RESUMO
A reverse genetic strategy was used to isolate Arabidopsis plants containing "knockout" mutations in AKT1 and AKT2, two members of a K+ channel gene family. Comparative studies of growth and membrane properties in wild-type and mutant seedlings were performed to investigate the physiological functions of these two related channels. The growth rates of plants supplied with rate-limiting concentrations of K+ depended on the presence of AKT1 but not AKT2 channels. This result indicates that AKT1 but not AKT2 mediates growth-sustaining uptake of K+ into roots, consistent with the expression patterns of these two genes. K+ -induced membrane depolarizations were measured with microelectrodes to assess the contribution each channel makes to the K+ permeability of the plasma membrane in three different organs. In apical root cells, AKT1 but not AKT2 contributed to the K+ permeability of the plasma membrane. In cotyledons, AKT1 was also the principal contributor to the K+ permeability. However, in the mesophyll cells of leaves, AKT2 accounted for approximately 50% of the K+ permeability, whereas AKT1 unexpectedly accounted for the remainder. The approximately equal contributions of AKT1 and AKT2 in leaves detected by the in vivo functional assay employed here are not in agreement with previous RNA blots and promoter activity studies, which showed AKT2 expression to be much higher than AKT1 expression in leaves. This work demonstrates that comparative functional studies of specific mutants can quantify the relative contributions of particular members of a gene family, and that expression studies alone may not reliably map out distribution of gene functions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/fisiologia , Proteínas de Plantas/fisiologia , Canais de Potássio/fisiologia , Potássio/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Arabidopsis/genética , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/fisiologia , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Elementos de DNA Transponíveis , Regulação da Expressão Gênica de Plantas , Mutação , Fenótipo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Canais de Potássio/genética , Proteínas Proto-Oncogênicas c-aktRESUMO
The alpha subunit of a prototypical heterotrimeric GTP-binding protein (G protein), which is encoded by a single gene (GPA1) in Arabidopsis, is a modulator of plant cell proliferation. gpa1 null mutants have reduced cell division in aerial tissues throughout development. Inducible overexpression of GPA1 in Arabidopsis confers inducible ectopic cell division. GPA1 overexpression in synchronized BY-2 cells causes premature advance of the nuclear cycle and the premature appearance of a division wall. Results from loss of function and ectopic expression and activation of GPA1 indicate that this subunit is a positive modulator of cell division in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/citologia , Arabidopsis/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ácido 2,4-Diclorofenoxiacético/farmacologia , Alelos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Tamanho Celular , Genes de Plantas , Genes Reporter , Glucuronidase/análise , Glucuronidase/genética , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Ácidos Indolacéticos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Luz , Sistema de Sinalização das MAP Quinases , Morfogênese , Mutação , Peptídeos/farmacologia , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Plantas Tóxicas , Subunidades Proteicas , Proteínas Recombinantes/metabolismo , Transdução de Sinais , NicotianaRESUMO
To directly address the function of a putative auxin receptor designated ABP1, a reverse genetic approach was taken to identify and characterize ABP1 mutant alleles in Arabidopsis. A homozygous null mutation in ABP1 confers embryo lethality. Null mutant embryos develop normally until the early stages of the globular embryo but are unable to make the transition to a bilaterally symmetrical structure because cells fail to elongate. Cell division was also aberrant both in the suspensor and embryo proper. Antisense suppression of ABP1 in tobacco cells causes slow proliferation and eliminates auxin-induced cell elongation and reduces cell division. The complete lack of auxin-inducible elongation in individual cells confirms the results observed in embryos, indicates a cell autonomous function, and, taken together with biochemical evidence that ABP1 binds auxins, suggests that ABP1 mediates auxin-induced cell elongation and, directly or indirectly, cell division.
