大鳞副泥鳅GPR30基因的克隆及EE2暴露对其表达的影响

Cloning of GPR30 gene and the effects of EE2 on its mRNA expression in Paramisgurnus dabryanus

【目的】克隆大鳞副泥鳅(Paramisgurnus dabryanus)G蛋白偶联受体30(G protein-coupled receptor30)基因GPR30,研究其在成鱼各组织中的表达状况;用EE2(17α-ethynylestradiol)处理大鳞副泥鳅雌性幼鱼后,检测GPR30、ERαmRNA在幼鱼肝脏和卵巢中的表达状况,探究EE2对雌激素膜受体GPR30和经典雌激素核受体ERα基因表达的影响,推测GPR30可能存在的生理功能。【方法】采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术,克隆大鳞副泥鳅GPR30,对其核苷酸序列及所编码的氨基酸序列信息和进化关系进行分析;采用实时荧光定量PCR(Real-time PCR)方法,研究GPR30mRNA在大鳞副泥鳅成鱼性腺、肠道、肌肉、肾脏、脑、肝脏、鳃、眼睛等组织中的表达状况,以及经不同质量浓度(1,5,25ng/L)EE2处理后雌性幼鱼肝脏和卵巢中GPR30和ERαmRNA的表达。【结果】克隆获得了GPR30,该基因全长2 152bp,阅读框全长1 062bp,编码353个氨基酸,该基因氨基酸序列与斑马鱼的GPR30氨基酸序列相似性最高,达92.9%。GPR30氨基酸序列的比对结果显示,大鳞副泥鳅与其他脊椎动物的GPR30结构域一致。荧光定量PCR分析表明,GPR30在大鳞副泥鳅成鱼卵巢中表达最高,在雄鱼肠道中表达最低,且GPR30mRNA在雌雄大鳞副泥鳅的性腺中差异性表达,在卵巢中的表达量较精巢中高702倍。EE2暴露试验显示,在5ng/L EE2处理组的卵巢及1和5ng/L EE2处理组的肝脏中,GPR30有略微增长;随着EE2质量浓度的增高,ERαmRNA在肝脏中的相对表达水平较对照组显著增长。【结论】成功克隆了大鳞副泥鳅GPR30,其与哺乳动物的GPR30有相似的结构和功能,可能与雌激素效应存在密切关系。在基因转录水平上,GPR30与ERα可能不存在直接的显著相关关系。

【Objective】GPR30 of Paramisgurnus dabryanus （Cobitidae,Cypriniformes） was cloned and its expression in different adult tissues was examined.Expression of ERα （also known as Esr1） and GPR30 mRNA during early developmental stages of female P.dabryanus exposing to EE2 was detected. Then we focused on a reasonable speculation based on the results about the functions of GPR30.【Method】GPR30 was cloned by reverse transcription PCR method and rapid amplification cDNA ends （RACE） method.Tissue distribution and the expression level of GPR30 mRNA were analyzed with real-time quantitative RT-PCR method.Tissues tested included ovary,testis,intestine,muscle,spleen,brain,liver,gill and eye.【Result】 The full length cDNA of GPR30 was 2 152 bp,including a 1 062 bp open reading frame （ORF） coding for （encode） 353 amino acids.Alignment of the PdGPR30 amino acid sequence with counterparts in other vertebrates showed that PdGPR30 shared high similarity with the other amino acid sequences,especially Danio rerio（92.9%）.The amino acid sequences of PdGPR30 were highly similar to other vertebrates,as well as the structural domain and functional domain of GPR30.Results of qRT-PCR suggested that the expression of GPR30 mRNA was dramatically different in gonad of P.dabryanus,which was about 702 times in ovaries than in testes.The highest expression of GPR30 mRNA was observed in ovary, and the lowest was in intestine of male P.dabryanus.With the treatment of EE2,GPR30 mRNA expression in ovary with 5 ng/L EE2 and liver with 1 and 5 ng/L EE2 slightly increased.However,the expression of ERα mRNA increased significantly as the increase of EE2 concentration.【Conclusion】GPR30 gene was successfully cloned.Due to the similar gene structure and function,PdGPR30 is related to estrogen effect,which has been confirmed by previous researches in mammal. It may have no direct and distinct interaction between PdGPR30 and ERα in transcriptional level.