Preliminary Study On The Regulating Mechanism Of Insulin-like Peptide And Its Receptor On Ovarian Development Of Sepiella Japonica

Sepiella japonica is a traditional cephalopod fishery resource in China.Since 1970 s,due to the transitional fishing and the deterioration of environmental conditions,its resources have declined seriously.In recent years,due to the enhancement and releasing,artificial breeding and other technologies,the resources of cuttlefish have been restored to a certain extent.However,there is a problem of precocious puberty in cultured cuttlefish,which leads to the small size of cuttlefish individuals.Therefore,it is imperative to elucidate the neuroendocrine regulation mechanism of the reproduction of Sepiella japonica under artificial breeding conditions.Insulin-like peptide and its receptor can regulate the growth,development,aging,metabolism and reproduction of invertebrates through a conservative insulin signaling pathway.But the regulation and mechanism of insulin-like peptide and its receptor in the reproductive development of cephalopods,such as Sepiella japonica,has not yet been clarified.In this paper,the insulin-like peptide and its receptor genes of S.japonica were cloned for the first time,and the sequence analysis,subcellular localization,tissue and reproductive cycle expression characteristics,reproductive development function and related signal pathway were studied.The main findings were as follows:The complete cDNA sequence of SjILP obtained from S.japonica(GenBank accession number MK611805)was 926 bp by using the RACE cloning technology.The sequence included 310 bp 5'UTR,486 bp ORF region encoding 161 amino acids,and 130 bp 3'UTR.It was predicted that SjILP had a similar genetic structure to vertebrate insulin precursor: signal peptide,B chain,C peptide and A chain,and contained conservative cysteine residues and two C peptide cleavage sites.Amino acid sequence homology alignments showed that SjILP had the highest similarity to Octopus bimaculoides ILP(39.02%).Phylogenetic analysis revealed that Sj ILP was clustered with other identified mollusc ILPs.The complete cDNA sequence of SjILPR obtained from S.japonica(GenBank accession number MK611806)was 5288 bp by using the 5' and 3' RACE.It consisted of 487 bp 5'UTR,4764 bp ORF region encoding 1587 amino acids,and 37 bp 3'UTR.SjILPR had the typical structural characteristics of the insulin receptor family: receptor L-domain(L1 and L2),cysteine-rich domain(Cys),fibronectin type III domain(Fn III)and protein kinase domain(TK).Among them,the ?L1-Cys-L2? structure was a ligand binding site,and the TK domain played an important role in intracellular signaling.The homology alignment found that the amino acid sequences of insulin-like peptide receptors and their homologues were highly conserved among different species,and the similarity between Sj ILPR and O.bimaculoides ILPR was the highest(66.27%).The phylogenetic tree analysis showed that SjILPR was clustered with other confirmed mollusc ILPRs.Immunofluorescence technology was used to perform subcellular localization analysis of insulin-like peptide and its receptor protein of S.japonica,and it was found that the HEK293 T cells successfully transfected with Sj ILP-EGFP had green fluorescence in the cytoplasm and cell membrane,indicating that SjILP was localized on the cytoplasm and cell membrane,which was consistent with the feature of insulin-like peptides as secreted proteins.While the HEK293 T cells successfully transfected with Sj ILPR-EGFP only found green fluorescence signal on the cell membrane,indicating that Sj ILPR was localized on the cell membrane.And this result was consistent with the existence of transmembrane domains in its sequence.The real-time quantitative PCR technology was used to study the tissue distribution characteristics and the variation of insulin-like peptide and its receptor genes during the reproductive cycle in S.japonica.The results showed that SjILP and SjILPR were expressed in various tissues of S.japonica,but their expression levels in pancreas,ovary and liver tissues were significantly higher than other tissues(P <0.05).At the same time,Sj ILP and Sj ILPR had the same expression pattern in the four stages of ovarian development of female S.japonica: the expression of SjILP and SjILPR in pancreas,ovary and liver gradually increased from the oogonium production period,and reached the highest level in the interstitial growth period,but decreased significantly in the trophoplasmic growth period(P < 0.05).These results suggested that insulin-like peptide and its receptor may play an important role in gonadal development and reproductive function regulation of S.japonica.To further elucidate the function of insulin-like peptide and its receptor in the reproduction and development of S.japonica,RNA interference technology was used to interfere the Sj ILP and Sj ILPR genes,and the differentially expressed genes between the interference group and the control group were analyzed by transcriptome sequencing.It was found that there were 6307 differentially expressed genes in SjILP interference group compared with the control group,including 2577 down-regulated genes and 3730 up-regulated genes.And the Sj ILPR interference group had 4314 differentially expressed genes compared with the control group,including 2586 down-regulated genes and 1728 up-regulated genes.These genes were enriched in signal pathways related to gonadal development,including cell cycle,oocyte meiosis,progesterone mediated oocyte maturation,gonadal development,gonadotropin releasing hormone signal pathway,etc.We selected four down-regulated genes directly related to gonadal development,including vitellogenin 1(Vg1),vitellogenin 2(Vg2),cathepsin L1-like(CtsL1-like)and follistatin(FST),and the effect of RNA interference of Sj ILP and SjILPR genes on the expression of these genes was further verified by real-time quantitative PCR.The results showed that RNA interference could effectively inhibit the expression of SjILP and SjILPR genes in ovary and liver(P <0.05),and it could also significantly reduce the expression levels of Vg1,Vg2,CtsL1-like and FST genes in ovary and liver(P < 0.05).This suggested that Sj ILP and SjILPR genes may affect the expression of downstream related genes,thereby regulating the reproductive development process of squid.This suggested that SjILP and Sj ILPR genes may regulate the reproduction and development of S.japonica by influencing the expression of related downstream genes.