Identification of Exportin Family and Its Expression in <i>Oreochromis niloticus</i>

ZHANG Qingqing; YOU Huiqin; DU Kang; TAO Wenjing

doi:10.13718/j.cnki.xdzk.2024.01.007

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Exportins (XPOs) are responsible for the export of proteins and RNAs in the nucleus, which play an important role in the regulation of gene expression.The evolution and the expression pattern of the XPOs remain unclear.In this study, we firstly characterized XPOs in 22 representative animal species and analyzed its evolution systematically.Then, we analyzed the expression level of XPOs in different tissues of Oreochromis niloticus by transcriptome analysis and qRT-PCR.Finally, we analyzed the cellular location of xpo1a, xpo1b and xpo5 in tilapia gonads by fluorescence in situ hybridization.The results showed that the number of XPOs changed slightly in species with the second and third rounds of whole genome duplication, but increased significantly in species with the fourth round of whole genome duplication, indicating an expansion of this gene family. xpos was mainly expressed in the testis, where the expression of xpo1a was the highest. xpos displayed sexual dimorphic expression at 90, 180 and 300 days after hatching (dah).Among them, xpo5, a gene responsible for miRNA export, expressed higher in testis than in ovary.No sexual dimorphic expression of xpos was found in the gonads of tilapia at 5 dah and 30 dah.Our results showed that xpo1a, xpo1b and xpo5 were expressed in oocytes and somatic cells. xpo1a and xpo5 were mainly expressed in spermatogonia and spermatocytes, xpo1b was mainly expressed in spermatocytes.Taken together, in this study, we analyzed the evolution of XPOs and their expressions in tilapia.Our results laid the foundation for elucidating the role of XPOs in the reproduction of vertebrates.

Corresponding author: TAO Wenjing

Received Date: 25/01/2022

Available Online: 20/01/2024

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出核转运是基因发挥功能的重要一环，因而对出核转运相关蛋白的研究一直是生物学研究的热点.输出蛋白(Exportins，XPOs)家族属于Karyopherin超家族，包含IBN-N和CRM-1结构域，对RNA和蛋白质的出核转运起重要作用^[1].在细胞核内，Exportins与GTP和RNA/蛋白质结合形成三聚体；出核后，伴随着GTP的水解，RNA/蛋白质被释放，Exportins在核转运因子作用下重回细胞核^[2-3].迄今为止，真核生物中共鉴定出8个Exportins，分别是XPO1，XPO2，XPOT，XPO4，XPO5，XPO6，XPO7和IPO13^[4].2011年，有报道发现斑马鱼中不存在XPO1，而人的XPO7有2个拷贝^[5].另一研究发现斑马鱼的XPO1有2个拷贝(XPO1a和XPO1b)，而人所有的Exportin家族成员都只有1个拷贝^[6].由此可见，Exportin家族在脊椎动物中的分离鉴定与系统进化存在争议，有待进一步开展研究.

Exportin的表达对RNA和蛋白质的调控至关重要.最早在酵母中鉴定出的Exportin家族成员是XPO1，又称CRM1.XPO1对多种RNA与蛋白质出核以及细胞内环境稳态的维持具有重要作用^[7-9].XPO2是输入蛋白α的出核载体，输入蛋白α将细胞质的货物输入到细胞核内，通过XPO2回到细胞质中^[10-11].XPO4是核转化起始因子5(AeIF-5A)的核输出受体，也可以作为其他底物的输出受体^[12].XPO5可以运输多种RNA，主要参与pre-miRNA的运输，在miRNA成熟过程中起重要作用^[13-14].XPO6介导肌动蛋白复合物的核输出^[15].XPO7可以运输蛋白^[16].IPO13专一输出翻译起始因子1(AeIF-1A)^[17].以上研究主要是在细胞系中鉴定了出核蛋白的成员及功能，然而在脊椎动物，特别是鱼类中的表达模式未见报道.

尼罗罗非鱼(Oreochromis niloticus)是世界性经济水产养殖鱼类，其性别决定系统为XX/XY，实验室前期已经建立了全雄和全雌的繁殖体系，可生产全雄和全雌单性鱼.此外，其基因组序列、多个组织转录组数据以及不同发育时期的性腺转录组已公布^[18-20]，有助于基因的分离鉴定与表达模式分析.本研究分析了Exportin家族成员在22种代表性动物中的系统进化，并探究了其在尼罗罗非鱼各组织和各时期性腺的表达水平及细胞定位，为深入解析Exportin家族在脊椎动物生殖发育中的作用奠定基础.

