RESUMO
Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.
Assuntos
Bacillus subtilis/genética , Herbicidas/farmacologia , Oryza/efeitos dos fármacos , Oryza/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/genética , Oxirredutases/metabolismo , Éteres Fenílicos/farmacologia , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência de Bases , Primers do DNA , Resistência a Medicamentos/genética , Vetores Genéticos , Éteres Difenil Halogenados , Cinética , Dados de Sequência Molecular , Oryza/fisiologia , Folhas de Planta/efeitos dos fármacos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Protoporfirinogênio Oxidase , Proteínas Recombinantes/metabolismoRESUMO
In an effort to develop transgenic plants resistant to diphenyl ether herbicides, we introduced the protoporphyrinogen oxidase (EC 1.3.3.4) gene of Bacillus subtilis into tobacco plants. The results from a Northern analysis and leaf disc assay indicate that the expression of the B. subtilis protoporphyrinogen oxidase gene under the cauliflower mosaic virus 35S promoter generated resistance to the diphenyl ether herbicide, oxyfluorfen, in transgenic tobacco plants.