expression analysis of dc antifreeze protein during...

CHAPTER 3

EXPRESSION ANALYSIS OF Dc ANTIFREEZE PROTEIN DURING

COLD STRESS BY SEMI-QUANTITATIVE REVERSE TRANSCRIPTASE

PCR (sq RT-PCR) AND QUANTITATIVE REAL TIME PCR (q RT-PCR)

3.0 ABSTRACT

The change in environmental temperature activates the array of genes that helps

the plants to maintain the cellular and physiological functions. The transcript abundance

of the AFP in carrot plants was investigated by semi-quantitative reverse transcriptase

PCR (sq RT-PCR) and quantitative Real Time PCR (q RT-PCR). The sq RT-PCR

revealed that the AFP gene is cold induced as transcript accumulation increased along

with the stress time. The expression analysis by q RT-PCR revealed that, the gene

expression increased 380 fold after 24 h of cold stress. The results of sq RT-PCR and q

RT PCR revealed that the carrot AFP could be an ideal candidate for producing cold

tolerant transgenic plants.

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3.1 INTRODUCTION

The fluorescence based reverse transcription polymerase chain reaction (RT-PCR)

is one of the very useful tools in the molecular biology for the detection of gene

expression (Bustin 2000). There are many factors, which contributed to the success of the

Real Time PCR due to the following advantages: (i) The use of fluorescent dye molecules

to monitor the exponential amplification of products during each cycle of the reaction and

the combination of both DNA amplification and detection steps into one homogeneous

assay obviates the requirement for post PCR methods (ii) a wide (>107 fold) dynamic

range allows easy comparison among different RNA molecules that differ in their

abundance widely (iii) the assay depends on the inherent quantitative potential of the

PCR, making it both quantitative and qualitative assay and (iv) The internal variation in

the real time assay is very small and hence, it helps to generate reliable and reproducible

results. The high-throughput reactions, together with recent introduction of novel dye

chemistries, reliable instrumentation and improved protocols have lead the development

of Real Time PCR based detection assays (Nashed et al., 2003; Bianchi 2004;

Niesters 2004). The q RT-PCR extensively used in the studies of functional genomics,

molecular medicine, forensics, virology and biotechnology.

3.1.1 Quantitative Real Time PCR (q RT-PCR)- The main principle of q RT-PCR is

relatively simple, following the Reverse Transcription of RNA into cDNA, it requires a

specific detection (dye chemistry) to report the amplification and presence of PCR

products, an instrument to monitor the amplification and appropriate software for

quantitative analysis (Wittwer et al., 1997). The q RT-PCR is characterized by, the point


in time during reaction step when amplification of a PCR product is first detected.

The higher the starting abundance of the target cDNA, the sooner a significant increase in

fluorescence signal occurs. The detection method can be specific or non-specific based

on the chemistry of the dye used for the reaction. The most widely used non-probe based

chemistry monitors and detects the binding of SYBR Green I to double stranded DNA

molecules (Morrison et al., 1998). In the free solution, the unbound dye exhibits little

fluorescence, but during the PCR, increasing amounts of dye binds to the double stranded

DNA/cDNA molecules. This results in an increase in the fluorescence signal as the

polymerization proceeds due to the exponential amplification of target molecule.

The PCR product can be confirmed by plotting fluorescence as a function of temperature

to generate a melting curve of the amplicon (Ririe et al., 1997). One of the main

advantages of SYBR Green I based chemistry is that, in most cases an optimized

conventional semi- quantitative PCR assays can be converted immediately into real time

assays. But the major drawback is their specificity remains dependent on the specificity

of the primers used for the assay. Unlike SYBR Green I, the probe based (specific)

chemistries make use of amplicon specific fluorescent probes and fluorescent signal is

produced only if the probe hybridizes with its complementary cDNA/DNA. Hence, probe

based dye (Taqman) introduce an additional level of specificity, in effect combining

RT-PCR with a validation step, previously carried out separately after the PCR.

