表达情况相反，达到了显著水平（P= 0.05）；Sry、Wt1及Wnt1雌雄间表达差异不显著，其余9个基因均为雌性特异性表达基因。Amberg 等[87]使用相同的测序技术构建了密西西比铲鲟雌雄性腺cDNA文库。值得一提的是，在通过荧光定量PCR技术检测筛选出的性别特异表达基因发现了两个仅存在于卵巢cDNA文库中的在Wnt 信号通路中的拮抗因子Dkk1（Dickkopf-1）和Dact1（Dapper-1），推测这两个因子可能在鲟鱼性别分化及性腺发育过程中起重要作用，可作为性别标记深入研究。Hagihara等[88]通过8尾性腺未分化的9月龄俄罗斯鲟构建了性腺cDNA文库，筛选出25个俄罗斯鲟性别分化相关候选基因[Gsdf、Sox9、Dmrt1（a、b）、Foxl2、Rspo1、Dax1、Sf1、Fsh、Lh、Amh、Igf1、Fshr、Lhr、Amhr2、Igf1r、Star、Cyp11a1、Hsd3b、Cyp17a1、Hsd17b1、Hsd11b2、Cyp19a1a、Ar、Era及Erb]，其中13个为鲟属上首次发现。Vidotto等[89]通过454测序方法构建了意大利鲟性腺和脑的雌雄cDNA文库，得到了55 000多个高质量的ESTs，筛选到32个性别相关基因，检测出21 791个与EST连锁的SNPs和5295个SSRs位点。
3 展望作为最古老的鱼类之一，鲟鱼具有较为特殊的进化地位，具有极高的经济价值和研究价值。开展鲟鱼性别决定及性别分化调控机制的研究是实现鲟鱼全雌化苗种培育的基础，是提高鲟鱼性别鉴定准确度、缩短性别鉴定年限，增加养殖经济效益的有效途径。目前性别决定及性别分化机制在鱼类上的研究较为广泛，大量与性别分化相关候选基因被筛选与鉴定，为性别决定及性别分化机制在鲟鱼上的深入研究提供了大量的研究思路及研究素材。
目前性别决定及分化基因在鲟鱼上的报道相对较少，主要集中在几个经典的与性别决定及分化的主效基因克隆与表达分析上，对功能方面的研究甚少。在鲟鱼上，Dmrt1及Foxl2基因保守性较高，其表达水平的研究结果与高等鱼类基本一致，而与精巢分化相关的Sox9基因在鲟鱼上与其他硬骨鱼不同，不存在基因复制现象，在性腺上的表达也不存在性别二态性[86]，推测其调控性别分化机制与高等鱼类不同。同时，根据鲟鱼cyp19a1基因的研究结果推测该基因的调控机制可能更接近于哺乳类动物，无脑型和性腺型的分化，但仍需进一步验证。随着新一代测序技术的开展，目前通过转录组测序技术已在鲟鱼上筛选到了更多的性别特异性表达候选基因，进一步检测及对候选基因的功能验证成为下一步的研究重点。相信随着研究的不断深入，鲟鱼性别决定及性别分化机制的谜团终将解开。
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