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Doctoral Thesis
DOI
https://doi.org/10.11606/T.11.2019.tde-20191220-121333
Document
Author
Full name
Irving Joseph Berger
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2005
Supervisor
Title in Portuguese
Transformação de genomas plastidiais de tomate e fumo visando a modificação do metabolismo de tocoferóis
Keywords in Portuguese
TRANSFORMAÇÃO GENÉTICA
EXPRESSÃO GÊNICA
FUMO
GENOMAS CLOROPLASTIDIAIS
METABOLISMO
TOMATE
VITAMINA E
Abstract in Portuguese
A transformação de genomas cloroplastidiais de tomate (Lycopersicon esculentum Mild.) no ano de 2001, mais de uma década após o primeiro resultado do desenvolvimento dessa tecnologia em fumo, ampliou a oportunidade para a introdução e expressão de transgenes em genomas plastidiais de plantas de valor alimentício. Dentre outras vantagens, a segurança ambiental e os altos níveis de expressão de transgenes, com conseqüente elevado acúmulo da proteína de interesse, são decorrentes da transformação de genomas cloroplastidiais. Uma vez que plastídeos estão envolvidos em diversos processos metabólicos celulares, a modificação de qualquer um desses processos pode ser viabilizada pela introdução de genes exógenos ou alteração de genes endógenos em plastídeos. Dadas as importantes propriedades terapêuticas associadas ao consumo de vitamina E, especialmente α-tocoferol, em doses superiores àquelas necessárias à nutrição humana (100 a 400 IU), este trabalho teve como objetivo a obtenção de plantas com alto conteúdo desta vitamina pela transformação de cloroplastos. Os genes s/r0089 e s/r0090 de Synechocystis sp. PCC6803, que codificam as enzimas γ-tocoferol metil transferase (y-TMT) e HPP dioxigenase (HPPDase) respectivamente, foram isolados e introduzidos em vetores de transformação de cloroplastos, resultando nos plasmídeos plJB30, plJB31 e plJB32, os quais contêm as regiões codificadoras dos genes s/r0089, s/r0090 e s/r0089+s/r0090, respectivamente. Experimentos de biobalística em explantes foliares de tomate e fumo foram conduzidos para a integração e expressão dos transgenes nos genomas plastidiais. Plantas de fumo contendo as três construções foram confirmadas serem homoplásmicas, possuindo os transgenes corretamente integrados. Desde que superexpressão de genes que codificam a enzima HPPDase em plantas podem levar à resistência aos herbicidas inibidores desta enzima, plantas transplastômicas de fumo foram analisadas e apresentaram resistência ao isoxaflutole em testes in vitro. Plantas de tomate homoplásmicas contendo os transgenes foram confirmadas somente para a construção plJB32, apresentando os transgenes corretamente integrados.
Title in English
Tomato and tobacco plastid genome transformation for engineering of the tocopherol metabolism
Abstract in English
Tomato (Lycopersicon esculentum Mild.) chloroplast transformation in 2001, more than one decade after the first results from the transplastomic technology in tobacco, has increased the opportunity to the introduction and expression of transgenes into plastid genomes of crop species. Among other advantages, environmental safety and high levels of transgene expression with consequent great accumulation of the introduced recombinant protein are results of the plastid transformation. Plastids take part in several metabolic cellular processes and so modification of any of the involved pathways could be viable by the introduction of exogenous genes or alteration of endogenous genes in chloroplast genomes. Since vitamin E, specially α-tocopherol, has important therapeutic properties when daily intakes exceed normal nutritional requirements (100 to 400 IU), the goal of this work was the production of vitamin E rich plants by chloroplast transformation. ln that way, the Synechocystis sp. PCC6803 s/r0089 and s/r0090 genes coding for the y-tocopherol methyl transferase (γ-TMT) and HPP dioxygenase (HPPDase) enzymes respectively were isolated and introduced in chloroplast transformation vectors resulting in the plJB30, plJB31 and plJB32 plasmids, which contain the s/r,0089, s/r,0090 and s/r0089+s/r0090 coding regions respectively. Biolistic experiments with tomato and tobacco leaf explants were performed for the integration and expression of the constructs into the plastid genomes. Transformed tobacco plants containing all the three constructs were confirmed to be homoplasmic and have the transgenes correctly integrated. Since overexpression of the HPPDase coding genes in plants can lead to resistance against HPPD inhibiting herbicides, transplatomic tobacco plants were analized and have shown resistance against isoxaflutole on in vitro tests. Tomato homoplasmic plants containing the transgenes were confirmed only for the plJB32 construct and have also the transgenes correctly integrated.
 
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Publishing Date
2019-12-20
 
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