Cloning from tissue surrogates: Antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs

AbstractThe defensive skin secretions of amphibians are a rich source of bioactive peptides. Here we describe a rapid technique for skin granular gland transcriptome cloning from a surrogate tissue—the secretion itself. cDNA libraries were constructed from lyophilized skin secretion from each of the Chinese frogs (Rana schmackeri, Rana versabilis, and Rana plancyi fukienensis) using magnetic oligo(dT) bead-captured polyadenylated mRNA as templates. Specific esculentin cDNAs were amplified by 3′-RACE using a degenerate primer designed for a consensus nucleotide sequence in the 5′ untranslated region of previously characterized ranid frog peptide cDNAs. The cloned cDNAs were found to encode the antimicrobial peptides esculentins 1 and 2 from each of the species examined. The presence of predicted peptide structures in skin secretions was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This experimental approach can thus rapidly expedite parallel transcriptome and peptidome analysis of amphibian granular gland secretions without harming or sacrificing donor animals