A Study On The Evolution Of The Intron Of Temporin Gene In The Black Spotted Frog

Amphibians use antimicrobial peptides(AMPs)to protect themselves from attacks of pathogenic microorganisms in the environment.The antimicrobial peptides are highly diverse in species and structure and driven by positive selection in evolution.However,our previous studies showed that Temporin of the black spotted frog(Pelophylax nigromaculata)is encoded by multiple loci but highly conserved through out different geographical populations,indicating that Temporin gene may be a fundimental and pillar member in the antimicrobial peptide immune system.However,it is not clear how Temporin gene evolves.The intron of Temporin gene is the main polymorphic and informative region to study evolution mode.We speculate that the intron would be functional to regulate gene experssion and subjected to positive or negetive selection rather than neutral as in nonfunctional regions.In this study,we collected 28 black spotted frogs from different geographic regions in mainland China.Nucleotide diversity,neutral selection,functional elements and positive selected codons were analyzed on the intron,and phylogeographic analysis was also carried out using mitochondrial D-loop sequences as a reference of population structure.Phylogeographic analysis confirmed that continent Chinese population of the frog in China fell into two major clades,A and B.Temporin genes sequenced from 28 individuals encoded only one mature peptide,while the intron showed 112 polymorphic nucleotide sites within 2880 bp fragment,(?= 0.00659),still far below the level of D-loop region(?= 0.03233).The overall selection of the intron is neutral with no significant geographical population differentiation.Further analysis revealed that it contains 31 basic elements potentially functional in regulating gene expression.These basic elements were highly conserved comparing with their flanking regions,indicating that the functional elements evolve under negative selection while the rest region under neutral,making the overall selection pattern on the intron close to neutral.These findings further surpported our spectation that Temporin gene evolves under negative selection and acts as the pillar of AMP system in this species.In addition,we also found the intron was susceptable to jumping during PCR amplification and Escherichia coli cloning,resulting in the missing of a 564 bp fragment between a 6 bp isochronous repeat sequence(5'-AGACCT-3').The mechanism of this event was also disccussed in this study.