isolation and characterization of cold stress...

CHAPTER 2

ISOLATION AND CHARACTERIZATION OF COLD STRESS

INDUCIBLE GENES IN CARROT BY SUPPRESSION SUBTRACTIVE

HYBRIDIZATION (SSH)

2.0 ABSTRACT

Daucus carota is cultivated widely in tropical and temperate regions, but grows

best in cool climates. Suppression Subtractive Hybridization (SSH) is a PCR based

method used to selectively amplify differentially expressed cDNAs and simultaneously

suppress non-target cDNAs. A subtraction forward library was constructed using total

RNA isolated from the leaves of cold stressed carrot plants to determine the genes

upregulated during cold stress. Out of the hundreds of clones obtained, randomly selected

clones were sequenced. From these sequences, 43 promising clones were submitted to the

NCBI EST database. Sequence analyses revealed the functions of these genes, which

were related to signal transduction, osmolyte synthesis and transport, regulation of

transcription, translation and protein folding. Sq RT-PCR analyses of Dc cyclin, Dc WD

and Dc profilin showed that, the first 2 genes were upregulated, where as Dc profilin

showed constitutive expression. However, the SSH analysis showed that all the 3 genes

were upregulated, as it is a much more sensitive technique.

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2.1 INTRODUCTION

A variety of molecular biological techniques are available to study the global gene

expression levels between two mRNA populations and to identify the differentially

expressed transcripts (Munir et al., 2004). The routine techniques used for the differential

expression studies are, representational difference analysis (RDA) (Lisitsyn et al., 1993),

Differential Display RT-PCR (DD RT-PCR) (Liang & Pardee 1992), Serial Analysis of

Gene Expression (SAGE) (Velculescu et al., 1995), Microarrays (Park et al., 2004) and

Suppression Subtractive Hybridization (SSH) (Diatchenko et al., 1996).

Suppression Subtractive hybridization (SSH) is a valuable tool for identifying the

differentially regulated genes, which are important for growth and differentiation of cells.

Many subtractive hybridization techniques have been developed in the last decade and

were used to isolate many genes from different systems (Sargent & Dawid 1983;

Hedrick et al., 1984; Hara et al., 1991; Wang & Brown 1991; Hubank & Schatz 1994).

Eventhough there are some advantages for each method, many of them require tedious

procedures, more amounts of starting material and are not cost effective, thereby reducing

their overall utility. SSH is a widely used technique to separate DNA molecules that

distinguishes two closely related DNA samples. The major applications of SSH are in

cDNA subtraction and genomic DNA subtraction studies. SSH is one of the common

technique for generating subtracted cDNA or genomic DNA libraries (Lukyanov et al., 1994;

Diatchenko et al., 1996; Gurskaya et al., 1996; Akopyants et al., 1998). In SSH, the

normalization step eliminates the intermediate steps of physical separation of single

stranded and double stranded cDNA molecules and requires only single round of

hybridization reaction, which results 1,000 fold enrichment of the differentially expressed


genes (Rebrikov et al., 2000). The subtraction process is based on suppression PCR effect and

it combines subtraction and normalization reaction in a single procedure. The normalization

step in subtraction hybridisation equalizes the abundance of different cDNA fragments in a

single round of subtraction (Diatchenko et al., 1996; Gurskaya et al., 1996; Jin et al., 1997).

However, the enrichment level of a particular cDNA depends on factors like, the original

abundance of particular cDNA, the ratio of its concentration in the samples used for

subtraction and the concentration of other differentially expressed cDNAs.

2.1.1 Principle of SSH- As the name indicates, the process includes two different

techniques in the isolation of uniquely expressed genes. The cDNA population in which

specific transcripts are to be found is called tester cDNA for the subtraction reaction and

the reference cDNA sample population for the subtraction reaction is called driver cDNA

(Diatchenko et al., 1996). The tester cDNAs and the driver cDNAs are digested with a

4 base cutters like RsaI or AluI to make blunt end cDNAs (Rebrikov et al., 2000).

The tester cDNA is then subdivided into two fractions (1 and 2) and each one is separately

ligated to different double stranded adapter molecules (adapters 1 and 2R respectively).

The ends of the synthetic oligonucleotides are designed in such a way that, they are not

phosphorylated at the 5‘end, so only one strand of each adapter becomes covalently

attached to the 5' end of the cDNAs. The samples are then heat denatured and allowed to

anneal with denatured driver cDNA population. During the first hybridization step, the

subset of single stranded tester molecules is normalized and hence concentrations of high

and low abundance transcripts become roughly equal. The normalization process occurs

because the annealing process generating homohybrid and heterohybrid cDNAs are more

rapid for more abundant molecules, (due to the second order of hybridization kinetics)


than less abundant cDNA, which remains single, stranded. By controlling the extent of

the hybridization reaction, the single stranded forms of highly abundant cDNAs can be

reduced to the same levels as those of less abundant cDNA populations, thereby

normalizing the representation of tester cDNA population (Hames & Higgins 1985).

At the same time, the population of type A molecules (fig. 2.1) is significantly enriched

for differentially expressed cDNA population, because it is common for tester and driver

samples non-target cDNAs known as type C molecules with the driver cDNA. During the

second step of hybridization, the two samples from the first set of hybridization reaction

are mixed and annealed further with additional freshly denatured driver. Under these

conditions, only single stranded A type tester cDNAs are able to re-associate and form

(B), (C), and new (E) hybrids. Type E hybrids are double stranded (ds) tracer molecules

with different ss ends, one of which corresponds to Adapter1 and another to Adapter 2R.

The entire cDNA population is then subjected to two rounds of PCR to selectively

amplify the differentially expressed sequences (Rebrikov et al., 2000). The first PCR is

performed with adapter specific primers. This ensures that those cDNAs with both the

adapter sequences are only amplified. Type A and D cDNA population lack primer

annealing sites and cannot be amplified. Type B cDNA population form stem-loop like

structures that suppress PCR amplification (Lukyanov et al., 1994; Siebert et al., 1995).

Type C cDNA population have only one primer-annealing site, hence it can be amplified

only at a linear rate. Only type E cDNA population, which have different adapter

sequences at their ends having two different primer annealing sites that can be amplified


exponentially. A nested PCR is followed by the first PCR to increase the specificity of

the reaction. The differentially expressed cDNA sequences are greatly enriched in type E

fraction from the subtracted cDNA pool.

