Mol Biol Rep (2012) 39:1011–1018
DOI 10.1007/s11033-011-0826-y
Identification of biotic and abiotic stress up-regulated ESTs
in Gossypium arboreum
Muhammad Younas Khan Barozai •
Tayyab Husnain
Received: 3 August 2010 / Accepted: 3 May 2011 / Published online: 10 May 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Asiatic desi cotton (Gossypium arboreum)
shows great potential against biotic and abiotic stresses. The
stress resistant nature makes it a best source for the identi-
fication of biotic and abiotic stress resistant genes. As in
many plants same set of genes show responding behavior
against the various abiotic and biotic stresses. Thus in the
present study the ESTs from the G. arboreum drought
stressed leaves were subjected to find the up-regulated ESTs
in abiotic and biotic stresses through homology and in-silico
analysis. A cDNA library has been constructed from
the drought stressed G. arboreum plant. 778 clones were
randomly picked and sequenced. All these sequences were
subjected to in-silico identification of biotic and abiotic
up-regulated ESTs. Total 39 abiotic and biotic up-regulated
ESTs were identified. The results were further validated by
real-time PCR; by randomly selection of ten ESTs. These
findings will help to develop stress resistant crop varieties for
better yield and growth performance under stresses.
Keywords cDNA library  Drought  Gene expression 
Gossypium arboreum  RT-PCR  Transcriptome
Electronic supplementary material The online version of this
article (doi:10.1007/s11033-011-0826-y) contains supplementary
material, which is available to authorized users.
M. Y. K. Barozai (&)
Department of Botany, University of Baluchistan, Sariab Road,
Quetta, Pakistan
e-mail: barozaikhan@gmail.com
M. Y. K. Barozai  T. Husnain
Center of Excellence in Molecular Biology, University of the
Punjab, 87-Canal Bank Road, Thokar Niaz Baig, Lahore 53700,
Pakistan
Introduction
The biotic and abiotic stresses adversely affect plant
growth and productivity. Abiotic stress like; cold, salinity,
drought, high temperature, oxidative stress and heavy
metal toxicity, in fact is the major cause of crop failure
worldwide, dipping average yields for most major crops by
more than 50% [1].
Cotton contribution is mainly to textile industry. It also
plays a part in oil and bio-energy production [2]. Two tet-
raploid species (G. hirsutum L. and G. barbadense L.) and
two diploid species (G. arboreum L. and G. herbaceum L.)
are in normal agriculture practices. Although the diploid
cotton species share just 2% to the world cottons, they are the
vital source of important biotic and abiotic resistant genes
with superior agronomic and fiber characters. They also
provide the best approach to study the Gossypium genome
response to various biotic and abiotic stresses through
advanced technique of molecular biology [3, 4]. Among the
diploid species, especially, desi cotton (G. arboreum L.) has
built in desirable resistant genes for various biotic and abi-
otic stresses like; drought, root rot, cotton leaf curl virus
(CLCuV) and insect pests (bollworms and aphids) [5, 6].
Orthologs based gene identification and analysis is a
valid and reliable approach in the current genomics era.
Barthelson et al. (2010) found hybridization of 9,562
G. arboreum genes to Arabidopsis thaliana microarray
platforms for comparative genomic studies. Similarly many
cotton micro-RNA genes were identified through in-silico
analysis using A. thaliana as a reference organism [7, 8].
Many studies suggest the expression of overlapping sets
of genes in response to biotic and abiotic stresses [9, 10].
Recently many researchers identified genes that showing
shared responses to major biotic and abiotic stresses in
plants [11–13].
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The expression databases and in-silico analysis tools are
the wealthy resources for comparative studies. One of such
expression-data analysis tool is the Genevestigator, a
publicly available and coupled with microarray database.
Many researchers have been reported ESTs expression
using Genevestigator in plants and animals [14, 15].
In the present study the G. arboreum ESTs from the
drought stressed leaves, having homology to A. thaliana,
were subjected to in-silico analysis for the identification of
the up-regulated ESTs under various abiotic and biotic
stresses. For this purpose a cDNA library has been con-
structed from the drought stressed leaves of G. arboreum.