3.1.2 Quantification Methods for the q RT-PCR- The quantification of the gene

expression or copy number can be either relative to an external standard curve or to one or

more co-amplified internal control mRNAs using normalisation reaction (Bustin 2000).


The absolute quantification is based on the use of a dilution series of an external standard,

which can be used to generate a standard curve of Ct (threshold cycle) against initial

target copy number (Ke et al., 2000). The copy number of unknown samples can be

determined from the linear regression of that standard curve, with the y-intercept giving

the sensitivity and the slope giving the amplification efficiency. The relative quantification to

internal standards compares the Ct values from target RNAs to those of one or more

internal reference genes and data are expressed as ratios of the target specific signal to the

internal reference. This gives a relative value for the target specific RNA products that

can be compared between samples and allows an estimate of the relative expression of

target mRNA in the specific samples. It is vital that, the PCR amplification efficiencies of

both the target and reference are similar, as this affects the accuracy of the results.

There are many models that use different algorithms for efficiency and claim to allow a

more reliable estimation of the real expression ratio (Pfaffl 2001; Liu & Saint 2002).

The expression of most reference genes is regulated and their levels vary significantly

with treatment or between individual samples. Furthermore, if the relative levels of the

reference and target RNAs vary by orders of magnitude, during amplification the former

may have entered its plateau phase by the time a Ct for the target becomes apparent.

Unless compensated, this may interfere with the precise quantification of the target RNA.

Therefore choosing specific reference samples is very critical for the real time reaction.

Besides being extremely powerful tool to quantify the gene expression, real time

PCR suffers from certain pitfalls, the main one being the normalization with a reference

gene. The expression profile of the reference gene/genes used for normalization reaction


in q RT-PCR analysis should remain constant between the cells of different tissues and

under different experimental conditions, or else it can mislead the results. The different

reference genes involved in basic cellular processes like18S rRNA, Ubiquitin, β-Actin

and Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) were used as internal

controls for the gene expression analysis, as they were presumed to have a uniform

expression. However, several reports revealed that the transcript levels of these genes

also vary considerably under different experimental conditions (Thellin et al., 1999;

Suzuki et al., 2000; Czechowski et al., 2005; Lee et al., 2010) and are considered

unsuitable for gene expression studies. Several statistical algorithms such as geNorm and

BestKeeper have been developed for the evaluation of best-suited reference gene(s) for

normalization of q RT- PCR data in a given set of biological samples. Recently, novel

reference genes were identified by genome wide analysis of whole-genome genechip data

for transcript normalization in Arabidopsis (Czechowski et al., 2005). Jain et al., (2006)

reported eukaryotic elongation factor 1-α (eEF1 α) was most stable across all the tissue

samples of rice examined and was used for the normalisation. α actin and EF1α showed

optimal expression in evaluated transgenic Eucommia ulmoides Oliver root lines

overexpressing IPPI or FPPS1 genes involved in isoprenoid biosynthesis (Chen et al., 2010).

Among the 4 common endogenous control genes studied, in Orobanche ramosa,

Or-actin1 and Or-ubiqutin1 were the stably expressed and therefore was used as the

reference for the normalisation (Verdejo et al., 2008). Among the different reference

genes tested in Lolium temulentum for normalisation, eEF-1 α and Ubq5 were the most

stable and Act1 was the least stable among the various genes tested (Dombrowski &

Martin 2009).


q RT-PCR was used to analyse the expression of 13 different genes under stress

of low temperature, drought, NaCl and plant hormones in Ammopiptanthus mongolicus

(Cao et al., 2009). Jiang et al., (2011) studied the expression of PP2C (protein phosphatase 2 C)

in rice during different abiotic stress by qRT-PCR and microarray analysis. The expression

pattern of COR15b and KIN1 genes in watermelon and pumpkin were determined by

qRT-PCR and was found that the expression increased during low temperature stress

(Guozhang et al., 2009). The transcript levels of Camellia sinensis COR1 was induced

after 7h of cold treatment, reached a maximum of 154 fold after 24h of low temperature

and then declined to 107-fold at 48h (Li et al., 2010).


