A Study On The Structure And Evolution Of Temporin-1N Gene In The Black Spotted Frog,Pelophylax Nigromaculata

Antimicrobial peptides (AMPs) is the first defense line of amphibian innate immunity characterized with extremely high diversity. There have been proposed three mechanisms to explain the diversity of AMPs:Duplication of AMP genes produces multiple novel genes; matured peptide region exhibites high mutation rate, and mutations are positively selected in favor of accumulation of synonymous mutations. However, how these mechanisms are realized has not yet explained.Our previous studies showed that Temporin is the most expressed AMPs in wild black spotted frog, Pelophylax nigromaculata and evolves under stong negative selection while other AMPs are under positive selection at different extent. We hypothesized that Temporin might play a umbrella-liked role in the whole AMP system and reduce selective pressure on other AMPs, allowing them evolve fastly though effective accumulation of mutations. In order to test the hypothesis, we chose a black spotted frog, cloned its Temporin genes using Genome Walking technology, and analyzed the structure and expression features of Temporin genes and their 5’-UTR regions. We observed that:1. Structurally, the Temporin-1N genes include 5’-UTR Exon 1 encoding signal peptide and partial acidic propiece Intron 1 Exon2 encoding the remaining part of acidic propiece and entire mature peptide followed with a 3’-UTR.2. Four Temporin-1N genes were identified in the genome of a single individual, and no nucleotide polymorphism was detected in mature peptide region, suggesting a extremely low diversity.3. cDNA based prediction showed Temporin-1N of black spotted frog is an a-helix alkaline and cationic peptide consisting of 15 amino acid residues. These features were similar to Temporin peptides of other amphibian species, suggesting multiple functions and broad antimicrobial spectrum as tested in enormous experiments.4. Neural Network Promoter Prediction proposed multiple promoter structure in both 5’-UTR and introns of Temporin-IN genes, suggesting that Temporin-1N had the potential of fast transcription.5. TRANSFAC prediction proposed a number of basic cis-acting elements of eukaryotes such as TATA-box, GC-box and CAAT-box, and transcription factor binding sites that are related to regulations of immunity specific and tissue specific expressions. These results imply that Temporin-1N would be involved in a variety of physiological processes.In general, the evidence that Temporin-IN has broad spectrum of antimicrobial activity, is encoded by multiple genes that are capable of fast transcription and is involved in a variety of physiological processes supported the hypothesis that it plays a key role in the entire antimicrobial peptide system, allowing other antimicrobial peptides accumulating mutations fastly under reduced selective pressure and achieving fast diversification.