Cloning And Expression Analysis Of The Immune-related Genes In The Early Developmental Stage Of Pacific Cod(Gadus Macrocephalus)

Pacific cod(Gadus macrocephalus) is an important economic fish species in the northern area of china. In recent years, due to fishing increase, leading to its wild resources continues to decline. The artificial breeding technology of Pacific cod continues to make breakthroughs. But it still appears sudden mass death during larval and juvenile stages. In order to solve this difficult problem, the own immune system of Pacific cod is considered from. The developmental characteristic of immune system at the early developmental stage of Pacific cod is measured through the transcription level of RAG1, Ig M and ISG15 genes in this experiment. This study provides a theoretical basis for targeted disease prevention during early stage of the artificial breeding.Firstly, the annex primers are designed according to the conserved regions of RAG1, Ig M and ISG15 in other teleost fish species. The head kidney and thymus of juvenile Pacific cod are used as the material; through gene cloning and sequencing the sequences of target genes are obtained. To study the mechanism of immune system formation at the early developmental stage of Pacific cod, the method of absolute quantification PCR is established in this study. The expression levels of target genes are detected in different tissues and during early developmental stage of Pacific cod separately. In addition, the expression of RAG1 in different tissues of Pacific cod is analyzed from the protein level by using Western Blot. The experimental results are as follows:The RAG1 c DNA sequence of Pacific cod is 3755 bp long. It includ an ORF of 1854 bp, encode 617 amino acid and predict its molecular weight is 69 k Da. Gen Bank accession number is KM408434. The alignment of RAG1 amino acid sequence between Pacific cod and other fish reveals. the homology is over 85%. The expression level of RAG1 in different tissues is detected by using absolute quantification PCR. The result shows, the liner regression equation and the regression coefficient(R2) of the standard curve are y =-3.2662 x + 33.772 and R2=0.9961. The RAG1 is only expressed in head kidney and thymus; but the signal is not detected in liver, spleen, intestine, gill and gonad. The expression level in thymus is far higher than that in head kidney. The expression level in thymus of juvenile is higher than that in thymus of adult. The result Western Blot from protein level shows the expression level of RAG1 in thymus is far higher than that in head kidney. In addition, the molecular weight of protein bands of RAG1 is 69 k Da. It verifies the accuracy of the obtained RAG1 sequence from protein level.The Ig M heavy chain c DNA sequence of Pacific cod is 1862 bp long. It includes an ORF of 1713 bp, encode 570 amino acids and predict its molecular weight is 63 k Da. Gen Bank accession number is KM408433. The analysis of amino acid sequence revealed, the homology of the amino acid is very high. The structure comprises a signal peptide, a variable region(VH), and 4 constant regions(CH) and a hinge region(Hinge); wherein the variable region includes 4 fragment regions(FR) and 3 complementarity-determining regions(CDR). This Ig M belongs to secretory immunoglobulin. The result of absolute quantification PCR of Ig M shows, the liner regression equation and the regression coefficient(R2) of the standard curve are y =-3.249 x + 32.27 and R2=0.998. The Ig M is expressed in thymus, head kidney, liver, spleen, intestine, gill and gonad. The expression level of Ig M in spleen of juvenile is higher than that in any other tissues of juvenile, and followed is in head kidney. While the expression level of Ig M in head kidney of adult is higher than that in any other tissues of adult.The ISG15 gene c DNA sequence of Pacific cod is 444 bp long, and encodes 85 amino acid. The nucleotide sequence alignment shows, it is only homology with the reported 3 kinds of the Atlantic cod ISG15, the highest homology is 90%. The result of absolute quantification PCR of ISG15 shows, the liner regression equation and the regression coefficient(R2) of the standard curve are y =-3.158 x + 33.85 and R2=0.998. The ISG15 is expressed in thymus, head kidney, liver, spleen, intestine, gill and gonad. The expression level of spleen is the highest in juvenile, and the expression level of gill is highest in adult. The expression level in tissues of juvenile is higher than that in tissues of adult. In order to analyss the expression rule of RAG1, Ig M and ISG15 during early developmental stage, the experiment uses the fertilized egg and larvae from 2 dpf to 95 dph. The result shows, the expression levels of RAG1 and Ig M begin to rise at 61 dph, and achieve the highest at 95 dph. While the expression level of Ig M is lower than RAG1. The expression level of ISG15 is low and stable before 61 dph, and swell at 95 dph. It is proposed that the “dangerous period” of low immunity and easy death of Pacific cod is before 95 dph.