3.2 MATERIALS AND METHODS

3.2.1 Plant Material and Growth Conditions- The seeds of D. carota were sowed

(in commercial soil mix in pots) in the growth chamber in the Laboratory of

Biotechnology and Plant Breeding, University of Evora, Portugal. The germinated plants

were maintained in controlled conditions at 25°C with, 75% humidity, 16/8h light/dark

cycle. The plants were nourished with 1/10 strength sterile MS salt solution.

3.2.2 Cold Stress Treatment- One month old carrot plants were exposed to 4°C for 24h

in a cooling incubator. Control samples were harvested immediately before giving the

cold stress treatment. The samples were collected after 10 min, 15 min, 4h, 6h and 24h of

stress. The plants were then transferred back to the normal conditions. Leaf samples were

also harvested after 24h and 48h after transferring the plants to the normal conditions

(recovery period). All the samples were immediately ground well to a fine powder with

liquid nitrogen and transferred to RNase DNase free microfuge tube and stored in -80°C

freezer until further processing.

For another experiment, one-month-old plants were also given stress for 6 days as

mentioned above. Samples were harvested after every 24h. All the samples were used for

sq RT- PCR.

3.2.3 RNA Isolation and DNase Treatment- The RNA were isolated from all the

samples using Qiagen RNase easy isolation Kit (Qiagen, Hilden, Germany) and on

column DNase treatment was performed using RNase-free DNase (Qiagen, Hilden,

Germany) according to manufacturer‘s instruction. The same protocol was followed as

mentioned in Chapter 2 2.2.2.1.


3.2.3.1 Quantification and Electrophoresis of RNA - The yield of the isolated RNA was

determined in Thermo scientific Nanodrop 2000c spectrophotometer (Wilmington, USA).

The 260/280 ratio was also determined. The integrity of RNA was checked in denaturing

agarose gel.

3.2.3.2 Synthesis of First Strand cDNA- The first strand cDNA was synthesised using

RETROscript® Kit, (Ambion Inc, USA). Same amount of RNA (2 μg) from all the

samples were used for the synthesis of cDNA. The reaction mix was made as follows in a

nuclease free PCR tube:

Total RNA- 2 μg

Oligo dT primer- 2μL

Nuclease free water was added to make the final volume to 12μL.

The samples were given a short spin and incubated at 72°C for 3 min to remove the

secondary structure of RNA and after the incubation period, the samples was immediately

placed in ice. Simultaneously a master mix was made with the following components:

RT PCR Buffer 10X - 2μL

dNTP mix (mM) - 4μL

RNase Inhibitor (1000U) - 1μL

MMLV RT (2000U) - 1μL

The master mix was mixed well and 8μL of mix was added to each tube.

The tubes were given a short spin and reverse transcription was performed at 42°C for 1h.

The enzyme was inactivated by incubating the samples at 92°C for 5 min. Aliquots of

5μL cDNAs were made and stored in -20°C freezer in nuclease free tube.


3.2.4 Sq RT-PCR- The cDNA was synthesised as mentioned above from all the samples

(0h to 6 days stress). cDNA (2μl) diluted 1:5 using nuclease water was used as template

for PCR using Dc AFP and Dc EF1α primers (table 3.1). PCR was performed using

Ready to Go PCR beads (GE healthcare, NJ, USA).

3.2.5 q RT-PCR- The transcript accumulation in different samples of carrot plants was

determined by qRT-PCR. Gene specific primers for Dc AFP were used for the q RT-

PCR. Two reference genes, Dc eukaryotic elongation factor 1 α (eEF1 α) and Dc β-

tubulin were used for normalization reaction.

