| Apostichopus japonicus is a species of sea cucumber in the phylum Echinodermata and family Stichopodidae.Which is an important marine Echinodermata species along the coast of China.The artificial breeding of A.japonicus has made a breakthrough since 1980 s,while a severely weight loss result from aestivation in high temperture has been noted to seriously affect its growth.The key to solve the problem is to reveal the regulatory mechanism of metabolism of the sea cucubmer during the status of aestivation.Serotonin receptor is a typical monoamine hormone receptor,which playing an important role in feed behavior regulation of animals.In consideration of its potential function in feed regulation process during aestivation,the molecular and activity features,as well as the transcriptional regulation in various tissues were analyzed in this study.The main results were as followed:The full-length cDNA sequence of Aj5-HT4 R was cloned using the rapid amplification of 5' and 3' cDNA ends and the sequence structure was analyzed.The results showed that the full-length cDNA sequence of Aj5-HT4R(KX583229)was 1,544 bp,which encoding 336 amino acid(aa)residues contained an open reading frame(ORF)of 1,011 bp,a 3'-UTR of 300 bp and 5'-untranslated region(UTR)of 233 bp.Like other G protein coupled receptors.The Aj5-HT4 R sequence contains a common seven-transmembrane(7TM)topology.The phylogenetic analysis revealed that there were basically six groups of 5-HTRs: 5-HT1 Rs,5-HT2 Rs,5-HTR4,5-HTR5,5-HTR6,and 5-HTR7.Aj5-HT4 R was clustered to a group with other identified 5-HT4 Rs and showing 40% identity with the predicated 5-HT4 R of Strongylocentrotus purpuratus.Real-time quantitative PCR assays were conducted to analyze the tissue distribution of Aj5-HT4 R mRNA in different aestivation phase of adult A.japonicas.The results showed that Aj5-HT4 R mRNA was ubiquitously detected in tissues of active and aestivated sea cucumbers include respiratory tree,intestine,and muscle.During the phase of aestivation,the Aj5-HT4 R expression was lower in intestine and muscle tissue.Despite this difference was not statistically significant,the extremely significant decrease in Aj5-HT4 R expression was observed in respiratory tree tissue.In addition,to further evaluate the possible physiological role of Aj5-HT4 R in the regulation of aestivation,the direct effect of 5-HT administration on the Aj PFK(Phosphofructokinase)mRNA expression was investigated.5-HT was administrated to the aestivated A.japonicas by intraperitoneal injection,and then real-time PCR was used to quantitatively analyze the transcriptional level of AjPFK in respiratory tree,intestine,and muscle tissues.The experimental group with 5-HT administration exhibited a signifcant increase in Aj PFK mRNA in the muscle than that of the control group with vehicle,which supposed that 5-HT may play an significance role in metabolism of the sea cucubmer.The characteristic of Aj5-HT4 R were further analyzed,and signifcant cell surface expression was observed under fluorescence microscopy,suggesting that C-terminal EGFP fusion did not affect Aj5-HT4 R expression and transportation.Upon activation by ligand,fluorescence of Aj5-HT4R-EGFP was rapidly and efciently internalised from cell surface into the cytoplasm.Our observation confrmed that Aj5-HT4 R is a complete and functional receptor.In order to have a look into the function of Aj5-HT4 R in aestivation regulation,we used HEK293 cells as a heterologous expression system to functionally characterize Aj5-HTR.For the frst time,we have demonstrated that Aj5-HT4 R is activated by 5-HT with EC50 value of the response in the nanomolar range.Our data provide evidence that Aj5-HTR is a Gs protein-coupled receptor,specifcally activating adenylyl cyclase to induce intracellular cAMP formation.It was the first report about the characteristics,potential functions of Aj5-HT4 R,which could provide theoretical basis for further researches on regulatory mechanisms of A.japonicus aestivation. |
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