778 clones were randomly picked and sequenced. All these
sequences were subjected to in-silico identification of
orthologs in A. thaliana and Genevestigator. Total 39 biotic
and abiotic up-regulated ESTs were identified through
in-silico analysis using A. thaliana as reference organism.
In these 39 ESTs, 24, showed up-regulation in both biotic
and abiotic stresses (category-A), 10 showed up-regulation
in only abiotic stresses (category-B) and five showed
up-regulation in only biotic stresses (category-C). The
results were further validated by RT-PCR; randomly
selected 10 ESTs from these 39 stress up-regulated ESTs.
Materials and methods
Plant material and stress treatment
Seeds of Asiatic desi-cotton (G. arboreum) variety FDH-
786 were obtained from local germplasm center (AARI,
Faisalabad). Concentrated H2SO4 was used for delinting of
seeds and washed five times with tap water to remove the
acid completely.
Seeds were grown in composite soil (peat, sand, soil,
1:1:1) in CEMB green house at temperature 25 ± 2°C, and
relative humidity near 50%. Metal halide illumination
lamps (400 W) were used to supplement natural radiation.
Light radiation reached a maximum of 1,500 lmpl m2 s-1
at the top of canopy at midday. The plants were drought
stressed according to Maqbool et al. [16]; briefly, the
volume of pure water added to the pots was calculated
periodically to maintain the pots of stressed treatments at
5% gravimetric humidity (GH) and non-stressed treatments
at 15% GH. For the comparative study of the plants in
responses to water stress, 40-days-old seedlings were
drought stressed for 15 days, until clearly showed the stress
symptoms.
Construction of cDNA library
Leaf samples were collected from stressed G. arboreum
plants in liquid N2 Jakola et al. [17] method with little
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Mol Biol Rep (2012) 39:1011–1018
modifications was used for total RNA extraction. The DNA
contamination from RNA was removed with the help of
Ambion’s DNAfreeTM Kit. The mRNA was isolated from
total RNA using the oligotex mRNA mini kit (Qiagen).
For the construction of cDNA libraries ‘‘CloneMinerTM
cDNA Library Construction Kit’’ (Invitrogen) was used.
Briefly, the mRNA was used for first strand cDNA synthesis
followed by second strand synthesis. The blunt ended
double strand cDNA proceed to attB1 adapter ligation. The
adapter ligated cDNA was subjected to size selection by gel
electrophoresis. Gel slice containing cDNA C1,000 bp
(1 kb) was excised. The 1.5 ll cDNA was taken and 50 ll
thawed ElectroMAX DH10B competent cells were added.
The contents were mixed gently and transferred to a cold
cuvette (0.1 cm) and electroporated the samples. Added
1 ml of SOC medium and transfer the entire solution to a
15 ml snap-cap. The electroporated cells were shaked for
1 h at 37°C at 225–250 rpm to allow expression of the
kanamycin resistance marker. After 1 h incubation at 37°C,
all cells were pooled into a 15 ml snap-cap tube and an
equal volume of sterile freezing media (60% SOC med-
ium:40% glycerol) was added to it.
PCR amplification of inserts
To confirm the transformation and check the insert size range,
the inserts were PCR amplified using the M13 universal
primers against the flanking regions. For this purpose 5 ll of
overnight grown culture was diluted in 50 ll water. Heat
shock was given at 95°C for 10 min and spin at 3,000 rpm for
5 min at 4°C. PCR reactions were performed to amplify insert
within the clone with 5 ll of diluted culture as template DNA
in 75 ll reaction mixture containing 0.8 ll M-13 forward and
0.8 ll M-13 reverse primers (20 lM), 7.5 ll of 109 PCR
buffer (200 mM Tris–Cl, pH 8.8, 100 mM (NH4)2SO4,
100 mM KCl, 20 mM MgSO4, 1 mg/ml BSA and 1%
Triton), 0.75 ll of 10 mM dNTPs and 2 U of Pfu DNA
polymerase, under the following conditions: initial denatur-
ation 94°C 5 min; followed by 40 cycles of denaturation at
95°C for 45 s, annealing at 52°C for 45 s, and extension at
72°C for 90 s and a final extension at 72°C for 15 min.
