Identificação de estirpes de rizóbios por seqüenciamento parcial dos genes 16S rDNA e nifH
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Data
2008-10-29
Autores
Toledo, Bethânia Figueiredo Barbosa de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A crescente demanda de alimentos causada pelo aumento populacional, aliado a alto custo de fertilizantes industrializados e impacto ambiental causado por eles leva, mundialmente, à utilização em grande escala de inoculantes de bactérias fixadoras de nitrogênio. Os inoculantes utilizados no Brasil (coleção SEMIA- IPAGRO) ainda não estão suficientemente caracterizados geneticamente. O objetivo deste trabalho foi avaliar e confrontar as seqüências parciais dos genes 16S rDNA e nifH de 26 estirpes padrões já classificadas, com 70 estirpes de rizóbios recomendadas e autorizadas para a produção de inoculantes no Brasil. Para esta finalidade, a partir das amostras de DNA extraídos destas bactérias foram realizadas reações de PCR com oligonucleotídeos iniciadores relativos à região codificadora do gene 16S rDNA e do nifH, sendo então seqüenciadas com o objetivo de detectar diferenças nucleotídicas entre as diferentes bactérias estudadas. Para a comparação dos resultados do seqüenciamento com a consulta de similaridade de nucleotídeos no BLASTn, foram geradas árvores filogenéticas através de ferramentas de bioinformática. Foi observado que a classificação taxonômica das estirpes SEMIA recomendadas para inoculação de leguminosas previamente disponível na FEPAGRO, com base em propriedades morfológicas e especificidade hospedeira, não foi confirmada em todas as estirpes pelos sequenciamentos parciais dos genes estudados. Sugerimos revisão da classificação destas estirpes. Concluímos também que a consulta de similaridade ao BLASTn pelo seqüenciamento parcial dos genes 16S rDNA e nifH é, na maioria dos casos, consistente com a classificação proposta pela construção de árvores filogenéticas destas sequências. Estas ferramentas apresentaram-se muito confiáveis para obtenção de classificação em nível de gênero das estirpes estudadas.
The growing demand for food caused by population growth, combined with high cost of fertilizers and industrial environmental impacts caused by them leading, worldwide, the large scale use of inoculants different nitrogen-fixing bacteria. The inoculants used in Brazil (SEMIA-IPAGRO collection) are not yet sufficiently characterized genetically. The purpose of this study was to evaluate and compare the sequences of partial 16S rDNA and nifH of 26 strains already classified, with 70 strains of rhizobia recommended and authorized for the production of inoculants in Brazil. For this purpose, from DNA samples taken from these bacteria were performed with PCR reactions with primers on the coding region of the gene 16S rDNA and nifH, and sequencing with the aim of detecting nucleotide differences between different bacteria studied. To compare the results of the consultation of similarity of sequences of nucleotides in BLASTn, phylogenetic trees were generated through bioinformatics tools. It was observed that the taxonomic classification of strains SEMIA recommended for inoculation of legumes previously available in FEPAGRO, based on morphological properties and host specificity, it wasn’t confirmed in all strains by partial sequencing of the genes studied. We suggest reviewing the classification of these strains. We concluded that the similarity of the consultation BLASTn by partial sequencing of 16S rDNA and nifH is, in most cases, consistent with the classification proposed by the construction of phylogenetic trees of these sequences. These tools were very reliable for obtaining classified in the genus level of strains studied.
The growing demand for food caused by population growth, combined with high cost of fertilizers and industrial environmental impacts caused by them leading, worldwide, the large scale use of inoculants different nitrogen-fixing bacteria. The inoculants used in Brazil (SEMIA-IPAGRO collection) are not yet sufficiently characterized genetically. The purpose of this study was to evaluate and compare the sequences of partial 16S rDNA and nifH of 26 strains already classified, with 70 strains of rhizobia recommended and authorized for the production of inoculants in Brazil. For this purpose, from DNA samples taken from these bacteria were performed with PCR reactions with primers on the coding region of the gene 16S rDNA and nifH, and sequencing with the aim of detecting nucleotide differences between different bacteria studied. To compare the results of the consultation of similarity of sequences of nucleotides in BLASTn, phylogenetic trees were generated through bioinformatics tools. It was observed that the taxonomic classification of strains SEMIA recommended for inoculation of legumes previously available in FEPAGRO, based on morphological properties and host specificity, it wasn’t confirmed in all strains by partial sequencing of the genes studied. We suggest reviewing the classification of these strains. We concluded that the similarity of the consultation BLASTn by partial sequencing of 16S rDNA and nifH is, in most cases, consistent with the classification proposed by the construction of phylogenetic trees of these sequences. These tools were very reliable for obtaining classified in the genus level of strains studied.
Descrição
Palavras-chave
Rizobio, Filogenia, Bradyrhizobium spp, Ensifer spp, Phylogeny
Como citar
TOLEDO, Bethânia Figueiredo Barbosa de. Identificação de estirpes de rizóbios por seqüenciamento parcial dos genes 16S rDNA e nifH. 2008. xii, 127 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2008.