Molecular Cloning,expression And Physiological Functions Of Leptin And Its Receptor Genes In Cynoglossus Semilaevis

In the present study,the full-length cDNA sequences of two leptin genes,lepa and lepb and its receptor gene lepr were cloned from Cynoglossus semilaevis using homologous cloning and RACE strategy.Then the spatial and temporal expression patterns of these three genes were investigated.Furthermore,the in vitro prokaryotic expression technology was established for tongue sole lepa and lepb and the recombinant proteins with biological activity was obtained.The results of this study provide basic data and theoretical support for comprehensively understanding of the physiological mechanism underlying growth and development of tongue sole.1.Cloning and tissue expression analysis of leptin and its receptor genesThe full-length cDNA sequences of two leptin genes,lepa and lepb and a receptor gene lepr were cloned.The full-length cDNA of tongue sole lepa and lepb were 1265 bp and 1157 bp in size,encoding for proteins of 160 aa and 158 aa,respectively.The three-dimensional structures modeling of tongue sole LepA and LepB showed strong conservation of tertiary structure with other vertebrates.The full-length cDNA of tongue sole lepr was 4576 bp,encoding a protein of 1133 aa which contained all functionally important domains conserved among vertebrate LepRs.Phylogenetic analysis showed that LepA is closely related to the lepA of the Acanthophylla,LepB and LepR are clustered together with LepB and LepR in the order Acanthoptera,respectively.lepa gene was highly expressed in ovary and brain.However,lepb mRNA is widely expressed in various tissues,suggesting that lepb may have a wide range of physiological roles.lepr mRNA is most abundant in ovary,and low in pituitary,gill and kidney.In addition,other tissues have low but detectable lepr levels,suggesting that ovary may be the main target organ for its physiological function.2.Dynamic expression patterns of leptin and its receptor genes during early life stagesThe expression characteristics of two leptin genes and one receptor gene in tongue sole in unfertilized eggs and during early life stages were investigated.Results showed that leptin ligand and receptor genes were detected in unfertilized eggs and embryos at all development stages,but the expression levels were vary with the development stage.lepa mRNA levels fluctuated slightly before stage 8?morula?and then increased significantly,followed by a reduction between stage 9?blastula?and stage 10?gastrula?.Subsequently,the expression increased again,reaching the maximal levels at stage 12?embryo encompassing yolk 70%?,just before hatching.On the other hand,lepb and lepr mRNAs showed similar expression profiles,with a peak level at stage 9 and stage 8,respectively,displaying a bell-shaped curve.In addition,the expressions of GH,IGF-I and IGF-II in unfertilized eggs and embryonic development stages were simultaneously detected.Partial correlation analysis showed that lepr gene expression level was negatively correlated with IGF-I and IGF-II gene expression level during embryonic development,and GH gene expression level was positively correlated with IGF-I gene expression level.During the larval and juvenile growth stages,the expression level of lepa mRNA gradually increased and peaked at 10 dph,and then decreased from 15 dpf to 40 dph.On the other hand,lepb mRNA expression level increased significantly at 3 dph and then gradually decreased until 15 dph.Subsequently,lepb mRNA expression level increased again from 20-25 dph and then decreased at 30 dph.The lepr mRNA level fluctuated slightly at 1-25 dph,peaked at 30 dph,and decreased significantly at 40 dph.In addition,the expression levels of GH,IGF-I and IGF-II mRNA were also investigated.Partial correlation analysis showed that lepa gene expression level was negatively correlated with lepb gene expression level,and positively correlated with lepr,GH and IGF-II gene expression level in larval and juvenile fish.At the same time,the expression of IGF-II was positively correlated with lepr and IGF-I.3.Physiological roles of leptin and its receptor genes during ovarian maturation cycleDuring annual ovarian maturation cycle,the levels of lepa and lepr mRNA in brain,pituitary and ovary were significantly higher than that of lepb mRNA.The level of lepa gene expression level in brain peaked at stage III,then decreased to the lowest level at stage IV.The level of lepb gene expression achieved the minimum level at stage IV,and then gradually increased to the highest level at stage VI.The lepr gene expression level was significantly higher at stage II,but significantly lower at stage III-V,and increased again at stage VI to the same level of stage II.The expression level of lepa in pituitary gland?Plepa?decreased to the lowest level at stage III,then peaked at stage IV.The expression level of lepb?Plepb?peaked at stage III,then decreased significantly and maintained a low expression level at stage IV-VI.The expression level of lepr mRNA?Plepr?decreased to the lowest level at stage III,then increased significantly at stage IV-V,and decreased again at stage VI with higher levels than that of stage II.The expression level of lepa gene?Olepa?decreased at stage II,and reached the lowest level at stage V,increased again at stage VI.The lepb gene?Olepb?reached a high expression level at stage III,but then increased significantly.The level of lepr gene expression?Olepr?decreased at stage II until the lowest level at stage V,but increased significantly at stage VI.Partial correlation analysis showed that Blepa was positively correlated with Blepb,Plepb,Olepb and Olepr,and negatively correlated with Plepa and Plepr.Blepb was positively correlated with Blepr,Olepa,Olepb and Olepr.Blepr was positively correlated with Plepr.Plepa was positively correlated with Plepr and negatively correlated with Olepa,Olepb and Olepr.The level of serum E2 increased significantly from stage II to stage IV,and then decreased to the lowest level at stage V.Serum T level increased gradually from stage II,peaked at stage V,and then decreased significantly at stage VI.The changes of serum FSH level and serum LH level and serum leptin level were generally the same.They increased gradually from stage II to stage IV,and then peaked at stage V and VI,and the expression level was relatively low.Partial correlation analysis showed that there was a significant or very significant positive correlation between E2 and T,FSH,LH.LH was positively correlated with T and FSH.Leptin was positively correlated with E2,T,FSH and LH.These results suggest that leptin and its receptors are involved into the ovarian development of the tongue sole in a synergistic or antagonistic manner at the transcriptional and serum levels.4.Recombinant expression and bioactivity analysis of two leptin genes from Cynoglossus semilaevisThe peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis.The prokaryotic expression vector pQE30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30,which were transformed into E.coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing His 6 at the N-terminus.The obtained LepA and LepB polypeptides expressed in form of inclusion bodies with molecular weight were both 16 kDa,and the optimum condition for highest expression of the target proteins were induced by 0.5 mM IPTG at 37°C for 4 hours.The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3mg/ml and 0.25 mg/ml,respectively.Western-blotting and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity.The proteins purification was used Ni²⁺-NTA affinity chromatography,and high-purity recombinant proteins of lepa and lepb were obtained.In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from Cynoglossus semilaevis indicated that they could significantly inhibit the expression of lepa and lepb mRNA in hypothalamus,which verified that the obtained recombinant proteins have biological activities.