Cloning and characterization of novel cyclotides genes from South American plants

Cyclotides are multifunctional plant cyclic peptides containing 28-37 amino acid residues and a pattern of three disulfide bridges, forming a motif known as the cyclic cystine knot. Due to their high biotechnological potential, the sequencing and characterization of cyclotide genes are crucial not only for cloning and establishing heterologous expression strategies, but also to understand local plant evolution in the context of host-pathogen relationships. Here, two species from the Brazilian Cerrado, Palicourea rigida (Rubiaceae) and Pombalia lanata (A.St.-Hil.) Paula-Souza (Violaceae), were used for cloning and characterizing novel cyclotide genes. Using 3' and 5' RACE PCR and sequencing, two full cDNAs, named parigidin-br2 (P. rigida) and hyla-br1 (P. lanata), were isolated and shown to have similar genetic structures to other cyclotides. Both contained the conserved ER-signal domain, N-terminal prodomain, mature cyclotide domain and a C-terminal region. Genomic sequencing of parigidin-br2 revealed two different gene copies: one intronless allele and one presenting a rare 131-bp intron. In contrast, genomic sequencing of hyla-br1 revealed an intronless gene-a common characteristic of members of the Violaceae family. Parigidin-br2 5' and 3' UTRs showed the presence of 12 putative candidate sites for binding of regulatory proteins, suggesting that the flanking and intronic regions of the parigidin-br2 gene must play important roles in transcriptional rates and in the regulation of temporal and spatial gene expression. The high degree of genetic similarity and structural organization among the cyclotide genes isolated in the present study from the Brazilian Cerrado and other well-characterized plant cyclotides may contribute to a better understanding of cyclotide evolution.