This document summarizes the use of reporter genes to study the ecology of rhizobia. It discusses several reporter genes including gusA, lacZ, xylE, luxAB, celB, and gfp. The gusA gene encodes beta-glucuronidase and is commonly used to track rhizobial strain competition and colonization. The lacZ gene encodes beta-galactosidase and is also used to study rhizobia. Reporter genes allow indirect quantification of rhizobia in nodules and soil. GFP has been used to visualize the mycoparasitism of Trichoderma against the plant pathogen Rhizoctonia solani. Reporter genes are powerful tools for understanding rh
Use of reporter genes in the process of selection of the transformants from the non transformants, and the current use of these reporter genes as the Desired genes.
this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers
Use of reporter genes in the process of selection of the transformants from the non transformants, and the current use of these reporter genes as the Desired genes.
this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
In this PPT You can learn briefly about Reporter Gene and Gene fusion And Gene manipulation method.
Reference From Microbiology.
Author - Brock. 12th Edition,
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
The presentation describes the advantages of plastid transformation over 'conventional' nuclear transformation, hurdles to plastid transformation, its advantages. The presentation also covers some successful plastid engineering and its potential.
Majority of agronomic traits are quantitative and are controlled polygenetically.Instead of producing transgenic plants through single gene transfer many researchers are attempting on multigene engineering. The simultaneous transfer of multiple genes in to plants will enable us to produce plants with more desirable characters. Engineering of genes coding for complete metabolic pathways, bacterial operons or biopharmaceuticals that require an assembly of complex multisubunit proteins etc are some of the successful examples of multigene engineering.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
In this PPT You can learn briefly about Reporter Gene and Gene fusion And Gene manipulation method.
Reference From Microbiology.
Author - Brock. 12th Edition,
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.
genetic engineering, principles, b pharma 6th sem, biotechnology
What is a gene ?
Definition
History
Process
Molecular tools of genetic engineering
Restriction enzymes
History of restriction enzyme
Mechanism of action
Types of restriction enzymes
Application of restriction enzymes
Blunt ends
Sticky ends
transgenic
cisgenic.
knockout organism.
Host organism vector
TRANSGENIC PLANTS
DOLLY THE SHIP
TRANSGENIC ANIMALS
The presentation describes the advantages of plastid transformation over 'conventional' nuclear transformation, hurdles to plastid transformation, its advantages. The presentation also covers some successful plastid engineering and its potential.
Majority of agronomic traits are quantitative and are controlled polygenetically.Instead of producing transgenic plants through single gene transfer many researchers are attempting on multigene engineering. The simultaneous transfer of multiple genes in to plants will enable us to produce plants with more desirable characters. Engineering of genes coding for complete metabolic pathways, bacterial operons or biopharmaceuticals that require an assembly of complex multisubunit proteins etc are some of the successful examples of multigene engineering.
Successful colonization of roots and Plant growth promotion of sorghum (Sorgh...Premier Publishers
Pseudomonas putida (P29) and Azotobacter chroococcum (Azb19) are the efficient promising strains selected from in vitro plant growth promoting studies. These two strains were tested for their ability to promote growth of sorghum and colonize sorghum roots. Seed bacterization with P29 and Azb19 resulted in increased plant height, shoot height, root volume, leaf area and total plant dry mass. Further, bacterial inoculation also significantly increased macro-and micro-nutrient uptake by sorghum plants. Using electroporation method, pure cultures of P29 and Azb19 were transformed with pHC 60 plasmid containing gfp gene. Transformants detected by colony PCR were used to study the colonization pattern on roots of sorghum. Confocal fluorescence scanning microscope (CLSM) was used to locate the inoculants on or inside roots. Root colonization in sorghum by P29 was internal whereas Azb19 was detected on root surface. GFP-tagged Pseudomonas was predominantly detected at the root differentiation zone. In case of Azb19 small aggregates of micro-colonies were observed on the surface of the roots. The efficient sorghum root colonization by these inoculants clearly demonstrated that the introduced strains could successfully inhabit the rhizosphere and thus resulting in increased nutrient uptake. Inoculation with P29 resulted in increased uptake of P (288.5%), K (179.1%), Fe (242.7%), and Zn (168.1%) as compared to Azb19 where the uptake of P, K, Fe, Mn, and Zn increased by 142.6%, 161.6%, 199.5%, and 121.9%, respectively. On the other hand, inoculation with Azb19 could enhance better uptake of N (163.6%) as compared to P29 (133.3%). The strains also differed in their mode of root colonization.
Gene silencing techniques for crop improvementJhilickBanerjee
Gene silencing is a technique that aims to reduce or eliminate the production of a protein from its corresponding gene. Gene silencing is the regulation of gene expression in a cell.
Gene silencing can occur during either transcription or translation.
Gene silencing is often considered as “Gene knockdown’ i.e their expression is reduced. In contrast , when genes are knocked out they are completely erased from the organism’s genome and thus have no expression.
