RESUMO
BACKGROUND: DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. METHODS AND RESULTS: In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6 days and the concentrations of 3 and 10 µM Azacytidine for 10 and 3 days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. CONCLUSIONS: Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.
Assuntos
Inativação Gênica , Nicotiana/genética , Azacitidina/farmacologia , Sequência de Bases , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , DNA de Plantas/genética , Dosagem de Genes , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Sulfitos , Chá/química , Nicotiana/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , TransgenesRESUMO
One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Óleos Industriais/análise , Poluentes do Solo/metabolismo , Bacillus subtilis/classificação , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Bacteriocinas/genética , Biodegradação Ambiental , Biologia Computacional , Mapeamento de Sequências Contíguas , Ontologia Genética , Lipopeptídeos/biossíntese , Lipopeptídeos/genética , Anotação de Sequência Molecular , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Filogenia , Solo/química , Microbiologia do Solo , Tensoativos/química , Tensoativos/metabolismo , Sequenciamento Completo do GenomaRESUMO
Hyaluronic acid (HA), a unique polysaccharide with excellent Physico-chemical properties, is broadly used in pharmaceutical, biomedical, and cosmetic fields. It is widely present in all vertebrates, certain bacterial strains, and even viruses while it is not found in plants, fungi, and insects. HA is naturally synthesized by a class of integral membrane proteins called Hyaluronic acid synthase (HAS). Thus far, industrial production of HA is carried out based on either extraction from animal sources or large-scale microbial fermentation. The major drawbacks to using these systems are contamination with pathogens and microbial toxins. Recently, the production of HA through recombinant systems has received considerable attention. Plants are eco-friendly ideal expression systems for biopharmaceuticals production. In this study, the optimized human hyaluronic acid synthase2 (hHAS2) sequence was transformed into Nicotiana tabacum using Agrobacterium rhizogenes. The highest rhHAS2 concentration of 65.72 ng/kg (wet weight) in transgenic tobacco hairy roots was measured by the human HAS2 ELISA kit. The HA production in the transgenic hairy roots was verified by scanning electron microscope (SEM) and quantified by the HA ELISA kit. The DPPH radical scavenging activity of HA with the highest concentration of 0.56 g/kg (wet weight) showed a maximum activity of 46%. Gel Permeation Chromatography (GPC) analyses revealed the high molecular weight HA (HMW-HA) with about > 0.8 MDa.
Assuntos
Produtos Biológicos/metabolismo , Hialuronan Sintases/metabolismo , Ácido Hialurônico/biossíntese , Nicotiana/genética , Nicotiana/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Agrobacterium/genética , Sequência de Bases , Produtos Biológicos/química , Cromatografia em Gel/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Hialuronan Sintases/genética , Ácido Hialurônico/química , Microscopia Eletrônica de Varredura/métodos , Peso Molecular , Plantas Geneticamente Modificadas , Transformação GenéticaRESUMO
Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 T (= LMG 29756 T = CFBP 8484 T) sp. nov., Pseudomonas inefficax JV551A3 T (= DSM108619 T = CFBP 8493 T) sp. nov., Pseudomonas persica RUB6 T (= LMG 29757 T = CFBP 8486 T) sp. nov. and Pseudomonas shirazica VM14 T (= LMG 29953 T = CFBP 8487 T) sp. nov.
Assuntos
Genoma Bacteriano/genética , Filogenia , Pseudomonas putida/classificação , DNA Bacteriano/genética , Genes Bacterianos/genética , Genes Essenciais/genética , Hibridização de Ácido Nucleico , Fenótipo , Pseudomonas putida/genética , Análise de Sequência de DNA , Microbiologia do Solo , Especificidade da EspécieRESUMO
Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of concatenated 16S rRNA, gyrB and rpoD sequences for 33 Pseudomonas isolates showed that 15 belonged to four clusters within the 'P. fluorescens' group, i.e. one corresponding to P. thivervalensis, two others including P. moraviensis or P. baetica, and the last one without closely-related established species. The 18 other isolates belonged to five clusters within the 'P. putida' group, one including P. mosselii and P. entomophila, another including strains currently described as P. putida, and three without closely-related species described. Ten isolates were selected based on in vitro inhibition of R. solani. Cellulase activity was identified in three pseudomonads, chitinase activity in two pseudomonads, extracellular protease activity in nine pseudomonads and hydrogen cyanide production in two pseudomonads. Genes coding for production of phenazine, pyoluteorin, pyrrolnitrin and 2,4-diacetylphloroglucinol were not found, whereas the 1-aminocyclopropane-1-carboxylate deamination gene acdS was present in three pseudomonads. The antagonistic acdS+ strain VKh13 from the 'P. putida' group effectively protected soil-grown bean from R. solani AG 4-HGI. Results show that pseudomonads from uncharacterized taxa were readily obtained from Iranian soils and displayed biocontrol potential against R. solani.
