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Amphibian skins are rich of bioactive material resources and contain a group of bioactive peptides called bradykinins which are similar to the mammalian hormones and neurotransmitters. More than ten bradykinin-related peptides and their cDNAs have been identified from amphibians, but their genes are unknown.In this study, skin cDNA library of Odorrana grahami was constructed successfully, and four cDNA sequences encoding bradykinin precursors were screened out from the library. All the four precursors share highly conserved signal peptides, acidic spacer peptides and mature bradykinins. They contain one, two, four and six copies of mature bradykinins respectively. According to the cDNA sequences data, the first gene sequences encoding the amphibian bradykinin were successfully obtained by using the total DNA from O. grahami as template, and by means of methods such as thermal asymmetric interlaced PCR (TAIL-PCR). The bradykinin gene is 7481 bp in length, and contains two exons and two introns. The first exon of the gene encoding a signal peptide, the second exon encoding acidic spacer peptide, N-extension, mature bradykinin and C-extension. An intron is inserted between the first and the second exons and the other intron is inserted between 5’-untranslated region of the gene. The gene was analyzed in transcription factor database, and many structural motifs for the binding of factors involved in the transcriptional control, such as SRY, LYF-1, GATA-1, DeltaE, S8, CdxA, MZF1, NKX2.5 and MIF1, were found in the promoter region of the gene. The roles of the binding sites in amphibian bradykinin gene are still unknown, but obtaining the gene may facilitate to understand the regulation and possible functions of amphibian skin bradykinin-related peptides. Particularly the immune defense-related transcription factor binding sites found in the gene suggest that amphibian bradykinin may have a defense function related with the regulation of immune, which was supported by the high similarity between the singal sequences of bradykinin precursor and antimicrobial peptides.At the same time, three bradykinin-related peptides, bradykinin, Thr6-bradykinin and Leu5Thr6-bradykinin, were deduced from these four bradykinin precursor sequences encoded by the amphibian cDNA sequence. In order to ascertain the difference between these three peptides and to help us understand the functions of amphibian skin bradykinin, two kinds of peptides included bradykinin (RPPGFSPFR) and Leu5Thr6-bradykinin (RPPGLTPFR) were synthesized for bioassay. These two peptides were found to elicit concentration-dependent contractile effects on isolated guinea pig ileum in the bioassay. Their activities had obvious difference although these two peptides have only two amino acid residues distinct. Under the same concentrations, bradykinin usually had stronger contractile activity on isolated guinea pig ileum than that of Leu5Thr6-bradykinin.
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