Clone sequencing
The sequencing reaction was performed with the ABI
prism Dye Terminator kit. 778 randomly selected clones
were sequenced with Dye terminator Chemistry on Applied
Biosystems Sequencer model 3100/3700. Raw EST
sequences were subjected to base assignment and quality
scores using Phred software [18, 19] the vector and poor
quality sequences were removed. These ESTs were sub-
mitted to Genbank EST database with the accession num-
ber GE653350-GE654127.
Mol Biol Rep (2012) 39:1011–1018
In-silico analysis
Blast search
The ESTs were subjected to BlastN analysis against the
non-redundant (nr) databases with E-value \1.0 e-7 and
selecting organism Arabidopsis at NCBI GenBank to
search for similarity. The gene code names (Atg) of
Arabidopsis orthologs were saved.
Gene ontology (GO) and functional annotation
The gene code names (Atg) of Arabidopsis orthologs,
identified by similarity search, were subjected to GO
functional categorization at TAIR web site publically
available at (http://Arabidopsis.org/tools/bulk/go/index.jsp),
[20] on the basis of cellular components, molecular
functions and biological processes. The genes annotation
list and charts were saved.
Biotic and abiotic responding ESTs identification
by Genevestigator response viewer
The Arabidopsis orthologs to G. arboreum identified by
similarity search, using as a reference were subjected to
Genevestigator Response Viewer [21], to find their
expression pattern by analyzing the Log2 ratios (stress/
control) in biotic and abiotic stresses. Three categories of
stress response ESTs viz: A (showing up regulation in both
biotic and abiotic stresses); B (showing up regulation only
in abiotic stresses) and C (showing up regulation only in
biotic stresses) were identified. The Arabidopsis orthologs
to G. arboreum having Log2 values (stress/control) either
C1.5 in one stressed condition (biotic or abiotic) or C1.0 in
second stressed condition (biotic or abiotic) were placed in
category ‘‘A’’. Similarly the Arabidopsis orthologs to
G. arboreum having Log2 values (stress/control) C1.5 in
abiotic and biotic stresses were placed in B and C cate-
gories, respectively.
Validation studies by quantitative real-time PCR
Real-time PCR reactions were carried out to validate the
results of in-silico identified abiotic (drought) responding
ESTs. Ten abiotic (drought) up-regulated ESTs were ran-
domly selected to confirm the in-silico identified ESTs. The
sequences of primers used in real-time PCR with Genbank
Acc: No. and product size are given in Supplementary
Table S1. The product size ranges from 80 to 116 bp.
Different concentrations in serial dilution of the PCR
product containing GE654076 were used as standard to
validate the iQ5 Cycler reaction and to determine the
1013
quantification range (standard curve). The G. arboreum
Histone-3 gene was used as house-keeping control. The
total RNA from the drought stressed and control (regularly
watered) leaves were extracted and single strand cDNAs
were constructed as mention earlier. 100 ng of cDNA was
used in each reaction. Each sample was used in triplicate
pattern. The reaction conditions were as follows: initial
denaturation at 95°C for 3 min followed by 35 cycles of
denaturation at 94°C for 30 s, annealing at 60°C for 30 s,
and extension at 72°C for 30 s and final elongation step at
72°C for 10 min. A melting curve analysis was carried out
by continuously monitoring fluorescence between 60 and
95°C with 0.5°C increments every 30 s.
Statistical analysis of the real-time results was per-
formed using iQ5 software (Bio-Rad) version 1.0 on the
basis of CT values of the gene in different samples con-
verted to their linear form using the mathematical term
2CT [22], normalized with Histone-3 gene. Analysis of
variance (ANOVA) was performed to analyze significant
difference in transcript expression in drought stressed and
control plants. The efficiency was observed in a range of
94–97%.
Results and discussion
cDNA library
The cDNA library inserts sizes were confirmed by expo-
nentially amplification through PCR and visualization on
agarose gel. The gel showed well distinct sharp bands
(Fig. 1). 96% bands clearly showed inserts sizes in a range
of 1–1.5 kb.
ESTs are the valuable resources for the identification of
genes, expressed at a particular stage of an organism. In the
present study, the EST sequences generated from the
drought stressed G. arboreum cDNA library, was ranges
from 133 to 976 bp with an average sequence length of
683 bp. The maximum number of ESTs were found from
501 to 600 bp followed by 601–700, 701–800, 401–500,
801–900, and 301–400 as shown in Fig. 2. These results
are in consistent with previous studies [23, 24].
