马氏珠母贝前列腺素受体EP4基因的克隆与表达分析

Cloning and Expression Analysis of Prostaglandin Receptor EP4 Gene from Pinctada fucata martensii

前列腺素受体EP4(prostaglandin E4 receptor,EP4)是前列腺素E2(prostaglandin E2,PGE2)的受体亚型之一,是一种G蛋白偶联受体(G-protein coupled receptor,GPCR),可通过调控环磷酸腺苷(cyclic adenosine monophosphate,cAMP)的浓度参与调控细胞的生理活动。为探讨EP4在马氏珠母贝(Pinctada fucata martensii)的功能分化和表达调控机制,本研究采用RACE技术获取马氏珠母贝两个EP4基因(PmEP4-1和PmEP4-2)的cDNA全长序列,应用实时荧光定量PCR(qPCR)技术检测它们在各组织中的表达差异,分析两个EP4亚型在马氏珠母贝不同组织中的功能分化特征,并利用生物信息学方法分析其表达调控机制,探讨其功能分化机制。结果显示,PmEP4-1基因cDNA序列全长1575 bp,开放阅读框(ORF)1098 bp,编码365个氨基酸;PmEP4-2基因的cDNA全长1976 bp,其中ORF长1533 bp,编码510个氨基酸;序列分析表明,PmEP4-1和PmEP4-2在胞内和胞外部分的氨基酸序列差异较大,并且蛋白质功能位点个数不同,说明其功能可能存在差异;进化树分析表明,PmEP4-1和PmEP4-2起源于不同的祖先基因。荧光定量PCR检测结果显示两者在各组织中均表达,但存在不同的表达模式,PmEP4-1在血液中表达量最高,PmEP4-2在肝胰腺中表达量最高,进一步说明两个基因亚型的功能差异。生物信息学分析说明PmEP4-1的表达受Mat1-Mc、XFD-2、Oct-1等转录因子调控,而PmEP4-2的表达受Oct-1、CF2-II、Barbie Box等转录因子调控。本研究为进一步探究EP4在马氏珠母贝中的生物学功能提供数据基础。

The prostaglandin E4 receptor(EP4)is one of the prostaglandin E2(PGE2)receptor subtypes,and is a kind of G protein coupled receptor(GPCR),which can participate in the regulation of cell physiological activities by regulating the concentration of cyclic adenosine monophosphate(cAMP).In order to explore the functional differentiation and expression regulation mechanism of EP4 of Pinctada fucata martensii,we obtained the full cDNA sequence of two EP4 genes of P.f.martensii(PmEP4-1 and PmEP4-2)by using RACE technology,and used Real-time quantitative PCR(qPCR)to detect their expression difference in various tissues and analyzed their functional differentiation in different tissues.And then,we used bioinformatics methods to analyze their expression regulation mechanism and explore their functional differentiation mechanism.The results showed that the full-length cDNA of PmEP4-1 was 1575 bp,including 1098 bp of open reading frame(ORF)that encoded 365 amino acids.While PmEP4-2 gene was 1976 bp,including 1533 bp of the ORF which encoded 510 amino acids.Sequence analysis shows that the amino acid sequence of PmEP4-1 and PmEP4-2 in the intracellular and extracellular parts are quite different,and the number of protein functional sites is also different,indicating that there may be differences in their functions.Phylogenetic tree analysis showed that PmEP4-1 and PmEP4-2 originated from different ancestral genes.qPCR results showed that both of PmEP4-1 and PmEP4-2 were expressed in all tissues,but showed different expression modes,PmEP4-1 had the highest expression in blood,while PmEP4-2 has the highest expression level in the hemolymph.Bioinformatics analysis shows that the expression of PmEP4-1 is regulated by transcription factors such as Mat1-Mc,XFD-2,and Oct-1,while the expression of PmEP4-2 is regulated by transcription factors such as Oct-1,CF2-II,and Barbie Box.This research provided the basis for the further study of EP4 in P.f.martensii.