DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
Organogenesis and somatic embryogenesis - In vitro mutant selection for bioti...Jyoti Prakash Sahoo
1. Direct embryogenesis
In direct somatic embryogenesis, the embryo is formed directly from a cell or small group of cells without the production of an intervening callus.
2. Indirect embryogenesis
In indirect somatic embryogenesis, callus is first produced from the explant.
Embryos can then be produced from the callus tissue or from a cell suspension produced from that callus.
DNA construct instability in bacteria used for Agrobacterium mediated plant t...iosrjce
The use of plasmid in the production of genetically modified (GM) crops is highly essential in
research and in commercial production of GM plants. However plasmid instability constitutes a major problem
in the use of recombined microorganisms in the production of GM crops. In this study we evaluated the stability
of p8114 carrying a gene coding for a transcription factor (TFIIIA) driven by Cassava Vein Mosaic Virus
(CsVMV) promoter and an nptII selectable marker driven by 35S promoter in the T-DNA. The plasmid was
amplified in E.coliDH5α strain on Luria Broth (LB)agar supplemented with 100 µg/ml kanamycin. The colonies
were confirmed by Restriction Fragment Length Analysis (RFLA) and by DNA sequencing. The confirmed
colonies were stored as glycerol stock at -80
0C and as DNA extracts in TE buffer at 40C. Agrobacterium strains
LBA4404, EHA 105 and AGL1 were also transformed with DNA from the confirmed colonies. Plasmid stability
was evaluated after 3 months. Sixteen to hundred percent level of instability was observed in E.colicolonies
stored at -80
0C and 50% level of instability in plasmid transformed into Agrobacterium strain LBA4404.
Agrobacterium strain LBA4404 showed a higher level of stability 75% compared to EHA 105 (0%) and AGL1 (50%).
Organogenesis and somatic embryogenesis - In vitro mutant selection for bioti...Jyoti Prakash Sahoo
1. Direct embryogenesis
In direct somatic embryogenesis, the embryo is formed directly from a cell or small group of cells without the production of an intervening callus.
2. Indirect embryogenesis
In indirect somatic embryogenesis, callus is first produced from the explant.
Embryos can then be produced from the callus tissue or from a cell suspension produced from that callus.
RNAi-DIRECTED SILENCING OF POTENT STRESS TOLERANT GENE(S) AND ITS EFFECT ON S...Indrani Baruah
Determination of role of potential stress tolerance genes by RNAi directed silencing with special outline for betaine aldehyde dehydrogenase gene in rice
abiotic stress is imposing heavy loses in crop production. management practices cannot be a permanent solution for this problem. development of varieties with stress tolerance is an cost effective way to overcome abiotic stresses. for that studying the genetic basis is necessary
this presentation intends to familiarize students with the basic concept of male sterility. this is deemed essential to proceed with the cytoplasmic male sterility.
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
• Gene optimization: genes synthesis, vector construction
• Vector construction
• Screening of expression strains
• Small-scale expression
• Protein expression optimization
• Large-scale expression and purification
• Protein folding, modification, renaturation, endotoxin detection and removal
• Protein identification
https://www.creative-biolabs.com/vaccine/virus-like-particles-based-vaccines.htm
In immunology, an antigen is a molecule capable of inducing an immune response in the host organism. High-quality antigens for vaccine development solution are supposed to cover viral antigens, parasitic antigens, bacterial antigens, tumor antigens and so on. https://www.creative-biolabs.com/vaccine/category-antigens-1.htm
Inability of flowering plants to produce functional pollen.
Male sterility is agronomically important for the hybrid seed production.
Onion crop provides one of the rare examples of very early recognition of male sterility cultivar Italian Red (Jones and Emsweller 1936)
Its inheritance and use in hybrid seed production (Jones
and Clarke 1943).
Since then male sterility is reported in a fairly large number of crops including vegetables.
A number of developments have been made in the molecular biology of oat (Avena spp.) in recent years. Many of these were recently described at the Fourth International Oat Conference, held on 18 to 23 October, in Adelaide, South Australia. These advances include a report of oat transformation and regeneration, the characterisation of J3-glucanase genes in oat, the further development of a molecular genetic map in oats, and the characterisation of genes encoding novel oat grain proteins. A technique for assessing pedigrees in the oat and other cereal crops has been reported using a modified electrophoretic technique.
