Studies About The Role Of Prolactin In Osmoregulation Of The Spotted Scat(Scatophagus Argus)response To Environmental Salinity Change

Scatophagus argus(also named spotted scat)is a euryhaline sea fish with breeding migration feature.Due to its ornamental and economic value,it has become one of the most popular mariculture species in the South China.According to the previous researches,S.argus has a wide tolerance to the salinity change.Therefore,we think that S.argus must have extremely efficient osmoregulatory mechanism to maintain homeostasis,which makes it become an ideal material for studying the molecular mechanism of salinity adaptation of fish.The endocrine system is one of the main ways for fish to osmotic regulation.In this system,prolactin(PRL)has been paid much attention to due to its significant function in helping fish adapt to freshwater.In this paper,we cloned the full-length c DNA sequence of prl from S.argus,named saprl,and two receptors of it,named saprlr1 and saprlr2.Additionally,the expression patterns of these three genes were analyzed.Afterwards,we designed specific primers with restriction sites to amplify the sa PRL target fragments,and successfully construct the recombinant plasmid,sa PRL-p ET28a(+),which can express PRL protein in vitro using the pET-28a(+)vector.After the abundant induced expression in E.coli,we purified and got the active protein sa PRL.Furthermore,we studied in vitro the interaction among saprl,and the downstream genes and ion transporters/channels,which are related to osmoregulation.This provides the theory basis for the further study of the molecular mechanism of PRL in osmotic regulation.The main results of this study are below:1.We successfully cloned the full-length c DNA sequences of saprl,saprlr1 and saprlr2 from S.argus.The c DNA length of saprl was 1550 bp,ORF(open reading frame)was 639 bp which encoded 212 amino acids(aa).The c DNA length of saprlr1 was 2384 bp,ORF was 1908 bp which encoded 635 aa.The c DNA length of saprlr2 was 2495 bp,ORF was 1680 bp which encoded 559 aa.Acccording to the multiple amino acid sequence alignment,we found that saprl contained the common conserved domain with other species.And it had four highly conserved cysteine residues(C)at the C-terminal,which formed two molecules of disulfide bond.saprl had a lack of 12-14 aa at the N-terminal,which involved one disulfide bond.The extracellular domains of saprlr1 and saprlr2 were very similar,which had highly conserved WS motif and four conserved cysteine residues(C).However,the intracellular domains were different.saprlr1 had conserved Box1 and Box2 region,while saprlr2 only had Box1 region.All these three genes in S.argus had high similarity with those of Acanthopagrus schlegelii.Phylogenetic analysis showed that saprl was clustered into a branch with prl of Perciformes,and seperated from that of mammals.saprlr1 and saprlr2 was clustered with prlr1 and prlr2 of A.schlegelii into a branch,respectively.Additionally,saprlr1 was clustered into a branch with other fish prlr1 clade,separating from non-fish vertebrates.Tissue distribution analysis showed that saprl just highly expressed in brain and pituitary.saprlr1 and saprlr2 highly expressed in the osmoregulartory organs,like gill,kidney and intestines,which illustrated that the main function of sa PRL in S.argus was osmoregulation.Moreover,sa PRL mainly located in the cerebral cortex of S.argus.Under different salinity environments,the expression levels of branchial and renal saprlr1 and saprlr2 of low salinity group were all higher than that of high salinity group,which demonstrated that sa PRL and its specific receptors played an significant role for S.argus in the adaptation to the low salinity environment.Our research was aimed at providing fundamental basis for the follow-up study.2.After the acute hypotonic shock,the expression level of brain sa PRL and serum PRL of S.argus were up-regulated significantly in a short period,which illustrated that PRL could rapidly synthesize and secrete under hypotonic stimulation.Thus,we suspected sa PRL acted as an acute response hormone for S.argus adapting to the low salinity environment.In addition,it's possible that the phenomenon that transferring S.argus to fresh water(FW)didn't affect its survival and vitality was related to the synergistic effect of PRL and GH.The expression levels of saprlr1 in gill and kidney were all significantly up-regulated under acute hypotonic stress,which demonstrated that this gene played a key role for the adaptation of S.argus to the low salinity environment.The expressive suppression of saprlr2 might be related to the maintenance of high-level PRL in S.argus.The study of this chapter verified Ras/ERK signal transduction pathway could in response to the osmotic stress in fish.Moreover,the study about the downstream ion transporters/channels showed that the expression of ncc was up-regulated under hypotonic shock,which hinted that NCC had an effect in reabsorbing Na+ and Cl-in low salinity environment.Branchial nkcc2 didn't reveal expressive difference under acute hypotonic shock.Howerver,the significant expressive promotion of renal nkcc2 hinted that this ion transporter had an effect in reabsorbing Na+ and Cl-under hypotonic stress as well.Thus,the expression of nkcc2 under salinity stress probably remains tissue-specific.The m RNA expression level of cftr gene was also significantly down-regulated for hypotonic stimulation,which illustrated that this gene might paly a role in inhibiting secretion of Cl-in the adaptation of S.argus to FW.The study of this chapter provided certain theoretical basis for further study of sa PRL in the adaptation of S.argus to acute hypotonic stress.3.We successfully constructed the prokaryotic expression plasmid,sa PRL-p ET28a(+).In the host bacteria that contained sa PRL-p ET28a(+),we added IPTG with 1 mmol/L final concentration.After 4-hour induction at 32 ?,massive recombinant proteins expressed,which was the optimum induced expression condition for sa PRL recombinant protein.After low temperature induced expression(IPTG 0.5 mmol/L,16 oC),we obtained soluble sa PRL recombinant protein,which laid the foundation for the follow-up study.4.In this chapter,we processed the primary cell of gill(SG cell)and kidney(SK cell)from S.argus using the purified sa PRL recombinant protein to further study the function of sa PRL to its specific receptor and the downstream ion transpoters/channels,NCC,NKCC2 and CFTR.The results showed that after processing the SG cells and SK cells for 3 h using 500 ng/m L sa PRL recombinant protein,the expression levels of the related genes changed.And after around 24 h,the expression levels would recovery to original.Processing with sa PRL recombinant protein could up-regulated the gene expression levels of saprlr1,saprlr2 and ncc in SG cells and SK cells.Additionally,when the final concentration of sa PRL recombinant protein was more than 100 ng/m L,they showed significant differences.Processing with sa PRL recombinant protein didn't have an effect on the expression of nkcc2 in SG cells.However,nkcc2 in SK cells was up-regulated significantly after processed with 500 ng/m L sa PRL recombinant protein.sa PRL recombinant protein could down-regulate the expression levels of cftr gene with no statistical difference both in SG and SK cells.Therefore,sa PRL may have direct function to NCC and NKCC2 when S.argus adapts to low salinity,which induces the fish to absorb Na+ and Cl-.CFTR may play a role in inhibiting the secretion of Cl-when S.argus adapts to FW environment.However,the results illustrated that functioning CFTR had no direct association with sa PRL.