2024, Vol. 31 
草鱼(Ctenopharyngodon idella)是我国淡水养殖重要的品种之一，因其肉质鲜美、营养丰富、适应力强、生长快、规格大等优点，被广泛养殖[1-2]。近年来，随着养殖密度和规模的扩大，草鱼养殖出现病害频发和生长性状退化等问题，造成巨大的经济损失[3-5]。因此，改良品系对草鱼健康养殖十分重要[6]。草鱼初次性成熟需要4—5年[7]，传统育种方法周期长，需要运用新技术加快草鱼的育种进程[8]。
生殖干细胞移植技术在缩短鱼类性成熟周期、种质资源保存、濒危物种保护等方面具有巨大的应用价值[9-10]。鱼类生殖干细胞移植技术是将供体鱼的生殖干细胞移植到受体鱼腹腔，供体鱼生殖干细胞迁移至受体鱼生殖嵴并嵌合，经过增殖分化，待受体鱼性成熟时产生供体鱼的功能性配子[11-12]。鱼类生殖细胞移植技术最先在斑马鱼(Danio rerio)中建立[13]，经过不断发展，在其他鱼类也取得突破性进展，如大西洋鲑(Salmo salar)[14]和金鱼(Carassius auratus)[15]。为了加快草鱼新品种创制，笔者建立了草鱼精原干细胞移植技术，草鱼和赤眼鳟(Squaliobarbus curriculus)的亲缘关系较近，赤眼鳟初次性成熟只需要2年[16]，以草鱼作为供体提供精原干细胞，以赤眼鳟作为受体代孕产生草鱼配子。供体与受体生殖细胞的鉴定是生殖干细胞移植的重要环节，然而，目前缺乏移植后鉴定草鱼和赤眼鳟生殖细胞的方法。
鱼类中，常见的生殖细胞标记基因有vasa[17-18]、dnd[19-20]、nanos[21-26]、dazl[27-28]等。研究表明，Nanos蛋白与生殖细胞发育相关[29-30]。nanos2基因是脊椎动物nanos基因家族的一员[22-23]，其在生殖细胞的表达特征已在多种鱼类中展开研究。斑马鱼和点带石斑鱼(Epinephelus coioides) nanos2基因被鉴定为生殖干细胞(germline stem cells, GSCs)的特异性基因[22,31]；青鳉(Oryzias latipes) nanos2基因在卵原细胞和精原细胞中表达[25]；在虹鳟(Oncorhynchus mykiss)中，nanos2的表达仅限于未分化的精原细胞亚群[23]；而银鲫(Carassius gibelio) nanos2基因除了在生殖干细胞表达外，还在精原细胞和初级精母细胞表达[32]。这些研究表明nanos2是一种可靠的生殖细胞标记基因。
本研究克隆鉴定了草鱼和赤眼鳟nanos2基因的全长cDNA序列，研究了它们在不同组织的表达特征；通过nanos2基因的序列比对，开发并验证了区分草鱼和赤眼鳟生殖细胞的PCR方法。本研究为进一步研究nanos2在草鱼和赤眼鳟性腺发育的分子机制提供了有价值的信息，也为后续监测供体草鱼生殖细胞在受体赤眼鳟性腺的嵌合及发育情况提供了依据。
1 材料与方法 1.1 样品采集本研究使用的草鱼和赤眼鳟分别从武汉市宏新渔业有限公司和梁子湖试验场获得。选择3尾雄性草鱼[(49.5±0.5) cm, (1.39±0.06) kg]、3尾雌性草鱼[(48.5±0.5) cm, (1.37±0.1) kg]、3尾雄性赤眼鳟[(37.83±0.29) cm, (0.43±0.05) kg]、3尾雌性赤眼鳟[(41.67±1.53) cm, (0.62±0.06) kg]作为实验鱼。放血后20 min内，分别采集鳃、心脏、肝脏、脾脏、肾脏、肠、脑、精巢和卵巢保存于RNA保存液(Vivacell，中国)，用于RNA提取，取部分精巢和卵巢置于Bouin’s固定液(Scientific Phygene，中国)用于石蜡切片。所有实验程序均按照中国水产科学研究院长江水产研究所制定的《实验动物养护和使用指导原则》实施。
1.2 RNA提取和反转录利用FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme，中国)提取总RNA，使用微量分光光度计(Thermo Scientific，美国，NanoDrop One)测浓度，通过琼脂糖凝胶电泳检测RNA质量，将高质量总RNA保存在−80 ℃备用。使用PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa，日本)将1 µg总RNA反转录成cDNA, 4倍稀释后分装保存于−20 ℃备用。
