Cloning, Expression And Functional Study Of Leptins And NPYs In Jian Carp (Cyprinus Carpio Var.Jian)

Jian carp(Cyprinus carpio var.jian)was artificially bred by Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences.Jian carp is a good stock of cultivated fish species with good economic traits and has been extended to most of the aquatic company in China.In recent years,we have focused on growth traits in jian carp genetic improvements and take some achievements.However,the growth trait will eventually reach a plateau of increasing and the direction of breeding will turn to other economic traits.So it is necessary to study on fish appetite factors,which provide theory information for jian carp breeding.The main results can be summarized as follows:1.Identification of housekeeping genes suitable for gene expression analysis in jian carp(Cyprinus carpio var.jian)In this report,we performed a systematic analysis to identify an appropriate housekeeping(HK)gene for the study of gene expression in jian carp.For this purpose,partial DNA sequences of four potential candidate genes(elongation factor 1 alpha(EF-1?),glyceraldehyde-3-phosphate(GAPDH),beta-actin(ACTS),18S ribosomal RNA(18S rRNA)were isolated and their expression were studied using RNA extracted from nine tissues(forebrain,hypothalamus,liver,fore intestine,hind intestine,ovary,muscle,heart,kidney)in juvenile and adult jian carp.Gene expression level was quantified by quantitative real time RT-PCR(qRT-PCR)and expression stability was evaluated by comparing coefficient of variation(CV)of the Ct value.The resulted showed that EF-1?was the most suitable HK gene in juvenile and adult jian carp all tissues.However,in different developmental stages,there was not a single gene for normalization of gene expression in all tissues.EF-1? was the most stable gene only in forebrain,hypothalamus,liver,heart,and kidney.These results provide data that can be expected to aid jian carp researches in gene expression analysis,but underlined the importance of identification HK gene for each new experimental paradigm.2.Cloning and spatiotemporal expression of leptins in jian carpLeptin is the product of obese gene and plays a major role in food intake regulation.RT-PCR and PCR method were used to isolate the partial sequence of jian carp lepitns including an open reading frame(ORF).Leptins expression was analyzed in juvenile and adult jian carp different tissues by real time PCR.The results showed that there were three homology leptins including jlLEP-A1,jlLEP-A2 and jlLEP-B.The sequence length of jILEP-A1 was 741 bp including an intron of 93 bp.The corresponding ORF was 516 bp in length,which encoded 171 amino acids with a predicted protein molecular weight of 19.3 kD.The sequence length of jlLEP-A2 was 792 bp including an intron of 102 bp.The corresponding ORF was 516 bp in length,which encoded 171 amino acids with a predicted protein molecular weight of 19.5 kD.The sequence length of jlLEP-B was 1194 bp including an intron of 687 bp.The corresponding ORF was 507 bp in length,which encoded 168 amino acids with a predicted protein molecular weight of 19.2 kD.The homology analysis showed that the sequence similarity of leptin protein was little.jlLEP-A1 and jlLEP-A2 shared 82.5%sequence identity,but only 29.5%,28.8%with jlLEP-B.There were highest value of 99.4%and a lowest value of 22.3%in sequence similarity between jlLEP-A1 and common carp leptin ?,and jlLEP-A1 and human leptin respectively.There were highest value of 98.8%and a lowest value of 22.1%in sequence similarity between jlLEP-A2 and common carp leptin ?,and jlLEP-A2 and pufferfish leptin respectively.There were highest value of 85.7%and a lowest value of 17.1%in sequence similarity between jlLEP-B and zebafish leptin-B,and jlLEP-A2 and medaka leptin-B respectively.Although the little sequence identity,jlLEP-A1,jlLEP-A2 and jlLEP-B all had two cysteine residues which were necessary for leptin biological function.Phylogenetic analysis of the leptin gene family indicated that there were two type of leptin.jlLEP-A1 and jlLEP-A2 were clustered to a branch and jlLEP-B to another branch.The leptins expression patterns were different between juvenile and adult jian carp.And the difference also existed between female and male jian carp.In juvenile jian carp,the relative expression level of jlLEP-A2 in different tissues was significantly lower than that of jlLEP-A1 and jlLEP-B at the 0.05 level.jlLEP-A1 and jlLEP-B were mainly expressed in gonad(ovary and testis)and liver.In female carp,jlLEP-A1 and jlLEP-A2 were mainly expressed in hypothalamus and liver.The relative expression level of jlLEP-Al in different tissues was significantly higher than that of jlLEP-A2 and jlLEP-B at the 0.05 level.However,the relative expression level of jlLEP-B in different tissues was lower.In male fish,hypothalamus was the most diverse expression tissue in jlLEP-Al,followed by liver.Muscle was the most diverse expression tissue in jlLEP-A2,followed by testis and liver.Hypothalamus was the most diverse expression tissue in jlLEP-B.3.Cloning and spatiotemporal expression of NPYs in jian carpNPY is a member of a peptide family and plays a major role in food intake regulation.RT-PCR and PCR method were used to isolate the partial sequence of jian carp NPYs including an open reading frame(ORF).NPYs expression was analyzed in juvenile and adult jian carp different tissues by real time PCR.The results showed that there were two homology NPYs including jlNPYa and jlNPYb.The sequence length of jlNPYa was 2084 bp including three introns,which were 316 bp with phage 0,897 bp with phage 2 and 178 bp with phage 2 in length respectively.The corresponding ORF was 291 bp in length,which encoded 96 amino acids with a predicted