Cloning and tissue expression of liver-expressed antimicrobial peptide Leap-2 in Ussuri cisco Coregonus ussuriensis exposed to bacterial infection
LIU Enhui,HUANG Tianqing,GU Wei,WANG Bingqian,ZHANG Yanping,LI Chunyu,XU Gefeng
1.Key Laboratory of Freshwater Aquatic Biotechnology and Breeding,Ministry of Agriculture and Rural Affairs,Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China;2.Gansu Fisheries Research Institute,Lanzhou 730030,China;3.Xinjiang Tianyun Organic Agriculture Company Limited,Yili 835708,China
Abstract: To understand the molecular structure and expression characteristics of liver-expressed antimicrobial peptide Leap-2 in Ussuri cisco Coregonusussuriensis Berg,the open reading frame (ORF) of the Leap-2 gene was amplified from the liver tissue of Ussuri cisco with body weight of (30.0±1.2)g by PCR,and the Leap-2 amino acid sequence was analyze by bioinformatics software.The relative expression levels were detected in healthy tissues and tissues of Ussuri cisco intraperitoneally infected by Aeromonashydrophila,Aeromonassalmonicida or Staphylococcusaureus at a dose of 100 μL/ind.of 1×106 CFU/mL by fluorescence quantitative real-time PCR (qRT-PCR).The results showed that Leap-2 gene (GenBank:MZ170050) had ORF region of 276 bp,which was cloned from the liver of Ussuri cisco by PCR.Leap-2 encoding 91 amino acids and its mature peptide region contained four highly conserved cysteine residues.Phylogenetic analysis showed that Ussuri cisco Leap-2 amino acid sequence was closely related to coho salmon Oncorhynchuskisutch,with the homology of 96.7%.Tissue expression showed that Ussuri cisco Leap-2 was the most abundant in the liver,followed by the intestine and kidney,with the minimal expression level in muscle.The up-regulated expression of Leap-2 was observed in all detected Ussuri cisco exposed to A.hydrophila,A.salmonicida,and S.aureus infection.Western blot revealed that the expression of Leap-2 protein was up-regulated within 24 h,indicating that Leap-2 was involved in the immune response of Ussuri cisco to bacteria.The finding may provide theoretical reference with understanding of the immune response of Ussuri cisco.
2022, Vol. 37 
