Caracterização in silico de genes da biossíntese de lipídeos e utilização de eixos embrionários e nós-cotiledonares na transformação genética de soja
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Data
2012-06-15
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Universidade Federal de Viçosa
Resumo
As atividades das enzimas oleoil dessaturase, 1,2-diacilglicerol colinafosfotransferase (CPT) e a lisofosfatidilcolina aciltransferase (LPCAT) regulam a biossíntese de lipídeos em plantas. Dessa forma, um melhor conhecimento dos genes que codificam essas enzimas é importante no processo de melhoramento genético da qualidade do óleo de soja. O objetivo do presente trabalho foi caracterizar in silico os genes que codificam as enzimas CPT e LPCAT em soja e avaliar a utilização de eixos embrionários e nós-cotiledonares como explantes em protocolos de cultura de tecidos para transformação genética, via RNA de interferência, com cassetes de silenciamento do gene da oleil dessaturase. Análises computacionais mostraram que o genoma da soja apresenta dois genes que codificam a CPT e dois genes que codificam a LPCAT, sendo que um desses genes apresentou um transcrito alternativo. Nas regiões promotoras foram identificados cis-elementos responsáveis pela expressão semente-específica e também de resposta a estresses biótico e abiótico. A análise in silico indica que os genes são expressos em diferentes condições de estresse, em embriões somáticos em diferenciação, em sementes imaturas, em tecidos de raiz e em calos. A análise das sequências aminoacídicas revelou a presença de domínios transmembrana e a busca da localização celular indicou que as enzimas CPT e LPCAT estão localizadas no retículo endoplasmático. Como esses genes se expressam em sementes, eles são possíveis candidatos à manipulação genética visando a melhoria da qualidade do óleo. Dois protocolos de transformação de soja foram avaliados para a possível obtenção de soja com óleo que apresente maior estabilidade oxidativa. Nesse sentido, as variedades comerciais CAC-1 (de ciclo tardio) e CD-201 (de ciclo precoce) foram submetidas à infecção via Agrobacterium tumefaciens da linhagem KYRT1. O protocolo utilizado para os eixos embrionários foi mais rápido, com meios de cultura menos elaborados em relação à composição e um maior número de plantas foi transferido para a casa de vegetação, onde permaneceram até a produção de vagens e sementes. Foi possível realizar todas as etapas, desde a infecção até regeneração e aclimatação das plantas, para as variedades utilizadas, pelos protocolos descritos.
The activities of the enzymes oleoyl desaturase, 1,2-diacylglycerol cholinephosphotransferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) regulate the biosynthesis of lipids in plants. Thus, a better understanding of the genes encoding these enzymes is important in the genetic manipulation of the soybean oil quality. Therefore, the first chapter presents the in silico characterization of the genes encoding the enzymes CPT and LPCAT in soybean. The second chapter presents an evaluation of the use of embryonic axes and cotyledonary nodes as explants in tissue culture protocols for genetic transformation, via RNA interference, with silencing cassettes of the oleyl desaturase gene. Computer analysis showed that the soybean genome has two genes encoding CPT and two genes encoding LPCAT, with one of these models showing an alternative transcript. Cis-elements in the promoter regions were identified that are responsible for seed-specific expression and also in response to biotic and abiotic stresses. In silico analysis suggested the expression of the genes in different stress conditions, in differentiating somatic embryos, in immature seeds, in root tissue and callus. The amino acid sequence analysis revealed the presence of transmembrane domains and searching for subcellular localization indicated that CPT and LPCAT are located in the endoplasmic reticulum. Since these genes are expressed in seeds, they are possible candidates for genetic manipulation aim the improvement of oil quality. Two soybean transformation protocols were evaluated for the possibility of obtaining soybean oil with greater oxidation stability. The commercial varieties CAC-1 (late cycle) and CD-201 (early cycle) were subjected to infection via Agrobacterium tumefaciens strain KYRT1. The protocol used for the embryonic axis transformation was faster, with less elaborate culture media in relation to composition and a greater number of plants were transferred to the greenhouse, where they remained until the production of pods and seeds. It was possible to perform all stages, from infection to regeneration and acclimatization of plants, for the varieties used with the protocols described.
The activities of the enzymes oleoyl desaturase, 1,2-diacylglycerol cholinephosphotransferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) regulate the biosynthesis of lipids in plants. Thus, a better understanding of the genes encoding these enzymes is important in the genetic manipulation of the soybean oil quality. Therefore, the first chapter presents the in silico characterization of the genes encoding the enzymes CPT and LPCAT in soybean. The second chapter presents an evaluation of the use of embryonic axes and cotyledonary nodes as explants in tissue culture protocols for genetic transformation, via RNA interference, with silencing cassettes of the oleyl desaturase gene. Computer analysis showed that the soybean genome has two genes encoding CPT and two genes encoding LPCAT, with one of these models showing an alternative transcript. Cis-elements in the promoter regions were identified that are responsible for seed-specific expression and also in response to biotic and abiotic stresses. In silico analysis suggested the expression of the genes in different stress conditions, in differentiating somatic embryos, in immature seeds, in root tissue and callus. The amino acid sequence analysis revealed the presence of transmembrane domains and searching for subcellular localization indicated that CPT and LPCAT are located in the endoplasmic reticulum. Since these genes are expressed in seeds, they are possible candidates for genetic manipulation aim the improvement of oil quality. Two soybean transformation protocols were evaluated for the possibility of obtaining soybean oil with greater oxidation stability. The commercial varieties CAC-1 (late cycle) and CD-201 (early cycle) were subjected to infection via Agrobacterium tumefaciens strain KYRT1. The protocol used for the embryonic axis transformation was faster, with less elaborate culture media in relation to composition and a greater number of plants were transferred to the greenhouse, where they remained until the production of pods and seeds. It was possible to perform all stages, from infection to regeneration and acclimatization of plants, for the varieties used with the protocols described.
Descrição
Palavras-chave
Eixos embrionários, Soja, Nós-cotiledonares, Embryonic axes, Soybean, Cotyledonary nodes
Citação
SOUSA, Cassiana Severiano de. In silico characterization of genes involved in lipid biosynthesis and use of embryonic axes and cotyledonary nodes in genetic transformation of soybean. 2012. 94 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2012.