Methods used to silence genes include RNAi, CRISPR or siRNA, these reduce the expression of the gene by 70% but do not completely eliminate it.
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Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
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Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
1. REPORTER GENES FOR THE STUDY
OF RHIZOBIAL ECOLOGY
Presented by-
Neha Sharma
Punjab Agricultural University
2. INTRODUCTION
Some beneficial genera Rhizobium, Bradyrhizobium and
Azorhizobium, forms nodule and fixes atmospheric nitrogen
A sequence of events allows the establishment of a
symbiotic state where nitrogen is fixed and assimilated into
organic nitrogenous compounds
Strains generally faced various abiotic and biotic stresses
The phenomenon of strain competition is poorly understood
The ecology of rhizobia in the soil is crucial to the
establishment and performance of the legume-rhizobium
symbiosis
3. ABIOTIC FACTORS
Temperature-
The elevated temperatures delay nodule initiation, nodule development,
and interfere with nodule functioning
Moisture:
Very low or very high soil moisture contents are harmful for rhizobia
Soil pH:
Extremes of pH frequently reduce the nodule formation by reducing the
colonization of soil and the legume rhizosphere by rhizobia
Soil salinity:
High external salts induce an increase in the intracellular pool of amino
acids helps in survival
Available nutrients:
Correlation between the soil organic matter levels and the size or strain
composition of populations of rhizobia have been reported
4. BIOTIC FACTORS
Competition:
The ideal rhizobial strain for inoculation should have the ability
to persist and grow in the soil or around plant roots. The highly
effective strains of rhizobia generally fail to grow under natural
soil condition may be due to inter-strain competition
Antagonism:
Many strains of rhizobia produce bacteriocins which inhibit the
growth of the related strains
Predation and parasitism:
Bdellovibrio bacteriovorus parasitizes certain rhizobia and
cause their lysis.
Exposure of sensitive rhizobia to phages leads to selection of
variants with reduced sensitivity to phage lysis.
5. APPROACHES TO STUDY THE ECOLOGICAL
FACTORS
Indirect methods-
i. By accessing the proportion of nodules by ELISA,
DNA hybridization.
ii. Identifying rhizobia by antibiotic resistance,
bacteriophage typing, PAGE, immunoblot
techniques.
Direct quantitative measurements-
Plant infection method, fluorescent antibody
technique, antibiotic marker technique, genetic
markers and non-radioactive DNA hybridization
6. REPORTER GENE
A gene which is attached to a regulatory sequence
of another gene of interest in bacteria, cell culture,
animals or plants
used to study the role of a microorganism in
ecosystem
to investigate the expression of other genes for
which functional assays are not available or are
difficult.
9. CONSTRUCTION OF MINI-TRANSPOSONS
Tn5 transposable element is used.
Herrero et al. (1990) and de Lorenzo et al. (1990)
constructed mini-transposons, for the insertion of reporter
gene and transferred from E. coli to Rhizobium through
conjugation.
An additional transposase gene was attached external to
the transposon to increases stability and acceptability in
Rhizobium .
10. Gene cassette- consists of the marker gene itself and
of sequences that regulate gene expression. Such
sequences are primarily known as promoters and
terminators that are essential for switching on and off
gene expression.
Promoters may be regulated, generally either by gene
products of other regulating sequences or environmental
signals.
11. 1. gusA
isolated from E.coli
gene product- glucuronidase
Substrate- X-glcA (5-bromo-4-chloro-3-indolyl-glucuronide),
Magenta-glcA (5-bromo-6-chloro-3-indolyl-β-D-glucuronide)
gusA-marked cells turn blue when the washed root is incubated in a
phosphate buffer containing a GUS substrate such as X-glcA
Useful for the study of Rhizobium, Bradyrhizobium, Agrobacterium,
Azospirillum, Pseudomonas, Streptomyces competition and plant
colonization
Advantage-
i. Easy to perform
ii. eliminates the time-consuming steps of picking nodules and
preparing bacterial isolates
iii. Can be detected by the visualization method
12. mTn5SSgusA10-
tac promoter regulate the expression, with the
repressor gene.
Advantage- marker gene is not induced without
inducer IPTG (isopropyl-ß-D-thiogalactoside)
mTn5SSgusA11-
the marker gene is constitutively expressed
It is used to detect rhizobial strain in soil and
rhizosphere
13. mTn5SS gusA31
used for symbiotic expression study of nodule
occupancy
promoters of the nifH gene codes for the Fe-
component of the enzyme nitrogenase
Gene expression occurs in symbiotic conditions
mTn5SSgusA20-
aph promoter- kanamycin resistance gene,
function in Gram -ve bacteria
constitutive gusA expression
Used for rhizosphere colonization
14. IN BACTERIA-LEGUME SYMBIOSIS
• gusA gene from Escherichia coli with plasmid
Tn5gusA1 & Tn5gusA2 to study the expression of
Rhizobium meliloti symbiotic genes (nod,nif,fix)
during the nodule organogenesis of alfalfa was used.
