Caracterização dos genes que codificam os inibidores de protease Bowman-Birk (BBI) e transformação de nós cotiledonares para o silenciamento gênico de BBI em sementes de soja

• Ano de defesa: 2010
• Autor(a) principal: Barros, Beatriz de Almeida
• Orientador(a): Barros, Everaldo Gonçalves de
• Banca de defesa: Fietto, Luciano Gomes , Santos, Marcelo de Oliveira , Guimarães, Valéria Monteze
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Genética e Melhoramento
• Departamento: Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
• País: BR
• Palavras-chave em Português: Inibidores Bowman-Birk; Transformação de soja; qRT-PCR; Promotor semente-específico; RNAi
• Palavras-chave em Inglês: Bowman-Birk inhibitors; Soybean transformation; qRT-PCR; Seed-specific promoter; RNAi
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS
• Link de acesso: http://locus.ufv.br/handle/123456789/1318

In addition to storage proteins the seeds of soybean (Glycine max L. Merrill) accumulate protease inhibitors such as Bowman-Birk inhibitors (BBI), which are regarded as antinutritional factors. The reduction of these inhibitors in soybean seeds could lead to increased bioavailability of amino acids obtained from soy foods. The genes encoding BBI in soybean constitute a multigene family with at least five members: BBI-A, B, BBI, BBI-CII, DII-BBI and BBI-EI. The aim of this study was to characterize the genes encoding Bowman-Birk (BBI) protease inhibitors and to transform cotyledonary nodes to silence the BBI gene in soybean seeds. Computer analysis showed that the soybean genome has 11 loci which potentially encode BBI proteins. Of these, six encode type A, CII and DII inhibitors which are only expressed in seeds. The expression profiles of these genes during seed development are similar. Transcripts of BBI-DII are the most abundant, followed by BBI-A and BBI-CII. The expression of the BBI-D gene was compared with that encoding subunit α of the storage protein β-conglycinin (α-βC) during seed development using quantitative RT-PCR. Expression of BBI-D was significantly higher than that of α-βC, indicating that the promoter of BBI-D might be an excellent candidate for direct expression of transgenes in soybean seeds. To reduce the levels of BBI in soybean seeds, cotyledonary nodes were transformed using Agrobacterium tumefaciens (KYRT1 strain) carrying the vector pBBIAi. In total 1800 explants were transformed, 16 shoots elongated, and of these 11 developed roots. Seven seedlings were established in the greenhouse. Small samples of leaf from these seedlings were collected for DNA extraction and three seedlings were shown to be transformed using PCR. The low transformation efficiency and high frequency of false positive plants are in agreement with information from the literature. It can be concluded that the system adopted for transformation is adequate, but in future works modifications related mainly with the system used for selection and shoot regeneration should be considered.