3.2.5.1 Primer Designing for Quantitative RT-PCR- The primers for the Dc AFP,

Dc eEF1 α and Dc β- tubulin were designed using the Primer Express Software ®,

Applied Biosystems 7500 Real Time PCR machine. The primers used for the reaction are

given in the following table 3.1:

Sl. No Gene Primers Synthesized

01 Dc AFP Fw: 5‘- CGA CAA GCA AGC TTT ACT CCA A -3‘

Rev: 5‘- CGT CTG ACA CCC ATG AG TCT GT -3‘

02 Dc eEF1α Fw: 5‘- TGG TGA TGC TGG TTT CGT TAA G -3‘

Rev: 5‘- ATG GGA GGG TAG GAC ATG AAG GT -3‘

03 Dc β- tubulin Fw: 5‘- GGG CTC TCT ATG TCT TCC ACA TTC -3‘

Rev: 5‘- AAA CTG TTC ACT AAC TCG TCG AAA CA -3‘

Table 3.1- The Sequence of Primers used for q RT-PCR Expression Analysis

3.2.5.2 Optimization of Primer Concentration- The reactions were performed in

Applied Biosystems Real Time PCR 7500 (ABI PRISM® 7500, Foster City, CA) Sequence


Detection System "SDS". Expression stability of two-reference gene eEF1α and

β- tubulin was also studied. Three different primer concentrations 300nm, 500nm and

900nm were used to optimise the reaction conditions.

3.2.5.3 AFP Expression Analysis in Carrot- The cDNA from all the samples were

diluted 1:5 ratio in nuclease free water. Each reaction mix of 15μL contained 1.5μL of

forward and reverse primers, 7.5μL MaximaTM

SYBR Green/ROX qPCR 2X master mix

(Fermentas Inc., Maryland, USA), 2μL of cDNA and 2.5μL of nuclease free water.

Two separate reactions were carried out, one using eEF1α primers for normalisation and

other with AFP specific primers.

3.2.5.4 Standard for q RT-PCR- For each gene, standard cDNAs was amplified along

with sample cDNAs in the same PCR run. The standard for the experiments were made

as follows, The cDNA from different samples were pooled and 10μL was aliquoted in a

tube and the cDNA mix was taken in another tube with 40μL of nuclease free water.

Four further serial dilutions were made and were used as standard cDNA.

3.2.5.5 Loading the Master Mix and cDNA in Microwell Plate- The 96 well plate from

Applied Biosystems (MicroAmp® Optical 96-well reaction plate, Applied Biosystems, USA)

was used for loading the reaction mix. The master mix (13μL) was added to each well in the 96

well plate. cDNA (2μL) was then added to all the wells and for each sample, a duplicate was

made. A non-template control (without cDNA) was made for both eEF1α and AFP. The

samples were mixed well and the plate was sealed with adhesive films (MicroAmp® Optical

Adhesive Film, Applied Biosystems, USA). The PCR condition was as follows, an initial

denaturation at 95°C for 10 min (2 min 50°C initial step was followed by a 10 min 95°C step to

activate the polymerase) followed by 40 cycles of denaturation at 95°C for 15s and


annealing/extension at 60°C for 1 min. After the amplification for 40 cycles, a dissociation

reaction was also performed in order to ascertain that only a single product was amplified and

the Applied Biosystems software were used for melting curve analysis. Each reaction was

repeated minimum of two times.

3.2.5.6 Calculation of the Ct Values- The experiments were analyzed with auto baseline and

manual threshold chosen from the exponential phase of the PCR amplification.

The comparative Ct method (using the 2-ΔΔCt

method) uses the arithmetic formula to calculate

the gene expression. The data obtained was presented as fold change in transcript level with

reference to the control (calibrator) and was calculated according to 2-ΔΔCt

method.

The relative transcript level of the target genes was normalized to the internal control gene.

