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1.
Enzyme Microb Technol ; 180: 110471, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38959818

RESUMO

Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5' extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.

2.
Biosci Biotechnol Biochem ; 85(8): 1789-1797, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34057172

RESUMO

Headspace solid-phase microextraction combined with gas chromatography/mass spectrometry is one of the strongest tools for comprehensive analysis of volatile compounds and has been used to analyze aromatic components of mango and investigate its varietal characteristics. In this study, profiling of aroma compounds in 17 mango cultivars, grown in the same green house to exclude the effect of environmental factors, was conducted and the patterns were subjected to principal component analysis (PCA) to identify the relationship between the aroma components and cultivars. Fifty-nine different volatile constituents were detected from the blends of these 17 mango cultivars. The cultivars were divided into 4 clusters using PCA based on the volatile components determined in the study. Aiko was found to mainly contain δ-3-carene and showed a composition more similar to its pollen parent, Irwin, than to its seed parent, Chiin Hwang No. 1.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Mangifera/química , Compostos Orgânicos Voláteis/análise , Análise de Componente Principal , Microextração em Fase Sólida/métodos
3.
J Pestic Sci ; 44(1): 33-40, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30820171

RESUMO

The purpose of this study is to isolate the beneficial microorganisms whose growth is promoted in the presence of charcoal materials. We successfully isolated strain IA, whose growth is promoted on an agar plate with charcoal materials, and identified it as a novel strain of the Bacillus sp. The growth of strain IA in the liquid medium was promoted by the addition of both activated charcoal (AC) and rice husk biochar (RHB). Moreover, the sporulation of strain IA in the RHB medium and the antifungal activity of the culture supernatant of the RHB medium were much higher than those with AC. HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A in the RHB medium was 8 times higher than that in the medium without RHB. This is the first paper to describe the positive effect of RHB on microbial metabolisms.

4.
J Biosci Bioeng ; 123(3): 376-381, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27789172

RESUMO

Mechanical damage is one of the unavoidable environmental stresses to plant growth and development. Plants induce a variety of reactions which defend against natural enemies and/or heal the wounded sites. Jasmonic acid (JA) and salicylic acid (SA), defense-related plant hormones, are well known to be involved in induction of defense reactions and play important roles as signal molecules. However, defense related metabolites are so numerous and diverse that roles of individual compounds are still to be elucidated. In this report, we carried out a comprehensive analysis of metabolic changes during wound response in citrus plants which are one of the most commercially important fruit tree families. Changes in amino acid, sugar, and organic acid profiles in leaves were surveyed after wounding, JA and SA treatments using gas chromatography-mass spectrometry (GC/MS) in seven citrus species, Citrus sinensis, Citrus limon, Citrus paradisi, Citrus unshiu, Citrus kinokuni, Citrus grandis, and Citrus hassaku. GC/MS data were applied to multivariate analyses including hierarchical cluster analysis (HCA), primary component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) to extract stress-related compounds. HCA showed the amino acid cluster including phenylalanine and tryptophan, suggesting that amino acids in this cluster are concertedly regulated during responses against treatments. OPLS-DA exhibited that tryptophan was accumulated after wounding and JA treatments in all species tested, while serine was down regulated. Our results suggest that tryptophan and serine are common biomarker candidates in citrus plants for wound stress.


Assuntos
Citrus/metabolismo , Metabolômica , Folhas de Planta/metabolismo , Estresse Fisiológico , Citrus/química , Ciclopentanos/análise , Ciclopentanos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Oxilipinas/análise , Oxilipinas/metabolismo , Folhas de Planta/química , Ácido Salicílico/análise , Ácido Salicílico/metabolismo , Cicatrização
5.
J Biosci Bioeng ; 122(2): 208-12, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26852089

RESUMO

Citrus plants are world widely cultivated as horticultural tree crops, and nowadays their pharmacological activities have been well studied. Since research of defense responses in citrus plants have been mainly focused on the post-harvested fruits because of their commercial importance, defense mechanisms during their developmental stages have not been well understood. In the present study, two wound-induced compounds were isolated from leaves of Citrus hassaku, and their structures were elucidated by high-resolution electron spray ionization mass spectra (HRESIMS) and nuclear magnetic resonance (NMR) analyses. One of these compounds was identified as a known flavanone, hesperetin. The other was characterized as a novel furofuran lignan, and was named 'biscitrusnin-A'. Their antimicrobial activities were also evaluated.


