title: Cloning and characterization of novel cyclotides genes from South American plants.
abstract: 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.
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Nicolau Brito da
authors.initials: NB
authors.lastname: Cunha
authors.affiliation: Universidade Federal de Uberlândia, Av. Getúlio Vargas, número 230, Centro 38, Patos de Minas, MG, 700-128, Brazil.
authors.forename: Aulus Estevão Anjos de Deus
authors.initials: AE
authors.lastname: Barbosa
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Renato Goulart
authors.initials: RG
authors.lastname: de Almeida
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: William Farias
authors.initials: WF
authors.lastname: Porto
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Mariana Rocha
authors.initials: MR
authors.lastname: Maximiano
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Luana Cristina Silva
authors.initials: LC
authors.lastname: Álvares
authors.affiliation: Departamento de Botânica, Instituto de Ciências Biológicas. Bloco D. Universidade de Brasília. Campus Darcy Ribeiro 70904-970, Asa Norte. Brasília, DF, Brazil.
authors.forename: Cassia Beatriz Rodrigues
authors.initials: CB
authors.lastname: Munhoz
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Chesterton Ulysses Orlando
authors.initials: CU
authors.lastname: Eugênio
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Antônio Américo Barbosa
authors.initials: AA
authors.lastname: Viana
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.|S-Inova Biotech, Pós Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil.
authors.forename: Octavio Luiz
authors.initials: OL
authors.lastname: Franco
authors.affiliation: Centro de Analises Proteômicas e Bioquímicas, Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916 Módulo B Avenida W5, Brasília, DF, 70790-160, Brazil.
authors.forename: Simoni Campos
authors.initials: SC
authors.lastname: Dias
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D010456:Peptides, Cyclic / Q000096:biosynthesis / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D019666:Rubiaceae / Q000235:genetics / Q000378:metabolism; D013020:South America; D013045:Species Specificity
chemical_list: D018076:DNA, Complementary; D010456:Peptides, Cyclic; D010940:Plant Proteins
keywords: cyclotides; palicourea rigida; pombalia lanata; rubiaceae; violaceae