1.3 草鱼和赤眼鳟nanos2基因全长cDNA的克隆采用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)获得草鱼和赤眼鳟nanos2基因全长cDNA序列。根据NCBI数据库(https://www.ncbi.nlm.nih.gov)中与草鱼和赤眼鳟亲缘关系较近的武昌鱼(Megalobrama amblycephala) nanos2基因(XM_048189299.1)的核苷酸序列设计得到两对引物，分别为Ci_nanos2 F1和Ci_nanos2 R1、Sc_nanos2 F1和Sc_nanos2 R1 (表1)，经过PCR和测序得到草鱼和赤眼鳟nanos2基因部分cDNA序列。根据草鱼和赤眼鳟nanos2基因部分序列，设计得到用于RACE实验的特异性引物(Gene-Specific Primers, GSPs)，分别应用于草鱼3ʹRACE (Ci_nanos2 3ʹ F1和Ci_nanos2 3ʹ F2)、草鱼5ʹRACE (Ci_nanos2 5ʹ R1和Ci_nanos2 5ʹ R2)、赤眼鳟3ʹRACE (Sc_nanos2 3ʹ F1和Sc_nanos2 3ʹ F2)、赤眼鳟5ʹRACE (Sc_nanos2 5ʹ R1和Sc_nanos2 5ʹ R2)(表1)。选择A260/A280比值在1.8~2.0，电泳条带28S∶18S rRNA亮度比值约为2的RNA样品用于RACE实验，利用SMARTer® RACE 5ʹ/3ʹ Kit (TaKaRa，日本)试剂盒合成第一链cDNA，使用SeqAmpTM DNA Polymerase (TaKaRa，日本)进行3ʹRACE和5ʹRACE的PCR扩增，利用PMD-19T载体(TaKaRa，日本)克隆纯化后的PCR产物，经过测序拼接得到草鱼和赤眼鳟nanos2基因的全长cDNA序列。草鱼和赤眼鳟nanos2基因全长cDNA已上传至BankIt，登录号分别为PP790588和PP790592。
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表1 本研究所使用引物 Tab. 1 Primers used in this study |
通过Vector NTI 11.5.1比对分析核苷酸序列；通过ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/)预测开放阅读框(ORF)；通过NetPhos 3.1 (https://services.healthtech.dtu.dk/services/NetPhos-3.1/)预测蛋白磷酸化位点；通过SignalP-4.1 (https://services.healthtech.dtu.dk/services/SignalP-4.1/)预测信号肽；通过SMART (https://smart.embl.de/smart/set_mode.cgi?NORMAL=1)预测蛋白质的结构域；通过Expasy-ProtParam tool (https://web.expasy.org/protparam/)分析蛋白质理化性质，包括分子量和等电点；利用NCBI数据库工具[Protein BLAST: Align two or more sequences using BLAST (nih.gov)]计算物种间Nanos2蛋白序列的一致性百分比；通过MEGA 11软件的Clustal W算法比对氨基酸序列，采用最大似然法构建系统发育进化树，每组分析设置1000个重复；通过MEGA11软件和ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi)[33]进行氨基酸序列多重比对和绘图(表2)。
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表2 不同物种Nanos2蛋白及登录号 Tab. 2 Accession number of different species for Nanos2 protein |
将部分性腺固定在Bouin’s固定液中，根据组织块大小固定1~4 h后，使用70%、80%、90%、95%、100%乙醇梯度脱水，二甲苯透明、石蜡浸渍、包埋。利用切片机(徕卡，德国，RM2245)对性腺石蜡样品连续切片，切片厚度固定为5 µm, 37 ℃烘干后进行苏木精–伊红(HE)染色(Biosharp，中国)，中性树脂(Biosharp，中国)封片。在显微镜(徕卡，德国，DM5000 B)下观察和拍照，分析性腺的组织学特征。
