Download Plant Transformation

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.
ß-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 ßglucuronides
• easily assayed for histochemical analysis, using Xgluc (5-bromo, 4-chloro, 3-indoyl ß–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 6567 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 protein
structure