Assuntos
Antibacterianos/química , Antibacterianos/isolamento & purificação , Citrus/química , Doenças das Plantas , Folhas de Planta/química , Furanos/química , Furanos/isolamento & purificação , Hesperidina/química , Hesperidina/isolamento & purificação , Lignanas/química , Lignanas/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , Ferimentos e Lesões
6.
J Biosci Bioeng ; 121(2): 235-41, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26188419

RESUMO

Citrus plants are well known as a rich source of VOCs, and several have important roles in defense responses. However, how VOCs are regulated in response to environmental stress is not yet well understood. In this study, we investigated dynamic changes of VOCs present in leaves of seven Citrus species (Citrus sinensis, C. limon, C. paradisi, C. unshiu, C. kinokuni, C. grandis, and C. hassaku) in response to mechanical wounding, jasmonic acid (JA), and salicylic acid (SA) as determined by gas chromatography/mass spectrometric analysis followed by multivariate analysis (principal component analysis, PCA, and orthogonal partial least squares-discriminant analysis, OPLS-DA). PCA and OPLS-DA suggested that changes in VOC profiles against stress stimuli were much diverse among Citrus species. OPLS-DA showed that C6 volatiles, such as hexanal and trans-2-hexenal, were induced in response to JA and SA stimuli in C. sinensis and C. grandis, while the other VOCs were decreased under all tested stress conditions. α-Farnesene was induced in all species except C. hassaku after wounding or JA treatment. In addition, α-farnesene was also induced in response to SA stimuli in C. unshiu and C. kinokuni. Therefore these volatiles can be candidates of the common stress biomarkers in Citrus. Our results will give a new insight into defense mechanisms in Citrus species.


Assuntos
Citrus/metabolismo , Meio Ambiente , Folhas de Planta/metabolismo , Estresse Fisiológico , Compostos Orgânicos Voláteis/metabolismo , Aldeídos/metabolismo , Citrus/efeitos dos fármacos , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Folhas de Planta/efeitos dos fármacos , Ácido Salicílico/metabolismo , Sesquiterpenos/farmacologia
7.
DNA Res ; 18(1): 65-76, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21149391

RESUMO

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ~95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.


Assuntos
Genoma de Planta , Jatropha/genética , Proteínas de Plantas/genética , Análise de Sequência de DNA
8.
Opt Express ; 18(13): 13708-19, 2010 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-20588505

RESUMO

We propose and demonstrate the use of subharmonically synchronized laser pulses for low-noise lock-in detection in stimulated Raman scattering (SRS) microscopy. In the experiment, Yb-fiber laser pulses at a repetition rate of 38 MHz are successfully synchronized to Ti:sapphire laser pulses at a repetition rate of 76 MHz with a jitter of <8 fs by a two-photon detector and an intra-cavity electro-optic modulator. By using these pulses, high-frequency lock-in detection of SRS signal is accomplished without high-speed optical modulation. The noise level of the lock-in signal is found to be higher than the shot noise limit only by 1.6 dB. We also demonstrate high-contrast, 3D imaging of unlabeled living cells.


Assuntos
Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Artefatos , Núcleo Celular/ultraestrutura , Parede Celular/ultraestrutura , Desenho de Equipamento , Microesferas , Mitocôndrias/ultraestrutura , Modelos Teóricos , Poliestirenos , Nicotiana/citologia , Vacúolos/ultraestrutura
9.
J Biol Chem ; 285(21): 16267-74, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20335168

RESUMO

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 A resolution. The TDH tetramer forms a central pore with dimensions of 23 A in diameter and approximately 50 A in depth. Pi-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.