1.6 nanos2基因表达特征分析利用荧光定量PCR (qPCR)技术分析nanos2基因在草鱼和赤眼鳟不同组织的表达特征，包括鳃、心脏、肝脏、脾脏、肾脏、肠、脑、精巢和卵巢。利用Primer Premier 5软件设计得到用于qPCR的引物，如表1所示，分别为草鱼nanos2 (Ci_nanos2 qPCR F1和Ci_nanos2 qPCR R1)、草鱼β actin (Ci_β actin F1和Ci_β actin R1)、赤眼鳟nanos2 (Sc_nanos2 qPCR F1和Sc_nanos2 qPCR R1)和赤眼鳟EF 1α (Sc_EF 1α F1和Sc_ EF 1α R1)，扩增效率分别为100%、96%、101%和97%。使用荧光定量染料PowerUpTM SYBRTM Green Master Mix (Applied Biosystems，美国)在ABI QuantStudioTM 6 Flex PCR系统(Applied Biosystems，美国)上检测基因mRNA水平。qPCR反应体系：10 µL PowerUpTM SYBRTM Green Master Mix、8.4 µL ddH20、1 µL cDNA、0.35 µL上游引物和0.35 µL下游引物。qPCR反应程序：预变性包括50 ℃ 2 min, 95 ℃ 10 min；三步法扩增40个循环，每个循环包括95 ℃ 15 s, 58 ℃ 15 s, 72 ℃ 20 s。熔解曲线：95 ℃ 15 s, 60 ℃ 60 s, 95 ℃ 15 s。每种样品设置3个生物学重复和3个技术重复。β-actin和EF 1α作为内参基因，结果用2−ΔCt表示。统计分析采用t检验，最终数据以平均值±标准差表示。当P<0.05, P<0.01, P<0.001时，表示差异显著。统计数据用GraphPad Prism 8.0绘制。
1.7 利用物种特异性引物区分草鱼和赤眼鳟生殖细胞利用Vector NTI 11.5.1对草鱼和赤眼鳟的nanos2基因核苷酸序列进行比较，根据其序列的差异部分设计相应的物种特异性引物(表1)，分别为草鱼特异性引物(nanos2 F1和nanos2 R1)、赤眼鳟特异性引物(nanos2 F2和nanos2 R2)和共同表达引物(nanos2 F3和nanos2 R3)，用于PCR和1.5%琼脂糖凝胶电泳分析，以区分草鱼和赤眼鳟生殖细胞。PCR体系为5 µL 2×Taq master mix, 3.5 µL ddH2O, 1 µL cDNA, 0.4 µL上游引物和0.4 µL下游引物。PCR反应程序为94 ℃预变性5 min后，进入35个扩增循环，每个循环包括94 ℃ 30 s、58 ℃ 30 s、72 ℃ 15 s，最后72 ℃延伸10 min。
2 结果与分析 2.1 nanos2基因序列分析利用PCR技术从草鱼精巢克隆得到442 bp的nanos2基因部分cDNA序列，通过RACE技术获得nanos2基因649 bp的全长cDNA序列，ORF为438 bp, 5ʹUTR为26 bp, 3ʹUTR为185 bp。ORF编码145个氨基酸，具有锌指结构域，包含23个磷酸化位点，无信号肽(图1a)。预测的氨基酸序列理论等电点为8.96，相对分子质量为16735.78。
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图1 草鱼和赤眼鳟nanos2基因cDNA序列和推导的氨基酸序列a. 草鱼nanos2基因；b.赤眼鳟nanos2基因. 起始密码子(ATG)和终止密码子(TAA)标记为粗体，加尾信号(AATAAA)标记为粗体和下划线；锌指结构域用黄色突出显示；预测的丝氨酸(S)、苏氨酸(T)和酪氨酸(Y)残基上的磷酸化位点用黑色方框显示. Fig. 1 The cDNA and deduced amino acid sequences of Ctenopharyngodon idella and Squaliobarbus curriculus nanos2a. C.idella nanos2; b. S. curriculus nanos2. The start codon (ATG) and stop codon (TAA) are in bold; the polyadenylation signal (AATAAA) is marked using bold underlined; Zinc Finger is shaded by bright yellow; the predicted phosphorylation sites of serine (S), threonine (T) and tyrosine (Y) residues are shown in black boxes. |
