Molecular Cloning And Transcriptional Analysis Of A NPY Receptor-like In Common Chinese Cuttlefish Sepiella Japonica

Sepiella japonica is an important marine economic species.Its artificial breeding has developed rapidly.But the relatively small form induced by the precocious puberty seriously affected its economic benefits.Thus research focusing on the squid growth and reproductive development mechanism is the key to solve this problem.Neuropeptide Y receptor(NPYR),a typical G protein-coupled receptor,plays an important role in the growth and development of breeding.Experimental methods such as molecular biology,cell biology,characteristics and sequence analysis were used to detect the Sepiella japonica NPYR-like(Sj NPYR-like)gene sequence characteristics,typical feature,subcellular localization,tissue distribution,and expression changes of varying regulation in growth cycle as well as of functional regulation in feeding.Main findings are as follows:The full-length c DNA sequence of Sj NPYR-like was acquired by using RACE clone technology.With analysis of its sequence,results showed that the full-length c DNA of Sj NPYR-like(KX683395)was 1,902 bp,which contained a 492 bp 5'-untranslated region(UTR),a 228 bp 3'-UTR,and a 1,182 bp open reading frame(ORF)encoding 393 amino acid(aa)residues.As similar with other GPCRs,Sj NPYR-like has the typical seven transmembrane(TM)domains of TM1(AA36-58),TM2(AA79-101),TM3(AA111-133),TM4(AA145-167),TM5(AA200-222),TM6(AA254-271)and TM7(AA286-308).Phylogenetic analysis showed that Sj NPYR-like was homologous with three subtypes(NPYR-1,NPYR-2,and NPYR-5)of neuropeptide Y receptors in vertebrates and got together with invertebrates NPFRs.Multiple sequence alignment showed that the homology between Sj NPYR-like and cephalopod Octopus neuropeptide Y receptor is up to 81%.Using cell biology research method,the cell location of Sj NPYR-like receptor was analyzed.Sj NPYR-EGFP fusion expression vector was constructed,meanwhile HEK293 cell membrane and nuclear were stained by fluorescent Di I and DAPI.Detected by confocal microscopy,results showed that the Sj NPFR-like was localized on the membrane which is consistent with the prediction of its seven transmembrane domains.This outcome showed that Sj NPYR-like is a membrane receptor which is laying the foundation for the further exploration.The tissue expression characteristic of Sj NPYR-like m RNA in different reproductive stages was analyzed by quantitative Real-time PCR.Results indicated that Sj NPYR-like gene had the higher expression in tissues of brain,gonad and liver,while the expression levels of other tissue are relatively low.The expression of Sj NPYR-like goes through a significantly variation during growth and development stages of cuttlefish.The consequence showed that the expression of Sj NPYR-like experiences a remarkable rise from oogonia stages to interstitial growth stages,while experiences a distinguished decline during trophoplasmic growth stages(P < 0.05).The characteristic of Sj NPYR-like varied expression demonstrates that Sj NPYR-like may play an important function during the development and reproduction of cuttlefish.In the present study,Sj NPYR-like was identified and preliminarily explored for sub-cellular localization and physiological function.These results could provide theoretical basis and reference for further in-depth research to slove the problem of S.japonica precocious puberty in artificial culture.