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Doctoral Thesis
DOI
10.11606/T.64.2013.tde-22042013-161909
Document
Author
Full name
Eveline Carla da Rocha Tavano
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2013
Supervisor
Committee
Martinelli, Adriana Pinheiro (President)
Monteiro, Mariza
Wulff, Nelson Arno
Coletta Filho, Helvecio Della
Mourão Filho, Francisco de Assis Alves
Title in Portuguese
Transformação genética de Citrus sinensis (L.) Osbeck para resistência a Candidatus Liberibacter ssp
Keywords in Portuguese
Citros
HLB
Peptídeo antibacteriano
Promotor tecido-específico
Resistência a doenças
Transgenia
Abstract in Portuguese
A doença Huanglongbing (HLB) associada a bactéria Candidatus Liberibacter spp., que coloniza os vasos do floema, é considerada uma das mais graves doenças de citros. Uma importante estratégia para o controle desta doença consiste na produção de plantas transgênicas, expressando genes que codificam peptídeos antibacterianos especificamente no local de colonização do patógeno. O objetivo deste trabalho foi obter plantas transgênicas de laranja doce, expressando o gene que codifica o peptídeo antibacteriano atacina A (attA) dirigido por promotores específicos para a expressão gênica no floema. O trabalho foi iniciado com a elaboração de construções gênicas contendo o gene attA (associado ou não ao peptídeo sinal), sob o controle dos promotores AtSuc2 (transportador de sacarose), AtPP2 (proteína de floema 2), clonados de Arabidopsis thaliana, ou CsPP2 (proteína de floema 2), clonado de Citrus sinensis. Os experimentos de transformação genética foram realizados com C. sinensis cv. 'Hamlin', 'Valência', 'Pêra' e 'Natal', via Agrobacterium tumefaciens, utilizando-se segmento de epicótilo como explante. A identificação de plantas transgênicas foi realizada por meio da análise de PCR. Plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação específica para o cultivo de plantas transgênicas. Análises de Southern e Northern blot foram realizadas em plantas aclimatizadas, confirmando-se a integração e transcrição do gene attA, respectivamente. A expressão do gene attA também foi confirmada pela análise de RT-qPCR. Plantas de laranja 'Hamlin' contendo o gene attA (associado ou não ao peptídeo sinal), sob o controle dos promotores AtSuc2 ou AtPP2 foram propagadas por enxertia, para futura avaliação da resistência a Candidatus Liberibacter asiaticus
Title in English
Genetic transformation of Citrus sinensis (L) Osbeck for resistance to Candidatus Liberibacter spp
Keywords in English
Antibacterial peptides
Citrus
Disease resistance
HLB
Tissue-specific promoters
Transgenic
Abstract in English
Huanglongbing (HLB) associated to Candidatus Liberibacter spp., which colonizes the phloem, is considered one of the most serious diseases of citrus. One important strategy to control this disease consists of producing transgenic plants expressing, in the bacteria colonization tissue, genes encoding antibacterial peptides. The objective of this work was to produce transgenic sweet orange plants expressing genes encoding the antibacterial peptide attacin A (attA) driven by phoem-specific promoters. The work started with the development of the gene constructs, containing the attacin A gene (with or without signal peptide) controlled by either sucrose transporter gene (AtSuc2) or phloem protein 2 gene promoters (AtPP2) from Arabidopsis thaliana, or phloem protein 2 gene promotor (CsPP2) from Citrus sinensis. The genetic transformation of C. sinensis 'Hamlin', 'Valencia', 'Pera' and 'Natal' cultivars was done via Agrobacterium tumefaciens. Epicotyls segments collected from in vitro germinated seedlings were used as explants. Transgenic plants were identified by PCR analyses. PCR positive plants were acclimatized and transferred to specific greenhouse. Integration and transcription of the attA gene was confirmed in acclimatized transgenic plants by Southern and Northern blot analysis, respectively. The attA gene expression was validated by RT-qPCR analysis. 'Hamlin' transgenic cultivars containing the AtSuc2 or AtPP2 promoters controlling the expression of attA (with or without signal peptide) were propagated by grafting, for future evaluation of Candidatus Liberibacter asiaticus resistance
 
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Publishing Date
2013-05-29
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • TAVANO, E. C. R., et al. Genetic transformation of Citrus sinensis cv. Hamlin with attacin A driven by a phloem tissue-specific promoter for resistance to Candidatus Liberibacter spp. Acta Horticulturae, 2013.
  • OLIVEIRA, C. R., et al. Genetic transformation of citrus sinensis cv. Natal for resistance to Huanglongbing. In 2013 In Vitro Biology Meeting, Providence, Rhode Island, USA, 2013. 2013 In Vitro Biology Meeting. : Society for In Vitro Biology, 2013. Abstract.
  • TAVANO, Eveline Carla da Rocha, et al. Genetic transformation of Citrus sinensis with attacin A gene driven by phloem tissue specific promoters for resistance to Candidatus Liberibacter spp. In XII International Citrus Congress, Valencia, 2012. XII International Citrus Congress., 2012. Abstract.
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