200 resultados para Rhizobium


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Tobacco plants were transformed with a chimeric transgene comprising sequences encoding β-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense ∼22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

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An important role of RNA interference (RNAi)-like pathways in plants is defense against viral infection. Viruses can overcome this defense by expressing proteins that suppress the pathway. A new study of Agrobacterium tumefaciens infection reveals that this plant pathogen, although a bacterium, also induces and then suppresses the host RNAi response. © 2006 Nature Publishing Group.

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The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms - for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

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Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

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We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

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Survey of rhizobium inoculation methods used in chickpeas of the northern grains region with aim to adopt technologies for future microbial inoculants.

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Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

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Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium, is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.

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El presente trabajo consistió en un estudio comparativo de rendimientos en el cultivo de frijol común (Phaseo/us vulgaris, L), estableciendo dos experimentos en la Estación Experimental "La Compañía", Carazo. El primer experimento se estableció en el período de postrera (octubre 96-enero 97), con el propósito de determinar el efecto de diferentes sistemas de labranza y la inoculación de la bacteria Rhizobium tropici UMR 1899 sobre el rendimiento del cultivo del frijol, así también sobre las concentraciones y extracciones de nitrógeno, fósforo y potasio en tos diferentes tejido de la planta (grano, follaje y raíz). La variedad evaluada fue OOR-364. El diseño utilizado fue un bloque completo al azar (BCA), con seis tratamientos y cuatro repeticiones. Los datos se analizaron por medio de ANDEVA y prueba de rangos múltiples de Tukey al 5 %. Se realizó una fertilización fosfórica y nitrogenada, esta última aplicada en dos momentos, a la siembra 45 kglha y 15 dds 45 kglha. Las variables evaluadas fueron peso seco de las malezas, peso seco del follaje, número de nódulos por planta, peso de nódulo por planta, peso seco de raíz y rendimiento del grano. El mayor rendimiento de grano se obtuvo en labranza cero más rastrojo, en el follaje las mayores concentraciones de nitrógeno y potasio fueron en labranza cero más rastrojo y de fósforo en labranza convencional, las mayores extracciones de estos tres elementos se dieron en labranza convencional. Las mayores concentraciones de fósforo y potasio en la raíz de frijol se encontraron en labranza cero más rastrojo más subsoleo y de nitrógeno en labranza mínima; en el mismo tejido las mayores extracciones de nitrógeno se presentaron en labranza convencional más rastrojo más subsoleo, de fósforo en labranza cero más rastrojo más subsoleo y de potasio en labranza mínima más rastrojo. Respecto al inoculante no se encontraron diferencias significativas en las concentraciones de nitrógeno, fósforo y potasio en la raíz, pero sí hubo diferencias significativas en las extracciones de estos tres elementos en el mismo tejido. No hubo efecto significativo del inoculante en el número de nódulo por planta, pero sí hubo efecto significativo en el peso del nódulo,. El segundo experimento se estableció en el periodo de postrera (octubre 2000- enero 2001). El propósito del experimento fue determinar el efecto de los sistemas de labranza y rastrojo sobre el crecimiento y rendimiento del cultivo después de tres años de barbecho. Se utilizó la misma variedad, el ensayo se estableció en bloque completamente al azar (BCA), con seis tratamientos y cuatro repeticiones. La fertilización consistió en 2.5 qqlmz de la fórmula 18-46-00 al momento de la siembra. Las variables evaluadas fueron altura de la planta, número de ramas por planta, área foliar, altura de inserción a la primera vaina, número de vainas por planta, número de granos por vaina, peso de cien granos y rendimiento. El mayor rendimiento de grano se presentó en labranza convencional más rastrojo, luego en labranza cero. La mayor altura de planta se presentó en labranza mínima más rastrojo, en el área foliar se notaron diferencias significativas a los 49 y 56 dds y altamente significativas a los 42 dds. En cuanto a la altura de inserción a la primera vaina no hubo diferencias significativas. Los sistemas de labranza mostraron efecto significativo en el número de vainas por planta, número de granos por vaina y peso de cien granos.