1. 材料

尼罗罗非鱼由西南大学淡水鱼类资源与生殖发育教育部重点实验室提供.饲养于室内循环水系统中，自然光周期，水温常年保持在26±1 ℃.XX单性鱼苗是由XX伪雄鱼(XX雌鱼用激素处理后转变为可产精子)与正常的XX雌性鱼交配获得，XY单性鱼苗由YY超雄鱼和正常XX雌性鱼交配获得.动物实验严格按照《实验动物饲养和使用指南》操作，并获得审委会批准.

2. 方法

2.1. 基因家族成员的鉴定与系统进化树的构建

将尼罗罗非鱼Exportin全序列作为query，在NCBI对果蝇(Drosophila melanogaster)、玻璃海鞘(Ciona intestinalis)、文昌鱼(Branchiostoma floridae)、象鲨(Callorhinchus milii)、矛尾鱼(Latimeria chalumnae)、非洲爪蟾(Xenopus tropicalis)、壁虎(Gekko japonicus)、鸡(Gallus gallus)、人(Homo sapiens)、小鼠(Mus musculus)、斑点雀鳝(Lepidosteus oculatus)、斑点叉尾鮰(Ictalurus punctatus)、黄颡鱼(Tachysurus fulvidraco)、斑马鱼(Danio rerio)、虹鳟(Oncorhynchus mykiss)、金鱼(Carassius auratus)、大西洋鳕鱼(Gadus morhua)、半滑舌鳎(Cynoglossus semilaevis)、红鳍东方鲀(Takifugu rubripes)、青鳉(Oryzias latipes)和斑马拟丽鱼(Maylandia zebra)的蛋白库进行比对.Bioedit软件用于氨基酸序列的编辑、保存，采用邻接法(Neighbor-Joining method，NJ)以MEGA X^[21]软件构建系统进化树，并用Adobe Illustrator CS6软件编辑系统进化树.

2.2. Exportin家族成员在尼罗罗非鱼中的表达模式

通过前期对尼罗罗非鱼成鱼8个不同组织(头肾、肾脏、肝脏、卵巢、肌肉、脑、心脏和精巢)^[20]和尼罗罗非鱼孵化后5，30，90，180，300 d性腺的转录组数据进行分析，获得xpos在各组织和性腺发育各时期的表达数据，基因表达以FPKM(Fragments Per Kilobase per Million)计量，用TBtools绘制heatmap，最终数据制图在GraphPad Prism 5(GraphPad Software，San Diego，CA)上完成.

2.3. 实时荧光定量PCR(Quantitative Real-time PCR，qRT-PCR)验证Exportin家族成员的表达模式

对孵化后180 d的尼罗罗非鱼脑、垂体、鳃、心脏、脾脏、肝脏、肠、卵巢、精巢、肾脏、头肾、肌肉进行取材，液氮速冻后加入RNAiso plus裂解并通过氯仿抽提的方式提取各组织总RNA(n=3).利用TaKaRa公司反转录试剂盒进行反转录获得各组织cDNA.将制备的cDNA稀释10倍后作为qPCR模板，按照SYBR Green Ⅰ Master Mix (TaKaRa，大连)说明书配制qPCR体系并通过Stepone Plus实时荧光定量PCR仪进行检测，qPCR引物见表 1.扩增参数：95 ℃预变性30 s，95 ℃变性15 s，60 ℃退火34 s(采集)，循环40圈.持家基因β- actin作为内参，基因的相对表达水平采用公式R=2^-ΔΔCt方法计算，结果以x±s表示，用GraphPad Prism 5软件中单因素方差分析(one-way ANOVA)方法进行差异显著性检验，p＜0.05表示差异有统计学意义.

