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Master's Dissertation
DOI
10.11606/D.64.2012.tde-18012013-145236
Document
Author
Full name
Ilse Fernanda Ferrari
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2012
Supervisor
Committee
Figueira, Antonio Vargas de Oliveira (President)
Maia, Ivan de Godoy
Souza, Silvana Aparecida Creste Dias de
Title in Portuguese
Caracterização de promotores de expressão especifica de cana-de-açucar (Saccharum ssp.) em sistema modelo Micro-Tom (Solanum lycopersicum L)
Keywords in Portuguese
'beta'-glucuronidase (GUS)
'Micro-Tom'
Expressão estável
Expressão transiente
Promotores de metalotioneínas
Promotores sintéticos
Saccharum ssp
Abstract in Portuguese
O emprego de novas tecnologias, como a transgenia, associadas ao melhoramento convencional de cana-de-açúcar apresenta grande potencial no combate a pragas e desenvolvimento de novas variedades. Entretanto, a falta de especificidade na expressão temporal e/ou local dos genes introduzidos tem se mostrado um fator limitante para o sucesso de produtos derivados da transgenia. Os promotores das metalotioneínas, por exemplo, podem representar uma alternativa ao uso de promotores constitutivos, particularmente, aqueles de metalotioneínas do tipo 1 (MT1) por apresentarem níveis elevados de expressão, em diferentes tecidos/órgãos e serem responsivos a estresses bióticos e abióticos. O uso de promotores sintéticos, contendo apenas elementos-cis, como GCG-like, W boxes e JERE, tem sido relevante por induzirem a expressão gênica local em resposta ao ataque de agentes bióticos. Este trabalho teve por objetivo a caracterização funcional de promotores de genes de metalotioneínas de cana-de-açúcar e o uso de elementos regulatórios sintéticos e, em paralelo, avaliou-se o uso de promotores sintéticos no controle da expressão gênica em cana-de-açúcar. Após as análises de expressão transiente, verificou-se que o promotor SoMT1b apresentou atividade GUS e GFP em epitélios de cebola e os promotores sintéticos, 4X Wbox, 4X GCC-like, 4X JERE e 4xW 4xS-box, e o promotor SoMT1b foram capazes de dirigir a expressão do gene repórter uidA (GUS) em calos embriogênicos de cana-de-açúcar. Em análise de expressão estável, o promotor SoMT1b foi capaz de dirigir a expressão do gene GUS para os frutos e sementes de tomate 'Micro-Tom', mas não foi responsivo a estresse por herbivoria, cádmio e cobre. Também foi realizada a transformação de plantas de cana-de-açúcar, as quais ainda estão sendo analisadas. Os resultados obtidos demonstram a funcionalidade do promotor SoMT1b
Title in English
Characterization of promoters specific expression of sugar cane (Saccharum spp.) In model systems Micro-Tom (Solanum lycopersicum L)
Keywords in English
"Micro-Tom'
'beta'-glucuronidase (GUS)
Metallothionein promoters
Saccharum ssp
Stable expression
Synthetic promoters
Transient expression
Abstract in English
New technologies, like genetic transformation, associated with conventional breeding of sugarcane have a large potential in developing new varieties tolerant to biotic and abiotic stress. However, the lack of specificity in the spatial and/or temporal expression of the introduced genes has been a limited factor for the success of products derived from transgeny. Metallothionein promoters, for instance, can represent an alternative to the use of constitutive promoters, particularly those from metallothionein type 1 (MT1) because they present high expression levels in different tissues / organs and are responsive to biotic and abiotic stress. Another alternative is the use of synthetic promoters which contain only cis elements, as GCG-like, W-box and JERE, which induces local gene expression in response to pathogen attack. In this work, we aimed to make the functional characterization of sugarcane metallothionein promoters and synthetic regulatory elements, in parallel, we evaluated the use of synthetic promoters in the control of gene expression in sugarcane. After transient expression analyzis, it was found that SoMT1b promoter was able to control the expression of the reporter genes to GUS and GFP in onion epithelium and GUS in sugarcane embryogenic calli. Additionally, synthetic promoters 4X Wbox, 4X GCC-like, 4X JERE and 4xW 4xS-box were able to direct the expression of the gene uidA (GUS) in embryogenic calli of sugarcane. In stable transformation analysis, SoMT1b promoter was capable of directing uidA expression in fruits and seeds of tomato cv. 'Micro-Tom', but it was not responsive to herbivory, cadmium and copper stress. It was also carried out the transformation of sugarcane plants with the construction containing the SoMT1b promoter, but these are still being analyzed. The results demonstrate the functionality of the SoMT1b promoter in sugarcane and tomato
 
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Publishing Date
2013-02-26
 
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