The results were analyzed using ABI PRISM® SDS software (version 2.0) and calculated

using the comparative threshold cycle Ct method according to the manufacturer‘s instructions

for data normalization. The Ct values of the target were subtracted from the Ct values of

calibrator to determine the ΔΔCt. The amount of target, normalized to an endogenous

reference and relative to a calibrator, is given by 2-ΔΔCt

. For the ΔΔCt calculation to be valid,

the efficiency of the target amplification and the efficiency of the reference amplification

must be approximately equal. The amplification efficiency was calculated from the slope of

the standard curve.

2-ΔΔCt

= relative expression

ΔCt (sample) = Ct (AFP) – Ct (EF1 α)

ΔΔCt = ΔCt (sample) –ΔCt (calibrator)


3.3 RESULTS AND DISCUSSION

3.3.1 RNA Isolation and cDNA Synthesis- The RNA from all the samples were

quantified (table 3.2) and the integrity of the RNA were checked in denaturing agarose

gel (fig. 3.1). The intact bands of 28S rRNA and 18S rRNA were seen along with the low

molecular weight rRNA.

Sl. No Sample at different

time point of stress

RNA concentration

(ng/µL) 260/280 ratio

01 Control 921.4 2.11

02 10 min 900.5 2.12

03 15 min 1572.4 2.14

04 4 hours 1263 2.08

04 6 hours 1649.2 2.14

05 24 hours 190.3 2.09

06 24 hour recovery 279.7 2.11

07 48 hour recovery 426.5 2.1

Table 3.2- The Yield of Total RNA from Carrot Leaf Quantified using Nanodrop

Quantification of RNA in the samples is very important, as it is a must to use

same amounts of RNA for cDNA synthesis for the comparative study of gene

expression/transcripts. The presence of plant genomic DNA is challenging for q RT-PCR

as the genomic DNA can potentially be co-amplified during the PCR reaction leading to

erroneous interpretation. It is also important to test for genomic DNA contamination and

to record the threshold cut-off criteria for the amounts of genomic DNA contamination

that are tolerable. Hence the DNase treatment was essential before synthesising the


cDNA. The overall yield of RNA was little reduced after the treating the samples with

DNase. In order to check the presence of genomic DNA in the RNA sample, the RNA

was used as template for PCR using eEF1α primers and the PCR was performed using

PCR beads. Synthesis of cDNA from the isolated RNA was only performed, when PCR

was negative which indicated that the genomic DNA was absent in the RNA sample.

Reports say, 3 different primer sets were used to check the genomic DNA contamination

in RNA samples from rice samples before q RT-PCR (Caldan et al., 2007).

Figure 3.1- Total RNA was Resolved in 1.2% Denaturing Gel using 1 X MOPS Buffer

Lane 1- Control sample, Lane 2- 10 min stress, Lane 3- 15 min stress,

Lane 4- 4h stress, Lane 5- 6h stress, Lane 6- 24h stress

Lane 7- 24h recovery sample, Lane 8- 48h recovery sample

The reverse-transcription step for the synthesis of first strand cDNA introduces

substantial variation into q RT-PCR assay (Stahlberg et al., 2004). The reverse transcription

step was carried out in duplicate and that the total RNA concentration used for the

synthesis of cDNA was made constant in every sample. Karrer et al., (1995) reported that

the efficiency of conversion of RNA to cDNA depend on template abundance.

The cDNA synthesis is significantly lower when target templates are less. RNA (2μg)

was used for the synthesis of cDNA from all the samples in the present study. cDNA can

be synthesised by using either random primers/oligo-dT or gene-specific primers.

1 2 3 4 5 6 7 8

120

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The majority of cDNA is from ribosomal RNA derived, when random hexamer used.