Figure 2.1 – SSH Principle (Clonetech Laboratories, CA, USA)


SSH was used to study differentially expressed transcripts in Arabidopsis with

response to ozone, bacterial and oomycete pathogens and the signalling compounds such

as salicylic acid (SA) and Jasmonic acid (Mahalingam et al., 2003). A substracted cDNA

library was constructed in the high altitude plant Lepidium latifolium to screen the cold

responsive genes (Aslam et al., 2009). A novel metallothionein like gene was identified

from the subtracted library of Cicer microphyllum, which responds to various abiotic

stresses (Singh et al., 2010). Kang et al., (2010) identified several genes by SSH, which

were induced by salt stress in seedlings of Medicago trunculata. An AP2 containing

protein was involved in response to metal stress in Physcomitrella patens was identified

by a modified SSH protocol (Cho et al., 2007). Xu et al., (2006) identified several

pathogen induced defense genes in Chestnut rose after constructing a cDNA library by

SSH. Gene expression in Fucus vesiculosus L. was investigated using cDNA library

generated by SSH for algae undergoing mild desiccation stress and identified many genes

upregulated (Pearson et al., 2001). A SSH library was constructed in Mitragyna speciosa

to identify the expression of genes involved in secondary metabolism after treatment with

Salicylic acid (Jumali et al., 2011).

In this study, a forward cDNA library was constructed to identify the cold

responsive genes in the vegetative tissues of Daucus carota.


2.2 MATERIALS AND METHODS

2.2.1 Plant Material, Growth Conditions and Cold Stress Treatment- D.carota cv Kuroda

seeds were procured from the Horticulture training centre, Ooty, India. The seeds were sowed

(in commercially available soil mix in pots) in the containment facility of Defence Institute of

Bio-Energy Research, Haldwani, India. The germinated plants were maintained in controlled

conditions at 25°C with 16/8h light/dark cycle. The plants were nourished with 1/10 strength

sterile MS salt solution. One month old plants were exposed to cold stress at 4°C for 24h in a

cooling incubator with 16/8h light/dark cycle. Plants maintained in the containment facility

with normal conditions were used as control for the experiment.

2.2.2 DEPC Treatment- Diethylpyro Carbonate (DEPC) is a strong inhibitor of RNases

that works by covalently modifying RNases. All the glasswares used for the RNA related

work were thoroughly rinsed with detergent and then with sterile distilled water.

The glasswares were baked in hot air oven at 240°C overnight and then immersed in 0.1%

DEPC solution overnight. The glasswares were autoclaved at 121°C for 1h. The

microfuge tubes and pipetteman tips used were of nuclease free polypropylene (sterile).

The non-disposable plastic wares were thoroughly rinsed with 0.1N NaOH, 1mM EDTA

followed by nuclease free water. Polycarbonate or polystyrene materials were cleaned by

immersing in 3% hydrogen peroxide solution for 10 min. Peroxide solution was removed

by rinsing with DEPC treated and with sterile water prior to use. Electrophoresis tanks,

gel casting trays and combs were cleaned with detergent solution (e.g., 0.5% SDS)

thoroughly rinsed with RNase-free water and was allowed to dry. All the solutions

(except Tris based solution as DEPC interferes with the activity of Tris) were

made/diluted using 0.1% DEPC.


2.2.2.1 RNA Isolation from Control and Cold Treated Plants- Young leaves from the

control and cold stress treated plants were harvested at the same time (in order to avoid

any physiological variation) and immediately frozen in liquid Nitrogen until further

process. The total RNA was isolated from control and test sample using Qiagen RNeasy

RNA isolation kit (Qiagen, Hilden, Germany) as per the manufacturer‘s instructions.

The leaves (approximately 100mg) were ground well with liquid Nitrogen. The powdered

leaves were then transferred to RNase free tube and 450µL of RLT buffer (containing

45µL of β-mercaptoethanol) were added and vortexed vigorously. The lysate was

transferred to a QlA shredder column (lilac) in a 2mL collection tube. The samples were

then centrifuged at 12000g for 2 min. Absolute ethanol (225µL) was added to the lysate

and mixed immediately. The samples were then transferred to an RNeasy mini column

(pink) that is placed in a 2mL collection tube and centrifuged for 15s at 12000g. Buffer

RW1 (700µL) was added to the RNeasy column and the samples were centrifuged for 15s

at 12000g. On column DNase digestion was performed to remove the genomic DNA

contaminants from RNA. DNase (30µL) was mixed with 70µL of RDD buffer and added

directly on to the membrane of the column. The samples were incubated at RT for

15 min. After the digestion, the column was washed with 500µL of RW1 buffer. To the

column, 500µL of RPE solution was added and centrifuged for 15s at 12000g twice.

The RNA was eluted by adding 40µL RNase free water to the column and centrifuged for

1 min at 12000g. The concentration of RNA was quantified and the integrity of RNA was

checked in formamide gel.


2.2.2.2 Purification of mRNA from Total RNA

2.2.2.2.a- Annealing of Probe- The mRNA was purified from the total RNA using

PolyATtract®

mRNA isolation system I (Promega, Madison, USA) according to

manufacturer‘s instruction. The RNA from the control and cold treated samples were

taken separately in an RNase free tube and the volume was made upto 2.43mL using

RNase free water. The tubes were placed in a heating block for 10 min at 72°C to

denature the secondary structure of RNA. Biotinylated oilgo dT (10µL) and 60µL of 20X

SSC were added to the tube and mixed gently by pipetting back and forth. The contents

were allowed to cool at RT.

2.2.2.2.b- Washing of Streptavidin-Paramagnetic Particles- The Streptavidin

MagneSphere® Paramagnetic Particles (SA-PMP‘s) requires BSA for stabilization,

which is present in the storage buffer. The SA-PMPs was provided at a concentration of

1mg/mL in PBS, 1mg/ml BSA and 0.02% Sodium azide. The tubes with SA-PMPs were

resuspended (one tube per sample) by flicking the bottom of the tube gently until they

were completely dispersed and captured by placing the tube in the magnetic stand, until

the SA-PMPs collected from the side of the tube. The supernatant was carefully removed

and the particles were washed thrice with equal volume of 0.5X SSC.

