RESUMO
The nitrated compounds 2,4-dinitrotoluene (2,4-DNT), 2,4,6-trinitrotoluene (TNT), and pentaerythritol tetranitrate (PETN) are toxic xenobiotics widely used in various industries. They often coexist as environmental contaminants. The aims of this study were to evaluate the transformation of 100 mg L-1 of TNT, 2,4-DNT, and PETN by Raoultella planticola M30b and Rhizobium radiobacter M109c and identify enzymes that may participate in the transformation. These strains were selected from 34 TNT transforming bacteria. Cupriavidus metallidurans DNT was used as a reference strain for comparison purposes. Strains DNT, M30b and M109c transformed 2,4-DNT (100%), TNT (100, 94.7 and 63.6%, respectively), and PETN (72.7, 69.3 and 90.7%, respectively). However, the presence of TNT negatively affects 2,4-DNT and PETN transformation (inhibition > 40%) in strains DNT and M109c and fully inhibited (100% inhibition) 2,4-DNT transformation in R. planticola M30b.Genomes of R. planticola M30b and R. radiobacter M109c were sequenced to identify genes related with 2,4-DNT, TNT or PETN transformation. None of the tested strains presented DNT oxygenase, which has been previously reported in the transformation of 2,4-DNT. Thus, unidentified novel enzymes in these strains are involved in 2,4-DNT transformation. Genes encoding enzymes homologous to the previously reported TNT and PETN-transforming enzymes were identified in both genomes. R. planticola M30b have homologous genes of PETN reductase and xenobiotic reductase B, while R. radiobacter M109c have homologous genes to GTN reductase and PnrA nitroreductase. The ability of these strains to transform explosive mixtures has a potentially biotechnological application in the bioremediation of contaminated environments.
Assuntos
Agrobacterium tumefaciens/fisiologia , Dinitrobenzenos/metabolismo , Enterobacteriaceae/fisiologia , Oxirredutases/genética , Tetranitrato de Pentaeritritol/metabolismo , Trinitrotolueno/metabolismo , Biodegradação Ambiental , Genoma Bacteriano , Filogenia , Sequenciamento Completo do GenomaRESUMO
Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.
Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.
Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.
Assuntos
Agrobacterium tumefaciens/fisiologia , Orchidaceae/crescimento & desenvolvimento , Técnicas de Embriogênese Somática de PlantasRESUMO
Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380456, 310372, 200240, 130156, and 100130 well-developed shoots in only 240270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.
Assuntos
Musa/crescimento & desenvolvimento , Musa/genética , Regeneração , Transformação Genética , Imuno-Histoquímica , Southern Blotting , Reação em Cadeia da Polimerase , Plantas Geneticamente Modificadas , Agrobacterium tumefaciens/fisiologia , Musa/microbiologia , Organogênese Vegetal , GlucuronidaseRESUMO
Phyllosticta citricarpa is the epidemiological agent of Citrus Black Spot (CBS) disease, which is responsible for large economic losses worldwide. CBS is characterized by the presence of spores (pycnidiospores) in dark lesions of fruit, which are also responsible for short distance dispersal of the disease. The identification of genes involved in asexual reproduction of P. citricarpa can be an alternative for directional disease control. We analyzed a library of mutants obtained through Agrobacterium tumefaciens transformation system, looking for alterations in growth and reproductive structure formation. Two mutant strains were found to have lost the ability to form pycnidia. The flanking T-DNA insertion regions were identified on P. citricarpa genome by using blast analysis and further gene prediction. The predicted genes containing the T-DNA insertions were identified as Spindle Poison Sensitivity Scp3, Ion Transport protein, and Cullin Binding proteins. The Ion Transport and Cullin Binding proteins are known to be correlated with sexual and asexual reproduction in fungi; however, the exact mechanism by which these proteins act on spore formation in P. citricarpa needs to be better characterized. The Scp3 proteins are suggested here for the first time as being associated with asexual reproduction in fungus. This protein is associated with microtubule formation, and as microtubules play an essential role as spindle machinery for chromosome segregation and cytokinesis, insertions in this gene can lead to abnormal formations, such as that observed here in P. citricarpa. We suggest these genes as new targets for fungicide development and CBS disease control, by iRNA.
Assuntos
Agrobacterium tumefaciens/fisiologia , Ascomicetos/fisiologia , Regulação Fúngica da Expressão Gênica , Interações Microbianas , Mutação , Reprodução Assexuada/genética , Transformação Genética , Citrus/microbiologia , DNA Bacteriano , Ordem dos Genes , Genes Reporter , Mutagênese Insercional , Doenças das Plantas/microbiologiaRESUMO
Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 µM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.
