| This paper focuses on the serotonin and dopamine signaling system in sea cucumber(Apostichopus japonicus).Research on the function of signal system based on the molecular characteristics and functional mechanism of receptor.The fusion expression vector of receptor was constructed by molecular and cellular biological methods,and the subcellular localization characteristics and cellular signal pathway characteristics of receptor protein were systematically studied by model cell expression technology;the temporal and spatial expression characteristics of receptor gene were analyzed by quantitative analysis of gene expression,and the expression of 5-hydroxytryptamine(5-HT)and dopamine(DA)were analyzed by immunohistochemistry.To study the regulation of 5-HT and DA signaling system on the physiological process of sea cucumber.The main results are as follows:1.The 5-HT receptor(Aj5-HT4/6R)of A.japonicus was cloned and characterized.The results showed that Aj5-HT4/6R coding gene contained 1290 bp open reading frame(ORF),encoding 429 amino acids;cluster analysis showed that Aj5-HT4/6R receptor was homologous with 5-HT4R and 5-HT6R families in other animals,and it was identified as 4/6 family of 5-HT receptor;through multiple sequence alignment and protein 2D and 3D model prediction analysis,it was proved that Aj 5-HT4/6R had a seven transmembrane structure.The membrane localization and ligand activated endocytosis of Aj5-HT4/6R were revealed by HEK293 expression analysis system.2.The Gas/PKA/MAPK signal transduction pathway mediated by Aj5-HT4/6R activation was identified by the detection of intracellular signal factors.The results showed that Aj5-HT4/6R had a high affinity for 5-HT.When Aj5-HT4/6R was activated by 5-HT,it could further activate adenylate cyclase to produce the accumulation of second messenger cAMP,which proved that Aj5-HT4/6R was activated and coupled with Gas protein.After Aj5HT4/6R was activated,.it-could-significantly mediate MAPK phosphorylation,showing a concentration and time gradient dependence of 5-HT ligand,and its EC50 value was 0.4203±0.07 μM in the nanomolar range Moreover,H89,an inhibitor of PKA,could inhibit the phosphorylation of MAPK mediated by Aj5-HT4/6R,which indicated that PKA was involved in intracellular signal transduction.3.The dopamine receptor(Aj-DAiR)of A.japonicus was cloned.Its sequence,molecular characteristics and functional activity were systematically analyzed and characterized.The results showed that Aj-DAiR coding gene contained 1170 bp open reading frame(ORF),encoding 389 amino acids;cluster analysis showed that Aj-DA1R was homologous with dopamine receptor type 1 family in other animals,and it was predicted that Aj-DA1R could be classified as dopamine receptor type 1;through multiple sequence ratio pair and protein 2D and 3D model prediction analysis,it was proved that Aj-DAiR had a completed seven transmembrane structure.The membrane localization of Aj-DA1R and its ligand activated endocytosis were revealed by HEK293 expression analysis system.4.By analyzing the distribution of 5-HT and DA ligands and the temporal and spatial changes of receptor gene expression in multiple tissues of A.japonicus,the physiological regulation functions of 5-HT and DA signaling system were preliminarily discussed.The results showed that 5-HT ligand had obvious distribution signals in muscle,respiratory tree,digestive tract and anterior part of sea cucumber;The results showed that the expression level of Aj5-HT4/6R gene in muscle and respiratory tree was high,and the expression of Aj5HT4/6R gene in muscle tissue of sea cucumber had obvious seasonal variation characteristics,and the expression level of Aj5-HT4/6R decreased significantly during aestivation;the data showed that the 5-HT signaling system was involved in the physiological activities of sea cucumber,such as energy metabolism and exercise;DA ligand was only distributed in the anterior nerve group of sea cucumber.And the Aj-DA1R gene was highly expressed in the nerve tissue of sea cucumber,and widely expressed in non-gonadal tissues.Its expression in muscle tissue of sea cucumber also had obvious seasonal variation characteristics,and the expression of Aj-DA1R was significantly lower than that in other months during aestivation.The data showed that DA signaling system was widely involved in the physiological regulation process of sea cucumber,and played an important role in nerve activity. |
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