2.2.2.2.c- Capture and Washing of Annealed Oligo (dT)-mRNA Hybrids- The entire

content of the reaction mix was carefully transferred to the tube containing the SA-PMPs

and incubated at room temperature for 10 min. The tubes were inverted in every 2 min to

ensure proper mixing of RNA and oligo dT. The tubes were then placed in the magnetic

stand and the supernatant was carefully removed without disturbing the SA-PMP

69

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particles. The particles were washed 4 times with 1.5mL of 0.1% SSC by gently flicking

the bottom of the tube until all particles were resuspended. The supernatant was removed

after the final wash (without disturbing the pellet).

2.2.2.2.d- Elution of mRNA- Finally SA-PMP particles were added to the 50µL of

RNase free water and gently resuspended by flicking the tube. The tubes were then

placed in the magnetic stand and the eluted mRNA was carefully separated from the

particles and transferred to a fresh RNase free microfuge. The microfuge was centrifuged

at 12000g for 1 min to pellet the magnetic particles and mRNA was transferred to a fresh

tube.

2.2.2.3 First Strand cDNA Synthesis - In a RNase free PCR tube, 2µg of poly A mRNA

and 1µL (10µM) oligo dT were added (for both tester and driver separately) and the

volume was made to 5µL using nuclease free water. The contents were briefly mixed and

incubated at 72°C for 2 min in a thermal cycler. The samples were snap cooled in ice

after the incubation period. To the tube 2µL of 5X RT buffer (Fermentas Inc., Maryland,

USA), 1µL of 10mM dNTP mix (Fermentas Inc., Maryland, USA) and 1µL of 20U/µL

M-MLV RT (Fermentas Inc., Maryland, USA) were added and the volume were made to

10µL using nuclease free water. The contents in the tube were briefly mixed in a mini

centrifuge. Revere transcription was performed at 42ºC for 60 min in a thermal cycler.

The tubes were immediately placed in ice to terminate the reaction (after the incubation

period) and samples were used for the synthesis of cDNA.

2.2.2.4 Second Strand cDNA Synthesis- The whole sample from the first strand cDNA

was used for the synthesis of second strand. In the same tube, 48.4µL of nuclease free

water, 16µL of 5X second strand synthesis buffer, 1.6µL of dNTPs (10mM each) and


4.0μL of 20X second strand enzyme were added. The final volume of the mix was made

to 80µL and incubated at 16°C for 2h in a thermal cycler. After the incubation period,

2μL (6 U) of Platinum Pfu DNA polymerase (Fermentas Inc., Maryland, USA) was

added and the contents were mixed well. The PCR was performed with the following

conditions, an initial denaturation at 95ºC for 3 min followed by 30 cycles of denaturation

at 95ºC for 15 s and extension at 66 ºC for 6 min. The reaction was terminated using 4μL

of EDTA/Glycogen mix. The sample was mixed thoroughly with equal volume of

Phenol: Chloroform: Isoamyl alcohol (25:24:1) and centrifuged at 12000g for 10 min at

RT. To the extracted upper aqueous layer, 25μL 4M NH4OAc and 187.5μL absolute

ethanol were added. The samples were then centrifuged at 14000g for 10 min at room

temperature, supernatant was removed, pellet was washed with 70% ethanol and air dried

and dissolved in 20μL sterile water.

2.2.3 RsaI Digestion- The purified product (second strand DNA) was used for the

digestion with RsaI (Fermentas Inc., Maryland, USA). Each tester (stress sample) and

driver (control sample) double stranded cDNA was digested with RsaI to generate

shorter, blunt ended ds-cDNA fragments that are optimal for subtraction and adapter

ligation. In a sterile PCR tube, 40μL of the purified PCR product, 5μL of 10X RsaI

buffer, 8μL of nuclease free water and 2μL (10U/μL) of RsaI were added. The samples

were briefly centrifuged and incubated in water bath for 2h at 37°C. The digestion

reaction was arrested using 4μL of 20X EDTA. The digestion products were purified

(as mentioned above) and used for adaptor ligation.

2.2.4 Suppressive Subtractive Hybridization and Construction of Subtracted cDNA

library- Suppressive Subtractive Hybridization (SSH) was carried out using Polymerase


Chain Reaction (PCR) Select cDNA Subtraction kit (Clonetech laboratories, CA, USA)

according to manufacturer‘s instruction. Double stranded cDNA was synthesized from

the 2μg of poly A+ purified mRNA.

2.2.4.1 Ligation of Adaptor to Tester cDNA Population- The purified RsaI digested

cDNA was used for adaptor ligation. The purified cDNA (tester) was diluted in nuclease

free water (1μL cDNA is mixed with 4μL of sterile water) and the samples were kept in

ice. A ligation mix was made as shown in table 2.1,

Sl. No Component Volume per

reaction

01 Nuclease free water 3μL

02 5X ligation buffer 2μL

03 T4 DNA ligase (400U/ μL) with 3mM ATP (final ATP

concentration in mix will be 300 μM).

1μL

Table 2.1- Ligation Mix for Adaptor Ligation 1.

The ligation mix was used for preparing the ligation reaction with two different adaptors

as mentioned in table 2.2,

Sl. No Component Tester

1-1

Tester

1-2

01 Diluted tester cDNA 2μL 2μL

02 Adaptor 1 (10 μM) 1μL -

03 Adaptor 2R (10 μM) - 1μL

04 Ligation master mix 7 μL 7 μL

Final Volume 10μL 10μL

Table 2.2- Ligation Mix for Adaptor Ligation 2


The components were briefly centrifuged and incubated in a thermal cycler for

overnight at 16°C. One microliter of EDTA was added to arrest the reaction and the

samples were incubated at 72°C for 5 min to inactivate the ligase.

2.2.4.2 First Hybridization Reaction- In the first hybridization, an excess of driver

cDNA was added to tester cDNA (ligated with adaptor) samples were then heat

denatured and allowed to anneal. The remaining ss cDNAs (which were available for the

second hybridization) was dramatically enriched for differentially expressed sequences

as non-target cDNAs present in the tester and driver cDNA forming hybrids.

The hybridization buffer was made to thaw in RT and hybridization mix was made

according to the following (table 2.3)

Sl. No Component Tester

1-1

Tester

1-2

01 RsaI digested Driver cDNA (excess) 1.5μL 1.5μL

02 Adaptor 1-ligated Tester 1-1 1.5μL -

03 Adaptor 2R-ligated Tester 1-2 - 1.5μL

04 4X Hybridization Buffer 1μL 1μL

Final Volume 4μL 4μL

Table 2.3- First Hybridization Mix Components

The samples were overlaid with a drop of mineral oil and were briefly centrifuged.

The samples were initially incubated at 95°C for 2 min and left for hybridization at 68°C

for 12h.

