RESUMO
Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.
Assuntos
Gossypium , Agrobacterium/genética , Animais , Gossypium/genética , Insetos , Melhoramento Vegetal , Plantas Geneticamente Modificadas/genética , Interferência de RNA , RNA de Cadeia Dupla/genéticaRESUMO
BACKGROUND AND AIMS: Pre-harvest sprouting susceptibility in grain sorghum (Sorghum bicolor) is related to low seed dormancy and reduced embryo sensitivity to inhibition of germination by abscisic acid (ABA). Intra-specific variability for pre-harvest sprouting might involve differential regulation of ABA signalling genes. METHODS: Sorghum genes encoding homologues for ABA signalling components from other species (ABI5, ABI4, VP1, ABI1 and PKABA1) were studied at the transcriptional and protein level (ABI5) during grain imbibition for two sorghum lines with contrasting sprouting phenotypes and in response to hormones. KEY RESULTS: Transcript levels of these genes and protein levels of ABI5 were higher in imbibed immature caryopses of the more dormant line. Dormancy loss was related to lower transcript levels of these genes and lower ABI5 protein levels in both genotypes. Exogenous ABA inhibited germination of isolated embryos but failed to prevent ABI5 rapid decrease supporting a role for the seed coat in regulating ABI5 levels. CONCLUSIONS: Several genes involved in ABA signalling are regulated differently in imbibed caryopses from two sorghum lines with contrasting pre-harvest sprouting response before - but not after - physiological maturity. A role for ABI5 in the expression of dormancy during grain development is discussed.
Assuntos
Ácido Abscísico/genética , Germinação/genética , Reguladores de Crescimento de Plantas/genética , Proteínas de Plantas/fisiologia , Sementes/genética , Sorghum/genética , Ácido Abscísico/análise , Ácido Abscísico/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Western Blotting , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Germinação/fisiologia , Reguladores de Crescimento de Plantas/análise , Reguladores de Crescimento de Plantas/fisiologia , Reação em Cadeia da Polimerase , Sementes/crescimento & desenvolvimento , Sorghum/crescimento & desenvolvimentoRESUMO
Asr1 and Asr2 are water stress-inducible genes belonging to the Asr gene family, which transcriptionally regulate a sugar transporter gene, at least in grape. Using an in situ RNA hybridization methodology, we determined that, in basal conditions, expression of Asr2 in tomato leaves is detected in the phloem tissue, particularly in companion phloem cells. When plants are exposed to water stress, Asr2 expression is contained in companion cells but expands occasionally to mesophyll cells. In contrast, Asr1 transcript localization seems to be sparse in leaf vascular tissue under both non-stress and stress conditions. The occurrence of Asr transcripts precisely in companion cells is in accordance with the cell type specificity reported for hexose-transporter protein molecules in grape encoded by the only Asr-target gene known to date. The results are discussed in light of the reported scarcity of plasmodesmata between companion cells and the rest of leaf tissue in the family Solanaceae.
Assuntos
Genes de Plantas , Folhas de Planta/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Proteínas de Plantas/análiseRESUMO
The Asr gene family is present in Spermatophyta. Its members are generally activated under water stress. We present evidence that tomato ASR1, one of the proteins of the family, accumulates in seed during late stages of embryogenesis, a physiological process characterized by water loss. In vitro, electrophoretic assays show a homo-dimeric structure for ASR1 and highlight strong non-covalent interactions between monomers prone to self-assemble. Direct visualization of single molecules by atomic force microscopy (AFM) confirms that ASR1 forms homodimers and that uncovers both monomers and dimers bind double stranded DNA.