克氏原螯虾前列腺素E_(2)受体EP4基因的序列特征及表达分析

Characterization and Expression Analyses of Prostaglandin E_(2) Receptor EP4 Gene in Procambarus clarkii

前列腺素E_(2)(PGE_(2))受体EP4在动物体的卵巢发育、排卵和生殖调控方面具有重要的作用。利用转录组测序的方法从克氏原螯虾卵巢中获得了PGE_(2)受体EP4(PcEP4)基因的具有完整开放阅读框的cDNA序列。为阐明该基因的序列特征及其在克氏原螯虾卵巢中的作用,对该基因的序列进行生物信息学分析,并利用实时荧光定量PCR方法对该基因的表达模式进行检测。生物信息学分析结果显示:PcEP4基因开放阅读框长2505 bp,编码835个氨基酸;PcEP4是一个典型的具有7个跨膜α-螺旋结构的G蛋白偶联受体蛋白;克氏原螯虾EP4氨基酸序列与凡纳滨对虾的相似性最高,亲缘关系最近。基因表达结果显示:PcEP4基因表达量在克氏原螯虾成熟卵巢中的表达量最高;在Ⅰ~Ⅵ期的卵巢中,PcEP4基因在Ⅴ期卵巢中的表达量最高;PGE_(2)注射后36、48、60 h,克氏原螯虾卵巢中PcEP4基因表达量与对照组相比显著升高(P<0.05),注射72 h后克氏原螯虾的抱卵率与对照组相比也升高。试验结果表明,EP4作为PGE_(2)的受体与PGE_(2)一起在克氏原螯虾的卵巢中具有促进卵子成熟和排卵的重要作用。

Prostaglandin E_(2)(PGE_(2))receptor EP4 plays an important role in the regulation of ovarian development,ovulation and reproduction in animals.The cDNA sequence of the prostaglandin E_(2)(PGE_(2))receptor EP4 gene with the complete ORF region was obtained from the ovary of Procambarus clarkii using transcriptome sequencing.In order to explore the sequence characteristics and the function of EP4 gene in ovary of P.clarkii(PcEP4),the gene sequence was analyzed by bioinformatics,and the gene expression pattern was detected by real-time fluorescent quantitative PCR.Bioinformatics analysis results showed that the ORF of PcEP4 was 2505 bp,encoding a deduced protein with 835 amino acids;PcEP4 is a typical G protein coupled receptor with 7 transmembraneα-helical structures;EP4 amino acid sequence of P.clarkii and Litopenaeus vannamei were the most consistent,and the genetic relationship was the closest.Gene expression analysis results showed that PcEP4 gene expression level was the highest in the ovary in P.clarkii;in theⅠ-Ⅵstage of ovaries in P.clarkii,PcEP4 gene expression level was the highest in stageⅤovary;At 36,48 and 60 h after PGE_(2) injection,the expression level of PcEP4 gene in the ovaries of P.clarkii was significantly higher than that in the control group(P<0.05),the egg holding rate of P.clarkii also increased compared with the control group at 72 h after PGE_(2) injection.The above results indicate that,as a PGE_(2) receptor,EP4 plays an important role in promoting oocyte maturation and ovulation of P.clarkii together with PGE_(2).