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

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 receptorβ,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,ERβ1cDNA全长为4262bp,其中包含239bp5′非编码区,2349bp3′非编码区和1674bp的开放阅读框,共编码557个氨基酸。ERβ2 cDNA全长为2506bp,包含5′非编码区393bp,3′非编码区109bp,阅读框为2004bp,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ERβ2,其中卵巢中ERβ1的表达量是ERβ2的164倍。注射雌二醇(17beta-estradiol,E2)24h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(7.5mg/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E2剂量(2.5mg/kg、5.0mg/kg)组均下调,而在E2高剂量(7.5mg/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、7.5mg/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。

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, Oneorhynehus 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 ERa/β1/β2 mRNA in hypothalamus, pituitary and testis by E2 indicated that various biological functions existed among these three ER subtypes in blue tilpia.