Induction Of Antimicrobial Peptides From Rana Dybowskii Under Rana Grylio Virus Stress And Bioactivity Analysis

Antimicrobial peptides (AMPs) are a kind of small molecules, what in vivo of many creatures play important roles in preventing the invasion of microorganisms and other pathogens, in particular the Ranidae of amphibians. The continual emergence of antibiotics-resistant pathogenic microorganisms has greatly stimulated researchers to seek novel antimicrobial agents with broad-spectrum antimicrobial activity. The skin glands of Ranidae are rich sources of antimicrobial peptides. This paper took the the Rana dybowskii as the research object to study the induction of antimicrobial peptides from Rana dybowskii under Rana grylio virus (RGV9506) stress and analyze the bioactivity.In this study, the genomic RNA of Rana dybowskii was extracted from its skin under Rana grylio virus stress. Five new cDNA sequences encoding five mature peptides Ranatuerin-2YJ (GLMDIFKVAVNKLLAAGMNKPRCKAAHC), Dybowskin-YJb (IIPLPLGYFAKKP), Dybowskin-YJa (IIPLPLGYFAKKKKKKDPVPLDQ), Temperin-YJa (VLPLLETCSMTC-WENNQTFGK) and Temperin-YJb (VLPLVGNLLNDLLGK) were obtained by RT-PCR with a pair of degenerate primers designed according to the conserved terminal sequences of cDNA encoding antimicrobial peptide precursors of genus Rana.The relative changes in gene expression of five antimicrobial peptides showed certain regularity: under virus stress, all the peptides showed significant reduction (8fold changes, p<0.05) in gene expression compared to the control at1～3h after stress; at3～12h after stress, all the peptides showed sustained up-regulated expression; after24h, gene expression of the five peptides showed down-regulation.The antimicrobial activities of the peptides were analyzed and results demonstrated that all these peptides showed a significant anti-Rana grylio virus activity. Especially the antiviral activity of the Ranatuerin-2YJ was significantly higher than those of the others peptides (p<0.05). And the virus was gradually cleared with the increase in gene expression.Among the five peptides obtained in this work, Ranatuerin-2YJ also showed a broad-spectrum anti-Gram-positive bacteria and anti-Gram-negative bacteria activity. It showed a strong antimicrobial activity against S. aureus, E. coli, S. marcescens and K. pneumoniae (MIC,22.5-35μg/mL), showed a moderate antimicrobial activity against S. enteritidis, B. cereus and Str. pyogenes (MIC,47.5-60μg/mL), while all of the bacteria used in work were insensitive to Dybowskin-YJa, Dybowskin-YJb, Temperin-YJa and Temperin-YJb (MIC,>100μg/mL).At the same time it showed7.64%hemolysis activity which were significantly lower than the others (p<0.05).Moreover, Ranatuerin-2YJ was widely distributed in the skin, liver, spleen and blood of R.dybowskii, while the other four peptides can only be cloned from the skin, indicating that the Ranatuerin-2YJ in vivo plays an important role in the protection against pathogens invasion.