RESUMO
Real-time PCR is a powerful technique used for quantification of defined nucleic acid sequences. Numerous applications of this method have been described including: gene expression studies, diagnosis of pathogens, and detection of genetically modified organisms or mutations. Here, we describe a simple and efficient protocol to determine gene expression in cereals, based on real-time PCR using SYBR® Green dye. This technique provide an inexpensive alternative, since no probes are required, allowing for the quantification of a high number of genes with reduced cost.
Assuntos
Perfilação da Expressão Gênica , Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de PlantasRESUMO
Circular RNAs (circRNAs) are a widespread class of endogenous noncoding RNAs and they have been studied in the past few years, implying important biological functions in all kingdoms of life. Recently, circRNAs have been identified in many plant species, including cereal crops, showing differential expression during stress response and developmental programs, which suggests their role in these process. In the following years, it is expected that insights into the functional roles of circRNAs can be used by cereal scientists and molecular breeders with the aim to develop new strategies for crop improvement. Here, we briefly outline the current knowledge about circRNAs in plants and we also outline available computational resources for their validation and analysis in cereal species.
Assuntos
Biologia Computacional , Grão Comestível/genética , Regulação da Expressão Gênica de Plantas , RNA Circular , RNA de Plantas , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Melhoramento Vegetal , Software , NavegadorRESUMO
Developing disease resistance is one of the most important components of any plant breeding program. Citrus traditional breeding methods (bud sport selection, crossbreeding, and other breeding channels) are a laborious task and often hampered by long juvenility, a high degree of heterozygosity, polyembryony, self-incompatibility, and abortion of reproductive organs. An interesting alternative to the classical breeding approach is the use of genetic transformation, which provides the means for adding a single agronomic trait to a plant without otherwise altering its phenotype. Agrobacterium tumefaciens-mediated transformation has been carried out with numerous hybrids and citrus species. This technique allowed us to introduce the Bs2 gene in Citrus, as well as to increase citrus canker resistance in transgenic Bs2 gene-expressing lines.
Assuntos
Citrus sinensis/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Resistência à Doença/genética , Resistência à Doença/imunologia , Vetores Genéticos/genética , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plasmídeos/genética , Xanthomonas/patogenicidadeRESUMO
Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.
Assuntos
Capsicum/genética , Citrus sinensis/genética , Citrus sinensis/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas campestris/patogenicidade , Agrobacterium tumefaciens/genética , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/genética , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Transformação GenéticaRESUMO
Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.
Assuntos
Citrus/parasitologia , Doenças das Plantas , Reação em Cadeia da Polimerase , Rhizobiaceae/patogenicidadeRESUMO
Huanglongbing (HLB), a devastating citrus disease caused by the bacterium Candidatus Liberibacter spp., is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of Ca. Liberibacter spp. in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.(AU)