Bioinformatics analysis
Homology search
Seven hundred seventy eight sequences (ESTs) were sub-
jected to homology search using BlastN analysis against
the non-redundant (nr)-database in NCBI GenBank. 78%
(78% = 605 sequences) clones sequences (ESTs) didn’t
show significant homology, 11% (11% = 84 clones)
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Fig. 1 PCR confirmation of clones. Randomly selected 96 clones
were exponentially amplified through PCR and resolved on 1.5%
agarose gel to confirm the transformation and the insert sizes. The
94% clones showed insert size ranges from 1 to 1.5 kb. Where, Lane
1–96, PCR amplified cDNA clones and M = 1 kb DNA ladder
Fig. 2 ESTs length distributions. The ESTs length ranged from 133
to 976 bp with an average sequence length of 683 bp. The maximum
number of ESTs were found from 501 to 600 bp followed by
601–700, 701–800, 401–500, 801–900, and 301–400
showed significant homology with Arabidopsis sequences,
6% (6% = 51 sequences) showed significant homology
with Gossypium species and 5% (5% = 38 sequences) had
significant homology with other plant species.
The Arabidopsis orthologs of G. arboreum were further
categorized on the basis of molecular functions, cellular
components and biological processes through Gene-
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Fig. 3 The G. arboreum ESTs Gene Ontology (GO) classification.
The bar charts showed the distribution of ESTs among three principal
GO categories. Where the GO molecular function revealed that the
majority of the ESTs were engaged in enzymatic activity followed by
binding, unknown molecular function, and transport activity (a). The
GO cellular components reveled that mostly ESTs are involved in
cytoplasmic organelles followed by membrane, nucleus, cell wall,
extracellular, and unknown cellular components (b). The GO
biological process annotation categorized the greater part of
G. arboreum homologs in cellular processes, followed by response
to biotic and abiotic stresses, transport, signalling, transcription, and
unknown biological processes (c)
Ontology (GO) annotation. The GO molecular function
revealed that the majority of the ESTs were engaged in
enzyme activity followed by binding, unknown molecular
function, and transport activity (Fig. 3a). The GO catego-
rization for cellular components reveled that mostly ESTs
were involved in cytoplasmic organelles followed by
membrane, nucleus, cell wall, extracellular, and unknown
cellular components (Fig. 3b). The GO biological process
annotation categorized the greater part of G. arboreum
homologs in cellular processes, followed by response to
biotic and abiotic stresses, transport, signaling, transcrip-
tion, and unknown biological processes (Fig. 3c). Zhang
et al. [24] reported almost similar results in the
G. arboreum.
Mol Biol Rep (2012) 39:1011–1018
Biotic and abiotic stress resistant ESTs
Arabidopsis orthologs to G. arboreum ESTs were subjected
to Genevestigator Response Viewer [21], to find their
expression under biotic and abiotic stresses. The analysis of
the Log2 ratios for Arabidopsis orthologs under biotic and
abiotic stresses resulted 39 biotic and abiotic up-regulated
ESTs, that is equal to 5% of total ESTs and 46.4% to the
Arabidopsis homologs.
Of 39 up-regulated ESTs, 24, belong to both biotic and
abiotic (A-category), ten belong to abiotic (B-category) and
five belong to biotic (C-category). In A-category 11 are
drought and biotic, five are heat and biotic, five are cold and
biotic and three are salt and biotic up-regulated ESTs. In
drought and biotic six are drought and CaLCuV, two are
drought and B. graminis, two are drought and P. syringae
and one is drought and nematode up-regulated ESTs. The
heat and biotic consist of four heat and nematode and one
heat and TuMV up-regulated ESTs. The cold and biotic
consist of two in each cold and TuMV and cold and nema-
tode and one is cold and P. syringae up-regulated ESTs. In
salt and biotic, salt and nematode, salt and CaLCuV and salt
and P. graminis have one up-regulated ESTs in each. The
B-category has five heat, three drought and two cold up-
regulated ESTs. The C-category has two Nematodes, two
CaLCuV and one TuMV up-regulated ESTs. The 39 up-
regulated ESTs with their Genbank Accession numbers,
functions, E-values with A. thaliana homologs and the Log2
expression ratios are shown in Supplementary Table S2.