Assuntos
Proteínas de Bactérias/química , Proteínas Hemolisinas/química , Multimerização Proteica , Vibrio parahaemolyticus/química , Fatores de Virulência/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Cristalografia por Raios X , Proteínas Hemolisinas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Vibrio parahaemolyticus/metabolismo , Fatores de Virulência/metabolismo
10.
J Nat Prod ; 73(1): 71-4, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20000453

RESUMO

The P450 cytochrome monooxygenase gene, ltxB, was cloned and overexpressed in Escherichia coli as a 6xHis-tagged protein. The resulting recombinant LtxB was purified by Ni-NTA affinity chromatography and characterized biochemically. Purified LtxB demonstrated typical cytochrome P450 spectroscopic properties including substrate-induced transition from a low-spin (lambdamax=414 nm) to high-spin state (lambdamax=386 nm) upon incubation with N-methyl-L-valyl-L-tryptophanol. The catalytic activity of LtxB was verified by demonstrating the oxidation/cyclization of N-methyl-L-valyl-L-tryptophanol to (-)-indolactam V. LtxB shows a relaxed specificity for analogue substrates in which the valyl group is substituted for other aliphatic groups. The relaxed substrate specificity of LtxB, along with the relaxed specificity of the prenyltransferase, LtxC, allowed for the enzymatic production of a series of (-)-indolactam V and lyngbyatoxin analogues.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/metabolismo , Lactamas/metabolismo , Toxinas de Lyngbya/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Indóis/química , Indóis/isolamento & purificação , Lactamas/química , Lactamas/isolamento & purificação , Toxinas de Lyngbya/isolamento & purificação , Toxinas de Lyngbya/metabolismo , Estrutura Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato
11.
J Biomed Opt ; 14(4): 040506, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19725711

RESUMO

We demonstrate that stimulated parametric emission (SPE) microscopy enables label-free, 3-D visualization of internal hemoglobin distribution of live mouse and chicken erythrocytes with high sensitivity. Change in hemoglobin distribution in chicken erythrocytes before and after ethanol fixation is clearly visualized.


Assuntos
Eritrócitos/metabolismo , Hemoglobinas/análise , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Animais , Células Cultivadas , Galinhas , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem , Distribuição Tecidual
12.
Appl Microbiol Biotechnol ; 84(2): 375-82, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19562333

RESUMO

Recent reports on high-speed affinity screening systems for yeast cells using flow cytometry have not been adapted to screening yeast cells that display hydrolyzing enzymes, since the fluorescent molecules which are released from fluoresceinated substrate diffuse into solution after enzymatic reaction. In this research, yeast cells displaying beta-glycosidase were individually captured in micro-sized calcium alginate beads by using the newly developed reverse micelle method to prevent diffusion of hydrolyzed fluorescent substrates. By adopting flow sorting to these captured cells, active cells were successfully enriched about 82-fold from a mixed suspension with negative controls. This system should be a useful method for high-speed screening of yeast cells that display various hydrolyzing enzymes and has potential application to screening randomized libraries of enzyme-displayed yeast cells with higher activities.


Assuntos
Alginatos/química , Citometria de Fluxo/métodos , Leveduras/enzimologia , beta-Glucosidase/metabolismo , Separação Celular/métodos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Microesferas , Octanos/farmacologia , Leveduras/citologia
13.
Opt Express ; 17(5): 3651-8, 2009 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-19259205

RESUMO

We theoretically show that the shot-noise-limited sensitivity of stimulated Raman scattering (SRS) microscopy, which enables high-contrast vibrational imaging, is similar to that of coherent anti-Stokes Raman scattering microscopy. We experimentally confirm that the sensitivity of our SRS microscope is lower than the shot-noise limit only by <15 dB, which indicates that the high-sensitivity of SRS microscopy is readily available.


Assuntos
Microscopia/instrumentação , Análise Espectral Raman/instrumentação , Microscopia/estatística & dados numéricos , Fenômenos Ópticos , Sensibilidade e Especificidade , Nicotiana/citologia
14.
Plant Cell Rep ; 28(5): 759-68, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19214515

RESUMO

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T(2) generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously.


Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Cromossomos Artificiais Bacterianos , Cinamatos/farmacologia , DNA de Plantas/genética , Expressão Gênica , Técnicas de Transferência de Genes , Genes de Plantas , Vetores Genéticos , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Microesferas , Poaceae/genética , Transgenes
15.
Planta ; 229(4): 931-43, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19148672

RESUMO

The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N-acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor.


Assuntos
Avena/metabolismo , Terpenos/análise , ortoaminobenzoatos/análise , Apoptose , Avena/citologia , Avena/efeitos dos fármacos , Cromatografia Líquida , Cinética , Microscopia de Fluorescência , Oligossacarídeos/farmacologia , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos , Espectrometria de Massas por Ionização por Electrospray , Terpenos/química , Terpenos/metabolismo , Fatores de Tempo , ortoaminobenzoatos/química , ortoaminobenzoatos/metabolismo , Fitoalexinas
16.
J Biosci Bioeng ; 106(2): 194-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18804064

RESUMO

In this paper, we report a novel method for delivering genes into chloroplasts of tobacco cells using laser microablation. The plasmid pLD200-GFP was introduced into chloroplasts of Nicotiana tabacum cv. Xanthi guard cells and transient GFP expression was detected in the chloroplasts after 2-3 d of incubation. The technique uses an argon fluoride (ArF) excimer laser to perforate the cell surface in a 4 mum(2) area in the presence of plasmid coated gold microparticles. Pretreatment of guard cells to promote stomatal closure prior to laser ablation resulted in a significant increase in the survival rate of cells and a transient expression rate of 2-3% in trial number basis was archived. Our method has unique advantages such as avoiding laborious pretreatments that adversely affect cell viability and specific delivery of transgenes into a desired cell in complex leaf tissue. This technique is a potential tool for cell specific transient gene expression studies for elucidation of gene regulation and expression.


Assuntos
Cloroplastos/metabolismo , Expressão Gênica , Lasers de Excimer , Nicotiana/ultraestrutura , Ouro/química , Tamanho da Partícula
17.
J Biosci Bioeng ; 105(1): 77-80, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18295726

RESUMO

The efficiency of bioactive-beads-mediated plant transformation was improved using DNA-lipofectin complex as the entrapped genetic material instead of naked DNA used in the conventional method. In the improved method, beads aggregated and formed clusters around the protoplasts resulting in a 4-fold higher transformation efficiency than that by the conventional method.


Assuntos
DNA/química , Técnicas de Transferência de Genes , Fosfatidiletanolaminas/química , Plantas/genética , Transformação Genética , Caulimovirus/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Plantas/química , Regiões Promotoras Genéticas , Protoplastos/química , Protoplastos/metabolismo
18.
J Biol Phys ; 34(1-2): 51-62, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19669492

RESUMO

Laser-scanning fluorescence microscopy for efficient acquisition of time-gated and spectrally resolved fluorescence images was developed based on line illumination of the laser beam and detection of the fluorescence image through a slit. In this optical arrangement, the fluorescence image was obtained by scanning only one axis perpendicular to the excitation line, and the acquisition time was significantly reduced compared with conventional laser-scanning confocal microscopy. A multidimensional fluorescence dataset consisting of fluorescence intensities as a function of x-position, y-position, fluorescence wavelength, and delay time after photoexcitation was analyzed and decomposed based on the parallel factor analysis model. The performance of the line-scanning microscopy was examined by applying it to the analysis of one of the plant defense responses, accumulation of antimicrobial compounds of phytoalexin in oat (Avena sativa), induced by the elicitor treatment.

19.
Biotechnol Bioeng ; 93(2): 325-31, 2006 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-16193516

RESUMO

A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.


Assuntos
Técnicas de Transferência de Genes , Lasers , Microinjeções/métodos , Plantas/efeitos da radiação , Caulimovirus/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Microscopia Eletrônica de Varredura , Cebolas/química , Cebolas/genética , Cebolas/efeitos da radiação , Plantas/ultraestrutura , Regiões Promotoras Genéticas/genética
20.
J Biosci Bioeng ; 102(6): 575-8, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17270726
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