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Selectable marker GENES

Selectable marker genes are essential in plant transformation systems, providing resistance to toxic substances, allowing for the survival of transformed cells. Various types of selectable marker genes exist, including antibiotic, antimetabolite, and herbicide resistance genes. Reporter genes, such as GUS and GFP, are used to assess plant transformation, with GFP being favored for its ease of use and non-destructive screening capabilities.

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11 views

Selectable marker GENES

Selectable marker genes are essential in plant transformation systems, providing resistance to toxic substances, allowing for the survival of transformed cells. Various types of selectable marker genes exist, including antibiotic, antimetabolite, and herbicide resistance genes. Reporter genes, such as GUS and GFP, are used to assess plant transformation, with GFP being favored for its ease of use and non-destructive screening capabilities.

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krittikabhowmik.2003
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SELECTABLE MARKER

GENES
SELECTABLE MARKER GENES
 The selectable marker genes are usually an integral part of plant
transformation system.
 They are present in the vector along with the target gene.

 In a majority of cases, the selection is based on the survival of the


transformed cells when grown on a medium containing a toxic substance
(antibiotic, herbicide, antimetabolite).
 This is due to the fact that the selectable marker gene confers resistance
to toxicity in the transformed cells, while the non- transformed cells get
killed.
 A large number of selectable marker genes are available and they are grouped
into three categories— antibiotic resistance genes, antimetabolite marker
genes, and herbicide resistance genes
SELECTABLE MARKER GENES

hptII gene codes for hygromycin phosphotransferase (HPT)


enzyme
NptII (neomycin phosphotransferase)gene resistance to
kanamycin in bacteria and geneticin in eukaryotes.

bar gene confers resistance to phosphinothricin in plants.


 Pmi gene(derived from E. coli) coding for phosphomannose
isomerase enzyme
REPORTER GENES:

A reporter gene may be regarded as the test gene whose


expression can be quantified.
The plant transformation can be assessed by the expression of
reporter genes (also called as screen able or scoreable genes).
In general, an assay for the reporter gene is carried out by
estimating the quantity of the protein it produces or the final
products formed.
SCREENING MARKER- GUS ASSAY

 GUS assay is a simple method for detection of transformed


cell without needing any complicated equipment.

 β-glucuronidase, an enzyme from the bacterium


Escherichia coli is utilized in this technique.
 This enzyme can transform colorless or non-fluorescent
substrates into coloured (Blue) after Histochemical gus
staining
GUS ASSAY
GREEN FLUORESCENT PROTEIN (GFP)

Green fluorescent protein (GFP), Gene for GFP has been isolated from jelly fish
Aequorea victoria which is a luminescent organism. The original gfp gene has been
significantly modified to make it more useful as a reporter gene. GFP emits
fluorescence which can be detected under a fluorescent microscope.

GFP is coded by gfp gene, is being widely used in recent years. In fact, in many instances,
GFP has replaced GUS since assays of GFP are easier and non-destructive. Thus,
screening of even the primary transplants can be done by GFP which is not possible
with other reporter genes.
OF THE LUCIFERASE (LUC) GENE
GLOWING TOBACCO TRANSFORMED WITH FIREFLY LUCIFERASE GENE.
LUCIFERASE
 Production and emission of light by a living organism
is known as Bioluminescence. Luciferases are
enzymes which emit light.
 The enzyme firefly luciferase, encoded by the gene
luc, catalyses the oxidation of D-luciferin (ATP
dependent) which results in the emission of light that
can be detected by sensitive luminometers.
 The firefly luciferase gene, however, is not widely used
as a marker gene since the assay of the enzyme is
rather cumbersome.
CHLORAMPHENICOL ACETYL TRANSFERASE (CAT GENE):

 The cat gene producing chloramphenicol acetyl transferase


(CAT) is a widely used reporter gene in mammalian cells.
 Due to the availability of GUS and GFP reporter systems for
plant trans- formants, CAT is not commonly used.
 However, some workers continue to use CAT by a sensitive
radioactive assay, for the detection of the reporter gene cat.
THANK YOU

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