We found that the G. arboreum ESTs GE653583,
GE653376, GE653653, and GE653923 showed up-regula-
tion in response to Turnip mosaic virus (TuMV), a member
of the Potyviridae. The infecting host range for TuMV is
very wide at least 318 plant species of 43 families [25]. The
TuMV up-regulated ESTs are the good source for identifi-
cation of resistant genes against TuMV [26].
We identified that the ESTs GE653702, GE653710,
GE653741, GE653950, GE653602, GE653395, GE653887,
GE654100, and GE653855 showed up-regulation to cab-
bage leaf curl virus (CaLCuV), a member of the Be-
gomovirus genus, belongs to the family Geminiviridae
[27]. It is among the large, diverse family (Geminiviruses)
of plant infecting viruses that cause severe crop losses
worldwide [28]. The research on plant viruses showed that
viral infection is accompanied by many changes in the
plant transcriptome. These changes are the responses to
limit the viral infection by the infected plants [29]. Num-
bers of virus responding genes during infections are
reported in many plants [30, 31]. The G. arboreum ESTs
responding to CaLCuV are the potential source of genes
that can protect the plant from such viruses.
In our findings the G. arboreum ESTs GE653401,
GE653455 and GE654020 showed up-regulation in response
1015
to Blumeria graminis infection. Blumeria graminis is an
economically important pathogen of cereals that causes
powdery mildew. The infection spread through the fungal
conidia dispersal. The plant receiving these conidia on leaves
developed the pathogen symptoms [32]. The result of the
infection in the cereal crops are the great yield losses [33].
Protection from this disease can be achieved by using spraying
foliar fungicides or development of resistant crop cultivars by
manipulating them with pathogen resistant gene(s). The
G. arboreum ESTs showing response to this pathogen are the
strong candidates for resistant varieties creation.
In our result ESTs GE653491, GE653379 and GE653450
showed up-regulation against Pseudomonas syringae.
P. syringae is a gram-negative plant pathogen that educes a
wide range of symptoms in plants, including blights (rapid
death of tissue), leaf spots and galls [34]. The best approach
to defend the plant from this pathogen is the use of pathogen
resistant genes. The ESTs from G. arboreum responding to
this pathogen are the potential source of defending genes in
plants against the P. syringae.
In this study the G. arboreum ESTs GE654069,
GE653362, GE653374, GE653410, GE653776, GE653952,
GE654027, GE653654, GE653433, and GE653706 showed
up-regulation against parasitic nematode infection. More
than $100 billion per year losses have been reported for the
parasitic nematode infection in plants. These complicated
soil-dwelling pests parasitize plant roots, seizing the
nutritional resources of their hosts while escaping from the
host defenses. Nematicides have been successfully used to
control nematodes but host manipulating with resistance
gene(s) is a preferable alternative because of the expense
and environmental toxicity of nematicides [35]. The ESTs
of G. arboreum showing response to parasitic nematode
infection are the potential candidates as resistant genes to
cope the infection.
The G. arboreum ESTs GE653702, GE653710,
GE653741, GE653950, GE653602, GE653395, GE653401,
GE653455, GE653491, GE653379, GE654069, GE654004,
GE654013, and GE653686 showed over expression in
drought stressed condition. The EST (GE653741) has sig-
nificant homology to stress-induced cysteine proteinase
(LtCyp1). Proteases have role in the elimination of
Mis-folded proteins. Misfolding proteins are continuously
generated by a variety of mechanisms such as, mutation,
biosynthesis errors, spontaneous denaturation, ROS dam-
age, biotic and abiotic stresses and disease. Correspond-
ingly, cysteine proteases are induced when tissues are
exposed to different abiotic stresses such as dehydration or
salinity in Arabidopsis leaves [36] and by low temperature
in tomato [37]. Usui et al. [38] found that cysteine protease
is up-regulated in response to oxidative stress and plays a
role in the maintenance of cell metabolism under oxidative
stress conditions in Chlamydomonas species.
