关键词: 棘胸蛙; 抗菌肽; Cathelicidin; 病原菌胁迫

Abstract:

Objective Cathelicidins are a family of endogenous antimicrobial peptides unique to vertebrates， which have potent antibacterial and anti-inflammatory activities. This study identified a novel Cathelicidin from Quasipaa spinosa and investigated its gene structure and function， thereby providing a basis for future research on the antimicrobial peptide gene family. Method The complete sequence of Cathelicidin gene was obtained through gene cloning. The physicochemical properties， domains， signal peptides， secondary and tertiary structures of the encoded protein were analyzed by bioinformatics methods， and the phylogenetic tree was constructed by Mega X software. The expression of Cathelicidin gene in various tissues of Quasipaa spinosa was detected by Real-time quantitative PCR， as well as the expression changes of Cathelicidin gene under the stress of Citrobacter freundii （C. freundii） and Citrobacter braakii （C.braakii）. Result The full length of the Cathelicidin gene sequence was 730 bp， the open reading frame was 465 bp， encoded 154 amino acids. The molecular mass of Cathelicidin was 137 254.64 u， and its isoelectric point （pI） was 5.20. The 1-20 amino acids were signal peptides， and there was a structural domain between the 21-116 amino acids， which contained four conserved cysteine residues and a mature peptide containing 18 amino acids at the terminal. The prediction of secondary structure showed that the proportions of alpha helix， beta sheet and random coil of the Cathelicidin protein were 34.42%， 21.43% and 44.16%， respectively. The homology modeling of the tertiary structure showed that it was formed by the winding of a single peptide chain， and its functional domain was composed of alpha helix and beta sheet. Phylogenetic analysis revealed that the Cathelicidin from Quasipaa spinosa was most closely related to that of Nanorana yunnanensis （family Dicroglossidae）， with the two forming a distinct clade. The results of Real-time quantitative PCR showed that Cathelicidin gene was expressed in all tissues of Quasipaa spinosa， with higher expression in liver and kidney. After infection with Citrobacter， the expression of Cathelicidin gene in the liver of Quasipaa spinosa showed a temporal expression change characteristic. After infection with C. freundii， compared with 0 h， its expression reached an extremely significant peak at 72 h. However， C.braakii infection caused a continuous downregulation of its expression. Conclusion The Cathelicidin gene exhibited a well-defined structure and a broad expression profile across tissues in Quasipaa spinosa. Its mRNA expression displayed distinct temporal patterns in response to different pathogenic bacteria. These findings laid a theoretical foundation for further exploration of the Cathelicidin potential applications in the aquaculture industry.

Key words: Quasipaa spinosa; antimicrobial peptide; Cathelicidin; pathogen stress