A new molecular tool for transgenic diatoms: control of mRNA and protein biosynthesis by an inducible promoter-terminator cassette.
Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen-free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms.
Biochemie I, Universität Regensburg, Germany.
Nicole
N
Poulsen
Nils
N
Kröger
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D009566:Nitrates; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009565:Nitrate Reductases; D050901:Nitrate Reductase