Cloning And Expression Analysis Of The Interferon Pathway-Related Genes In The Early Developmental Stage Of Gadus Macrocephalus

Pacific cod(Gadus macrocephalus) is an important economic fish species in china. The quantity of wild Pacific cod has declined drastically during the past decades due to overfishing and infectious diseases. Efforts have been made to maintain the stock via artificial breeding. A big challenge in such endeavor is high mortality in the larvae, caused by multiple factors including infections. To explore this phenomenon, a preliminary study on the signal pathway of interferon system has been reported in this experiment. In order to provide a theoretical basis for the prevention and treatment of disease of Pacific cod early artificial breeding, the study measured the expression pattern of the interferon system in the early stage of the development of Pacific cod by determining the transcription levels of the interferon system related genes such as TLR3, MDA5, IPS1, IRF3, IRF7, and ATG5.The annex primers of TLR3, MDA5, IPS1, IRF3, IRF7, and ATG5 were designed based on the partial sequences of the transcriptome information of Pacific cod. The kidney of juvenile Pacific cod was used as the material; through gene cloning and sequencing the sequences of target genes are obtained. To study the interferon system formation at the early developmental stage of Pacific cod, the method of absolute quantification PCR was established in this study. The expression levels of 6 target genes were detected in different tissues and larvae stage of Pacific cod separately. In addition, the differential expression of the same gene were analysised through the expression of target gene was induced which 2dph and 12 dph larvae were immersed in Poly(I:C) in this study. The experimental results are as follows:The TLR3 cDNA sequence of Pacific cod is 3322 bp long. It do not includ a full open reading frame(ORF) and the longest ORF encode 377 amino acid. The expression level of TLR3 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-3.1475 x + 36.756 R2 = 0.9962. The transcription of TLR3 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of liver, heart and thymus were higher than other tissues and the expression levels of gill, brain and muscle were less.The MDA5 cDNA sequence of Pacific cod is 2604 bp long. It do not includ a full ORF and the longest ORF encode 844 amino acid. The expression level of MDA5 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-3.2154 x + 36.154 R2 = 0.9949. The transcription of MDA5 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of kidney, heart, intestine and thymus were higher than other tissues and the expression levels of brain and muscle were less.The ATG5 cDNA sequence of Pacific cod is 895 bp in length with a whole ORF, and encoded 275 amino acids residues. The molecular weight of the encoded protein was about 32.2kDA. The results of nucleotide sequence alignment showed that the similarity between Pacific cod and Oreochromis niloticus, Pacific cod and Cynoglossus semilaev was 87.27%, respectively. The phylogenetic tree constructed using the ATG5 nucleotide sequences of Gadus macrocephalus and some other animals reflected a genetic relationship which conformed to the viewpoint of traditional taxonomy. The result of amino acid sequence analysis showed a high conservation. The expression level of ATG5 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-2.8646X+31.989 R2 = 0.998. The transcription of ATG5 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of liver,thymus and kidney were higher than other tissues.The IPS1 cDNA sequence of Pacific cod is 1539 bp long. It do not includ a full ORF and the longest ORF encode 161 amino acid. The expression level of IPS1 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-3.1287 x + 34.206 R2 = 0.9868. The transcription of IPS1 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of kidney, liver and thymus were higher than other tissues and the expression levels of brain and gill were less.The IRF3 cDNA sequence of Pacific cod is 1878 bp in length with a whole ORF which was 1377 bp, and encoded 459 amino acids residues. The molecular weight of the encoded protein was about 51 kDa. The results of amino acids sequence alignment showed that the amino acids sequence included a DNA binding domain(DBD) containing five conserved tryptophan residues and a conserved C-terminal IRF association domain(IAD). The phylogenetic tree showed that the IRF3 of G. macrocephalus was clustered with the IRF3 of other species, far from IRF1 and IRF2. The expression level of IRF3 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-3.3449X+41.001 R2 =0.9896. The transcription of IRF3 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of gonad, liver and thymus were higher than other tissues.The IRF7 cDNA sequence of Pacific cod is 2032 bp in length. It contains an open reading frame of 1323 bp long, encoding 440 amino acids, a DNA-binding domain(DBD), an IRF association domain(IAD), and a serine-rich domain(SRD). In the DBD, the tryptophan cluster consists of only four tryptophans. In the phylogenetic tree, Pacific cod IRF7 was more closely related to Gadus morhua IRF7 than to others. The expression level of IRF7 in different tissues was detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve were y =-3.2881 x + 43.399, R2=0.9904. The transcription of IRF7 gene was expressed in various tissues in the Pacific cod. The results showed that the expression levels of spleen, kidney and thymus were higher than other tissues.In order to analyss the expression rule of TLR3, MDA5, IPS-1, IRF3, IRF7, and ATG5 in larvae stage, the experiment uses the fertilized egg and larvae from 1 dpf to 33 dph by using absolute quantification PCR. The results showed that the expression of TLR3,IRF3,IRF7 were both significantly up-regulated in 25 dph and the expression of TLR3,MDA5,IPS1 were both significantly up-regulated in 1dph. The expression of 6 target genes were both expressed stability in other dph. This result showed that the 2dph and 12 dph were both the critical time for the immune response of Pacific cod. And the IFN play an important role in the prevention of the disease.In order to analyss the expression rule of TLR3, MDA5, IPS-1, IRF3, IRF7, and ATG5 with the Poly(I:C) challenge, the study collected the 3H,6H,12 H,24H,48 H,72H larvae of 2dph and 12 dph after Poly(I:C) immersion. The results showed that the expression of TLR3,IRF3,IRF7 were both significantly up-regulated in different degree.The highest expression level almost appear at 48 H and 72 H.