Study On Screening And Functional Characterization Of Neuropeptides Related To Reproductive Control Of Sea Cucumber

This study focuses on the screening and functional identification of neuropeptides related to reproductive control of sea cucumber Apostichopus japonicus,through bioinformatics analysis,molecular cloning,function evaluation in vivo and in vitro.Based on the data from the sea cucumber genome and transcriptome,four neuropeptide coding genes with predicated functions related to reproductive development of sea cucumber were screened.Focusing on the sea cucumber seapeptin Kisspeptin neuropeptide,the molecular structure,activity characteristics and physiological functions were initially clarified.The main results are listed as follows:1.Based on the A.japonicus genomic and transcriptomic data,4 neuropeptide-like gene sequences related to reproduction regulation of sea cucumber(AjKisspeptin,NPS/CCAP type peptide(NG),F-likeSALMF(FS),L-likeSALMF(LS))were screened out.Further analysis on AjKisspeptin indicates that this gene encodes 180 amino acid kisspepetin precursor with 543 bp ORF,including 23 aa signal peptide and mature pepetides: AjKiss1a(AGSLDc<CLEASC>EDVERRGRQPNRNAHYRTLPF-NH2)and AjKiss1b(SAVKNKNKSRARPPLLPF-NH2).The precursor peptide was deduced with an isoelectric point(pI)of 4.90 and a molecular weight(Mw)of 20.283 kD.The qPCR assays were further conducted and expressional variation of these four screened genes at different stages of reproductive development of A.japonicus was analyzed.The results provide a reference for biological function identification on these neuropeptides in future.2.Using the constructed fusion expression vector of A.japonicus Kisspeptin receptor(AjGPR54-EGFP),we testified the functional activity of an artificially synthesized Kisspeptin mature peptide(AjKiss1b-10).Results indicated that the receptor expressed in HEK293 was activated inducing significant receptor endocytosis.The intracellular calcium mobilization and ERK1/2 phosphorylation was also detected providing evidences for the interaction between AjKiss1b-10 and AjGPR54.The results indicate the functional acitivity of the predicted A.japonicus Kisspeptin and support the further identification of the physiological functions of this neuropeptide system.3.Further physiology experiments in vivo and in vitro were conducted to reveal the physiological functions of AjKiss1b-10.Through gene expression analysis and immunofluorescence histochemical detection,it was found that A.japonicus Kisspeptin was expressed with significantly high level during reproductive process,and the precussor protein was mainly distributed in the testis,and nerve ring.In vitro physiological experiments,AjKiss1b-10 can activate the Kisspeptin receptor in cultured ovarian cells mediating ERK1/2 phosphorylation.It was further confirmed that AjKiss1b-10 was involved in the functional regulation of respiratory tree and digestive tract of sea cucumbers leading to weight loss through pharmacological experiments.