RNAi-DIRECTED SILENCING OF POTENT STRESS TOLERANT GENE(S) AND ITS EFFECT ON S...Indrani Baruah
Determination of role of potential stress tolerance genes by RNAi directed silencing with special outline for betaine aldehyde dehydrogenase gene in rice
abiotic stress is imposing heavy loses in crop production. management practices cannot be a permanent solution for this problem. development of varieties with stress tolerance is an cost effective way to overcome abiotic stresses. for that studying the genetic basis is necessary
this presentation intends to familiarize students with the basic concept of male sterility. this is deemed essential to proceed with the cytoplasmic male sterility.
This PPT has described how to produce soluble anf high amount of recombinant protein in E.coli host. This PPT has mentioned different expression vectors, different E.coli Expression host strain and other strategies for getting high expression of desired gene.
• Gene optimization: genes synthesis, vector construction
• Vector construction
• Screening of expression strains
• Small-scale expression
• Protein expression optimization
• Large-scale expression and purification
• Protein folding, modification, renaturation, endotoxin detection and removal
• Protein identification
https://www.creative-biolabs.com/vaccine/virus-like-particles-based-vaccines.htm
In immunology, an antigen is a molecule capable of inducing an immune response in the host organism. High-quality antigens for vaccine development solution are supposed to cover viral antigens, parasitic antigens, bacterial antigens, tumor antigens and so on. https://www.creative-biolabs.com/vaccine/category-antigens-1.htm
Inability of flowering plants to produce functional pollen.
Male sterility is agronomically important for the hybrid seed production.
Onion crop provides one of the rare examples of very early recognition of male sterility cultivar Italian Red (Jones and Emsweller 1936)
Its inheritance and use in hybrid seed production (Jones
and Clarke 1943).
Since then male sterility is reported in a fairly large number of crops including vegetables.
A number of developments have been made in the molecular biology of oat (Avena spp.) in recent years. Many of these were recently described at the Fourth International Oat Conference, held on 18 to 23 October, in Adelaide, South Australia. These advances include a report of oat transformation and regeneration, the characterisation of J3-glucanase genes in oat, the further development of a molecular genetic map in oats, and the characterisation of genes encoding novel oat grain proteins. A technique for assessing pedigrees in the oat and other cereal crops has been reported using a modified electrophoretic technique.
Cellular signal transduction pathways under abiotic stressSenthil Natesan
Abiotic stresses, especially cold, salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Plants have stress-specific adaptive responses as well as responses which protect the plants from more than one environmental stress. There are multiple stress perception and signaling pathways, some of which are specific, but others may cross-talk at various steps (Knight & knight ,2001).Many cold induced pathways are activated to protect plants from deleterious effects of cold stress, but till date, most studied pathway is ICE-CBF-COR signaling pathway (Miura and Furumoto,2013 ) . The Salt-Overly-Sensitive (SOS) pathway, identified through isolation and study of the sos1, sos2, and sos3 mutants, is essential for maintaining favorable ion ratios in the cytoplasm and for tolerance of salt stress (shi .et al ,2002). Both ABA-dependent and -independent signaling pathways appear to be involved in osmotic stress tolerance (Nakashima and shinozaki, 2013) .ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules and the ROS signaling networks can control growth, development, and stress response ( Mahajan,s and Tuteja, 2005) .
1 Objectives Genetically transform bacteria with for.docxmercysuttle
1
Objectives
Genetically transform bacteria with
foreign DNA and induce
expression of genes encoded on
DNA to produce novel
Isolate chromosomal DNA from
Introduction
In this portion of the lab, you will perform a
procedure known as genetic
transformation. that a gene is
a piece of DNA that provides the
instructions for making (codes for) a
protein. This gives an organism a
particular trait. Genetic transformation
literally means change caused by genes,
involves the insertion of a gene into an
organism in order to change the organism’s
trait. transformation is used in
many areas of biotechnology. In
agriculture, genes coding for traits such as
pest, or spoilage resistance can be
genetically transformed into plants. In
bioremediation, bacteria can genetically
transformed with genes enabling them to
digest oil spills. In medicine, diseases
caused defective genes are beginning
to be treated by gene therapy; that is, by
genetically transforming a person’s
cells with healthy copies of the defective
gene that causes the
You will use a procedure to transform
bacteria with a gene that codes for Green
Fluorescent (GFP). The real-life
source of this gene is the bioluminescent
jellyfish Aequorea victoria.
