Cloning from tissue surrogates: antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs.
The 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.
Chen
Tianbao
T
Institute of Biotechnology, Fuzhou University, Fuzhou 350002, Fujian Province, People's Republic of China.
Zhou
Mei
M
Chen
Wei
W
Lorimer
Joanne
J
Rao
Pingfan
P
Walker
Brian
B
Shaw
Chris
C
D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011898:Ranidae / Q000235:genetics*; D020539:Sequence Analysis, Protein; D012867:Skin / Q000378:metabolism*
D029845:Amphibian Proteins; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; C092874:esculentin protein, Rana esculenta