Cloning,Expression And Function Of Ovarian Development Related Genes ActR In Common Chinese Cuttlefish,Sepiella Japonica

Sepiella japonica is one of the most important Marine cephalopods with the high economic and nutritional value in China.Although a breakthrough has been made in the artificial breeding of S.japonica,the prevalence of precocious puberty has led to the miniaturization of cuttlefish individuals,which seriously affects its commercial value and breeding results.It is essential for solving the problem to study the regulatory mechanism of reproduction and development of the cuttlefish under the condition of breeding.As one receptor related to sexual maturation,activin receptor(ActR)plays an important role in sexual maturation and reproductive development of many animals.In view of the potential involvement of ActR in the regulation of cuttlefish reproductive development,the full cDNA sequence of ActR gene of S.japonica was cloned for the first time in this paper.And the function of ActR in the ovarian development of S.japonica was explored by analyzing its sequence characteristics,subcellular localization,receptor type identification and temporal and spatial expression of reproductive axia as well as using RNA interference technology.The main results were as followed:1.The full cDNA length sequence of SjActR was 2264 bp(MH249268)which been obtained by 5'RACE and 3'RACE.It consists of 211 bp 5'UTR,535 bp 3'UTR and 1518 bp ORF,and can encode 505 amino acids.The predicted molecular weight(Mw)and isoelectric point(pI)of protein were 58.06 kDa and 8.88 respectively.Similar to other receptor of serine/threonine kinase family members,the structure of SjActR also includes three domians : activin type I and type II receptor domains(hydrophilic ligand domain,66 aa),transmembrane domain(31 aa)and serine/threonine kinase domain(292 aa).In homology comparison,SjActR1 sequence was highly conserved in activin type I and II receptor domain,transmembrane domain and serine/threonine kinase domain,with 94%,100% and 99% similarities with ActR1 of Sepia latimanus,respectively.The phylogenetic analysis of ActR revealed that SjActR was clustered with ActR1 from the mollusk branch,which suggests that the obtained activin receptor belongs to ActR1.2.The properties and localization of SjActR1 receptor were analyzed.The SjActR1-EGFP expression vector was constructed and transfected into HEK293 T cells.The green fluorescence was mainly distributed on the membrane with staining by Dil and DAPI staining.It was confirmed that SjActR1 was localized on the cell membrane,which was consistent with the transmembrane domain of SjActR1 sequence.This suggests that SjActR1 is a membrane receptor and plays an important role in activin signaling.3.The expression specificity of SjActR1 in different tissues of female S.japonica was studied by real-time quantitative PCR.The results showed that SjActR1 gene was expressed in different organs of female S.japonica.The expression level of SjActR1 gene in reproductive development-related tissues such as brain,ovary and liver were high,while the expression level in other tissues was relatively low.The expression of SjActR1 fluctuated significantly during the reproductive developmental cycle of female S.japonica.From oogonium stage to interstitial growth stage,the expression of SjActR1 increased significantly with the development of oocyte and decreased significantly during the vegetative growth stage.These results suggest that ActR1 may play an important role in the development of cephalopod ovaries.4.In order to reveal the specific function of SjActR1 in the reproductive development of cephalopods,we investigated the correlation between SjActR1 and reproductive development of female cuttlefish by injecting estradiol and GnRH,and RNA interference of SjActR1 gene.The results showed that 24 h after injection of estradiol and GnRH,the SjActR1 gene was significantly up-regulated in the ovary and liver tissues of female S.japonica,and vitellogenin gene was also up-regulated in the ovary and liver.In vivo RNA interference experiment,it was found that Sj ActR1 was significantly down-regulated in ovary and liver tissues in both experimental groups,and vitellogenin gene was significantly downregulated in ovary in the experimental group.This indicates that SjActR1 gene can be upregulated by estrogen and GnRH stimulation during the gonadal development of female S.japonica and can regulate the synthesis and secretion of vitellogenin.The results of the research on activin receptor in this study lay a foundation for further study on the regulation mechanism of female reproductive development of S.japonica and the role of steroid hormones in cephalopod species formation.It also provides a theoretical basis for illustrating the mechanism of sexual precocity of cephalopods such as S.japonica under artificial breeding conditions.