Plant genome editing identify the plant transgenic or non transgenic using some of the reporter gene plant biology.

1. SEMINAR PRESENTED
ON
REPORTER GENE
IN
SESSION 2021-2022
Department of Biotechnology
School of Biotechnology & Genetic Engineering
Bharathidasan University, Tiruchirapalli-620024.
SUBM
ITTEDBY:
M.Manikandan
II.M.Sc.Biotechnology
SUB:Plant Biotechnology
REGNO:20BT20.

2. Introduction:
 A Reportergenesmayberegardedasthetestgenewhoseexpressioncanbe
quantified.The planttransformationcanbeassessedbytheexpressionof
reportergenes(alsocalledasscreenableorscoreablegenes).
 Ingeneral,anassayforthereportergeneiscarriedoutbyestimatingthe
quantityoftheproteinitproducesorthefinalproductsformed.

3. An ideal reporter gene:
 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.

4. Reporter gene used for gene transfer in plants
their sources, and detection assays :

5. Opine synthase (ocs,nos genes):
The common opines present in T-DNA of Ti or Ri plasmid of Agarobacterium are
octopine and nopaline, respectively produced by the synthase genes ocs and nos.
The transformed status of the plant cells can be easily detected by the presence
Of these opine.
Opines can be separated by the electrophoresis and identified.
Alternately, the enzyme activities responsible for the production of opines can
also be assayed.

6. Green Fluorescent Protein(gfp):
 From Jellyfish Aquoria victorica, glows in blue light 395 nm giving green fluorescence
(510 nm).
 Allows non-destructive imaging of plants and sub cellular localization of GFP by
Microscope.
 GFP is a small protein of 238 amino acids.
 Can be detected in vivo (non destructively) by using fluorescence microscope.
 Different variants like EGFP, Red GFP,EYFP,etc available.
 Does not require any substrate, can be detected directly.

7. β- Glucuronidase(GUS):
 The bacterial enzyme β- glucuronidase (GUS) , which is coded by the E,COLI gene,uidA
(also known as gusA).
 GUS is probably the most widely used reporter gene in plants,and low endogenous activity
In plant.
 Codes for the b-glucuronidase which breaks x-gluc (5-bromo-4 chloro-3 indol β-D glucuroni
de) into blue colour, can be used for histochemical analysis of gene expression.
 Converts 4MUG (4-methylumbellifery –β-D glucuronide) into a fluorescent compound 4MU
(Methyl Umbeliferone); can be used quantification by fluorescent measurement.
 Can also be quantified spectrophotometrically by
Using p-nitrophenyl galactoside as substrate.

8. GUS GENE (uidA):

9. β- Galactosidase:
 β- galactosidase which is encoded by the bacterial gene lacZ.
 In bacteria, β- galactosidase cleaves the disaccharide lactose (sugar found in milk)
into glucose and galactose.
 β- galactosidase cleaves the colorless substrate x-gal (5-bromo-4-chloro-3-indolyl-
β- galactopyranoside) into galactose and a blue insoluble product of the cleavage.
 Because most genomes do not contain lacZ, it can be used as a reporter gene
(E.g: blue white selection).

10. β- Galactosidase:

11. Luciferase gene luc or lux:
 From firefly (luc) or bacteria (lux)
 These enzyme catalyzing a light- emitting-reaction.
 Converts luciferin into oxyluciferin in the presence of
Oxygen and ATP.
 Oxyluciferin is highly unstable,single excited compound which emits
Fluorescence upon relaxation to ground state.
 Light emission can be monitored visually or photographically.
 Detected in tissue extracts or even in the intact plant after watering
with luciferin.
 Yellow-green light will be emitted.

12. Alkaline phosphatase pho A:
 From Bacteria E.COLI
 Claves compounds containing phosphate group like o-nitrophenyl phosphate, x-phos
(5 bromo-4 chloro-3 indolyl phosphate)
 Can be use for colorimetric or histochemical detection (with o-nitrophenyl phosphate
orX-phos) or by fluorescence measurement (with 4-MUP).
 Forms yellow nitrophenol with o-nitrophenyl phosphate.
 Gives dark blue coloured precipitate with x-phos in the same manner as
Does β-glucuronidase with x-gluc.

13. Chloramphenicol Acetyl transferase (cat gene):
 The cat gene producing chloramphenicol acetyl transferase (CAT) is a widely
used reporter gene in mammalian and plant 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.

14. Application:
 Normalize for changes in cell physiology. Events associated with cell physiology can affect
Reporter gene expression.
 Monitor RNA interference..
 Assess cell signalling pathways.
 Examine nuclear receptors

15. BIBLIOGRAPHY:
 https://link.springer.com/protocol/10.1385/1-59259-827-7:203
 https://www.biologydiscussion.com/genetics/engineering/select
able-marker-genes-and-reporter-genes/10752
 https://www.slideshare.net/SACHINHALAPPA/reporter-genes-80479
096
 Text book of Biotechnology- Dr,RC.Dubey.

16. THANK YOU