If the target mRNA is present at low levels, it may not be primed proportionately and hence

the subsequent amplification process may not be quantitative (Zhang & Byrne 1999). Hence,

oligo dT primers were used to synthesise the cDNA. Bustin et al., (2005) reported that,

oligo dT is an ideal choice to obtain a faithful cDNA population from the mRNA pool.

The two most commonly used reverse transcriptase enzymes are from Avian

Myeloblastosis Virus (AMV) and Moloney Murine Leukaemia Virus (M-MLV).

The M-MLV-reverse transcriptase enzyme has significantly less RNAse H activity than

AMV-RT (Gerard et al., 1997). As it was reported, reduced RNAse H activity can

interfere with the synthesis of long amplicons (De Stefano et al., 1991), M-MLV-RT

along with oligo dT will be the best combination of reagents for first strand synthesis to

obtain full-length cDNA molecules. The influence of amplicon size on real-time PCR

results using artificially degraded RNA from a breast cancer cell line were studied by

Antonov et al., (2005). It was found that the amplification efficiency was more when the

amplicon size was small and vice versa.

3.3.2 sq RT- PCR- The conditions for PCR (before starting q RT-PCR) were optimised

for the primer pairs and reactions were repeated twice. The optimal length of amplicon

for qRT-PCR should be less than 100bp (Bustin et al., 1999). The shorter amplicons

amplify more efficiently than the longer one. This is because, the amplicons are more likely

to be denatured during the denaturation step of the PCR, allowing the probes and primers to

compete more effectively for binding to their complementary targets. The amplicon size of Dc

AFP, Dc eEF1α and β- tubulin were 78bp and 80bp respectively (fig. 3.2).

121


Figure 3.2- sq RT PCR of AFP, β- Tubulin and eEF1 α

Lane M- DNA marker

Lane 1- Carrot AFP, Lane 2- Carrot β- Tubulin, Lane 3- Carrot eEF1 α

The carrot cDNA was used as the template for PCR using Ready to go PCR beads and the

products were separated in 2% agarose gel.

Although a low level of AFP transcript was observed in the non-acclimated

sample, the transcript was clearly more abundant in cold treated sample. The transcript

accumulation was very high after 24h of stress at 4°C. The transcript accumulation was

slightly increased thereafter (2 day stress) and remained same until 6 days (fig. 3.3).

The expression of AFP in carrot was not altered in control experiments (without cold

treatment). The eEF1α used as a reference for the RT-PCR showed constitutive

expression pattern. The transcript accumulation of AFP was reported to be more in roots

of carrot than in stem and leaf during cold stress (Smallwood et al., 1999).

M 1 2 3

80bp

10kb

100bp


Figure 3.3- AFP Expression Analysis in Carrot During Cold Stress

Lane 1- Control sample, Lane 2- 1 day stress, Lane 3- 2 days stress,

Lane 4- 3 days stress, Lane 5- 4 days stress, Lane 6- 5 days stress

Lane 7- 6 days stress.

Total RNA was resolved in 1.4% denaturing gel using 1XMOPS buffer. Total RNA (1μg) was

loaded in each well to check the integrity. The carrot cDNA was used as the template for PCR,

using Ready to go PCR beads and the amplicons were separated in 2% agarose gel. eEF1α was

used as the reference gene for the sq RT-PCR.

The transcription factors and ice binding proteins like AFP are induced rapidly during

the early phase of the response to low temperature or freezing stress, the transcript abundance

reach maximal levels after several hours of stress and the expression either remains same or

decrease (Thomashow 2001; Yamaguchi-Shinozaki & Shinozaki 2006). Arabidopsis

CBF1 and CBF3 showed peak induction after 6h of stress and CBF2 showed peak induction

after 3h of stress treatment (Gong et al., 2002). The enhanced transcription of four different

CBF genes, VvCBF1, VvCBF2, VvCBF3 and VvCBF4 in grapevine in response to low

temperature stress was reported by Xiao et al., (2008). It was found that the expression of AFP

is induced by low temperature stress; however, it was not clear with the data from sq RT-PCR,

how many folds the expression was increased. Therefore q RT-PCR was performed to

determine the exactly when the gene is induced in response to low temperature stress.

