RESUMO
Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.
Assuntos
DNA de Plantas/isolamento & purificação , Extração Líquido-Líquido/métodos , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Arabidopsis/química , Arabidopsis/genética , Avicennia/química , Avicennia/genética , Cacau/química , Cacau/genética , Clorofórmio/química , DNA de Plantas/química , DNA de Plantas/genética , Epigênese Genética , Frutas/química , Frutas/genética , Cloreto de Lítio/química , Paspalum/química , Paspalum/genética , Fenol/química , Folhas de Planta/química , Folhas de Planta/genética , Raízes de Plantas/química , Raízes de Plantas/genética , Caules de Planta/química , Caules de Planta/genética , RNA de Plantas/química , RNA de Plantas/genética , Plântula/química , Plântula/genética , Sorghum/química , Sorghum/genéticaRESUMO
Quantitative and qualitative relationships were found between secreted proteins and their activity, and the hyphal morphology of Moniliophthora perniciosa, the causal agent of witches' broom disease in Theobroma cacao. This fungus was grown on fermentable and non-fermentable carbon sources; significant differences in mycelial morphology were observed and correlated with the carbon source. A biological assay performed with Nicotiana tabacum leaves revealed that the necrosis-related activity of extracellular fungal proteins also differed with carbon source. There were clear differences in the type and quantity of the secreted proteins. In addition, the expression of the cacao molecular chaperone BiP increased after treatment with secreted proteins, suggesting a physiological response to the fungus secretome. We suggest that the carbon source-dependent energy metabolism of M. perniciosa results in physiological alterations in protein expression and secretion; these may affect not only M. perniciosa growth, but also its ability to express pathogenicity proteins.
Assuntos
Basidiomycota/fisiologia , Cacau/citologia , Cacau/microbiologia , Carbono/farmacologia , Proteínas Fúngicas/metabolismo , Micélio/efeitos dos fármacos , Micélio/fisiologia , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Biomassa , Cacau/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Hifas/citologia , Hifas/efeitos dos fármacos , Meristema/efeitos dos fármacos , Meristema/microbiologia , Necrose , Fenótipo , Nicotiana/efeitos dos fármacos , Nicotiana/microbiologiaRESUMO
The genome sequence of the hemibiotrophic fungus Moniliophthora perniciosa revealed genes possibly participating in the RNAi machinery. Therefore, studies were performed in order to investigate the efficiency of gene silencing by dsRNA. We showed that the reporter gfp gene stably introduced into the fungus genome can be silenced by transfection of in vitro synthesized gfpdsRNA. In addition, successful dsRNA-induced silencing of endogenous genes coding for hydrophobins and a peroxiredoxin were also achieved. All genes showed a silencing efficiency ranging from 18% to 98% when compared to controls even 28d after dsRNA treatment, suggesting systemic silencing. Reduction of GFP fluorescence, peroxidase activity levels and survival responses to H(2)O(2) were consistent with the reduction of GFP and peroxidase mRNA levels, respectively. dsRNA transformation of M. perniciosa is shown here to efficiently promote genetic knockdown and can thus be used to assess gene function in this pathogen.
Assuntos
Agaricales/fisiologia , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Doenças das Plantas/microbiologia , RNA de Cadeia Dupla/genética , Cacau , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Viabilidade Microbiana , Peroxirredoxinas/metabolismo , RNA de Cadeia Dupla/metabolismoRESUMO
During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and beta-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source-sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey.
Assuntos
Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Hymenaea/genética , Chuva , Plântula/crescimento & desenvolvimento , Plântula/genética , Árvores/genética , Xilanos/metabolismo , Actinas/metabolismo , Transporte Biológico/efeitos da radiação , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas , Glucosiltransferases/genética , Glicosiltransferases/genética , Hymenaea/enzimologia , Hymenaea/crescimento & desenvolvimento , Hymenaea/efeitos da radiação , Ácidos Indolacéticos/metabolismo , Luz , Filogenia , Plântula/efeitos da radiação , Árvores/enzimologia , Árvores/crescimento & desenvolvimento , Árvores/efeitos da radiação , beta-Frutofuranosidase/genética , beta-Galactosidase/genéticaRESUMO
Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcane's ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.
RESUMO
Expressed sequence tags (ESTs) have proven to be a valuable tool to discover single nucleotide polymorphism (SNP) in human genes but their use for this purpose is still limited in higher plants. Using a database of approximately 250,000 sugarcane ESTs we have recovered 219 sequences encoding alcohol dehydrogenases ( Adh), which tagged 178 distinct cDNAs from 27 libraries, constructed from at least four different cultivars. The partitioning of these ESTs into paralogous genes revealed three Adh genes expressed in sugarcane, one Adh2 and two Adh1. The soundness of the partition was carefully checked by comparison to external data, especially from the closely related sorghum. Analysis of polymorphism in the alignments of EST sequences revealed a total of 37 highly reliable SNPs in the coding and untranslated regions of the three Adh genes. In the coding regions, the mean occurrence of SNPs was one for every 122 base pair. A total of eight insertion-deletions was observed, their occurrence being limited to untranslated regions. These results show that EST data constitute an invaluable source of sequence polymorphism for sugarcane that is worth carefully collecting for the future development of new marker tools.
Assuntos
Álcool Desidrogenase/genética , Etiquetas de Sequências Expressas , Genes de Plantas/genética , Polimorfismo de Nucleotídeo Único/genética , Saccharum/genética , Sequência de Bases , DNA Complementar/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize alpha-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix alpha-coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5' Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.