RESUMO
PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.
Assuntos
Brassicaceae/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Primers do DNA , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Inibidores de Proteases/classificação , Inibidores de Proteases/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5'RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5' to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5' region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.
Assuntos
DNA Mitocondrial/genética , DNA de Plantas/genética , Dosagem de Genes , Genes de Plantas/genética , Helianthus/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Sequência de Bases , Éxons/genética , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência de Lisina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sítio de Iniciação de TranscriçãoRESUMO
Three proteinase inhibitor genes have been identified in the rapeseed (Brassica napus) genome. They are highly homologous to other genes of the mustard inhibitor (MSI) family of proteinase inhibitors characteristic of Cruciferae. In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti-2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI-2 protein strongly inhibits (Ki=44 nM) a glutamyl proteinase from Streptomyces griseus.
