Plant Transformation SRM University植物转化 SRM大学.pptVIP

Lecture 5 Reporter genes Reporter Genes A gene encoding an enzyme medium modification is added along with your gene nucleic acid sequences encoding easily assayed proteins Reporter genes include ?-galactosidase (encoded by lacZ), ?-glucuronidase (encoded by uidA), chloramphenicol acetyltransferase, luciferase and green fluorescent protein (GFP) . Novel Reporter Genes Luciferase - gene from fireflies with causes the glow - add a substrate to tissue that has been transformed and it lights up Green Fluorescent Protein - from jellyfish - under lights and filter the transgenic plants will fluoresce GUS - glucuronidase gene will convert added substrate to blue color. ?-glucuronidase (GUS) GUS is probably the most widely used reporter gene in plants low endogenous activity in plant stable enzyme which hydrolyses wide range of ?-glucuronides easily assayed for histochemical analysis, using X-gluc (5-bromo, 4-chloro, 3-indoyl ?–glucuronide). After cleavage, oxidation of the indole derivative causes dimerisation and the production of an insoluble indigo dye GUS expression The GUS gene creates blue coloration of transformed tissue when transformed cells or tissues are provided with the appropriate substrate. Luciferase (Luc)  enzyme from firefly (Photinus pyralis) produces flashes of light in the presence of luciferin and ATP detected in tissue extracts or even in the intact plant after watering with luciferin allows non-destructive imaging of plants  ) Green fluorescent protein (GFP) from the jelly-fish Aequoria victoria intrinsically fluorescent due to a chromophore in the protein by cyclisation and oxidation of the amino acids Ser-Tyr-Gly at positions 65-67 in the polypeptide allows non-destructive imaging of plants and sub cellular localization of GFP by microscopy several variants of GFP to give different colours – YFP (yellow), BFP (blue), CFP (cyan) produced by alteration in the chromophore (Tyr66), or residues close to the chromophore in the 3-D