1 2 3 4 5 6 7

RNA

AFP

eEF1α


3.3.3 q RT- PCR- The Ct is critical for precise and accurate quantification using q RT-PCR

(Higuchi et al., 1993). Fluorescence values are detected and recorded during each cycle during

the reaction. This value represents the amount of product amplified at that specific point in the

amplification reaction. If the transcript abundance is more at the beginning of the reaction, the

less number of cycles it takes to reach a point in which the fluorescent signal is initially

recorded as statistically significant (above background fluorescence) (Gibson et al., 1996).

This point is defined as the Ct and occurs during the exponential phase of amplification. Hence,

a low Ct value indicates the transcript abundance is more and vice versa.

To determine the time dependent transcript accumulation of the low temperature

induced AFP in carrot, the plants leaves were given stress at 4°C. The expression of AFP was

normalised with the expression of eEF1α. The stability of expression of β- tubulin and eEF1α

were studied. The Ct values of the β- tubulin were found to vary in carrot cDNA samples

(cold treated sample) whereas the Ct values of eEF1α were found to be stable. Hence, eEF1α

was used as the reference gene for q RT-PCR in carrot. eEF1α was top ranked as the

reference gene in tomato during Nitrogen and cold stress (Lovdal & Lillo 2009). eEF1 α and

YT521-B (YT521-B-like protein family protein) were reported as the best combination of

genes for normalisation of gene expression in perennial ryegrass (Lee et al., 2010).

q RT-PCR results revealed that AFP transcript level was very low under normal

conditions (without stress). The Ct for q RT-PCR, representing basal levels for transcript

was 32 for AFP under normal conditions, whereas the Ct for the reference gene EF1α

was 21 (table 3.2). The AFP expression rapidly increased to 3.14 fold after 15 min of

cold stress compared to the control plants. The transcript levels continued to increase and

were 386 fold after 24h of cold stress at 4°C. The transcript accumulation in the recovery

124


phase was also studied. The expression was reduced to 262 and 46 fold after 24h and 48 h

recovery period respectively (fig. 3.4). The slope of the standard curve and the regression

values were in the acceptable range (fig. 3.5). The efficiency of PCR reactions for AFP

and eEF1α were between 92-98% (table 3.3). The low transcript levels in control

conditions and the rapid induction of AFP transcripts in cold stress plants suggests that

transcription of AFP is very sensitive at low temperature and is likely to play important

role in providing cold tolerance in plants. Further, its turnover appeared to be very rapid

and the transcript accumulation remains at a high steady state level during the cold

acclimation period. The AFP transcripts in carrot was reported to be maintained at steady

state levels in leaves, stems and roots as long as they were exposed to cold and short day

(Smallwood et al., 1999). In our experiments, we found that the expression of gene was

downregulated slowly, once the plants are returned back to the normal conditions.

The present result strongly suggests that, cold induced AFP accumulation play an

important role in freezing tolerance in carrots. These results are consistent with a report

of Zhang et al., (2009) where the accumulation of LpIRI and LpIRI transcripts were

correlated with cold acclimation and increased freezing tolerance in perennial

ryegrass. In Lolium perenne, q RT-PCR analysis indicated that LpIRI-a was upregulated

approximately 40-fold while LpIRI-b was upregulated 7 fold after 1h of cold acclimation

and after one week of cold acclimation, the transcript level increased 8,000 fold for

LpIRI-a and 1,000 fold for LpIRI-b (Zhang et al., 2010). Both sq RT-PCR and q RT-PCR

results suggest that AFP is induced in response to low temperature stress in carrot. To the

best of our knowledge, this is the first study reporting the expression analysis of AFP in

carrot by q RT-PCR.