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Las dos grandes fuentes de alimentación animal son: energética que comúnmente esta dada por las gramíneas y proteica, la cual puede ser suplementada por diferentes vías, pero las mas simple y que la naturaleza la provee es a través de las leguminosas, producto de la asociación con bacterias del género Rhizobium, que les permite fijar nitrógeno atmosférico. Los estudio hechos de leguminosas al momento han sido muy pocos, y cuando se han hecho se ha centrado únicamente en zonas del caribe con alta precipitación y suelos ácidos. Por lo que con el objeto de conocer el comportamiento de adaptación agronómico y productivo de 9 especies de leguminosas consideradas como forrajeras en el municipio de Muy Muy, se realizó el presente estudio en la finca “La Cruz” propiedad del señor Santiago Espino, ubicada en el km 153 de la carretera Muy Muy-Matiguás, en la comunidad de “Aguas Calientes”, zona baja del municipio de Muy Muy, Matagalpa, localizado en las coordenadas geográficas 12º 45 ́48” latitud Norte y 85º 37 ́36”longitud Oeste, a una altitud de unos 286msnmy con una temperatura promedio anual de 25.0°C. se estableció el experimento de campo el 4 de Junio del 2007. Utilizándose un Diseño de Bloques Completos al Azar (BCA), con nueve tratamientos (especies evaluadas: Centrosema plumieri cv DICTA, Clitoria ternatea cv CEVAS, Canavalia brasiliensis CIAT 1700, Clitoria ternatea CV DICTA, Canavalia ensiformis, Lablab purpureus, Vigna unguiculata CIAT9611, Vigna unguiculata CIAT390-2 y Stylosanthes guianensis CIAT2243). Se midieron 9 variables: sobrevivencia, altura, vigor, cobertura, cobertura de maleza, suelo descubierto, incidencia de plagas, incidencia de enfermedades y producción de biomasa seca, además se realizó análisis de la composición química a ocho de las especies en estudio. S.guianensis 2243 presentó el mejor comportamiento agronómico, superando al resto de especies en casi todas las variables en estudio, salvo el caso de altura donde fue superada por C. brasiliensis 1700 con 95.5 cm y en suelo descubierto por C. plumieri DICTA con 4.38%. En sobrevivencia Stylo alcanzó un 100%, con cobertura de 76.25%, menor incidencia de plagas y enfermedades (0%) y ponderación en vigor de 4.9. Además S.guianensis 2243 presentó el mejor comportamiento en producción de biomasa seca con 3717.02 kg ha-1, seguida de C. plumieriDICTA con 1486.38 kg ha-1. La especie de menor comportamiento adaptativo fue V. unguiculata 9611, y en producción de biomasa L. purpureus con 206.82 kg ha-1. C. ensiformis presento mejor contenido proteico con 26.20% y C. ternateaCEVAS en la porción digerible con 85.71%, la de menor calidad fue S.guianensis 2243 con 13.26% de proteína y 54.59% de porción digerible. Aun con los resultados de calidad se considera que S. guianensis2243 es una especie promisoria para estas condiciones por su a daptabilidad y producción de biomasa, y calidad y a las especies C. plumieriDICTA y C. ternateaCEVAS por su calidad nutritiva, adaptación y producciónde biomasa.

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Cadmium (Cd) is a toxic, biologically non-essential and highly mobile metal that has become an increasingly important environmental hazard to both wildlife and humans. In contrast to conventional remediation technologies, phytoremediation based on legume rhizobia symbiosis has emerged as an inexpensive decontamination alternative which also revitalize contaminated soils due to the role of legumes in nitrogen cycling. In recent years, there is a growing interest in understanding symbiotic legume rhizobia relationship and its interactions with Cd. The aim of the present review is to provide a comprehensive picture of the main effects of Cd in N-2-fixing leguminous plants and the benefits of exploiting this symbiosis together with plant growth promoting rhizobacteria to boost an efficient reclamation of Cd-contaminated soils.