Fluorescent Protein causes the jellyfish to
fluoresce and glow in the dark.
LAB TOPIC 10: Nucleic Acids and Genetic Transformation
Following the procedure,
the bacteria express their newly acquired
jellyfish gene and produce the fluorescent
which causes them to glow a
brilliant green color under ultraviolet
In this activity, you will learn about the
process of moving genes from one organism
to another with aid of a plasmid. In
nature, bacteria can transfer plasmids back
and forth allowing them to share
beneficial genes. This natural mechanism
allows bacteria to adapt to new
environments. The occurrence of
bacterial resistance to is due to
the transmission of
Genetic transformation involves
insertion of some new DNA into the E.
cells. In addition to one large
bacteria often contain one or more
circular pieces of DNA called
Plasmid DNA usually contains genes for
than one trait. Scientists can use a
called genetic engineering to insert
coding for new traits into a plasmid.
In case, the pGLO plasmid carries the
GFP that codes for the green
fluorescent protein and a gene (bla) that
codes for a protein that gives the
resistance to an antibiotic. The genetically
engineered plasmid can then be used to
genetically bacteria to give them
this new
Figure 10.1 Bacterial cell undergoing genetic transformation with the pGLO
plasmid
Exercise 10.1
Bacterial Transformation
2
Pre-lab exercises:
Since scientific laboratory investigations
are designed to get information about a
question, our first might be to
formulate some questions for this
Can we genetically transform an organism?
Which organism is
1. To genetically tra ...
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdfamzonknr
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile el...Kalyani Rajalingham
Comparative mitochondrial zygomycetes: bacterial-like Rnase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperm,
By
Kalyani Rajalingham
Somayeh Haji Kazem Nili
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
1. Unlike the warm climates
that rule Asian countries,
North America is at the
mercy of harsh climates,
and frost. Productivity, and
yield is as such limited not
only by the crop's
production capacity, but
also temporal factors. In
particular, major crops
such as potatoes – freezing
sensitive – are cultivated in
cold climates which
substantially affects yield.
Between impossibility, and
actuality lies the potential
to genetically alter crops to
serve a defined function.
Where traditional breeding
has failed, biotechnology
has offered much potential
in the generation of crops
that are freezing tolerant.
Typically, this is realized
by conferring a gene -
AtCBF - from an organism
to a selected crop species.
AtCBF1-3, the CBF genes
found in Arabidopsis
thaliana, regulate the
activation of a myriad of
genes responsible for
freezing tolerance. In
particular, CBF genes
(AtCBF1, AtCBF2, and
AtCBF3), a transcription
factor found in Arabidopsis
thaliana, ciphers proteins
that bestows freezing
tolerance upon the plant
via induction of COR (cold
responsive genes).
Constitutive expression of
AtCBF was however found
to be associated with
adverse side effects.
Introduction of a transgene
is at times insufficient for
both resistance, and
productivity. The first
potential solution is to
inspect alternative
transgenes; the second
solution requires
modifications to the
existing system. In this
2. issue, Pino et al., (2007)
replaced a constitutive
promoter by a stress
inducible promoter to
enable production of the
transgene only under
stressful conditions. By
substituting a constitutive
CaMV 35S promoter with
a stress inducible rd29A
promoter, the authors
manage to bestow upon
Solanum tuberosum both
resistance, and
productivity.
It has been noted that
constitutive expression of
AtCBF – that confer
freezing resistance – leads
to retarded growth,
lowered biomass/foliar
mass, late flowering, and
abolished tuber formation.
The simplest solution to
this problem would be to
substitute the current
transgene with another.
However, it is quite
probable that the novel
transgene will result in the
same problem. In fact,
Kasuga et al. (1999)
utilized the DRE-binding
protein DREB1A, and the
CaMV 35S constitutive
promoter to confer
resistance to freezing,
drought, and salt stresses.