利用PCR技术从赤眼鳟精巢中克隆得到420 bp的nanos2基因部分cDNA序列，通过RACE技术获得nanos2基因636 bp的全长cDNA序列，ORF为435 bp, 5ʹUTR为25 bp, 3ʹUTR为176 bp。ORF编码144个氨基酸，具有锌指结构域，包含25个磷酸化位点，无信号肽(图1b)。预测的氨基酸序列理论等电点为8.86，相对分子质量为16387.32。
2.2 Nanos2氨基酸同源性和系统进化关系分析草鱼与其他脊椎动物的氨基酸序列比对结果显示(图2)，草鱼与赤眼鳟Nanos2蛋白的同源性最高(91.67%)，与斑马鱼(65.49%)、电鳗(50%)和太平洋蓝旗金枪鱼(47.69%)具有较高同源性，与黄喉拟水龟(44.53%)、青鳉(41.32%)、人类(40.98%)、小鼠(40.98%)和大西洋鲑(40%)的同源性较低。Nanos2系统发育进化树(图3)显示该树主要分为两大分支，一个分支由硬骨鱼类组成，草鱼和赤眼鳟聚在一起，其遗传距离最近；另一分支由四足类组成。
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图2 草鱼与赤眼鳟及其他脊椎动物Nanos2蛋白序列多重比对若某一列的相似性大于70%，则认为残基高度相似，用红色字母和蓝框显示；若100%相同，则用白色字母和红色背景标出. Fig. 2 Multiple amino acid sequence alignments of Nanos2 of Ctenopharyngodon idella and Squaliobarbus curriculus and other vertebratesIf the similarity score assigned to a column is greater than 70%, residues are considered as highly similar and are colored in red and framed in blue, and they are highlighted in white letters with red background in case of 100% identity. |
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图3 草鱼与赤眼鳟及其他脊椎动物Nanos2系统进化树分析 Fig. 3 Phylogenetic tree of Nanos2 of Ctenopharyngodon idella and Squaliobarbus curriculus and other vertebrates |
利用石蜡切片及HE染色分析了草鱼和赤眼鳟性腺的组织学特征，发现草鱼精巢只含有大量的精原细胞(图4a)，而赤眼鳟精巢含有少量的精原细胞和大量的初级精母细胞(图4d)；草鱼和赤眼鳟卵巢均以初级卵母细胞为主(图4b, 4e)。
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图4 草鱼与赤眼鳟nanos2在不同组织的表达水平分析a. 草鱼精巢；b. 草鱼卵巢；c. nanos2在草鱼不同组织的mRNA水平分析；d. 赤眼鳟精巢；e. 赤眼鳟卵巢；f. nanos2在赤眼鳟不同组织的mRNA水平分析. SG：精原细胞；PSC：初级精母细胞；OG：卵原细胞；POC：初级卵母细胞. 比例尺：50 µm. Fig. 4 Expression levels of nanos2 gene in different tissues of Ctenopharyngodon idella and Squaliobarbus curriculusa. testis of C. idella; b. ovary of C. idella; c. mRNA levels of nanos2 were detected in gonad and other tissues in C.idella; d. testis of S. curriculus; e. ovary of S. curriculus; f. mRNA levels of nanos2 were detected in gonad and other tissues in S. curriculus. SG: spermatogonia; PSC: primary spermatocytes; OG: oogonia; POC: primary oocyte. Scale bar: 50 µm. |