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植物与微生物之间的共生体(如菌根和根瘤)使宿主植物从生理、生态功能方面获得利益,其抗逆性得到提高,从而使植物的生存竞争能力得到提高。近二十年来,对于菌根或根瘤这样两位一体的共生体系的研究相当活跃,并取得显著的进展。但是对于“固氮植物、根瘤菌或放线菌、菌根真菌”这三者之间形成的共生体的研究还较少,而且基本上停留在对现象的描述上,缺少对三者之间关系的探讨及对共生机理的揭示。因此,研究木本固氮植物的联合共生体,对总结树木与微生物共生生态的基本规律、提示生物间的朴素协调关系、科学地利用共生资源、发挥共生优势以及促进生态物脆弱地带的生态恢复、提高土地生产力、丰富生态学理论都具有重要意义。通过对固氮树木沙棘和刺槐联合共生体人工构建的研究,得出如下结论:联合共生体的确认:通过显微观察和形态解剖特征观察,确认了在人工纯培养条件下,两种典型的固氮树种均能形成根瘤、菌根联合共生体。非豆科固氮树种沙棘与弗兰克氏放线菌共生形成根瘤的同时,还能与内生菌根菌共生形成内生菌根。豆科固氮树种刺槐在与根瘤菌共生形成根瘤的同时,还能与内生菌根真菌、外生菌根真菌共生形成内生菌根和外生菌根。菌根菌对固氮树种生长的促进作用:接种菌根真菌明显地促进了固氮树种的生长,但不同的菌种及组合促生效果不同。相比较而言,VAH较适合沙棘,而VAR则更适合刺槐,毛边华锈伞(H.mesophaseum)是比较适合剌槐的外生菌根真菌,这说明了共生菌种选择的必要性以及选择土著菌种的重要意义。内生菌根真菌和外生菌根真菌的混合接种对刺槐的生长具有增效作用,但不同菌种搭配的效果不同,尤其以VAR+毛边华锈伞(H.mesophaseum)接种组合效果最佳。固氮菌对固氮树种生长的促进作用:接种固氮菌能明显地提高结瘤固氮能力,从而促进宿主植物的生长。HR16是沙棘较理想的共生固氮菌株,接种HR16的沙棘植株的生理指标及生长指标均明显提高。接种根瘤菌336的刺槐小苗也明显地改善了其生长状况。联合共生体中菌根菌和固氮菌之间存在着相互依存,互相促进的关系:这是人工构建联合共生体的前提。沙棘联合共生体中,VA菌根菌对Frankia的结瘤固氮能力具有明显的促进作用,而同时Frankia对菌根的发育也具有促进作用。刺槐联合共生体中菌根菌和根瘤菌之间也表现出相同的规律,菌根真菌对根瘤菌的结瘤固氮能力具有明显的促进作用,尤其是内、外生菌根真菌混合接种,效果更佳。根瘤菌对菌根的发育也同时具有促进作用。菌根菌与根瘤菌之间的协同增效作用以及联合共生体人工构建的最佳组合:联合共生体中菌根菌和固氮菌之间的关系决定着二者对宿主植物的生长存在着协同增效作用。混合接种固氮菌和菌根菌,明显的提高了宿主植物生长的综合指标,但不同的菌种搭配表现出不同的作用。对沙棘而言,VAH+HR16(Frankia)是最佳的组合。对刺槐来说,根瘤菌(Rhizobium 336)+VAR+毛边华锈伞(H.mesophaseum 870191)是最佳的组合。无论是豆科固氮树木还是非豆科固氮树木,促进结瘤固氮能力的最佳组合、促进菌根发育的最佳组合以及促进植株生长的最佳组合是一致的。共生资源的调查及菌种培养条件:通过对刺槐林下菌根真菌资源的调查、分离和培养,进一步了共生资源,摸索出了一套较理想的适合外生菌根真菌的培养条件及培养基,为人工构建联合共生体提供了技术保障。综上所述,本文在木本固氮植物联合共生体人工构建的研究中得出具有重要意义的结论:从殂态解剖特征上确诊了木本固氮植物联合共生体人工构建的可能性;定量研究了菌根菌和固氮菌对宿主植物的促进作用以及两者之间的联合增效作用,选择出了最佳的联合共生体构建接种组合;对宿主植物、菌根菌、固氮菌之间的相互关系及机理进行了初步的探讨,为联合共生体人工构建应用于生产提供了理论基础和技术依据。

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Linhagens de B. subtilis têm sido bem estudadas como agentes de controle biológico, e também como produtoras de antibióticos. Uma linhagem de B. subtilis 0G isolada de solos supressivos a Fusarium solani foi avaliada quanto à utilização conjunta com Rhizobium phaseoli em feijoeiro, visando aumento de biomassa e nodulação de raízes. Em condições de casa de vegetação, a aplicação de B. subtilis, via sementes, promoveu um aumento na nodulação, em mais de 100%, em solo natural quando aplicado isoladamente ou em conjunto com R. phaseoli e em mais de 160% em solo esterilizado, quando comparado com o tratamento onde o Rhizobium foi aplicado isoladamente. Em solos esterilizados, a aplicação de B. subtilis, em conjunto com Rhizobium, promoveu um aumento significativo no peso da matéria seca das raízes (89%) e da parte aérea (83%). O tratamento de sementes com B. subtilis não afetou a emergência de plântulas em nenhum dos solos estudados. Pelos resultados obtidos pode-se concluir que a utilização de B. subtilis é bastante promissora para aumentar a nodulação de raízes e promover o crescimento de plantas de feijoeiro.