Assuntos
Arabidopsis/embriologia , Proteínas de Plantas/fisiologia , Receptores de Superfície Celular/fisiologia , Alelos , Arabidopsis/genética , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Replicação do DNA , DNA de Plantas/genética , Deleção de Genes , Marcação de Genes , Genes Letais , Genes de Plantas , Teste de Complementação Genética , Ácidos Naftalenoacéticos/farmacologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas de Plantas/genética , Plantas Tóxicas , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/fisiologia , Sementes/crescimento & desenvolvimento , Nicotiana/citologia , TransfecçãoRESUMO
The sequencing of the Arabidopsis plant genome is providing a fuller understanding of the number and types of plant genes. However, in most cases we do not know which genes are responsible for specific metabolic and signal transduction pathways. Analysis of gene function is also often confounded by the presence of multiple isoforms of the gene of interest. Recent advances in PCR-based reverse genetic techniques have allowed the search for plants carrying T-DNA insertions in any gene of interest. Here we report preliminary screening results from an ordered population of nearly 60,470 independently derived T-DNA lines. Degenerate PCR primers were used on large DNA pools (n = 2,025 T-DNA lines) to screen for more than one gene family member at a time. Methods are presented that facilitated the identification and isolation of isoform-specific mutants in almost all members of the Arabidopsis H(+)-proton ATPase gene family. Multiple mutant alleles were found for several isoforms.
Assuntos
Arabidopsis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Arabidopsis/genética , Sequência de Bases , Primers do DNA , Genoma de Planta , Mutagênese InsercionalRESUMO
The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.
Assuntos
Arabidopsis/química , Arabidopsis/fisiologia , Tilacoides/química , Membrana Celular , Cloroplastos/metabolismo , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Luz , Peptídeos , Fosforilação , Fotossíntese , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Treonina/química , Fatores de Tempo , Tripsina/farmacologiaRESUMO
A major question in plant physiology is how the large amount of sucrose made in leaves is transported to the rest of the plant. Although physiological, biochemical, and anatomical investigations have been performed in this field, to date there have been very few genetic studies. Using a reverse genetic screen, we have identified mutant Arabidopsis plants containing transferred DNA insertions in the gene encoding a phloem-specific sucrose transporter, SUC2. SUC2 is thought to function in loading sugar from the apoplast into the conducting sieve tubes. In the homozygous state, these mutations resulted in stunted growth, retarded development, and sterility. The source leaves of mutant plants contained a great excess of starch, and radiolabeled sugar failed to be transported efficiently to roots and inflorescences. These data provide genetic proof that apoplastic phloem loading is critical for growth, development, and reproduction in Arabidopsis and that SUC2 is at least partially responsible for this step.
Assuntos
Arabidopsis/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Plantas/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Primers do DNA , Proteínas de Membrana/genética , Mutação , Fenótipo , Proteínas de Plantas/genéticaRESUMO
Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.
Assuntos
Oligonucleotídeos/química , Sequência de Bases , Luz , Hibridização de Ácido Nucleico , FotoquímicaRESUMO
A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.
Assuntos
Proteínas de Arabidopsis , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Canais de Potássio/metabolismo , Potássio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Sódio/farmacocinética , Arabidopsis , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mutagênese Insercional/fisiologia , Proteínas de Plantas/genética , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Canais de Potássio/genética , Rubídio/farmacocinéticaRESUMO
Plant genomes encode a variety of protein kinases, and while some are functional homologues of animal and fungal kinases, others have a novel structure. This review focuses on three groups of unusual membrane-associated plant protein kinases: receptor-like protein kinases (RLKs), calcium-dependent protein kinases (CDPKs), and histidine protein kinases. Animal RLKs have a putative extracellular domain, a single transmembrane domain, and a protein kinase domain. In plants, all of the RLKs identified thus far have serine/threonine signature sequences, rather than the tyrosine-specific signature sequences common to animals. Recent genetic experiments reveal that some of these plant kinases function in development and pathogen resistance. The CDPKs of plants and protozoans are composed of a single polypeptide with a protein kinase domain fused to a C-terminal calmodulin-like domain containing four calcium-binding EF hands. No functional plant homologues of protein kinase C or Ca2+/calmodulin-dependent protein kinase have been identified, and no animal or fungal CDPK homologues have been identified. Recently, histidine kinases have been shown to participate in signaling pathways in plants and fungi. ETR1, an Arabidopsis histidine kinase homologue with three transmembrane domains, functions as a receptor for the plant hormone ethylene. G-protein-coupled receptors, which often serve as hormone receptors in animal systems, have not yet been identified in plants.
Assuntos
Arabidopsis/enzimologia , Plantas/enzimologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Membrana Celular/enzimologia , Histidina Quinase , Transdução de SinaisRESUMO
Proton pumps (H+-ATPases) are the primary active transport systems in the plasma membrane of higher plant cells. These enzymes are encoded by a large gene family expressed throughout the plant, with specific isoforms directed to various specialized cells. While their involvement in membrane energetics has been suggested by a large body of biochemical and physiological studies, a genetic analysis of their role in plants has not yet been performed. We report here that mutant Arabidopsis thaliana plants containing a phloem-specific transgene encoding a plasma membrane H+-ATPase with an altered carboxy terminus show improved growth at low pH during seedling development. These observations provide the first genetic evidence for a role of the plasma membrane H+-ATPase in cytoplasmic pH homeostasis in plants.