Assuntos
Antibiose , Fabaceae/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Pseudomonas/isolamento & purificação , Pseudomonas/fisiologia , Rhizoctonia/fisiologia , Irã (Geográfico) , Raízes de Plantas/microbiologia , Pseudomonas/classificação , Pseudomonas/genética , Rizosfera , Microbiologia do SoloRESUMO
In contrast to conventional antibiotics, which microorganisms can readily evade, it is nearly impossible for a microbial strain that is sensitive to antimicrobial proteins to convert to a resistant strain. Therefore, antimicrobial proteins and peptides that are promising alternative candidates for the control of bacterial infections are under investigation. The MAP30 protein of Momordica charantia is a valuable type I ribosome-inactivating protein (RIP) with anti-HIV and anti-tumor activities. Whereas the antimicrobial activity of some type I RIPs has been confirmed, less attention has been paid to the antimicrobial activity of MAP30 produced in a stable, easily handled, and extremely cost-effective protein-expression system. rMAP30-KDEL was expressed in Nicotiana tobacum hairy roots, and its effect on different microorganisms was investigated. Analysis of the extracted total proteins of transgenic hairy roots showed that rMAP30-KDEL was expressed effectively and that this protein exhibited significant antibacterial activity in a dose-dependent manner. rMAP30-KDEL also possessed thermal and pH stability. Bioinformatic analysis of MAP30 and other RIPs regarding their conserved motifs, amino-acid contents, charge, aliphatic index, GRAVY value, and secondary structures demonstrated that these factors accounted for their thermophilicity. Therefore, RIPs such as MAP30 and its derived peptides might have promising applications as food preservatives, and their analysis might provide useful insights into designing clinically applicable antibiotic agents.
Assuntos
Fármacos Anti-HIV , Antineoplásicos Fitogênicos , Expressão Gênica , Nicotiana/genética , Raízes de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Inativadoras de Ribossomos Tipo 2/genética , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sequência Conservada , Escherichia coli/efeitos dos fármacos , Ordem dos Genes , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Plantas Geneticamente Modificadas , Plasmídeos/genética , Matrizes de Pontuação de Posição Específica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 2/química , Proteínas Inativadoras de Ribossomos Tipo 2/isolamento & purificação , Proteínas Inativadoras de Ribossomos Tipo 2/farmacologia , TemperaturaRESUMO
Nitric oxide (NO) is one of the main signal molecules, which is involved in plant growth and development and can change regular physiological activity in biotic and abiotic stresses. In this study, the role of NO in induced resistance with Pseudomonas fluorescent (CHA0) and basal resistance against Rhizoctonia solani in bean plant was investigated. Our results revealed that P. fluorescent and R. solani can increase NO production at 6h post inoculation (hpi). Also, using the NO donor S-nitroso-N-acetyl D-penicillamine (SNAP) led to increase NO and bean plant resistance against R. solani. Utilizing the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide (cPTIO), not only decreased basal resistance but also reduced induced resistance. In continue, the activity of antioxidant enzymes was studied in the former treatments. SNAP, CHA0 and R. solani increased the activity of peroxidase (POX), catalase (CAT) and ascorbate peroxidase (APX) at 6, 12 and 24h post inoculation (hpi). In contrast, using cPTIO and R. solani simultaneously (cPTIO+R) showed reduction in activity of POX and APX at 6 hpi. The cPTIO+R treatment increased POX, APX and CAT activity at 12 and 24 hpi. Hydrogen peroxide (H2O2) monitoring in the leaf discs clarified that SNAP can increase H2O2 production like CHA0 and R. solani. On the other hand, SNAP increased the resistance level of leaf discs against R. solani. Treating the leaf discs with cPTIO led to decrease resistance against the pathogen. These leaf discs showed reduction in H2O2 production at 6 hpi and suddenly enhanced H2O2 generation was observed at 24hpi. This study showed that CHA0 can increase NO level in bean plants. NO induced H2O2 generation and regulated redox state of the host plant. This interaction resulted in significant defense against the pathogen.
Assuntos
Resistência à Doença , Óxido Nítrico/metabolismo , Phaseolus/imunologia , Doenças das Plantas/imunologia , Pseudomonas fluorescens/fisiologia , Rhizoctonia/fisiologia , Antioxidantes/metabolismo , Ascorbato Peroxidases/metabolismo , Benzoatos/farmacologia , Agentes de Controle Biológico , Catalase/metabolismo , Peróxido de Hidrogênio/metabolismo , Imidazóis/farmacologia , Doadores de Óxido Nítrico/farmacologia , Peroxidases/metabolismo , Phaseolus/citologia , Phaseolus/fisiologia , Doenças das Plantas/microbiologia , Folhas de Planta/citologia , Folhas de Planta/imunologia , Folhas de Planta/fisiologia , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.