Fusion with fluorescent proteins for subcellular localization of enzymes involved in plant alkaloid biosynthesis.
To establish the role in alkaloid metabolism of candidate genes identified in silico or by Omics approaches, it may be essential to determine the subcellular localization of the encoded proteins. The fusion with fluorescent proteins (FP) may now be used as a quite effective and reliable tool to investigate this question. The methodology involves the choice of the FP, the design and production of the appropriate FP fusions, and the use of a transient or stable transformation protocol applied to a homologous or heterologous plant system. This chapter describes the application of this methodology to an enzyme involved in indole alkaloid biosynthesis, with general considerations on the development of the approach.
Duarte
Patrícia
P
IBMC - Institute of Cellular and Molecular Biology, Porto University, Porto, Portugal.
Memelink
Johan
J
Sottomayor
Mariana
M
D000470:Alkaloids / Q000096:biosynthesis*; D017360:Arabidopsis / Q000166:cytology; D019677:Artificial Gene Fusion / Q000379:methods*; D030008:Catharanthus / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006371:Helium; D042541:Intracellular Space / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009195:Peroxidase / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014414:Tungsten
D000470:Alkaloids; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006371:Helium; D011092:Polyethylene Glycols; D009195:Peroxidase; D014414:Tungsten