2.2.4.3 Second Hybridization Reaction- The two samples from the first hybridization

reaction were mixed together and freshly denatured driver cDNA was added to further


enrich differentially expressed genes and for the formation of new hybrid molecules that

consists differentially expressed cDNAs with different adaptors on either ends.

The hybridization mix for the second round of hybridization was made as mentioned in

table 2.4,

Sl. No Component Volume per reaction

01 Driver cDNA 1μL

02 4X Hybridization Buffer 1μL


Final volume 4μL

Table 2.4- Second Hybridization Mix Components

The samples were briefly mixed by centrifugation and overlaid with 1μL of mineral

oil. The sample was denatured at 98°C for 2 min in a thermal cycler. The following step is

important to ensure that both the hybridization samples mixed with the denatured driver

at the same time. The pipette tip was gently touched to the mineral oil/sample interface of

the tube containing hybridization sample 2 and the entire sample was drawn from the

tube. The tip from the tube was removed and a small amount of air was drawn into the tip

creating, a slight air space below the droplet of sample. The same procedure was repeated

to take the freshly denatured driver cDNA in the same tip. At the end, the tip contained

both the hybridized sample 2 and the freshly denatured driver cDNA. The entire sample

was transferred to the tube containing first hybridization mix and it was mixed

immediately by pipetting up and down. The samples were centrifuged and left for second

hybridisation at 16°C for overnight in a thermal cycler. At the end of the hybridization

the samples were diluted using 200μL of dilution buffer and incubated at 68°C for 7 min.


2.2.5 Amplification of cDNA Inserts by PCR and Nested PCR- The resultant

subtractive product was amplified by PCR using primers that were complimentary to

sequence of adapters 1 and 2R. The diluted cDNA was used as the template for the first

PCR reaction. The PCR mix was made as follows (table 2.5)


01 Nuclease free water 18.5μL

02 Taq buffer 10X 2.5μL

03 dNTPs (10mM) 1μL

04 Diluted template 1μL

05 PCR primer 1 (10μM) 1μL

06 Platinum Taq DNA polymerase (2.5 U/μL) 1μL

Final Volume 25μL

Table 2.5- Components of Master Mix for First PCR

The samples were briefly centrifuged and overlaid with mineral oil. The reaction

mixture were incubated at 75°C for 5 min in a thermal cycler to extent the adaptors and

the PCR reaction was immediately commenced. The cycling conditions for the first PCR

was as follows, denaturation at 94°C for 30s followed by annealing at 66°C for 30s and

extension at 72°C for 90s for 27 cycles. At the end of the PCR, 8μL of the PCR product

was analyzed in agarose gel. The PCR product from the first reaction was diluted in water

in the ratio of 1:9 (3μL of the PCR product with 27μL of nuclease free water) and was

used as the template for the second reaction with nested primers.


A master mix for the second PCR was made as follows table 2.6,


01 Nuclease free water 17.5μL

02 Taq buffer 10X 2.5μL

03 dNTPs (10mM) 1μL

04 Diluted template 1μL

05 Nested PCR primer 1 (10μM) 1μL

06 Nested PCR primer 2R (10μM) 1μL

07 Platinum Taq DNA polymerase (2.5 U/μL) 1μL

Final Volume 25μL

Table 2.6- Components of Master Mix for Nested PCR

The samples were briefly centrifuged and overlaid with mineral oil. The cycling

conditions for nested PCR was as follows, at 94°C for 30s followed by annealing at 66°C

for 30s and extension at 72°C for 90 s for 12 cycles. PCR product (8μL) was analyzed in

agarose gel.

2.2.6 Transformation of the Subtracted Library to E.coli

2.2.6.1 RsaI Digestion of Subtracted Library and Purification- The subtracted cDNA

library product was purified using Genei PureTM

Quick PCR purification kit, GeneITM

,

India. The purified product was digested with RsaI. In a sterile PCR tube, 20μL of the

purified PCR product (subtracted forward cDNA library), 2.5μL of 10X RsaI buffer,

1.5μL of nuclease free water and 1μL (10U/μL) of RsaI were added. The samples were

briefly centrifuged and incubated in thermal cycler for 2h for digestion. The digested

product was purified and eluted in a final volume of 10μL twice.


2.2.6.2 Isolation of Plasmid from E.coli Harbouring pBluescript KS- A single colony

of E.coli. was inoculated in 50mL of LB containing 50μL of Ampicillin (100mg/mL) and

incubated overnight at 37°C in orbital shaker. The overnight grown culture was used for

the isolation of plasmid DNA (pBS). The plasmid DNA was used for restriction enzyme

digestion.

2.2.6.3 Digestion of the pBS Plasmid with SmaI- The pBS plasmid was digested with

SmaI enzyme inorder to obtain blunt ends. The digestion mix contained plasmid DNA

20μL, 10X SmaI digestion buffer (wih BSA) 2.5μL, nuclease free water 1.5μL and SmaI

1μL (10U/μL). The samples were briefly centrifuged and incubated at 37°C in a thermal

cycler for 3h.

2.2.6.4 Cloning of Subtracted cDNA Library to pBS Plasmid- The digested cDNA

library was cloned to linearised pBS plasmid by blunt end ligation. The components of

the ligation mix were made as shown in table 2.7.

Sl. No Component Volume per

reaction

01 Digested plasmid DNA (200ng) 2μL

02 Digested cDNA 2μL

03 5X ligation buffer 2μL


05 T4 DNA ligase (400U/ μL)

(with 3mM ATP, final ATP concentration in mix will

be 300μM)

1μL

Total volume 10μL

Table 2.7- Components of the Ligation Mix


The contents were briefly mixed and centrifuged. The samples were incubated in

a thermal cycler overnight at 16°C. Ligated product (2μL) was used for transformation to

E.coli Dh5α.

2.2.6.5- Transformation of Ligated Product to E. coli - The ligated product was

transformed into E. coli Dh5α cells (Genotype: F Φ80lacZΔM15 Δ(lacZYAargF) U169

recA1 endA1 hsdR17 (rk , mk ) phoA supE44 thi-1 gyrA96 relA1 tonA). The E. coli

competence cells were prepared according to Sambrook & Russell (2001) with minor

modification. LB medium (Himedia Laboratories, India) was routinely used to culture

E. coli. Single colony was inoculated in 25mL LB and incubated at 37°C for 6h.

Inoculum (1%) was transferred to 50mL LB and incubated in an orbital shaker until it

reaches 0.5-0.6 OD600. The culture was chilled in ice for 30 min. The cells were pelleted

by centrifugation at 1500g for 10 min at 4°C. The pellet was dispensed in 4mL of ice cold

sterile 0.1M CaCl2 and centrifuged at 1500g for 10 min at 4°C. The cells were finally

resuspended in 1mL 0.1 M CaCl2 and 50μL aliquots were made and stored at -80°C until

transformation.