Slope -1/slope 10^-1/slope -1/slope -1 E*100 (%) R2 Gene

-3.38688 0.295257 1.97359 0.97359 97.4 0.995945 Dc EF1α

-3.55565 0.281243 1.910921 0.910921 91.1 0.994154 Dc AFP

-3.39845 0.294252 1.96903 0.96903 96.9 0.99576 Dc EF1α

-3.38729 0.295222 1.97343 0.97343 97.3 0.9961 Dc AFP

Table 3.3- The Slope of the Standard Curve and PCR Efficiency for Dc AFP and Dc

eEF1α

Figure 3.4- The Temporal Expression of AFP during Cold Treatment at 4°C in One

Month Old Carrot Plants

The relative transcript level of the target gene was normalized to the reference gene Dc EF1α.

The fold in expression was calculated by 2-ΔΔCt

.

Time

Fold

increase

126


Time point AFP

Ct 1

EF1α

Ct 1 ΔCt 1

AFP

Ct 2

EF1α

Ct 2 ΔCt 2

AFP

Ct 3

EF1α

Ct 3 ΔCt 3

AFP

Ct 4

EF1α

Ct 4 ΔCt 4

Mean

ΔCt SD SE

ΔΔCt

(ΔCt of

sample

– ΔCt of

control)

2^-

ΔΔCt

(fold

increase)

0 hour 33.22 21.71 10.51 31.77 21.63 10.14 32.2 22.08 10.12 32.79 21.93 10.86 10.40 0.418 0.209 0 1

10 min 30.82 20.79 10.03 30.57 20.36 10.21 31.22 20.9 10.32 31.14 20.88 10.26 10.20 0.299 0.149 -0.20 1.15

15 min 30.47 21.63 8.84 30.62 22.02 8.6 30.79 22.09 8.7 30.31 21.38 8.93 8.84 0.205 0.102 -1.64 3.11

4 hour 30.47 22.46 9.83 30.66 22.47 8.19 30.76 22.55 8.21 30.82 22.41 8.41 8.01 0.153 0.038 -2.20 4.60

8 hour 28.77 22.71 6.06 28.98 22.59 6.39 28.5 22.01 6.49 27.91 22.14 5.77 6.06 0.463 0.076 -4.23 18.76

24 hour 23.18 21.27 1.91 23.24 21.15 2.09 22.86 21.04 1.82 22.82 21.39 1.43 1.91 0.215 0.107 -8.59 386.68

24 hour

recovery 24.38 21.48 2.9 24.38 21.54 2.84 24.47 22.29 2.18 24.26 22.69 1.57 2.9 0.086 0.043 -8.03 262.28

48

hourrecovery 24.44 21.65 2.79 24.6 21.63 2.97 30.03 23.65 6.38 30.81 23.49 7.32 2.79 3.42 1.71 -5.54 46.60

Table 3.2 – The Ct Values of Dc AFP and Dc eEF1α are shown. The ΔCt is Calculated by Subtracting the Ct of Sample with Ct

of Reference Gene. Each Reaction is Repeated Four Times


Figure 3.5- q RT-PCR of Carrot AFP

a & b- Dissociation curve of Dc AFP and Dc eEF1α (absence of multiple peak shows

amplification of single product). c & d - Amplification plot of Dc AFP and Dc eEF1α.

e & f - Standard curve for Dc AFP and Dc eEF1α

Sample

b

e f

a

d c

Reference gene


3.4 CONCLUSION

The expression analyses revealed that AFP gene in carrot is cold induced and

transcript abundance increased as cold stress duration is prolonged. It is a fact that,

agricultural production in many parts of the world is limited due to cold stress.

Hence, higher yield in many crops could be achieved by improving the freezing

tolerance. The carrot AFP gene was isolated and genetically transformed to tobacco and

tomato in an attempt to enhance and at the same time, to study the cold tolerance

a

c


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