RESUMO
The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.
Assuntos
Técnicas de Transferência de Genes , Plantas/genética , Pólen/genética , Eletroporação , Técnicas Genéticas , ProtoplastosRESUMO
GPD1 (encoding glyceraldehyde-3-phosphate dehydrogenase) is a constitutively expressed gene in Cochliobolus heterostrophus that produces a single transcript. The steady state level of GPD1 mRNA is 14-fold greater than that of the constitutively-expressed TRP1 gene (encoding a tryptophan biosynthesis enzyme) indicating that GPD1 has a stronger promoter and/or a more stable mRNA. A set of lacZ translational fusion vectors was constructed to compare the gene expression signals of GPD1, TRP1 and PRO1 (a C. heterostrophus genomic fragment selected for promoter activity) in C. heterostrophus as single copies at the same site in the chromosome. Under conditions that repressed endogenous beta-galactosidase expression, beta-galactosidase activity in transformants was constitutive and required the GPD1, TRP1 or PRO1 expression signals. In-frame GPD1::lacZ activities were 6-fold greater than in-frame TRP1::lacZ and PRO1::lacZ activities, indicating that GPD1 has more efficient expression signals.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Proteínas Fúngicas/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
A single gene (GPD1) encoding glyceraldehyde-3-phosphate dehydrogenase (GPD) was found in Cochliobolus heterostrophus. Homology with other fungal GPD-encoding genes was substantial at both the nucleotide and amino-acid levels. Positions of four introns found in GPD1 were conserved in the corresponding Aspergillus nidulans gpdA gene (which is known to have three additional introns absent in GPD1). The size (approximately 1300 nucleotides) of the single GPD1 transcript was consistent with the length (1011 bp) of the open reading frame. Several transcription initiation sites were identified, including major ones 45 and 40 bp upstream of the start codon. Conserved regulatory sequences were found in both the 5' and 3' flanking regions of GPD1.
Assuntos
Ascomicetos/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , Códon , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The utilization of electrofusion and electroporation techniques has had a major impact on the genetic manipulation of plants within the last decade. This review of the development of electrofusion and electroporation, as it applies to plants, highlights major developmental aspects of this technology. These include mechanisms for cell fusion, molecular exchange, and parameters that affect the efficiency of fusion and electroporation.
