Caracterização in silico de genes envolvidos na biossíntese de oligossacarídeos de rafinose em soja e construção de cassetes de silenciamento do gene da estaquiose sintase, via interferência por RNA

• Ano de defesa: 2011
• Autor(a) principal: Soares, Ana Paula Gomes
• Orientador(a): Guimarães, Valéria Monteze
• Banca de defesa: Oliveira, Luiz Orlando de , Damasceno, Cynthia Maria Borges
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Oligossacarídeos; Estaquiose sintase; RNA
• Palavras-chave em Inglês: Oligosaccharides; Stachyose synthase; RNA
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/313

Soybean (Glycine max L. Merrill) seed accumulates raffinose oligosaccharides (ROs), mainly raffinose and stachyose, which are considered antinutritional factors. The reduction of these carbohydrates in soybean seeds could result in decreased gastrointestinal problems generated from soy products and provide a positive impact on acceptance of these products. The biosynthesis of ROs is well characterized and involves the sequential action of a group of synthases. However, genes and the pattern of expression of these synthases have not been fully described in soybean. The aim of this study was to characterize and analyze in silico the expression of genes involved in biosynthesis of ROs and construction expression cassettes for gene silencing of stachyose synthase via RNA interference in soybean. Computer analysis showed that the soybean genome has 10 loci which potentially encode for synthases ROs. Of these, six encode galactinol synthase, three encode raffinose synthase and one encodes stachyose synthase. The expression profiles of candidate genes for galactinol synthase and raffinose synthase suggest that these genes are expressed in seeds, leaves and roots stressed. Stachyose synthase transcripts showed to be more abundant in leaves of stressed plants than in seeds. The genome of soybean also showed seven loci which potentially encode for seed inhibition proteins or alkaline α-galactosidases expressed mainly in seeds. To reduce the ROs content in soybean seeds were constructed expression cassettes for silencing of the gene encoding stachyose synthase, via RNA interference. The fragments isolated contain sequences corresponding to two distinct regions of stachyose synthase gene that show high and low identity with the raffinose synthase gene from soybean. The sense and antisense fragments of each gene region were cloned into the vectors pKANNIBAL and pBKN for simultaneous synthesis of inverted repeat regions, resulting in RNA hairpin spaced by an intron. Four constructions were obtained in which two are directed by the promoter 35SCaMV and two are directed by the promoter of the subunit of storage protein β-conglycinin. These constructs were transferred for binary vector pCAMBIA 3301 for further soybean genetic transformation.