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Master's Dissertation
DOI
10.11606/D.11.2015.tde-11092015-082205
Document
Author
Full name
Renata Beatriz Cruz
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2015
Supervisor
Committee
Mourão Filho, Francisco de Assis Alves (President)
Caldas, Danielle Gregorio Gomes
Harakava, Ricardo
Title in Portuguese
Transformação genética de laranja doce com o gene codificador de defensina de Citrus sinensis, sob controle dos promotores 35S (Cauliflower mosaic virus) ou AtSuc2 (Arabidopsis thaliana)
Keywords in Portuguese
Citros
Defensina
Huanglongbing
Promotor específico de floema
Transgenia
Abstract in Portuguese
A citricultura brasileira é a maior produtora e exportadora de citros e tem sido afetada por doenças que causam sérios prejuízos a produção e a qualidade dos frutos. No entanto, a cultura apresenta grandes problemas, entre eles, os fatores fitossanitários, que vem dizimando milhares de plantas e afetando a produtividade e a competitividade do setor. Atualmente, o huanglongbing (HLB), associado às bactérias de floema Candidatus Liberibacter spp., é considerado uma das mais destrutivas doenças de citros. A inexistência de cultivares de laranja doce resistentes ao HLB torna a transformação genética de citros uma ferramenta importante no controle desta doença. Para se defender do ataque de pragas e patógenos as plantas desenvolveram, durante o processo evolutivo, uma série de mecanismos de defesa, no qual pode-se incluir a produção de peptídeos com atividade antimicrobiana. As defensinas vegetais são peptídeos pequenos relacionadas à patogênese (PR), que possuem atividade antimicrobiana associada aos mecanismos de defesa das plantas. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas de laranja doce (Citrus sinensis L.) cvs. 'Hamlin', 'Natal', 'Valência' e 'Pera', via Agrobacterium tumefaciens, superexpressando o gene codificador de defensina (def), isolado de Citrus sinensis cv. 'Valência', dirigido pelo promotor com expressão preferencial no floema AtSUC2 (transportador de sacarose, clonado de Arabidopsis thaliana) ou pelo promotor constitutivo CaMV 35S (clonado do vírus do mosaico da couve-flor). Os explantes utilizados na transformação genética foram segmentos de epicótilo obtidos de plantas germinadas in vitro. A identificação das plantas transgênicas foi realizada por meio da análise da PCR, utilizando-se primers para a detecção do fragmento do gene de seleção nptII. As plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação. A análise de Southern blot confirmou a integração do transgene em 36 plantas. Foram obtidas 7 plantas transgênicas da cultivar 'Hamlin', 9 da cultivar 'Natal', 1 da cultivar 'Pera' e 9 da cultivar 'Valência' contendo a construção gênica pC35S/def, e 3 plantas transgênicas da cultivar 'Hamlin', 6 da cultivar 'Natal' e 1 da cultivar 'Valência' contendo a construção gênica pcAtSUC2/def. Os resultados obtidos neste trabalho serão importantes para futura avaliação e estudo visando o controle de Candidatus Liberibacter spp..
Title in English
Genetic transformation of sweet orange with the gene that encodes Citrus sinensis defensin under the control of 35S (Cauliflower mosaic virus) or AtSUC2 (Arabidopsis thaliana) promoters
Keywords in English
Citrus
defensin
Huanglongbing
phloem specific promoter
transgenic
Abstract in English
The Brazilian citrus industry is the world's largest producer and exporter of citrus, however, it has been affected by diseases that cause serious production losses and damages to fruit quality. However, the culture faces problems, namely phytosanitary issues that have been damaging thousands of plants, affecting yield and competitiveness of the sector. Currently, Huanglongbing (HLB), associated with phloem bacteria Candidatus Liberibacter spp., is considered one of the most destructive citrus diseases. The lack of sweet orange cultivars resistant to HLB makes genetic transformation an important tool in the disease control. To defend from pest and pathogen attack, plants developed a series of defense mechanisms during the evolutionary process, which may include the production of peptides with antimicrobial activity. Plant defensins are small peptides related to pathogenesis (PR) which have antimicrobial activities, associated with plant defense mechanisms . The objective of this study was to obtain transgenic plants of sweet orange (Citrus sinensis L.) cultivars 'Hamlin', 'Natal', 'Valência' and 'Pera' with Agrobacterium tumefaciens overexpressing the defensin gene (def), isolated from Citrus sinensis cv. 'Valência', controlled by the promoter with preferential expression in the phloem AtSUC2, (sucrose transporter, cloned from Arabidopsis thaliana) or by the constitutive promoter CaMV 35S (cloned from the mosaic virus of cauliflower). The explants used in genetic transformation were epicotyl segments obtained from germinated plants in vitro. The identification of transgenic plants was accomplished by PCR analysis using primers for the detection of nptII gene fragment. The PCR+ plants were acclimatized and transferred to greenhouse. The analysis of Southern blot confirmed the transgene integration in 36 plants. Seven transgenic plants were obtained for the cultivar 'Hamlin', nine for 'Natal', one for 'Pera' and nine for 'Valência' containing the gene construct pC35S/def and three transgenic plants for 'Hamlin', six for 'Natal' and one for 'Valência' containing the gene construct pcAtSUC2/def. The results obtained in this work are important for future evaluation of the plants for resistance to Candidatus Liberibacter spp..
 
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Publishing Date
2015-09-25
 
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