RESUMO
Transient expression is the nonstable expression of a transgene whereby the transgene does not integrate into the host's genomic DNA. Transient expression assays have 20 years of history in plant molecular biology research, being used to answer a variety of questions. The method described here allows the ability to test promoter activity for seed-specific expression by quantifying reporter protein production in immature seed tissues. This method is especially suited to test vector activity for stable expression, test promoter activity, and discern regions of a promoter that is necessary for transcription in seed tissues. The transient expression assay is a tool that has aided a great deal of molecular biology research.
Assuntos
Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Zea mays/genética , Zea mays/metabolismo , Biolística , Expressão Gênica , Técnicas Genéticas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sementes/genética , Sementes/metabolismo , Zea mays/embriologiaRESUMO
Efficient methods to characterize transgenic plants are important to quickly understand the state of the transformant. Determining transgene copy number is an important step in transformant characterization and can differentiate between complex and simple transformation events. This knowledge can be extremely useful when determining what future experiments and uses the transgenic lines can be utilized for. The method described here uses real-time quantitative PCR to determine the transgene copy number present in the genome of the transformant. Specifically, this method measures the relative transgene copy number by comparing it with an endogenous gene with a known copy number. This method is a quick alternative to the Southern blot, a method that is commonly used to determine gene copy number, and is effective when screening large numbers of transformants.
Assuntos
Dosagem de Genes , Técnicas Genéticas , Reação em Cadeia da Polimerase/métodos , Zea mays/genética , Sequência de Bases , Southern Blotting , Primers do DNA/genética , DNA Recombinante/genética , DNA Recombinante/isolamento & purificação , Plantas Geneticamente ModificadasRESUMO
Chimaeric promoters contain DNA sequences from different promoters. Chimaeric promoters are developed to increase the level of recombinant protein expression, to precisely control transgene activity or to combat homology-based gene silencing. Sets of chimaeric promoters, each containing different lengths of DNA from maize (Zea mays) 27zn (27 kDa gamma-zein) endosperm-specific promoter and the Glb1 (Globulin-1) embryo-specific promoter were created and tested in a transient expression assay of GFP (green fluorescent protein). Promoter fragments with the highest activity were combined to create the chimaeric promoter A27znGlb1. In the context of the chimaeric promoter, the selected Glb1 promoter fragment was necessary and sufficient to activate expression in embryo tissue and was functionally equivalent to the native Glb1 promoter. Similarly, the selected 27zn promoter fragment in the chimaeric promoter was necessary and sufficient to activate expression in endosperm tissue and was functionally equivalent to the native 27zn promoter. Maize transgenic plants containing the A27znGlb1 chimaeric promoter fused to GFP were produced to characterize this promoter in vivo. Quantitative reverse-transcriptase PCR was used to determine that the promoter was active in the embryo, endosperm, pericarp and immature leaf tissues. GFP activity in plants containing the chimaeric promoter was not significantly different in endosperm than the activity of GFP fused to the full-length 27zn promoter, nor was it different in embryo from the activity of GFP fused to the full-length Glb1 promoter. Transgene copy numbers were shown to be between 4 and 12 copies in different events.