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奥利亚罗非鱼雌激素β受体两种亚型cDNA的克隆、组织分布及雌激素对其表达的影响

Molecular cloning, tissue distribution of two estrogen receptor β cDNAs of blue tilapia (Oreochromis aureus) and effects of estrogen on their expression

    摘要
  • 摘要: 采用RT-PCR和RACE技术，克隆了奥利亚罗非鱼β雌激素受体基因（estrogen receptor β, ERβ）两种亚型的cDNA全序列（ERβ1和ERβ2）。荧光定量PCR分析雌雄奥利亚罗非鱼两种亚型的组织分布，并观察注射外源雌激素对雄性奥利亚罗非鱼下丘脑-垂体-性腺轴雌激素受体ERα和β（β1/β2）基因表达的影响。序列分析表明，ERβ1 cDNA全长为4 262 bp，其中包含239 bp 5′非编码区，2 349 bp 3′ 非编码区和1 674 bp的开放阅读框，共编码557个氨基酸。ERβ2 cDNA全长为2 506 bp，包含5′非编码区393 bp，3′非编码区109 bp，阅读框为2 004 bp，共编码667个氨基酸。氨基酸序列同源性分析显示奥利亚罗非鱼ERβ1与尼罗罗非鱼的相似性高达99.1%，而与鲈形目其它鱼类的相似性为82.6%～94.2%。ERβ2氨基酸序列与尼罗罗非鱼的相似性为98.7%，与大口黑鲈、虹鳟、底鳉及斑马鱼的相似性分别为81.8%、76.3%、64.7%和55.0%。在系统进化树上奥利亚罗非鱼的ERβ1和ERβ2分别与尼罗罗非鱼的相应受体聚类。奥利亚罗非鱼ERβ1/β2基因在所检测的10种组织中均有表达。ERβ1基因在卵巢、精巢、肝脏、肾脏和肠等组织的表达量均显著高于其他组织（P<0.05），以卵巢的表达量为最高。ERβ2基因在精巢、肝脏及肾脏组织的表达量较高（P<0.05）。ERβ1在卵巢、精巢、肠与下丘脑中的表达量显著高于ER〖WTB1〗β〖WTB1〗2，其中卵巢中ERβ1的表达量是ERβ2的164倍。注射雌二醇（17beta-estradiol, E2）24 h后雄性奥利亚罗非鱼下丘脑ERα/β1 基因的表达上调，其中以ERα的升幅最大，且存在剂量依赖效应。在E2高剂量组（7.5 mg/kg）下丘脑ERα的表达量显著升高（P<0.05），ERβ2 基因的表达则没有显著变化。垂体中3个基因的表达在中低E2剂量（2.5 mg/kg、5.0 mg/kg）组均下调，而在E2高剂量（7.5 mg/kg）组则上升，其中ERα的表达量显著升高（P<0.05）。精巢中ERβ2基因的表达量升高，以中、高剂量组（5.0、7.5 mg/kg）的升幅最大（P<0.05），而ERα/β1两个基因的表达量则没有明显变化。ERβ1/β2的不同组织分布以及雌激素对下丘脑、垂体及性腺ERα/β1/β2的不同的表达调控，提示奥利亚罗非鱼3种ER亚型存在着不同的生理功能。

     

    Abstract: Two sub-type estrogen receptor β (ERβ1/β2) cDNAs were obtained from blue tilapia, Oreochromis aureus by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RACE. The full length cDNA sequence of ERβ1 was 4 262 bp in length, containing a 5′ Untranslated Regions (UTR) of 239 bp, 3′ UTR of 2 349 bp and an open reading fame (ORF) of 1 674 bp which encoded a putative protein of 557 amino acids with an estimated molecular weight of 62 ku. The ERβ2 cDNA was 2 506 bp in length with 393 bp of 5′ UTR, 109 bp of 3′ UTR and an ORF of 2 004 bp which encoded a putative protein of 668 amino acids with an estimated molecular weight of 74 ku. The deduced amino acid sequence of ERβ1 of blue tilpia possessed 99.1% similarity with the counterpart of nile tilapia, O. niloticus, and similarities with other Perciformes fishes were from 82.6% to 94.2%. The ERβ2 amino acid sequences of blue tilpia shared 98.7%, 81.8%, 76.3%, 64.7% and 55.0% identities with nile tilapia, Micropterus salmoides, Oncorhynchus mykiss, Fundulus heteroclitus and Danio rerio respectively. Blue tilpia ERβ1/β2 were clustered with nile tilapia ERβ1/β2 respectively in the phylogenetic tree. ERβ1/β2 mRNA expressions between male and female were compared by Real-Time PCR (RT-PCR). ERβs were expressed ubiquitously and differently in ten tested tissues. ERβ1 was significantly expressed in ovary, testis, liver, kidney and intestinal tissues compared with other tissues (P<0.05). ERβ2 was highly expressed in testis, liver and kidney (P<0.05). In ovary, testis, intestine and hypothalamus, expression of ERβ1 was significantly higher than that of ERβ2, and it was 164 times of ERβ2 in ovaries. After 17 beta-estradiol (E2) was injected for 24 h, ERα/β1 mRNA in hypothalamus of male blue tilpia was increased, of which ERα increased significantly. Expression of ERα in hypothalamus increased significantly in 7.5 mg/kg E2 treated group, while no significant change was observed for ERβ2. In pituitary, expression of ERα/β1/β2 in the low and middle E2 concentrations (2.5, 5.0 mg/kg) was down-regulated, but up-regulated in high E2 concentration group (7.5 mg/kg), and significant difference was found for ERα(P<0.05) . ERβ2 expression was increased in testis significantly (P<0.05) in middle and high E2 concentration groups (5.0, 7.5 mg/kg). No significant change was observed for ERα/β1 expression. The different distribution of the ERβ1/β2 mRNAs and different regulation of ERα/β1/β2 mRNA in hypothalamus, pituitary and testis by E2 indicated that various biological functions existed among these three ER subtypes in blue tilpia.

     

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