reporter gene for functional genomics study

1. Ritasree Sarma
Types and relevance of
Reporter genes in plant
functional genomics studies

2. Reporter genes
• Gene whose products are easily detected or
monitered
• A gene that is used to `tag' another gene or
DNA sequence of interest
Identifying whether a certain gene has been
taken up by cell
Measurement of gene expression

3. • Easily quantifiable
• Relatively rapid degradation of the enzyme
• Lack of endogenous activity in the concerned cell
• Should not be toxic to cells
• Assay should be sensitive and reliable

5. Types of Reporter gene
Scorable reporter genes
• Expression of this results in quantifiable
phenotype
• Easily detected through highly sensitive enzyme
assays
Selectable reporter genes
• Expression of resistance to a toxin
• Selection of tranformants from nontransformants in
growth media containing selective agent

6. Scorable reporter gene
Green Fluorescent Protein
• Derived from jellyfish Aequorea victoria
• Formed by nucleophilic reaction between C-ter of S with N-ter of G,
formed imidazoline heterocyclic ring which oxidise with Y to yield
floroscence
 Variants of GFP
Yellow Fluorescent Protein
• Formed by mutation of Thr 203 residue to tyrosine
Blue Fluorescent Protein
• Modification of tyr66 to his
Cyan Fluorescent Protein
Modification of tyr66 to tryptophan
• Derived from Discosoma striata (Ds Red)
• Alternation of >30 amino acid to yield RFP-1
Shaner N.C et al, Nature Biotech 22,1562-1572 (2004)

7. Fruit series
Cubitt, A., R. Heim, S. Adams, A.
Boyd, L. Gross, R. and R. Tsien
(1995) Understanding, improving
and Using Green Fluorescent
Proteins. Trends in Biochemical
Sciences, 20:448-455.

8. Floroscent
protein
Excitation
wavelength
(nm)
Emission
wavelength
(nm)
GFP 488 525
YFP 514 590
RFP 561 575
CFP 442 480
Chem. Soc. Rev., 2009, 38, 2887–2921

9. GUS
• Derived from E.Coli
• uid A gene code for 12- -glucuronidase enzyme
• Enzymatic cleavage of X-Gluc (5-bromo-4-chloro-3-indolyl -D-
glucuronide) undergoes an oxidative dimerization to yield an indigo blue
precipitate
Luciferase
• Bacterial luciferase : Vibrio harveyi (luxA/luxB genes)
• Firefly (Photinus pyralis) luciferase (luc gene)
β-galactosidase (LacZ)
• Derived from:E.coli
• catalyzes the hydrolysis of X-Gal producing a blue precipitate

10. Selectable reporter gene
Antibiotic Resistance Genes
Neomycin phosphotransferase II (npt II gene)
• Derived from the transposon Tn5 code foraminoglycoside 3`
phosphotransferase
• Resistance to the antibiotic kanamycin neomycin by
phosphorylation
Hygromycin phosphotransferase (hpt gene)
• Derives from E. coli
• Resistant against hygromycin by phosphorylation

11. Herbicide Resistance Markers
Phosphinothricin acetytransferase (pat/bar gene)
• Derived from Streptomyces hygroscopicus
• Converts herbicides into acetylated forms
Resistant against Bialophos, phosphinothricin and glufosinate
Enolpyruvylshikimate phosphate synthase (epsps/aroA genes)
• Derived from Agrobacterium sp CP4
• Resistance against glyphosate which blocks the activity of EPSP synthase,
a key enzyme involved in the biosynthesis of aromatic amino acid
Bromoxynil nitrilase (bxn gene)
• Derived from Klebsiella pneumoniae
• The herbicide bromoxynil inhibits photosynthesis (photosystem II)
• Encode a specific nitrilase that converts bromoxynil to its primary
metabolite 3,5-dibromo-4-hydroxybenzoic acid

12. Reporter gene for functional genomics
 Identify a promoter, to study the expression pattern and strength
of the promoter
• Reporter gene is simply placed under the control of the target
promoter
 Gene expression assays
• Reporter is directly attached to the gene of interest to create
a gene fusion
• The two genes are under the same promoter elements and are
transcribed and then translated into protein
 Transformation and transfection assays
• Reporter genes expressed under their own promoter independent
from that of the introduced gene of interest
• Reporter gene can be expressed constitutively or inducibly

13. T. Weber, R. Köster / Methods 62 (2013) 279–
291

14. Protein-protein interaction??

16. Schematic diagrams of the PtMCP promoter::GUS and CaMV35S promoter::GUS construct. RB, right
border; LB, left border; NOSpro, nopaline synthase promoter; NPT-II, neomycin
phosphotransferase (II) coding region; NOS-T, nopaline synthase terminator; GUS,
β-glucuronidase gene; PtMCPpro, PtMCP promoter; CaMV35Spro, cauliflower mosaic
virus (CaMV) 35S promoter. The insertion position of the PtMCP promoter in the vector is
indicated by the restriction enzyme sites SacI and KpnI

17. (C and G), positive
controls (CaMV35S
promoter::GUS)
(D and H), negative
controls (wild-type
plants

20. • XPR1 as Pi exporter
• Homolog with arabidopsis PHO1
• Has 2 domain SPX and EXS
• SPX Domain is not required for Pi export
• Mostly localised in Golgi and trans golgi network using GFP as
marker
XPR1
TMX
2X CaMV SPX 4TM EXS GFP
2XCaMV 4MT EXS GFP

21. Co-expression of XPR1-GFP with different
subcellular markers in tobacco epidermal
cells.
Tobacco leaves were infiltrated with A.
tumefaciens strains harboring free GFP as
a control

22. Co-expression of XPR1-GFP
with different subcellular
markers in tobacco
epidermal cells.
Tobacco leaves were
infiltrated with A.
tumefaciens strains
harboring free GFP as a
control

23. T
Thank you