The Effect Of α₂ Adrenergic Receptor And 5-hydroxytryptamine 2A Receptor On The Metamorphosis Of Mytilus Coruscus

As important neurotransmitters,epinephrine and serotonin are widely distributed in the organisms,playing an important messenger role in the chemical synapses between neurons and receptors,participating in various complex life activities,and playing an important role in regulating homeostasis of the internal environment.Previous studies have shown that epinephrine and serotonin induce metamorphosis in a variety of molluscs,including mussel larvae with Mytilus coruscos,but their molecular pathways are unclear.In this study,M.coruscus was selected as the research object to investigate the proteomics of the larvae before and after metamorphosis,and to investigate the function of adrenergic receptor and serotonin receptor in the metamorphosis of mussels with M.coruscus.The main results are as follows:1.Analysis of proteomic difference before and after metamorphosis of M.coruscus larvae induced by epinephrine.In this study,label-free technology was used to isolate and screen differentially expressed proteins in the process of metamorphosis of M.coruscus larvae,and PRM technology was further used to identify key proteins related to development in the process of metamorphosis of larvae,and a total of 458 differentially expressed proteins were screened.The involved signaling pathways include epinephrine signaling pathway,G protein-coupled receptor signaling pathway,and protein phosphorylation signaling pathway,and are involved in basic cell structure and physiological activities such as cytoskeleton,energy metabolism and transcriptional translation.This study further revealed the important role of epinephrine signaling pathway in the metamorphosis process of M.coruscus,and provided a basis for the comprehensive analysis of the protein response mechanism of the metamorphosis process,as well as a reference for the understanding of the metamorphosis mechanism of Marine bivalves.2.Cloning and expression analysis of the alpha 2 adrenergic receptor and the 5-hydroxytryptamine 2A receptor genes in M.coruscus.Based on the transcriptome analysis results of M.coruscus,the alpha 2 adrenergic receptor and the 5-hydroxytryptamine 2A receptor,named asα₂AR and 5-HT2AR,were preliminarily screened and cloned.Both receptors are G-protein-coupled-receptors（GPCRs）,which contain a seven-fold transmembrane domain.The full-length c DNA sequence ofα₂AR was obtained using RACE.It was consisted of a 1359-bp ORF,a 73-bp 50-untranslated region（UTR）and a 66-bp 30-UTR.The ORF was predicted to encode a 452 amino acid protein.Theα₂AR gene expression was found in both male and female of M.coruscus,and the hemocytes expression was higher.Theα₂AR gene was also expressed in each developmental stage of M.coruscus larvae,and the expression level ofα₂AR gene was significantly increased in the larvae from pediveliger to post-larvae,suggesting thatα₂AR gene may be involved in regulating the metamorphosis and development of the M.coruscus larvae.The full length of 5-HT2AR gene was 2636 bp and 2124 bp of open reading frame（ORF）encoding 707amino acids.This study showed that 5-HT2AR m RNA expression was detected in all tissues and the highest expression was found in gill and female gonad,indicating that5-HT2AR gene may play an important role in perception of the external environment and promotion mature oocytes.The 5-HT2AR m RNA was expressed at all developmental stages,but there was no significant difference,indicating that 5-HT2AR may play a role in the whole development process of M.coruscus larvae.3.Study on the role ofα₂AR gene in the metamorphosis of M.coruscus larvaeIn this study,the role of AR gene in the metamorphosis of mussel larvae was detected by RNA interference technique.Mcα₂AR knockdown resulted in a profoundly reduction in the Mcα2AR transcript abuandance,and the expression level of the interference group was 84%lower than that of the control group,and subsequently caused a significant inhibiting activity on larval metamorphosis of M.coruscus,the metamorphosis rate of the interference group was 38%lower than that of the control group,but there was no significant difference in the survival rate of larvae among each group.This finding provides new insight into molecular basis of the AR gene modulating larval metamorphosis in this species.