利用qRCR研究了nanos2基因在不同组织的表达水平。草鱼中，nanos2基因主要在卵巢和精巢表达，且精巢的表达水平显著高于卵巢(P<0.001)，在肝脏有少量表达，其他组织不表达(图4c)；赤眼鳟nanos2基因主要表达于卵巢和精巢，且精巢的转录水平显著高于卵巢(P<0.01)，心脏有少量表达，其他组织不表达(图4f)。
2.4 物种特异性引物区分草鱼和赤眼鳟生殖细胞通过比对分析草鱼和赤眼鳟nanos2核苷酸序列，分别设计了草鱼nanos2特异性引物(nanos2 F1和nanos2 R1)、赤眼鳟nanos2特异性引物(nanos2 F2和nanos2 R2)和两个物种的通用引物(nanos2 F3和nanos2 R3)(表1)。通过PCR技术研究发现，草鱼nanos2特异性引物只在草鱼性腺扩增得到179 bp的目的条带，而赤眼鳟nanos2特异性引物只在赤眼鳟性腺扩增得到265 bp的目的条带，两个物种的nanos2通用引物在草鱼和赤眼鳟性腺都能扩增得到251 bp的目的条带(图5b)。由于目前鱼类中的研究表明nanos2为生殖细胞特异表达基因，因此本研究通过物种特异nanos2引物的PCR可以有效鉴定供体和受体生殖细胞。
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图5 草鱼和赤眼鳟nanos2基因特异引物的设计和PCR验证a. 草鱼和赤眼鳟nanos2基因序列比对和引物位置；b. nanos2基因的物种特异性PCR扩增.相同的碱基由白色字母和红色背景显示；起始密码子(ATG)和终止密码子(TAA)用黑框标出；nanos2 F1和nanos2 R1为草鱼特异扩增引物，nanos2 F2和nanos2 R2为赤眼鳟特异扩增引物，nanos2 F3和nanos2 R3为草鱼和赤眼鳟通用引物；M代表DL2000分子标记；N表示阴性对照. Fig. 5 Specific primers design and PCR validation of nanos2 gene in Ctenopharyngodon idella and Squaliobarbus curriculusa. Sequences alignment and primers of nanos2 of C. idella and S. curriculus; b. the species-specific PCR amplification of nanos2. Identical bases are shown with white letters and red background; the start codon (ATG) and stop codon (TAA) are in black boxes; nanos2 F1 and nanos2 R1 belong to C. idella-specific PCR amplification primers, nanos2 F2 and nanos2 R2 belong to S. curriculus-specific PCR amplification primers, nanos2 F3 and nanos2 R3 is the universal primers; M represents DL2000 molecular markers; N represents negative control. |