2.4. xpo1和xpo5在性腺中的细胞定位

通过荧光原位杂交(Fluorescence in situ hybridization，FISH)检测xpo1a，xpo1b和xpo5在孵化后120 d尼罗罗非鱼性腺中的细胞定位.石蜡切片经二甲苯脱蜡、梯度酒精复水后，放入1×PBS缓冲液中洗3次，每次5 min.经蛋白酶K消化(10 μg/mL)和4%多聚甲醛再固定后(每步10 min，室温)，将片子依次放入2 mg/mL甘氨酸，0.1 mol/L三乙胺盐酸盐(Sigama，USA)和0.25%乙酰酐(Sigama，USA)溶液中，每步5 min.之后将片子放入66%甲酰胺/2×SSC缓冲液中60 ℃预杂交2 h.随后，配制杂交缓冲液并加入地高辛标记的反义探针(1 000 ng/mL)在60 ℃湿盒中杂交14~16 h，探针引物序列见表 2.杂交完成后，将片子依次放入50%甲酰胺/2×SSC，2×SSC(60 ℃)以及DIG Ⅰ(RT)缓冲液中各洗2次，每次20 min.经1% BSA封闭后(30 min，室温)，加入anti-DIG-POD(Roche，Germany，1∶500)室温孵育30 min.DIG Ⅰ室温洗涤4次后，DIG Ⅱ封闭，孵育VASA抗体37 ℃ 30 min，1×PBS缓冲液洗4次后，孵育绿色荧光二抗，稀释比例参考厂家说明书(abcam)，37 ℃ 30 min后用1×PBS缓冲液洗4次，再孵育DIPI(1∶1 000) 37 ℃ 15 min，后用1×PBS缓冲液洗5次并用水溶性封片剂封片，最后在FV3000激光共聚焦显微镜(OLYMPUS，日本)下拍照.

4. 讨论与结论

信号分子的出核转运是多种信号通路的基础^[22]，输出蛋白(Exportins)扮演了重要的角色.全基因组复制一般被认为是基因家族扩张的原因^[23]，本研究通过对22种代表物种Exportin家族成员的鉴定分析，发现Exportin家族基因的数量在经过第2轮基因组复制和第3轮基因组复制^[24]后整体数量变化不大，而在经过第4轮基因组复制^[25-26]的鱼类，如虹鳟和金鱼数量显著扩张.核糖体蛋白家族在脊椎动物进化中也出现了类似的扩张情况^[23]，表明输出蛋白在蛋白质调控过程中起重要作用.我们还发现Exportin家族在进化过程中出现了部分基因的复制和缺失，本研究中xpo1最先在象鲨中出现复制，表明xpo1作为最主要的输出蛋白，在核转运中扮演重要角色^[27].在硬骨鱼中由于第3轮基因组复制，出现了ipo13的复制，但半滑舌鳎的xpo1和ipo13都没有产生复制，可能是由于发生了次生性丢失或者基因组测序和组装不完整.斑马鱼和红鳍东方鲀丢失了xpot，有研究报道，斑马鱼较其他硬骨鱼更容易出现重复基因的丢失^[28]，红鳍东方鲀的基因组小且紧密可能是xpot丢失的主要原因^[29].xpot与xpo5在四足动物、斑马鱼和果蝇中分别聚在一起^[6]，也有研究报道在人、海葵、大豆疫霉菌和拟南芥中xpo2与其他转运蛋白聚在一起，xpo4和xpo7聚为一支，xpo6单独一支^[5].血吸虫中输出蛋白xpo1，xpot和xpo5聚为一支^[30].我们通过Exportin家族系统进化分析发现，xpo1和ipo13聚为一支，xpot和xpo5聚为一支，xpo2，xpo4，xpo6和xpo7聚为一支.

输出蛋白通过运输的调节可能影响动物生殖. xpo1核运输的调节对哺乳动物生发泡的维持和卵母细胞减数分裂的恢复有重要作用^[31].小鼠中，xpo1在卵母细胞整个发育过程中都有表达，且主要在细胞质中表达^[32].与此一致的是，本研究发现xpo1a和xpo1b主要在尼罗罗非鱼卵原细胞、卵母细胞的细胞质中表达，表明xpo1在卵子发生中可能有重要作用.此外，xpo1a和xpo1b在尼罗罗非鱼精巢生殖细胞中表达，且在精巢中的表达高于卵巢，表明xpo1可能在精子发生中也有作用，有待进一步功能研究.众所周知，miRNAs广泛参与动物生殖调控^[33]，而xpo5对miRNA转运出核和成熟至关重要^[34]. xpo5在卵原细胞、卵母细胞和体细胞中表达，说明xpo5对miRNA调控广泛存在.在三疣梭子蟹中，xpo5的表达随着卵巢发育逐渐升高，在精巢中表达高于其他组织，说明xpo5可能通过调控miRNA合成来影响其性腺发育^[35]. xpo5在尼罗罗非鱼精巢中的表达比卵巢高，这与三疣梭子蟹表达一致.

本研究系统分析了输出蛋白在脊椎动物中的进化，探究了其在尼罗罗非鱼性腺中的表达模式，为进一步研究输出蛋白在脊椎动物生殖发育中的作用奠定了基础.

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Identification of Exportin Family and Its Expression in Oreochromis niloticus

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