| 其他摘要 | During the past century, numerous researches have been carried on the amphibian skin secretions for they form the first line of host defense. To date, hundreds of bioactive substances have been isolated from them. Odorrana grahami is a specific species of China. Preliminary tests showed that the skin secretions of Odorrana grahami had strong antimicrobial, hemolytic, and protease inhibitory activities. In this thesis, peptidomics and genomics analyses were used to study the anti-infection array of peptides of Odorrana grahami skin. 21 novel antimicrobial peptides were isolated from the skin secretions of O. grahami by a three-step protocol including one step Sephadex G-50 and two steps of RP-HPLC, and they belong to 17 different antimicrobial families. Among the 21 novel antimicrobial peptides, 8 of which belong to five of the known antimicrobial families previously identified in the skins of other species of Ranid frogs: Brevinin-1E (2 peptides), Brevinin-2E (1 peptide), Esculentin-1 (1 peptide), Esculentin-2 (1 peptide), and Nigrocin (3 peptides). The other 13 peptides show little structural similarity towards other known antimicrobial peptides and so are classified into 12 new families: Odorranain-A (1 peptide), Odorranain-B (1 peptide), Odorranain-C (1 peptide), Odorranain-G (1 peptide), Odorranain-H (2 peptides), Odorranain-J (1 peptide), Odorranain-L (1 peptide), Odorranain-M (1 peptide), Odorranain-N (1 peptide), Odorranain-O (1 peptide), Odorranain-Q (1 peptide), Odorranain-T (1 peptide). By cDNA cloning, 372 cDNA sequences of antimicrobial peptides were obtained from a single individual skin of this frog, encoding 107 novel antimicrobial peptides. This contribution almost doubles the number of currently reported amphibian antimicrobial peptides. These peptides can be organized into 30 divergent families, including 24 novel families. The diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal. The patterns of diversification, suggest that point mutations, as well as insertion, deletion, and “shuffling” of oligonucleotide sequences have been responsible. The diversity of antimicrobial peptides may have resulted from the diversity of microorganisms. The precursors of the 30 antimicrobial peptide families share similar signal and propiece peptide sequences, which show that they might origin from the same ancestral gene. The rate of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions were calculated in seven antimicrobial peptide families, and they displayed different nucleotide substitution patterns, indicating they might undergo different selecting pressures. These diverse peptides exhibit both diverse secondary structure and “host-defense” properties. Such extreme antimicrobial peptide diversity in a single amphibian species is amazing. This makes us have to reconsider the strong capability of innate immunity and molecular genetics of amphibian ecological diversification and doubt the general opinion that 20-30 different antimicrobial peptides can protect an animal because of the relatively wide specificity of the peptide antibiotics. Our studied also show synergy exists among the skin antimicrobial peptides of O.grahami. The cytoplasmic-membrane depolarization activities were determined on 7 antimicrobial peptides from different families. The results demonstrated that all the peptides studied here could depolarize the cytoplasmic membrane of S. aureus but with different potential. Their antimicrobial mechanisms were investigated. They exert their antimicrobial functions by various means, including forming lamellar mesosome-like structures, peeling off the cell walls, forming pores, and inducing DNA condensation. In conclusion, with respect to the development of antibiotics, these peptides provide potential new templates to further explore. |
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