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Master's Dissertation
DOI
10.11606/D.64.2011.tde-09022012-100212
Document
Author
Full name
Aline Borges
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2011
Supervisor
Committee
Caldas, Danielle Gregorio Gomes (President)
Rubiano, Luciana Benchimol
Silva, Marcio José da
Title in Portuguese
Análise da expressão de genes relacionados à interação incompatível Phaseolus vulgaris/Colletotrichum lindemuthianum
Keywords in Portuguese
Antracnose
Bibliotecas de ESTs
Feijoeiro comum
Interação incompatível
Quantificação relative
RT-qPCR
Abstract in Portuguese
A cultura do feijoeiro comum (Phaseolus vulgaris L.) se destaca como fonte protéica e mineral para as populações humanas, principalmente, para populações de baixa renda. Dentre os agentes patogênicos, o fungo Colletotrichum lindemuthianum (Sacc. & Magnus) Lams. Scrib., causador da antracnose, é considerado o de maior importância devido aos seus efeitos destrutivos para o feijoeiro. Este estudo teve como objetivo avaliar a expressão de genes candidatos a atuarem na defesa da planta durante a interação incompatível do feijoeiro SEL 1308 e raça 73 do C. lindemuthianum, considerando diferentes tecidos (folhas, epicótilo e hipocótilo) e períodos de interação (24, 48, 72 e 96 horas). Além disso, o trabalho visou a validação de bibliotecas de ESTs (Expressed Sequence Tags) de feijoeiro (MELOTTO et al., 2005) e a análise da estabilidade de possíveis genes de referência para as análises de expressão por RT-qPCR. Foram utilizados 20 genes no estudo: 12 possivelmente responsivos ao patógeno e oito candidatos a genes de referência. A eficiência média das curvas de amplificação e valores dos ciclos e de quantificação (Cq) foram definidos no programa LinRegPCR. A estabilidade dos genes de referência foi avaliada nos programas geNorm e NormFinder. A expressão relativa dos genes alvos e normalizadores e as análises estatísticas foram determinadas no programa REST. As combinações de genes de referência Act2/Ukn2 e InsDeg/Act2 foram utilizadas para normalização dos genes-alvo por RT-qPCR. Os dados de expressão relativa dos transcritos entre as bibliotecas de ESTs foram validados pelo RT-qPCR. Os genes-alvo analisados foram todos responsivos ao sistema fitopatogênico avaliado, revelando perfil transcricional específico nos tecidos e período de interação com o patógeno. Os dados permitiram a disponibilização de genes de referência importantes para análise de expressão por RTqPCR em feijoeiro sob estresse biótico; e indícios para melhor entendimento dos processos envolvidos nos mecanismos de defesa do feijoeiro ao patógeno da antracnose
Title in English
Expression analysis of genes related to incompatible interaction Phaseolus vulgaris/Coletotrichum
Keywords in English
Anthracnose
Common bean
ESTs libraries
Incompatible interaction
Relative quantification
RT-qPCR
Abstract in English
Common bean crop (Phaseolus vulgaris L.) highlight as protein and mineral source to the human population, especially, for low-income populations. Among the pathogens, Colletotrichum lindemuthianum (Sacc. & Magnus) Lams. - Scrib., which causes anthracnose, is considered the most important due to its destructive effects to common bean. This study aimed to evaluate the expression of candidate genes to act in defense of the plant during the incompatible interaction between common bean SEL 1308 and race 73 of C. lindemuthianum, considering different tissues (leaves, epicotyl and hypocotyl) and periods of interaction (24, 48, 72 and 96 hours). Also, this study attempted the validation of ESTs (Expressed Sequence Tags) libraries of common bean (MELOTTO et al., 2005) and the evaluation of candidate reference genes stability for expression analysis in RT-qPCR. Twenty genes were used: 12 candidate genes responsive to the pathogen and eight candidate reference genes. The average efficiency of amplification curves and the values of quantification cycles (Cq) were defined in LinRegPCR software. The stability of reference genes was evaluated using geNorm and NormFinder softwares. Relative expression of target and normalizer genes and statistical analysis were determined by REST software. The Act2/Ukn2 and InsDeg/Act2 reference genes combinations were used for normalization of target genes by RT-qPCR. The data of relative expression of transcripts between the libraries of ESTs were validated by RT-qPCR. All target genes analyzed were responsive to the pathogenic system evaluated, revealing specific transcriptional profiles in tissues and periods of interaction with the pathogen. The data provided the availability of normalizers important for expression analysis by RTqPCR in beans under biotic stress; and evidences to understand the processes involved in defense mechanisms of common bean against the anthracnose pathogen
 
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Publishing Date
2012-02-09
 
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