The 50µL aliquots of competence cells stored at -80°C were thawed using ice for

30 min and ligated product (5µL) was directly pipetted over competent cells. The cells

were mixed gently by tapping 4-5 times, incubated on ice for 30 min, which was

followed by a heat shock treatment at 42°C for 60 s. After the heat shock, the cells were

immediately placed in ice for 2 min. SOC media (250µL) was added to the tubes.

The tubes were finally incubated at 37°C for 1h at 225 rpm in orbital shaker. The cultures

were appropriately diluted in LB medium and 50-100µL of each culture was spread on


selection media (LB agar supplemented with 50mg/L Ampicillin). Isopropyl β-D Thio

Galactoside (8µL of 0.2 M IPTG) and 40µL of 20mg/mL X-Gal (5-bromo 4-chlororo

3-indolyl β- D galactoside) were spread on the plates prior to plating of the transformed

cells for blue/white selection to identify the recombinant clones. The plates were sealed

with parafilm and incubated upside down at 37°C overnight.

2.2.7 Differential Screening of the Subtracted Clones- The white transformed colonies from

the subtracted library was screened by colony PCR using M13 universal primers. The colonies

were patch streaked in fresh LB agar plates with 100mg/L Ampicillin and the tip containing the

colony was used as the template source for PCR. The PCR mix contained, 5μL 10X Taq

buffer, 2.5μL MgCl2 (25 mM), 1μL dNTP mix (10 mM each), 1μL each of M13 forward

primer (5‘-GTA AAA CGA CGG CCA GT-3‘) and M13 reverse primer (5‘-AAC AGC TAT

GAC CAT G-3‘) (10 pM), 39.3μL of PCR grade water and 0.2μL Taq DNA polymerase

(5U/μL). PCR was carried out with an initial denaturation at 95°C for 5 min, followed by

30 cycles of 95°C for 30s, 55°C for 60s, 72°C for 90s and a final extension of 5 min at 72°C.

The PCR products were resolved in 2% agarose gel and stained with ethidium bromide.

2.2.8 Plasmid Isolation and Sequencing of the Subtractive Clones- Clones were

selected randomly and inoculated in LB broth containing 50mg/L Ampicillin. The tubes

were incubated at 37°C for overnight in an orbital shaker and the plasmid DNA was

isolated from the selected clones. The plasmid DNA was sequenced in Chromous

Biotech, Bangalore, India using M13 forward primer.

2.2.9 EST Sequence Analysis - The vector sequences were first removed from the

obtained sequence using the Vec Screen online software (www.ncbi.nlm.nih.gov/

VecScreen/VecScreen.html). The EST sequences were analyzed for homology using

79


Basic Local Alignment Search Tool (BLASTN) from the National Center for

Biotechnology Information database (www.ncbi.nlm.nih.gov/blast/). Homology searches

were performed against non-redundant nucleotide sequence using BLASTN of BLAST

programme. The unique EST sequences were submitted in the EST GenBank database.

2.2.10 Semi quantitative (sq) RT-PCR Analysis- The transcript accumulation of

3 genes was studied after giving cold stress to carrot plants. One month old carrot plants

were given stress for 5 days at 4°C. Samples were then harvested every 24h and were

immediately frozen in liquid nitrogen and stored in -80°C until further process. RNA was

isolated from all the samples using Qiagen RNeasy RNA isolation kit (Qiagen, Hilden,

Germany) and on column DNase digestion was performed to remove the genomic DNA

from the RNA sample as mentioned in the section 2.2.2.1. The RNA was quantified in

Nanodrop (Thermo Scientific, USA) and the integrity was checked in formamide gel.

RNA (2µg) was used for the synthesis of cDNA using RETROscript® Kit- Reverse

Transcription for RT-PCR (Ambion Inc, USA) according to manufacturer‘s instruction.

Primers were designed for Dc WD protein, Dc Profilin 4 and Dc Cyclin 2b. The first

strand cDNA was diluted 10 times using nuclease free water and was used as template for

PCR. PCR was performed using Ready to go PCR beads (GE healthcare, NJ, USA).

The primer sequences are shown in the table 2.8. Dc Elongation factor 1 alpha (EF1α)

was used as the internal control for sqRT PCR. The PCR cycle consists of 28 cycles with

an initial denaturation at 94°C for 5 min followed by denaturation at 94°C for 30s,

annealing at 60°C for 30s and extension at 72°C for 30s. A final extension was 72°C for

10 min. The PCR products were separated in 2% agarose gel and stained with ethidium

bromide (80ng/μL). The sq RT-PCR was repeated twice to verify the results.


Sl. No Gene Primers

01 Dc WD Fw: 5‘- TGT CAA TGG CCT CCA CAA ATT-3‘

Rv: 5‘- TTT ACA GCT GAA GTG TGT TCT TCC A-3‘

02 Dc Profilin 4 Fw: 5‘- AAG CTC TGG TGT TTG GAG T-3‘

Rev: 5‘- TAA TCT CCA AGC CTC TCA AC-3‘

03 Dc Cyclin 2b Fw: 5‘- ACA ATT CGA GTG CTG TTT CT-3‘

Rev: 5‘-CTG TGG GTC ATC AAA TTT CT-3‘

04 Dc EF1α Fw: 5´- TGG TGA TGC TGG TTT CGT TAA G -3´

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

Table 2.8- Primers used for the sq RT-PCR Analysis

81


2.3 RESULTS AND DISCUSSION

2.3.1 Construction of Subtraction cDNA Library- The young rosette of the carrot

leaves of control and cold stressed plants were used for RNA isolation. The reliability of

the SSH method mainly depends on the quality and quantity of RNA used for the

subtraction procedure. The clear bands of both the 18S and 28S rRNA in the formamide

gel showed that the RNA is intact (fig. 2.2). High quality of mRNA was separated from

the total RNA and was used for the synthesis of cDNA. The forward library was

constructed using the stress-induced sample as the tester population and the control

sample as the driver population to determine the genes upregulated during cold stress.

