Identification,Expression,and Reproductive Regulation Of Neuropeptide FMRFamide Receptors Of Sepiella Japonica

Sepiella japonica is an important economic cephalopod in coastal areas of China.Due to excessive manual fishing and unreasonable resource consumption in the 1970s,the production of S.japonica rapidly declined and was on the verge of extinction.In recent years,with the promotion of techniques such as artificial breeding and multiplication and exile in many coastal areas,the resources of S.japonica have also been significantly restored.However,the artificially cultivated S.japonica is prone to problems such as precocious puberty,small size,and few eggs.Therefore,it is imperative to clarify the molecular regulation mechanism of reproductive development of S.japonica in artificial breeding.Neuropeptides play an important role in physiological reproduction,and SjFMRFamide gene and its receptors are currently involved in the regulation of reproductive development.However,there are still no clear research results about the role and mechanism of FMRFamide gene and its receptor in the regulation of reproductive development of cephalopods such as S.japonica.This thesis focuses on the two receptor genes of the neuropeptide FMRFamide:the Na+channel receptor of FMRFamide was cloned and identified and the tissue expression specific study of the receptor gene was carried out;and the mature FMRFamide peptide was mapped by immunohistochemistry;the G protein-coupled receptor and Na+channel receptor of FMRFamide were analyzed for mRNA level localization,subcellular localization,reproductive development function and other aspects.The main results are as follows:1.The full-length cDNA of S.japonica FMRFr-NaC gene was obtained using RACE(Rapid-Amplification of cDNA Ends)technology and named SjFMRFr-NaC.The full length of SjFMRFr-NaC is 3004 bp,including 262 bp 5’-UTR,930 bp 3’-UTR and 1812 bp ORF.It encodes 603 amino acid residues;the predicted MW is 67.58 KDa and the pI is 5.51;the predicted SjFMRFr-NaC contains 5 glycosylation sites and 69 phosphorylation sites;its amino acid sequence has no signal peptide at the N-terminus and has two transmembrane regions;The phylogenetic tree results show that SjFMRFr-NaC and the FMRFr-NaC of Sepia pharaonis,Octopus bimaculoides,O.vulgaris and other cephalopods are clustered together;the results of amino acid alignment showed that the sequence similarity of SjFMRFr-NaC compared to FMRFr-NaC in S.pharaonis,O.bimaculoides and O.vulgaris was 93.54%,74.48%and 71.79%,respectively;the results of RT-PCR and qRT-PCR showed that in female S.japonica with different gonadal development stages,the expression of SjFMRFr-NaC gene in optic lobe was significantly higher than those in other tissues(p<0.05),and the expression of SjFMRFr-NaC gene was the highest in stage Ⅳ andⅥ(p<0.05).SjFMRFr-NaC gene was also significantly expressed in brain of stage Ⅲ,brain,liver and nidamental gland of stage Ⅳ,pancreas of stage Ⅴ,and brain and nidamental gland of stage Ⅵ(p<0.05).2.The results of ISH showed that:in the optic lobe of S.japonica,the positive signals of the two receptors SjFMRFr-NaC and SjFMRFr-GP appeared in the medulla and deep retina,in which,SjFMRFr-GP was mainly expressed in the medulla area,while SjFMRFr-NaC was mainly expressed in the deep retina.In the brain of S.japonica,the positive signals of the two receptors can be observed in both the supraesophageal mass and the subesophageal mass.3.The results of IHC showed that immunologically active regions of FMRFamide were located in the deep retina and plexiform radial nerve fibers of the optic lobe,and FMRFamide immune signals were also detected in both the supraesophageal mass and the subesophageal mass of the brain;the results showed that SjFMRFamide-EGFP were localized in the HEK 293 cells cytoplasm,while SjFMRFr-GP-EGFP and SjFMRFr-NaC-EGFP are located on the cell membrane.