• To detect the expressed gene activity histochemical
assay of β-glucronidase was done
15. The nod genes, involved in nodule formation
expressed in the early phase (after 36 hour of
inoculation) on root surface
The nif ,fix genes were expressed 10 days after
inoculation in the active symbiotic zone of nodules.
16. Similar study in pigeon pea roots-
inoculation of pigeon pea root by a marked strain
Rhizobium sp.234 :gusA31,
unmarked sp. of Rhizobium sp.234:gusA11
Result- Marked strains of gusA31 showed activity
in the central region (nitrogen fixing zone) of the
nodule.
Rhizobium sp. marked with mTn5SSgusA11
showed gus activity to the outer region of the
nodule where new infection take place.
17. NODULE OCCUPANCY COMPETITION ASSAY
Pigeon pea plant inoculated with-
Rhizobium sp.NGR234 (unmarked)
Rhizobium sp. NGR234 :gusA31(marked)- 7:1.
Again, 1:15.
Result- The proportion of blue nodules increased with an
increased proportion of the gus marked strain.
18. gus reporter gene in the study of PGPR in
rhizosphere
To study the Azospirillum on wheat roots in colonization
assays pFAJ31.13-
Inserting A. brasilense gene upstream of a promoter
less reporter gene.
Result-
i. After inoculation, the bacterial cells were found in the
root hair zones and sites of lateral root emergence.
ii. A. brasilense mutants , impaired in motility,
chemotaxis and plant root attachment
It was concluded that some key compound are secreted
by the root hair zone of the wheat roots to which
Azospirillum is attracted and colonized.
19. 2. lac Z reporter gene
Codes for ß-galactosidase enzyme involved in the
metabolism of lactose in to glucose and galactose.
Colonies with lacZ gene can be identified on X-Gal
plates gives blue colour and can also be identified
on ONPG (ortho-nitrophenyl-β-galactoside) and
MUG (4 methylumbelliferyl-β-D-glucuronide)
substrates, which can be measured by yellow color
spectrophotometrically.
It is used to study rhizobial competition, root
colonization, gene expression, survival.
21. XYLE REPORTER GENE
• Xyl E gene encodes for 2,3catechol dioxygenase .
• Convert catechol ( colorless ) to 2- hydroxymuconic semi
aldehyde, a bright yellow pigment that can be visualized
or measure spectrophotomitrically.
• The gene is originally found on TOL plasmid in P. putida.
• Colony grown on media sprayed with catechol, positive
clones turn bright yellow.
• Product is soluble.
• The xylE gene used to study in situ gene expression .
22. LUXAB(BIOLUMINESCENCE)
luxAB gene code for the enzyme luciferase.
Bacterial luciferase catalyze a light emitting oxidation
reaction using O2 ,FMNH₂ and a long chain aliphatic
aldehyde (decanel).
Both o₂ and FMNH₂ are present in most bacterial cell so any
bacterium can produce light if contain lux AB gene if decanel
provide exogenously.
Photinus pyralis(firefly) have the gene luc that also use as a
reporter gene.
24. CEL B GENE
• Encode for thermostable β-galactosidase activity.
• It allow detection of the marker strain after heat-
inactivation of the endogenous enzyme.
• Isolated from thermophilic archeaon Pyrococcus furiosus.
• Half life 85 h at 100 0C
• Assay for detection:
• The washed roots are incubated in phosphate buffer at
700C in order to destroy endogenous enzymes.
• Roots are incubated in the presence of a chromogenic
substrate such as X-gal. Nodules containing the celB
marked strain turn blue
25. GREEN FLUORESCENT PROTEIN(GPF)
GFP is isolated from Pacific jellyfish Aequorea victoria,
and Sea pansy Renilla reniformis.
GFP is a protein composed of 238 amino acid residues
(26.9 kDa) that exhibits bright green fluorescence when
exposed to light in the blue to ultraviolet range.
GFP protein is coded by gfp reporter gene.
27. USE OF GFP TO STUDY THE TRICHODERMA-PATHOGEN-
PLANT INTERACTION
Trichoderma atroviridae strains are use as biocontrol
(biofungicide) agent for Rhizoctonia solani.
The antagonistic activity is attributed by nutrient competition,
antibiosis, the activity of cell wall lytic enzymes.
To study the antagonistic activity of Trichoderma we use gfp
activity.
For this experiment they use:
gfp tagged mutants of T. atroviride.
28. MYCOPARASITISM BY T.ATROVIRIDE IN COCULTURE
(A)T. atroviride hyphae grew towards the host hyphae.
(B)T. atroviride hyphae aligned with R. solani hyphae.
(C) R.solani broken hyphae during attack by T.
atroviride. This mode of attack was not observed with
the other fungal host.
29. (d) T. atroviride hyphae grew alongside with hyphae &
branched towards adjacent hyphae.
(e) T. atroviride spores adhered to the host hyphae &
germinated after attachment.
(f) The young T. atroviride hyphae parasitized the host
mycelium & also the T. atroviride hyphal tips swelled and form
papilla like structures.