The cDNAs samples, which were differentially expressed, were obtained after two

rounds of hybridization procedure followed by two rounds of suppression PCR (fig. 2.3).

The cDNAs thus obtained were enriched with genes upregulated during cold stress.

Figure 2.2 RNA Isolated from Control and Cold Stressed Carrot Plant

Lane M- Molecular weight marker; Lane 1- RNA from control plant;

Lane 2- RNA from cold stressed carrot

28S rRNA

18S rRNA

M 1 2

250bp

5 kb


Figure 2.3- Result of SSH using cDNA from Cold Stressed Leaves as Tester and

Control Leaves as Driver.

Lane M- Molecular weight marker, Lane 1 & 2- Smear after first round of PCR

Lane 3 & 4- Forward subtracted library

Figure 2.4- Screening of Insert Size by Colony PCR using M13 Primers

Lane M- DNA ladder

Lane 1-15 (upper panel) and lane 1- 19 (lower panel) - clones from forward library

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250bp

500bp

10kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

250bp

500bp

10kb

10kb

250bp

500bp

M 1 2 3 4

Subtracted forward library

250bp

10kb


2.3.2 Cloning and Differential Screening of the Subtracted cDNA Library- The PCR

product of the second round of PCR reaction that represented the subtracted cDNA

library was ligated to pBlue script KS (+) vector and transformed to E. coli cells.

The white colonies were screened using M13 primers and randomly selected clones were

used for sequencing (fig. 2.4)

2.3.3 Sequence Analysis- Around 75 randomly selected clones were sequenced. Around,

62 clones produced readable sequences. After removing the redundant or vector

backbone sequences 43 sequences of the potential clones were submitted in the public

EST database. The accession number of 43 potential clones with their putative function is

summarized in the table 2.9.

2.3.4 sq RT-PCR- The sq RT-PCR results indicate that cyclin and WD protein

upregulated after stress, whereas there was no change in expression level of profilin

(fig. 2.5). Cyclin is one of the major proteins that regulate different stages of the cell

cycle by its concerted expression and degradation. They along with cyclin dependent

kinases achieve by phosphorylating different targets. Interaction of Arabidopsis cyclin

D2 expressed in transgenic rice with endogenic cyclin-dependent kinase enhanced

seedling growth (Oh et al., 2008). Cell cycle activities involved in stress responses are

mediated by transcription factors (Morano et al., 1999). Transgenic rice expressing

OsMYB3R-2 enhanced low temperature tolerance that has been shown to be mediated by

alteration in cell cycle (Ma et al., 2009). The transcript level of cyclin D2, cyclin B2-2

and Cyclin Dependent Protein Kinase (CDPK) were upregulated during drought stress in

Arabidopsis, wheat and rice respectively (Kamal et al., 2010). In the present study, the

cyclin2b was found to be upregulated in carrot during cold stress.


The protein with WD repeats play a key role in signal transduction, cytoskeletal

dynamics, ribosomal RNA biogenesis (Neer et al., 1994; Smith et al., 1999), cytokinesis,

apoptosis, floral development and meristem organisation (Nocker & Ludwig 2003).

They have been classified based on sequence similarity (Nocker & Ludwig 2003).

Recently, a WD 40 in B.napus (BnSWD1) was reported to play a major role during salt

stress (Lee et al., 2010). SNF1 kinase is a key enzyme in plant steroid biosynthesis and it

phosphorylates the 3-hydroxy-3methylglutaryl-CoA reductase. Its activity is regulated by

PRL1, a conserved nuclear WD-protein that is implicated in cold tolerance in

Arabidopsis (Bhalerao et al., 1999). It was 2-16 folds repressed in leaves/shoots of cold,

high-salinity stressed chickpea (Mantri et al., 2010); on the other hand it was upregulated

in carrot during low temperature stress, suggesting that it may have a role in cold stress

tolerance.

The expression of profilin was unaffected under cold stress as seen from the sq

RT-PCR results (fig. 2.5), although SSH analyses show upregulation (table 2.9). SSH is a

powerful technique that can detect even minor changes in transcript levels as in the

present case. Profilins are a group of low molecular weight ubiquitous actin binding

eukaryotic proteins, which are involved in the remodelling of actin cytoskeleton

(Huang et al., 1996) and also involved in various signalling cascades in yeast and plants

(Vojtek et al., 1991; Machesky et al. 1993). Swoboda et al., (2001) reported that

remodelling architecture of cytoplasm is a normal process in cells to maintain the

membrane integrity during various environmental stresses and in carrot profilins might

also be contributing to the cold tolerance.

85


Figure 2.5- Expression Profile of 3 Genes of D. carota by sq RT- PCR (Dc Cyclin, Dc

WD and Dc Profilin)

Lane- C-Control sample, 1 to 5 Samples from 1st to 5

th day after stress.

2.3.5 Functional Classification of Cold Stress Induced ESTs- The unique ESTs were

annotated based on their similarities with existing sequences in GenBank using BLAST

based on their functions into 6 groups. They are cold, salt, drought, UV stress responsive

genes, genes involved in transcription/translation, genes involved in sugar/protein/lipid

metabolism, genes involved in maintaining structural integrity, genes involved in signal

transduction and genes of unknown functions (fig. 2.6).

C 1 2 3 4 5

RNA

Cyclin 2b

WD gene

Profilin

EF1α


Fig 2.6- Grouping of Cold Responsive Gene Sequences based on their Functions

Derived from SSH Technique

2.3.6 Comparative Analysis of the Sequences with Cold Regulated Transcriptome of

Plants- A comparative analysis of the cold upregulated transcriptome was carried out, to

correlate and understand the mechanism of low temperature tolerance in plants.

The upregulation of several genes of varied functions suggests that, the unique ESTs may

play role in complex biological processes. Wang et al., (2007) have reported that all the

upregulated genes may not have a role in stress tolerance, but could be in response to

damages caused by the stress. The largest number of genes belongs to the groups that are

upregulated during cold, drought or salinity stress. Apart from this, the upregulation of

genes of unknown functions and those involved in transcription/translation suggests that

the responses to cold stress are rather complex and multigenic as it was reported by

(Sun et al., 2007; Zhang et al., 2009; Kang et al., 2010).