However, constitutive
expression of the transgene
resulted in growth
retardation as well. The
alternative is to modify the
existing system; in this
case, the latter is done by
inducing the transgene
only when required or
under cold stress. In fact, it
has been shown multiple
times that a transgene
associated with a stress
inducible promoter
compared to a constitutive
promoter possess fewer
negative traits such as low
yield, or biomass.
Replacing a constitutive
promoter with a stress-
inducible promoter results
in transgenic Solanum
tuberosum plants that are
both highly productive,
and resistant.
The experiments
conducted by Pino et al.,
(2007) are akin to those
conducted by Kasuga et al.
(1999). Kasuga et al.
(1999) attempted to
compare the phenotype of
the 35S:DREB1A, and the
rd29A:DREB1A lines.
They showed that the
35S:DREB1A line had
fewer seeds, and showed
stunted growth. The
rd29A:DREB1A lines had
mild growth retardation.
Further, Kasuga et al.
(1999) showed that 96.2%
of the rd29A:DREB1A
lines, and 77.9% of the
35S:DREB1A lines
survived after exposure to
cold temperatures. Oddly
enough, the
rd29A:DREB1A lines
outperformed the
35S:DREB1A lines for
many a stress (drought
stress:
35S:DREB1A=39.7-69.2%
survival,
rd29A:DREB1A=76.7%
survival; salinity:
35S:DREB1A=29.4%
survival,
rd29A:DREB1A=78.6%
survival).
Much like Kasuga et al.
(1999), Pino et al., (2007)
altered the genomic unit by
replacing the constitutive
CaMV 35S promoter with
the rd29A stress inducible
promoter. Constitutive
over-expression of AtCBF
is responsible for the side
effects; use of a stress
inducible promoter such as
rd29A can reduce adverse
side effects. Following
substitution of the
constitutive CaMV 35S
promoter for the stress
inducible rd29A promoter,
and transformation using
agrobacterium into S.
tuberosum cv. Umatilla,
explants – derived from
transformed plants – were
propagated in vitro. Tissue
culture was utilized to
regenerate the callus.
Relative to the 35S:AtCBF
lines (constitutive
promoter), the
prd29A:AtCBF lines
showed higher plant, and
tuber mass. Foliar biomass
in the prd29A:AtCBF lines
3. were unaffected by the
construct.
The paper by Pino et al.,
(2007) implies that
transgenic systems can be
further altered to obtain
desired features instead of
resorting to novel
transgenes. This also
implies that transgenes
alone do not control the
system, but rather that by
modifying a subsection of
a unit (that controls
transcription/translation),
one can tweak the plant’s
genome to perform in a
particular manner. In other
words, this also implies
that when generating
transgenic plants,
introduction of a novel
gene or alteration of
existing genes is one way
of modifying the system;
however, modifications
can be carried out on the
promoter, and the polyA
signal amongst others. In
this paper, the authors
attempt to modify the
promoter in addition to the
transgene, however, it is
quite possible that with
further modifications, this
system can be tweaked.
For instance, it is quite
well known that there
exists a negative
relationship between
resistance, and growth. The
Resource allocation theory
states that due to a
limitation in the resources
available, the plant must
partition said resource
between resistance, or
growth. In such cases, the
activation of a particular
gene under a particular set
of circumstances would
enable the plant to bypass
this limitation. If such is
the case, then one might
add that reducing energy
expended for production of
proteins might aid the
plant. In other words, the
turn-over rate, the efficacy
of the transgene, and the
time of production would
affect the phenotype of the
plant.
References
Kasuga, M., Liu, Q.,
Miura, S., Yamaguchi-
Shinozaki, K., and
Shinozaki, K. (1999).
Improving plant drought,
salt, and freezing tolerance
by gene transfer of a single
stress-inducible
transcription factor. Nature
Biotechnology 17, 287-
291.
Pino, M., Skinner, J., Park,
E., Jeknić, Z., Hayes, P.,
Thomashow, M., and
Chen, T. (2007). Use of a
stress inducible promoter
to drive ectopic AtCBF
expression improves potato
freezing tolerance while
minimizing negative
effects on tuber yield. Plant
Biotechnology J 5, 591-
604.