| Amphibians have developped the complete antimicrobial peptides defense system when they cope with a variety of microbial invasion in the long-term evolution. It is the antimicrobial peptides diversity of sequence, structure, and biological activity that provides a quick and effective defensive barrier when amphibians survive in a complex habitat. The components and abundance of antimicrobial peptide can be regulated by the surrounding environmental factors, so understanding of these environmental factors and their influences on antimicrobial peptides is critical as to assess the immune status of amphibians. Unfortunately, we know little not only the interaction between the active peptides of the amphibian and environmental microorganism but also the expression pattern of antimicrobial peptides when amphibians are challenged by microorganism.Rana dybowskii was choosed as the research object which is the typical species of amphibians distributed in the Northeast of China. By analyzing the dynamic changes of antimicrobial peptide components and abundance in Rana dybowskii when the frogs were challenged by Rana grylio virus, the expression changes pattern of amphibians and its significance of immune defence were discussed when they resist the invasion of phthogenic microorganisms. RP-HPLC differencial profiles of skin secretions were constructed by high performance liquid chromatography technology when the frogs were challenged by RGV after1h,3h,6h,12h,24h,48h,72h(the seven time points). After that, the elution peak were collected and their antibacterial activity were detected, and the active components were isolatted and purified secondly. Skin secretions in enzymatic or non-enzymatic state of Rana dybowskii were separated and characterized by LC-MS/MS and MALDI-TOF/TOF. The high purity of the active peptide components were sequenced by N-terminal sequencing method. Finally, the recombinant antimicrobial peptides dybowskin-1ST which was obtained by the eukaryotic expression system was used as model to prove the role of antimicrobial peptides on wounds healing of mice.The results showed that the antimicrobial peptide components and their abundance of Rana dybowskii skin secretions occur significant changes and appear obvious laws when frogs were challenged by RGV after1h～72h. The skin secretions of concentration show the variation of the "V" pattern, lowest at24h; The profiles of RP-HPLC showed most antimicrobial peptide components of the skin secretions were significantly reduced after24h while frogs exposed to RGV, however, little effect on the antimicrobial peptide components.9family of14peptide sequences were obtained by Mass spectrometry and N-terminal sequencing. The four were full-length sequences, the others were non-full-length peptides with high credibility. All of that, the number of antimicrobial peptide is13which respectively are Brevinin-lDYa, Brevinin-1DYb, Brevinin-1Pb; Brevinin-2DYd, Brevinin-2DYe, Brevinin-2DYa; Ranatuerin-2Vb, Ranatuerin-2Amb; Japonicin-1CDYa, Dybowskin-1CDYa, Palustrin-2Isc, Temperin-1DYa, Temperin-2ST. The other is Bradykinin RS-14. The skin antimicrobial peptides of Rana dybowskii show a high degree of diversity and are rich of Brevinin-1and Brevinin-2family with broad-spectrum antimicrobial activity. Rana dybowskii skin antimicrobial peptides dybowskin-1ST not only has antibacterial activity but also can improve the growth of granulation tissue and the formation of collagen fiber, finally promote wound healing.The diversity of Rana dybowskii skin antimicrobial peptides reveals the frogs have faced the challenges of complex microbial flora in the evolutionary process. Rana dybowskii owned a strong antimicrobial peptide spectrum tell that the species has a strong protective immunity ability. In this study, the exploration in the antimicrobial peptide components and their antibacterial spectrum of Rana dybowskii contribute to amphibian species protection and disease prevention. |
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