There are several classes of transcription factors such as, AP2/ERF, DREB,

YABBY and Trihelix families, which are unique to plants and act as molecular switches

(Xie et al., 2009). Ramamoorthy et al., (2008) have identified that, some of these

transcriptional factors are involved in transcriptional regulation during environmental

stresses. Liu et al., (2011) reported that, the low osmotically responsive gene 2 (LOS 2)

from Poncirus trifoliate act as a transcriptional activator for various cold-responsive

genes. In carrot during cold stress expression of fas and DREB were upregulated. Fas is

involved in regulating flowering through YABBY in tomato (Cong et al., 2008). In carrot

normally vernalization is required for flowering and we propose that, the cold treatment

lead to the expression of fas, which in turn might initiate the flowering. The Dehydration

Responsive Element Binding (DREB) proteins have been found to confer tolerance to

cold/drought stress in Arabidopsis (Kasuga et al., 2004), wheat (Andeani et al., 2009),

rice (Dubouzet et al., 2003), cotton (Shan et al., 2007), and could be functioning similarly

in the carrot also. Similarly, the zinc finger CCCH protein enhanced in response to salt

stress in Arabidopsis (Sun et al., 2007). Other zinc finger proteins like Zat 12 and Gh ZFP 1

have been found to confer tolerance to oxidative stress and salt stress (Guo et al., 2009)

respectively.


Sl.

No

Accession

Number Predicted gene family Putative Function

E-

value

1 GW316731 A. thaliana cyclin2b Regulator of cyclin dependent

kinases (Cell cycle regulator)

3e-33

2 GW342890 R. communis WD protein Signal transduction/cell cycle

regulation

5e-04

3 GW314857 Dc 4 profilin Actin binding protein 3e-24

4 GW343024/

GW343026

Solanum lycopersicum- FAS

protein

Encodes transcription factor 4e-12

5 GW315340 Chrysanthemum vestitum-

DREB

Cold tolerance 1e-15

6 GW342888 A. thaliana Zinc finger CCCH

protein

Cold/salt stress 2e-06

7 GW343025 Talaromyces stipitatus- Rop

GTPase activator

Cold/salt/drought stress 9e-06

8 GW316484 Dc RNA polymerase Transcription 7e-14

9 GW316488 Hypochaeris megapotamica-

maturase K

Intron splicing 6e -19

10 GW316489 Ribosomal rRNA Translation 1e-13

11 GW342893 Peltandra virginica- tRNA-Lys

(trnK) gene

Translation 2e-14

12 GW276092 tRNA Leu trnL - trnF IGS Translation 9e 75

13 GW342886 Beta macrocarpa-

mitochondrial genome

DNA synthesis/transcription and

Translation

1e-06

14 GW316482 A. thaliana- sec 61 beta-

subunit

Protein translocation to ER 3e-09

15 GW342894 GABA permease Translocation of GABA 8e-10

16 GW276089 O.sativa- E3 ubiquitin ligase UV B light response/low

temperature tolerance

9e-48

17 GW343027 P. trichocarpa- shikimate

kinase

Cold stress 9e-07

18 GW316730 Candida albicans- choline

kinase

Salinity stress 0.070

19 GW276087 Levan sucrase Bacillus subtilis Osmotic stress 2e-74

20 GW314859 Chlamydia trachomatis-

Phosphoglucoisomerase gene

Involved in synthesis of

galactomanan (osmoprotectant)

6e-43

21 GW276091 Talaromyces stipitatus- Sugar

transporter protein

Transport of sugar across the

membranes

1e- 24


Sl.

No

Accession

Number Predicted gene family Putative Function

E-

value

22 GW343023 Cucumis melo- catalase 2 Oxidative stress 6e-08

23 GW342891 A. thaliana- Lipid associated

protein (fibrillin like proteins)

Cold/photooxidative stress 0.003

24 GW342892 Neosartorya fischeri-Integral

membrane protein

Cell structure maintenance 1e - 05

25 GW314854 A. thaliana- 60 α chaperonin sub

unit CCT family

Cold tolerance 6e-43

26 GW316481 Ricinus communis- ATP

binding protein

Osmotic stress 1e-22

27 GW342889 Cyanate hydratase Nitrogen metabolism 1e-12

28 GW316732 E.coli- serine deaminase

activator gene

Aminoacid metabolism 2e-21

29 GW316733 E.coli- BioH gene Lipid metabolism 6e-11

30 GW316485 S receptor kinases Self incompatibility 1e-18

31 GW276090 Populus sp- Drought stress

related protein

Drought stress tolerance 1e-38

32 GW314853 Populus trichocarpa- stress

protein

Stress tolerance 0.002

33 GW342887 Hydra magnipapillata- Putative

signal tranduction

Signal transduction 0.014

34 GW315344 A. thaliana genome Unknown -

35 GW316487 B. rapa genomic DNA clone Unknown -

36 GW314858 Oryza sativa genomic DNA-

chromosome 4, BAC clone

Unknown -

37 GW314852 Vitis vinifera clone Unkown -

38 GW315342 Populus trichocarpa clone Unknown -

39 GW316483 Oryza sativa japonica BAC

clone

Unknown -

40 GW316728 Populus trichocarpa- predicted

protein, mRNA

Unknown -

41 GW314856 Unknown Unknown -



Table 2.9- The Cold Stress Responsive Genes Isolated from Carrot using SSH


The G protein function as molecular switches to regulate numerous cellular

responses such as proliferation, differentiation, responding to external environmental

signals, etc. Rho-related GTPase in plants (Rop) play an important role in plant growth

and development by acting as a signalling protein and also confers abiotic stress tolerance in

Arabidopsis (Shin et al., 2009) and upon cold stress in rice (Hashimoto & Komatsu 2007).

The enhanced level of similar proteins was observed in carrot that may be playing same

role in signalling mechanism in response to low temperature treatment.

Regulation of transcription and translation plays an important role in stress

alleviation (Miranda et al., 2003). RNA polymerase and maturase K are not only

involved in transcription and post-transcriptional modification, but have also been

implicated in regulation of gene expression through miRNA/siRNA formation in

response to stress in plants (Sunkar et al., 2007). There are reports of enhanced levels of

RNA polymerases and maturase K and a corresponding increase in the miRNA levels in

various plants exposed to biotic and abiotic stresses (Lee et al., 2005; Zhou et al., 2008;

Garavaglia et al., 2010).

Translation is regulated at the level of stability of transcripts and initiation of

translation (Prabu et al., 2011). Transcript stability under stress is enhanced by the formation

of polysomes (Arendt & Weidner 2011). In our study, carrot system upregulation of rRNA

was observed similarly that could have helped in the maintaining the transcript stability.