Assuntos
Agrobacterium tumefaciens/genética , Ascomicetos/genética , Citrus/microbiologia , Técnicas de Transferência de Genes , Mutagênese Insercional/métodos , Doenças das Plantas/microbiologia , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismoRESUMO
Numerous chromosomal virulence genes (chv) have been shown to play an important role in the ability of Agrobacterium tumefaciens to transform plants. The A. tumefaciens chvH gene encodes a protein similar in sequence to the Escherichia coli elongation factor P (EF-P). In A. tumefaciens this factor is required for tumor formation and for full expression of the vir genes, exerting its activity at a post-transcriptional level. Cross-complementation assays suggest that the chvH gene and the efp gene of E. coli are functionally homologous. We have cloned and characterized the efp homolog gene in Brucella abortus which has 45% identity to A. tumefaciens chvH and 35% identity to E. coli efp. The gene complemented detergent sensitivity and virulence in the chvH A. tumefaciens mutant, suggesting that both genes are functionally homologous; the growth rate in complex medium also increased to wild type levels. An efp mutant in B. abortus 2308 grew slower in complex media and showed more sensitivity to detergents. Infection assays in J774 macrophage like cells revealed no significant differences between the wild type and the efp mutant strains. The recovery of this mutant from spleens of inoculated mice was equivalent compared to that of the parental strain suggesting that B. abortus efp is not required for virulence in an animal model. However the efp mutant revealed significant differences at 1 h-4 h post-infection in HeLa infection assays compared to the wild type strain, indicating that cellular internalization was affected in non-professional phagocytes. Double immunofluorescence assays for detecting extracellular and intracellular bacteria, demonstrated that the mutant attaches to HeLa cells as the wild type but is deficient in the internalization process, thus indicating that efp is involved in the penetration of Brucella in non-professional phagocytes.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Brucelose/microbiologia , Fatores de Alongamento de Peptídeos/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Brucella abortus/química , Brucella abortus/genética , Brucella abortus/patogenicidade , Feminino , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Kalanchoe/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Alinhamento de Sequência , VirulênciaRESUMO
Most boreal and temperate forest trees form a mutualistic symbiosis with soil borne fungi called ectomycorrhiza (ECM). In this association both partners benefit due to nutrient exchange at the symbiotic interface. Laccaria bicolor is the first mycorrhizal fungus with its genome sequenced thus making possible for the first time to analyze genome scale gene expression profiles of a mutualistic fungus. However, in order to be able to take full advantage of the genome sequence, reverse genetic tools are needed. Among them a high throughput transformation system is crucial. Herein we present a detailed protocol for genetic transformation of L. bicolor by means of Agrobacterium tumefaciens with emphasis on critical steps affecting the success and efficiency of the approach.
Assuntos
Agrobacterium tumefaciens/fisiologia , Laccaria/fisiologia , Agrobacterium tumefaciens/metabolismo , Laccaria/metabolismoRESUMO
Winged-helix transcriptional factors play important roles in the control of gene expression in many organisms. In the plant pathogens Xylella fastidiosa and Agrobacterium tumefaciens, the winged-helix protein BigR, a member of the ArsR/SmtB family of metal sensors, regulates transcription of the bigR operon involved in bacterial biofilm growth. Previous studies showed that BigR represses transcription of its own operon through the occupation of the RNA polymerase-binding site; however, the signals that modulate its activity and the biological function of its operon are still poorly understood. Here we show that although BigR is a homodimer similar to metal sensors, it functions as a novel redox switch that derepresses transcription upon oxidation. Crystal structures of reduced and oxidized BigR reveal that formation of a disulfide bridge involving two critical cysteines induces conformational changes in the dimer that remarkably alter the topography of the winged-helix DNA-binding interface, precluding DNA binding. This structural mechanism of DNA association-dissociation is novel among winged-helix factors. Moreover, we demonstrate that the bigR operon is required for hydrogen sulfide detoxification through the action of a sulfur dioxygenase (Blh) and sulfite exporter. As hydrogen sulfide strongly inhibits cytochrome c oxidase, it must be eliminated to allow aerobic growth under low oxygen tension, an environmental condition found in bacterial biofilms, xylem vessels, and root tissues. Accordingly, we show that the bigR operon is critical to sustain bacterial growth under hypoxia. These results suggest that BigR integrates the transcriptional regulation of a sulfur oxidation pathway to an oxidative signal through a thiol-based redox switch.
Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Sulfeto de Hidrogênio/metabolismo , Oxigênio/metabolismo , Plantas/microbiologia , Fatores de Transcrição/metabolismo , Xylella/metabolismo , Agrobacterium tumefaciens/crescimento & desenvolvimento , Agrobacterium tumefaciens/fisiologia , Animais , Proteínas de Bactérias/química , Biofilmes/crescimento & desenvolvimento , Sequência Conservada , Cisteína , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dioxigenases/metabolismo , Dissulfetos/química , Sulfeto de Hidrogênio/toxicidade , Camundongos , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Óperon/genética , Oxirredução , Estrutura Secundária de Proteína , Fatores de Transcrição/química , Transcrição Gênica , Xylella/crescimento & desenvolvimento , Xylella/fisiologiaRESUMO
The gene uidA, codes for beta-glucuronidase, which is one of the reporters more frequently utilized in transgenic plants. However, this can only be use if the selected organism does not present endogenous GUS-like activity. In tissues of C. chinense we found a GUS-like activity showing different levels of intensity. Histochemical screening showed that endogenous GUS-like activity decreased, or reduced significantly, in almost all tissues with exception of stament, when phosphate buffer was adjusted to pH 8. Subsequently, C. chinense zygotic embryo explants were transient transformed with Agrobacterium tumefaciens LBA4404 (pCAMBIA2301) and plantlets regenerated were histochemically stained in phosphate buffer pH 8. Observations of incubated tissues of C. chinense regenerants showed blue staining, suggesting expression of uidA. Incubated tissues of non-transformed regenerants did not show blue staining in phosphate buffer pH 8. The results show that for transformation experiments of C. chinense with uidA gene, pH 8 is recommended for histochemical staining.
Assuntos
Capsicum/fisiologia , Capsicum/genética , Glucuronidase , Agrobacterium tumefaciens/fisiologia , Regulação da Expressão Gênica de Plantas , Genes Reporter , Histocitoquímica , Concentração de Íons de Hidrogênio , Plantas Geneticamente Modificadas/genética , Regeneração , Transformação GenéticaRESUMO
In the present study, genotypic variation of Agrobacterium-mediated transformation of Korean Italian ryegrass has been evaluated. Mature seed-derived calli of seven cultivars were inoculated and co-cultured with Agrobacterium tumefaciens carrying the binary vector pCAMBIA1301, which contains a reporter gene (gus) and a plant selectable marker gene conferring resistance to hygromycin (hpt) in the T-DNA region. The effects of several factors such as callus type and callus age on transformation effectiveness and the expression of the GUS gene were investigated. The highest transformation effectiveness (6.7 percent) was obtained with the Hwasan 101 cultivar when 9-week-old calli (type-I) were inoculated with Agrobacterium. The overall transformation rates of the examined cultivars ranged from 0.4 percent to 6.7 percent. GUS histochemical assays, PCR, and southern analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of Italian ryegrass. Thus, evaluation of transformation effectiveness and selection of a suitable cultivar of Italian ryegrass may improve molecular breeding of this species.
Assuntos
Variação Genética , Lolium/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium tumefaciens/fisiologia , Transformação Genética , Genes Reporter , Marcadores Genéticos , Genótipo , Histocitoquímica , Seleção Genética , Sementes/genéticaRESUMO
AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.
Assuntos
Agrobacterium tumefaciens/genética , Metarhizium/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Aderência Bacteriana , DNA Bacteriano/genética , Metarhizium/fisiologiaRESUMO
An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20 degrees C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.
Assuntos
Agrobacterium tumefaciens/fisiologia , Hevea/embriologia , Sementes/crescimento & desenvolvimento , Transformação Bacteriana , Sequência de Bases , Southern Blotting , Primers do DNA , Glucuronidase/metabolismo , Hevea/crescimento & desenvolvimento , Hevea/microbiologia , Hevea/fisiologia , Plantas Geneticamente ModificadasRESUMO
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Basidiomycota/genética , DNA Bacteriano/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Basidiomycota/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Farmacorresistência Fúngica/genética , Genes Sintéticos , Fleomicinas/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Schizophyllum/genética , Seleção GenéticaRESUMO
The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.
El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Basidiomycota/genética , DNA Bacteriano/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Basidiomycota/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Farmacorresistência Fúngica/genética , Genes Sintéticos , Reação em Cadeia da Polimerase , Fleomicinas/farmacologia , Regiões Promotoras Genéticas/genética , Seleção Genética , Schizophyllum/genéticaRESUMO
A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.