Cloning and expression analysis of Cathelicidin gene in Mauremys mutica

【Objective】 The Cathelicidin gene of Mauremys mutica (MmCath) was cloned and its tissue expression pattern was analyzed, so as to provide theoretical foundation for further studying the potential function of MmCath gene in the innate immunity of M. mutica.【Method】 The cDNA sequence of the MmCath gene of M. mutica was cloned by RACE. Sequence analysis was conducted by bioinformatics software. The expression patternof MmCath gene in different tissues of M. mutica and its expression change after bacterial challenge were identified by real-time fluorescence quantitative PCR. 【Result】 The full length of the cDNA sequence of MmCath gene was 777 bp, including a 5' noncoding region of 63 bp, an open reading frame(ORF) of 483 bp, and a 3' noncoding region of 231 bp. The MmCath gene encoded 160 amino acid residues, which contained the signaling peptide region at the N terminus, the Cathelin peptide region with 4 conserved cysteine (Cys) residues, and the mature peptide region at C terminus, in accordance with the typical family characteristics of Cathelicidins protein family. MmCath precursor protein had a relative molecular weight of 17.74 kDa and theoretical isoelectric point(pI) of 5.27. In its secondary structure, α-helix accounted for 31.25%, random coil 20.63% and β-turn 48.12%. The amino acid sequence of MmCath precursor protein shared a highest sequence similarity(80.63%) and closest genetic relationship with the amino acid sequence of Cathelicidin in Chrysemys picta bellii. Expressions of MmCath gene were different in various tissues in M. mutica. Its higher relative expressions were shown in spleen and liver, while the relative expressions were lower in the tissues like skin, heart, kidney, lung, brain, intestine, and muscle. Up regulation of expression was shown in MmCath gene after the challenge of Aeromonas hydrophila. The relative expression levels at 3 and 6 h were extremely significantly higher than that at before challenge(0 h) (P<0.01, the same below), and then decreased. It markedly increased again at 36 h post challenge and decreased afterwards.【Conclusion】 The MmCath gene cloned from M. mutica belongs to the Cathelicidins gene family. It involves in the process of resisting attack of invading pathogen of M. mutica and can provide new ideas for effective control of bacterial diseases of M. mutica.