Translation initiation factor 4α also showed upregulation on exposure to cold stress and

similar results were also observed in plants like pea (Pham et al., 2000; Vashisht et al., 2005),

wheat (Kamal et al., 2010). Sec-β 61, which was upregulated in cold stressed carrot,

showed a similar response to wounding stress in Arabidopsis (Pnueli et al., 2003) and it


is also essential for translocation of proteins to ER and for the cell viability as reported in

many of the organisms (Leroux & Rokeach 2008). In carrot, Sec β 61 might be

responsible for the translocation of different cold tolerant proteins to ER for processing.

Apart from that GABA permeases that translocate GABA to combat stresses, which was

also enhanced in the cold stressed carrot. GABA, a non-protein amino acid has been found to

increase in response to various stresses (Cholewa et al., 1997; Serraj et al., 1998; Bouche &

Fromm 2004).

Ubiquitin ligases are the group, whose expression was enhanced during the cold

stress in carrot. Similar E3 ubiquitin ligases (SIZ1) resulted in the accumulation of SUMO

protein conjugates that provided cold tolerance in Arabidopsis (Chinnusamy et al., 2007).

Sumoylation is a post-translational modification that protects the target protein from

proteosomal degradation by preventing ubiqutination.

Shikimate, derived compounds have a major role in plant response to biotic and

abiotic stresses (Hamberger et al., 2006), mainly due to shikimate kinase, the first

enzyme in the pathway is reported to play a key role in providing stress tolerance to

Arabidopsis (Fucile et al., 2008) and maize (Zheng et al., 2006). Thus, the upregulation

of shikimate kinase may probably increase the synthesis of metabolites, which confer

tolerance to cold stress in carrot. Cold stress has been reported to mimic water deficit

conditions similar to salinity stress (Mahajan & Tuteja 2005). Multiple mechanisms seem

to confer tolerance to the osmotic stress. Choline kinase catalyses the synthesis

of phosphatidylcholine responsible for maintaining the osmolority of the plant cell

(Tasseva et al., 2004) and upregulation of this gene in carrot may also have an important

role in cold tolerance.


Cold stressed carrot showed upregulation of a transcript that had similarity with

the bacterial leavansucrase. Levansucrases are hexosyltransferases, mainly involved in

the metabolism of starch and sucrose and their expression in tobacco have been reported

to enhance the plant‘s osmotic tolerance (Park et al., 1999). Hence, we presume that the

carrot levansucrase might also be playing a similar role during low temperature stress.

Phosphoglucoisomerase catalyse the biosynthesis of galactomannans in plants

(Lee et al., 1984), which apart from their primary role as storage reserves in endosperm,

also provide osmoprotection during seed germination in response to external factors like

drought and low temperature in leguminous seeds (Mulimani & Prashanth 2002). Low

molecular weight sugars play a role as cryoprotectants as well as help to maintain

osmotic potential in different plant tissues. Sugar transporter proteins are involved in

distribution of sugars to various cells and tissues (Williams et al., 2000). In Arabidopsis,

ERD6, a putative sugar transporter protein (Kiyosue 1998) and tonoplast monosaccaharide

transporter proteins (Eckardt 2006) expressed in response to dehydration stress and during

general stress respectively.

The low temperature stress induced the generation of reactive oxygen species

(ROS), which have strong adverse effect on biomolecules and plant cell membranes

(Chaitanya et al., 2001). In plants, ROS are mitigated by the antioxidant enzymes like

catalase, peroxidase, and superoxide dismutase (Yong et al., 2008; Mallik et al., 2011).

Hence, these parameters were studied in cold stressed carrot, which showed that only

catalase is upregulated, which could be for combating the ROS generation. The prominent

role of catalase during low temperature stress has been already reported in wheat

(Apostolova et al., 2008) and maize (Prasad 1997). Fibrillins are a group of lipid binding

93


proteins in plastids, which are induced during various types of abiotic stress responses,

like a combination of photooxidative and cold stress in Arabidopsis (Youssef et al., 2010),

low temperature stress in rice (Lee et al., 2007). In carrot, upregulation of fibrillins may

confer the stability of the plastids upon cold treatment.

Maintenance of membrane stability is important for stress tolerance (Gulen et al., 2008).

The low temperature stress affects the normal functioning of integral membrane proteins

and the activity of these proteins mainly depends on the fluidity of the cell membranes.

Xv SAP1 an integral protein, isolated from Xerophyta viscosa show high homology to

WCOR413, a cold responsive protein from wheat (Garwe et al., 2003). This shows that

the integral proteins are not only involved in maintaining the cell structure, they are also

associated with low temperature stress.

Chaperonin 60 and other kind of chaperonins are chloroplast proteins, thought to

play a key role in assembly and folding of proteins. As in the case of cold stressed carrot,

it has been reported to be cold induced in yeast (Somer et al., 2002), high temperature

stress in rice (Han et al., 2009). In cold stressed carrots the upregulation of chaperonins

and other chaperons could help in the correct folding of proteins expressed during the

cold stress. The proper folding of the membrane proteins would be a paramount

importance for the cold tolerance function. ATP binding proteins are group of membrane

proteins, involved in the transport of solutes across the cell membranes. Similar to the

results in carrot, they have been found to have a role in cold stress tolerance in rice

(Cui et al., 2005) as well as drought stress in Arabidopsis (Valliyodan & Nguyen 2006)

and for Ca dependent ATPases in response to cold stress in maize (Jian et al., 1999).


The role of genes like cyanases, serine deaminases, S receptor kinase, etc. during

cold stress is not very clear yet. Though they have shown to have different roles in the

plant metabolic pathways, the upregulation might have direct/indirect role during the cold

stress, which has to be studied further. The comparative analysis of the transcriptome

showed that, most plants have a common mechanism to tolerate the low temperature

stress. From the current study, it is clear that the cold regulated transcriptome is

conserved in different plants species, though the level of up/down regulation vary among

different plant species.


2.4 CONCLUSION

In this study, we have identified around 50 genes, which might play a role in

diverse function during the cold stress in carrot. Confirmation of the specific function of

each differentially expressed gene (from the forward library in carrot during cold

acclimation) will further require biochemical, molecular, physiological and genetic

analyses. Future studies on this aspect will help in increasing our understanding of the

complex mechanisms of abiotic stress response, in particular cold stress, which will

ultimately lead to the development of effective cold tolerant transgenic crops.


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