RESUMO
Erythropoietin (EPO) is a hormone belonging to a group of hematopoietic growth factors that control the proliferation and differentiation of bone marrow cells. It induces the production of erythrocytes, thereby increasing the amount of circulating hemoglobin and oxygen. Previous attempts to transgenically express human EPO in plants failed to succeed because the plants exhibited abnormal morphology and infertility. In the present work, we describe the generation of fertile transgenic tobacco plants able to express a synthetic version of human EPO. A 582-bp fragment of the human EPO gene was synthesized using a PCR-based method and ligated into pCR-Blunt. After sequencing, the human EPO fragment was transferred to pWUbi.tm1 and the expression cassette was then transferred to the binary vector pWBVec4a. After Agrobacterium-mediated transformation of Nicotiana tabacum SR1 plants, integration of the transgene into T(0) and T(1) plant genomes was confirmed by PCR. The human EPO gene was found to be expressed in tobacco leaves at the mRNA and protein levels. Self-crossing allowed us to obtain T(1) plants exhibiting Mendelian segregation of the transgene. None of the plants presented any kind of malformation or deformity.
Assuntos
Agrobacterium tumefaciens/genética , Eritropoetina , Nicotiana/genética , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes , Agrobacterium tumefaciens/metabolismo , Clonagem Molecular , Eritropoetina/biossíntese , Eritropoetina/genética , Humanos , Dados de Sequência Molecular , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismo , Nicotiana/microbiologia , Transformação Genética , Ubiquitina/química , Ubiquitina/genéticaRESUMO
Tuberculosis remains the major cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. The molecular mechanisms of infection and persistence have not been completely elucidated for this pathogen. Studies involving nucleoid-associated proteins (NAPs), which have been related to the control and influence of virulence genes in pathogenic bacteria, can help unveil the virulence process of M. tuberculosis. Here, we describe the initial characterization of an ORF for an M. tuberculosis putative NAP. The Rv3852 gene was cloned and expressed, and its product purified to homogeneity. A qualitative protein-DNA binding assay was carried out by gel-retardation and the protein affinity for specific DNA sequences was assessed quantitatively by surface plasmon resonance (SPR). A stoichiometry of 10 molecules of monomeric protein per molecule of DNA was determined. The monophasic apparent dissociation rate constant values increased to a saturable level as a function of protein concentration, yielding two limiting values for the molecular recognition of proU2 DNA. A protein-DNA binding mechanism is proposed. In addition, functional complementation studies with an Escherichia coli hns mutant reinforce the likelihood that the Rv3852 protein represents a novel NAP in M. tuberculosis.