Assuntos
Ácidos/farmacologia , Arabidopsis/enzimologia , Arabidopsis/genética , Genes de Plantas , Plantas/enzimologia , Plantas/genética , Transgenes/genética , Arabidopsis/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/genética , Resistência a Medicamentos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
In plants, potassium serves an essential role as an osmoticum and charge carrier. Its uptake by roots occurs by poorly defined mechanisms. To determine the role of potassium channels in planta, we performed a reverse genetic screen and identified an Arabidopsis thaliana mutant in which the AKT1 channel gene was disrupted. Roots of this mutant lacked inward-rectifying potassium channels and displayed reduced potassium (rubidium-86) uptake. Compared with wild type, mutant plants grew poorly on media with a potassium concentration of 100 micromolar or less. These results and membrane potential measurements suggest that the AKT1 channel mediates potassium uptake from solutions that contain as little as 10 micromolar potassium.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Meios de Cultura , DNA Bacteriano/genética , Genes de Plantas , Potenciais da Membrana , Mutação , Técnicas de Patch-Clamp , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Canais de Potássio/genéticaRESUMO
More than 11 different P-type H(+)-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H(+)-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1-193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H(+)-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H(+)-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.
Assuntos
Arabidopsis/enzimologia , Compartimento Celular , Membrana Celular/enzimologia , Isoenzimas/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Epitopos , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/imunologia , Microscopia Imunoeletrônica , Microssomos/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Plantas Geneticamente Modificadas , ATPases Translocadoras de Prótons/classificação , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Frações Subcelulares/enzimologia , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificaçãoRESUMO
The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.
Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Elementos de DNA Transponíveis , DNA Bacteriano/metabolismo , Genes de Plantas , Sequência de Bases , Transporte Biológico , Linhagem Celular Transformada , Primers do DNA , DNA Bacteriano/genética , DNA de Plantas/análise , DNA de Plantas/genética , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Deleção de Genes , Família Multigênica , Mutagênese Insercional , Reação em Cadeia da Polimerase , Transdução de SinaisRESUMO
A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.
Assuntos
Arabidopsis/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Genes de Plantas , Arabidopsis/enzimologia , Clonagem Molecular , DNA Complementar/genética , Isoenzimas/genética , Dados de Sequência Molecular , Peso Molecular , Família Multigênica , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Terminologia como AssuntoRESUMO
In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.
Assuntos
ATPases Translocadoras de Prótons/análise , ATPases Translocadoras de Prótons/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Microscopia Eletrônica , Microscopia Imunoeletrônica , Modelos Estruturais , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-myc/análise , Proteínas Proto-Oncogênicas c-myc/biossíntese , ATPases Translocadoras de Prótons/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Sensibilidade e Especificidade , Sitios de Sequências RotuladasRESUMO
The Arabidopsis thaliana KAT1 cDNA encodes a voltage-gated inward-rectifying K+ channel. A KAT1 genomic DNA clone was isolated and sequenced, and a 5' promoter and coding sequences containing eight introns were identified. Reporter gene analysis of transgenic plants containing the KAT1 promoter fused to bacterial beta-glucuronidase showed robust beta-glucuronidase activity primarily in guard cells.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Plantas/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/biossíntese , Arabidopsis/citologia , Proteínas de Arabidopsis , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA de Plantas/metabolismo , Genes de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Canais de Potássio/genética , Proteínas Recombinantes/biossíntese , Mapeamento por RestriçãoRESUMO
Protoplasts were isolated from H89, an embryogenic sweet orange (Citrus sinensis (L.) Osbeck cv. Hamlin) suspension culture, and electroporated with p35S-GFP, a plasmid carrying the gene for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. p35S-GFP was constructed by replacing the GUS coding sequence of pBI221 with a functional GFP gene, thereby placing the GFP gene under the control of the CaMV 35S promoter. Protoplasts were viewed by incident-light fluorescence microscopy twentyfour h after electroporation. 20-60% of the protoplasts emitted an intense green light when illuminated with blue (450-490 nm) light.