| Pacific cod(Gadus macrocephalus)is an important marine fish in northern area of China.In recent years,the number of wild Pacific cod has declined gradually due to the deterioration of living environment and overfishing.Our laboratory has committed to the study of artificial breeding of Pacific cod,and achieved initial results.Many marine fishes can survive in the polar and northern coastal waters,due to the antifreeze proteins in their body can secrete antifreeze proteins.These proteins can effectively reduce the harm of low temperature to fish in a certain extent.In this study,transcriptome technology was used to study the antifreeze genes in Pacific cod.In this study,Illumina2000 sequencing technique was used to analyze the sequencing of the liver tissue in Pacific cod.For the transcriptome analysis,using an Illumina sequencing platform,61,778,789 raw reads were acquired.After a de novo assembly,77,561 unigenes were obtained.Unigenes length distribution ranges from 201 bp to 16,370 bp with an average length of 1,118 bp.Antifreeze glycoprotein(AFGP)and type IV antifreeze protein(AFP-IV)genes were obtained after analysis of the transcriptome data.Compared with Atlantic cod(Gadus morhua),Pacific cod AFGP sequencing results were similar.The sequence of the coding region was not found,due to repeated sequence was automatically removed in the sequencing process.Pacific cod AFP-IV was cloned and analyzed.The results showed the AFP-IV cDNA is 689 bp in length(GenBank accession number: KX430014.1),including a complete open reading frame(ORF)of 375 bp,encoding 125 amino acids.The predicted molecular weight(MW)is 13.98 kDa and the theoretical isoelectric point(PI)is 4.68.Sequence alignment revealed that the coding sequence of the AFP-IV gene in Pacific cod was similar to that in other known species.Sequence similarity between Pacific cod and other species ranges from 40% to 70%,and the homology of Pacific cod with Atlantic cod reached 99%.Full legth of AFP-IV has a signal peptide sequence at the N-terminus.Hydrophilic analysis of the mature peptide revealed a transmembrane region between 30(Met)-85(Glu)region.The secondary structure prediction revealed that the structure of AFP-IV is similar to apolipoprotein.The expression patterns in samles of larvae and various tissues from adult were analyzed using real-time PCR technique respectively,and β-actin was used as reference gene.In larval stage,the expression level of AFP-IV was the highest at 10 dph.In adult tissues,the expression level of AFP-IV was the highest in the liver.The recombinant plasmid pET-32a-AFP-IV was constructed and the recombinant protein was induced by IPTG.The recombinant protein was purified by ProteinISO Ni-NTA Resion.The polyclonal antibody was prepared by immunization with New Zealand long ear rabbit with purified recombinant protein.The polyclonal antibody titer reached 1: 1,600,000 using ELISA detection.The immunogenicity of the fusion protein was detected by western-blot,indicating the recombinant protein has a high affinity.Antifreezing activity of recombinant AFP-IV was detected in Escherichia coli,and the results showed that the recombinant protein had antifreezing activity,but not strong.In this study,transcriptome technology was used to study AFGP and AFP-IV.The results in this study provide information of antifreeze genes in Pacific cod. |
