RESUMO
In latex of rubber tree (Hevea brasiliensis), prohevein, homologous to potato win gene-encoded proteins, is processed to yield mature hevein. This mature hevein is composed of one chitin-binding domain and the C-terminal polypeptide homologous to pathogenesis-related proteins such as tobacco PR-4 and tomato P2 proteins. In contrast, prohevein was poorly cleaved to form the C-terminal polypeptide in transgenic tomato plants expressing hevein gene (HEV1)-driven polypeptides. However, mature hevein, the N-terminal cleavage form, was not found in this system. Immunoblot analysis of extracellular and intracellular fluid proteins showed that HEV1-encoded polypeptides accumulated intracellularly. In addition, retardation of growth of Trichoderma hamatum was observed in transgenic tomatoes constitutively expressing HEV1-encoded proteins.
Assuntos
Alérgenos , Peptídeos Catiônicos Antimicrobianos , Quitina/fisiologia , Lectinas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Precursores de Proteínas/metabolismo , Solanum lycopersicum/genética , Trichoderma/fisiologia , Antígenos de Plantas , Lectinas/metabolismo , Lectinas de PlantasRESUMO
Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Bases , Western Blotting , Quitina/metabolismo , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Proteínas de Plantas/genética , Biossíntese de Proteínas , Precursores de Proteínas/genética , Mapeamento por Restrição , Transcrição Gênica , ÁrvoresRESUMO
Hevein is a chitin-binding protein that is present in laticifers of the rubber tree (Hevea brasiliensis). A cDNA clone (HEV1) encoding hevein was isolated by using the polymerase chain reaction with mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea latex cDNA library as a template. HEV1 is 1018 base pairs long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187-amino acid polypeptide. This polypeptide has two striking features. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino termini of wound-inducible proteins in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems, and latex but not in roots.