protein molecular weight of 10.96 kD.The sequence length of jlNPYb was 2140 bp including three introns,which were 315 bp with phage 0,929 bp with phage 2 and 166 bp with phage 2 in length respectively.The corresponding ORF was 291 bp in length,which encoded 96 amino acids with a predicted protein molecular weight of 10.97 kD.The homology analysis showed that the sequence similarity of leptin protein was high.jlLNPYa and jlNPYb shared 97.9%sequence identity,above 60%with human,mouse,chicken and other fish NPY,but only 54.3%and 53.2%with pufferfish NPY respectively.Phylogenetic analysis of the NPY gene family indicated there were two types of NPY.jlNPYa and jlNPYb was clustered with a branch of NPY I.NPYs were mainly expressed in fore brain,hypothalamus,testis and liver.It was also expressed in other tissues with a lower level.In juvenile jian carp,the relative expression level of jlNPYb in hypothalamus was significant higher than that of jlNPYa at the 0.05 level.In adult jian carp,the NPYs expression pattern was different between the female and male.In female hypothalamus,the relative expression level of jlNPYa was significant higher than that of jlNPYb at the 0.05 level.However,the relative expression level of jlNPYb was significant higher than that of jlNPYa at the 0.05 level in male hypothalamus.4.Influence of fasting on leptins and NPYs relative expression level in jian carpFasting experiments were carried out to investigate the possible function of leptins and NPYs genes in food intake and energy metabolism.Total RNA was immediately extracted from female and male fore brain,hypothalamus,liver and testis respectively after food deprivation in both short-term(7 days)and long-term(45 days).The influence of fasting on gene expression of leptins and NPYs was studied by real time PCR.The results showed the leptins expression levels in fore brain,hypothalamus and liver significant changed in fasting at the 0.05 level.The hypothalamus was the most diverse expression tissue in jlLEP-B?The relative expression level of jlLEP-B increased and declined in femal and male jian carp during fasting period respectively,which implied that jlLEP-B may played an important role in hypothalamus.The liver was the most diverse expression tissue in jlLEP-A1 and jlLEP-A2.The relative expression level of jlLEP-A1 and jlLEP-A2 increased in femal jian carp during fasting period.In male jian carp,the relative expression level of jlLEP-A1 increased,and that of jlLEP-A2 declined,which implied that jlLEP-B may played an important role in liver.NPYs expression level was also changed in fore brain,hypothalamus,liver and testis at the 0.05 level during fasting period.The fore brain was the most diverse expression tissue in jlNPYa and jlNPYb.The relative expression level of jlNPYa declined and and that of jlNPYb increased in femal jian carp during the fasting period.However,the relative expression level of of NPYs increased in femal jian carp,which implied NPYs played an important role mainly fore brain.The relative expression level of jlNPYb was increased by fasting time,which implied jlNPYb played an important role in regulation of fish reproducing.5.The correlation between leptins SNPs and growth trait in jian carpThree SNPs(single nucleotide polymorphisms)were confirmed through comparing leptins sequences from 8 jian carp individuals,respectively.They were A1-T113C in jlLEP-A1,A2-G415A and A2-G427A in jlLEP-A2 respectively,which were all located in gene exon region.557 jian carp(294 male and 263 female)was detected by PCR-RFLP method to find SNPs.The correlation of SNPs genotypes and individual weight gain in juvenile and adult stages was analyzed.The results showed that A1-T113C loci was significantly associated with female juvenile weight gain.The individual with genotype of TC grew significantly faster than that of TT and CC at the 0.05 level and 0.01 level respectively.In adult stage,A1-T113C loci was also significantly associated with weight gain.The individual with genotype of TC grew significantly faster than that of TT at the 0.05 level.A2-G415A loci was significantly associated with male juvenile weight gain.The individual with genotype of AA and AG grew significantly faster than that of GG at the 0.05 level.In adult stage,the individual with genotype of AA and AG grew significantly faster than that of GG at the 0.01 level.A2-G427A loci was significantly associated with male juvenile weight gain.The individual with genotype of AA grew significantly faster than that of GA at the 0.05 level.In adult male stage,the individual with genotype of AA and AG grew significantly faster than that of GG at the 0.05 level.The results provided useful information in jian carp molecular marker assisted breeding.6.The prokaryotic expression of leptins in jian carpThe signal peptide cleavage sites were identified according to the jian carp leptins by SignalP-4.0 software.And pairs of primers were designed to amplify the leptins ORF region not including the signal peptide.The products were ligated into the expression plasmids of pET-32a(+)and pGex-4T-1 respectively,which constructed the recombination plasmid.The plasmid sequence was confirmed by sequencing and transfer into the host bacteria,Escherichia coli BL21(DE3).The recombinant proteins of pET-32a(+)/leptins and pGex-4T-1/leptins were expressed by induction with IPTG.The optimum concentration of IPTG was 1 mM for 5 h at 37?.However,the recombinant proteins of pET-3 2a(+)/jlLEP-A2 and pGex-4T-1/jlLEP-A2 were not expressed in the same condition.The recombinant proteins mainly existed in soluble form by SDS-PAGE electrophoresis analysis.The results provided useful information for leptins function research by injection recombinant proteins of leptins to jian carp.