Molecular Cloning And Expression Analysis Of C-type And G-type Lysozyme Gene In ChangFeng Carassius Auratus Gibelio During Bacterial Infection

ChangFeng crucian carp(ChangFeng Carassius auratus gibelio)(Variety registration number : GS-04-01-2015)has the advantages of tender meat,fast growth and genetic stability.The market prospect is very broad.However,with the increasing scale of aquaculture,bacterial disease is more and more serious in large-scale breeding process about crucian carp(Carassius auratus).Bactericidal activity of Lysozyme(lys)is broad spectrum,so lysozyme has an important position in fish non-specific immunity.In this study,the c-lys and g-lys cDNAs of ChangFeng Carassius auratus gibelio were cloned,the expression of c-lys and g-lys gene in ChangFeng Carassius auratus gibelio and Carassius auratus were detected.After infection,Gene expression in the liver,kidney,spleen and gills of ChangFeng Carassius auratus gibelio and Carassius auratus were also detected after injected by Edwardsiella tarda,Aeromonas hydrophila and Staphylococcus aureus.Meanwhile,the morphological changes of the four tissues were evaluated,and their immune defense function was evaluated.To explore the ability of anti-bacterial disease of ChangFeng Carassius auratus gibelio to provide certain theoretical basis.The results of this study are as follows:1.Cloning of c-lys and g-lys genes in ChangFeng Carassius auratus gibelioThe c-lys of ChangFeng Carassius auratus gibelio was 698 bp in length and 438 bp in open reading frame,encoding 145 amino acids.Containing Glu 53 and Asp 69 two conservative amino acid catalytic sites,eight conservative Cys,can form four pairs of disulfide bonds.With a N-terminal signal peptide,it is a secretory protein;g-lys length of ChangFeng Carassius auratus gibelio was 861 bp in length and 558 bp in open reading frame,encoding 185 amino acids.Containing three conserved catalytic sites of Glu(position 73),Pro(position 86)and Asp(position 97).There is a Cys residue,but it cannot form disulfide bonds,there is no N-terminal signal peptide,it is an intracellular protein.Amino acid sequence similarity and phylogenetic tree analysis showed that the c-lys and g-lys genes of ChangFeng Carassius auratus gibelio were conserved in the evolution of Cypriniformes.2.Expression of lysozyme gene in ChangFeng Carassius auratus gibelio and Carassius auratus were injection by three bacteriaC-lys and g-lys genes were expressed ubiquitously in ChangFeng Carassius auratus gibelio and Carassius auratus and abundantly in kidney.The expression of c-lys and g-lys genes in ChangFeng Carassius auratus gibelio and Carassius auratus were up-regulated in the liver,kidney,spleen and gill tissues after infection with bacteria.But the higherdegree and the peak of time of the two genes in the ChangFeng Carassius auratus gibelio and Carassius auratus are different.3.Pathological changes of the tissues in ChangFeng Carassius auratus gibelio and Carassius auratus after bacterial infectionAfter the illness,scales of ChangFeng Carassius auratus gibelio and Carassius auratus fall off,accompanied by varying degrees in areas such as the surface,the anus,and fin hyperemia and hemorrhage phenomenon,but two different fish species in the body color and the autopsy color change organizations exist a certain differences after bacterial infection.After stimulated by three kinds of bacteria,ChangFeng Carassius auratus gibelio and Carassius auratus showed similar symptoms: liver cells are abnormal arrangement,uneven distribution,nuclei size is inconsistent,shrinkage,fragmentation and necrosis,cell swelling,hepatic sinusoids and central venous congested,hepatocytes had vitreous changes;kidney showed glomerular enlargement,renal tubular epithelial cell shedding,granular changes,mononuclear macrophages increased,renal interstitial infiltrated by inflammatory cell;In spleen,red pulp and white pulp had a clear demarcation,nuclear fragmentation and even necrosis,splenic artery around has a large of reticular cells,lymphopenia.respiratory epithelial cells of gill lamellae floating,gill wire vasodilator,gill filament thicker short,gill lamellae has edema phenomenon,part of the diseased fish gill lamellae of tissue cells disintegrated and shedding.