本研究克隆鉴定了草鱼和赤眼鳟nanos2基因，并进行了序列分析。对于许多物种来说，nanos2编码氨基酸的个数在100~200之间，如草鱼和赤眼鳟nanos2分别编码145和144个氨基酸(图1a、1b)；银鲫、大黄鱼(Larimichthys crocea)和半滑舌鳎(Cynoglossus semilaevis) nanos2分别编码141、175和187个氨基酸[32,34-35]；奶山羊和水牛(Bubalus bubalis) nanos2分别编码138和155个氨基酸[36-37]；然而，海胆(Mesocentrotus nudus) nanos2编码230个氨基酸[38]。这些研究表明，nanos2编码氨基酸数目具有物种差异性。Nanos蛋白具有2个进化保守的Cys-Cys-His-Cys锌指基序(zf-nanos)，它们是潜在的锌结合位点[39-40]。在草鱼和赤眼鳟Nanos2氨基酸序列的C末端发现了典型的zf-nanos结构域(图1a, 1b)，这与在其他鱼类的研究一致[25,35-36]。对不同物种Nanos2氨基酸同源性分析(图2), zf-nanos结构域外的同源性相对较低，zf-nanos结构域高度保守，说明其对nanos2功能至关重要。已有研究表明，3′UTR在nanos2基因功能的发挥也起着至关重要的作用。nanos 3′UTR中的保守基序通过与Pumilio相互作用发挥功能[41-42]。在小鼠nanos2 3′UTR突变体中，发现nanos2 3′UTR通过调节Nanos2蛋白水平从而调控精子发生[43]。海胆中，nanos2 3′UTR对生殖细胞的发育具有关键作用[44]。草鱼和赤眼鳟nanos2基因3′UTR在生殖细胞的功能值得进一步研究。
利用qPCR研究了nanos2在草鱼和赤眼鳟性腺及其他组织的表达特征，结合性腺组织切片结果，表明nanos2在草鱼和赤眼鳟性腺发育时期高表达，在精巢的表达水平显著高于卵巢(图4)，这与半滑舌鳎、虹鳟、银鲫和海胆的研究结果一致[23,32,35,38]；在大黄鱼、条石鲷(Oplegnathus fasciatus)和尼罗罗非鱼(Nile tilapia Oreochromis niloticus)性腺中，检测到nanos2在精巢特异性表达，卵巢不表达[34,45-46]。这些研究结果表明nanos2在不同物种的表达模式具有一定的物种特异性。Nanos是一种RNA结合蛋白，它在动物生殖系的形成、发育和维持等方面起重要作用[47-50]。在小鼠和罗非鱼中，敲除nanos2导致性腺生殖细胞缺失[26,51]，结果表明nanos2基因在草鱼和赤眼鳟性腺发育过程中发挥重要作用。
草鱼和赤眼鳟Nanos2蛋白相似性高达91.67% (图2)，表明nanos2在这两种鱼中高度保守，该基因在两种鱼生殖细胞发育过程中的功能可能十分相似。建立生殖干细胞移植技术需考虑供体与受体的亲缘性，亲缘关系越近，越易获得供体的配子，目前报道了许多鱼类生殖细胞移植成功的案例，如鱼类属间移植，鱼类属内移植、鱼类种内移植等[8]。草鱼和赤眼鳟同属鲤形目鲤科雅罗鱼亚科，分属草鱼属和赤眼鳟属，亲缘关系很近[52]，系统发育树显示草鱼和赤眼鳟Nanos2聚为一支(图3)，其遗传距离最近，提示了在草鱼和赤眼鳟之间开展生殖干细胞移植的可行性。受体性腺中供体生殖细胞的鉴定是鱼类生殖干细胞移植的重要环节。Saito等[15]在vasa 3′UTR设计物种特异性引物，在受体斑马鱼中检测到供体闪电斑马鱼(Danio albolineatus)的生殖细胞；类似的，通过vasa物种特异性引物证实了在受体黄条鰤(Seriola lalandi)中存在来源于供体南方蓝鳍金枪鱼(Thunnus maccoyii)的生殖细胞[53]；根据amh基因第一个内含子序列设计特异性引物，成功的检测出供体来源的精子[54]；利用vasa、dnd和nanos2物种特异性引物能准确区分供体大黄鱼和受体黄姑鱼(Nibea albiflora)的生殖细胞[55]。本研究通过分析草鱼和赤眼鳟nanos2基因核苷酸的差异性，设计得到草鱼和赤眼鳟物种特异性引物，通过PCR快速有效地区分了它们的生殖细胞(图5a、5b)，为鉴定草鱼和赤眼鳟生殖干细胞移植成功与否提供了关键技术支撑。
4 结论本研究克隆鉴定了草鱼和赤眼鳟nanos2基因全长的cDNA序列，草鱼和赤眼鳟nanos2氨基酸序列同源性高达91.67%，系统发育进化树显示草鱼和赤眼鳟的亲缘关系很近；荧光定量分析显示，nanos2基因主要在草鱼和赤眼鳟性腺表达，且精巢的表达量显著高于卵巢；根据草鱼和赤眼鳟nanos2基因序列的差异，设计并验证了获得物种特异性引物，建立了有效鉴定草鱼和赤眼鳟生殖细胞的方法。本研究为进一步探究nanos2在草鱼和赤眼鳟性腺发育机制提供参考，也为后续草鱼生殖干细胞移植技术的建立奠定基础。
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