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The ESTs GE653602 has significant homology with
gamma-tocopherol methyltransferase. The products of
these genes act as antioxidant enzymes playing a key role
in conferring abiotic and biotic stress tolerance by scav-
enging reactive oxygen species (ROS) [39]. The EST
GE653455 showed significant homology with lipid transfer
protein (LTP). Trevino and Connell [40] have reported the
induction of three nsLTPs (non-specific lipid transfer pro-
teins) in Lycopersicon pennellii by drought conditions. The
EST GE653491 showed homology to heat shock protein
(Hsp). From various studies it has been demonstrated that
plant Hsps are not only produced in response to high
temperature but also under oxidative, salinity, drought, and
at freezing temperature stresses [41]. The EST GE653379
is identified as homolog of a kinase protein. The kinases
are involved in the cell signaling to create tolerance in
plants under biotic and abiotic stress conditions [42]. The
ESTs GE654013 showed similarity to zeaxanthin epoxi-
dase (ZE). Wang et al. [43] suggested that over-expression
of ZE impaired the function of the xanthophyll cycle and
aggravated PSII photoinhibition in tomato under high light
and low temperature stresses.
The ESTs GE653362, GE653374, GE653410,
GE653776, GE653583, GE653487, GE653633, GE654082,
GE653728 and GE654012 showed response against heat
stress. The EST Ge653362 has significant homology with
the plastid lipid-associated protein (PAP). The PAP role
under various stresses is well elucidated in plants [44].
The EST GE653410 has significant homology to cy-
clophilin protein (CyP). Plant cyclophilins are stress-
responsive proteins, and up-regulated gene expression have
been reported in response to environmental stresses such as
high temperature, chilling, salinity, wounding, as well as
virus infection and during chemically induced defense [45].
The EST GE653776 showed significant homology with
sorbitol related enzyme. Plants have the ability to tolerate
stress by a mechanism called as osmotic regulation by
osmoprotectant like; sorbitol [46]. The EST GE653583 has
significant homology with Arabidopsis Glyceraldehyde-3-
phosphate dehydrogenase that is found over-expressed in
many plants [24].
Many researchers have found that heat shock proteins
have very important role in the protection of plants under
various stress condition like; cold, drought and heat
[24, 47]. Our identified EST GE653336 showed homology
with DNAJ heat shock protein.
The ESTs GE653376, GE653653, GE653952, GE654027,
GE653450, GE653696, and GE654038 showed response to
cold stressed. The EST GE653653 showed homology to
temperature-induced lipocalin (TIL). Lipocalins and lipoca-
lin-like proteins are associated with the plant’s capacity to
create low temperature tolerance [48]. According to Zwenger
and Basu [49], the Arabidopsis terpene synthase gene was
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over expressed in response to various stresses, including low
temperature, dehydration, osmotic stress, nutrient, ozone and
mechanical stress. The EST GE654038 showed significant
homology with Arabidopsis terpene synthase gene.
The ESTs GE653654, GE653887 and GE654020
showed over-expression under salt stress. The EST
GE653887 showed significant homology with ATTAP2
transporter protein. Similar findings were reported in
Catharanthus roseus [50]. The EST GE654020 showed
homology with Adenylyl-sulfate reductase protein. Phart-
iyal et al. [51], observed the over-expression of the same
protein under cold stressed condition.
Validation studies
The G. arboreum abiotic and biotic stress resistant poten-
tial candidate ESTs, resulted from in-silico analysis were
further confirmed by quantitative real-time PCR. Ten biotic
and abiotic ESTs (GE653395, GE654069, GE654004,
GE653491, GE654013, GE653702, GE653741, GE653602,
GE653710, and GE653455) that identified as drought up-
regulated ESTs through in-silico analysis were randomly
selected for real-time RT-PCR validation studies under
drought (water stressed) condition. The G. arboreum
Histone gene was used as the reference gene to normalize
the expression levels. All transcripts showed different level
of up-expression in drought (water stressed) plants as com-
pared to control (well watered) plants. These results con-
firmed the in-silico identified stress resistant ESTs (Fig. 4).
Fig. 4 Relative fold expression of the selected G. arboreum stress-
tolerance ESTs, GE653395 (Dormancy-associated protein),
GE654069 (Starch synthase), GE654004 (Chalcone-flavanone isom-
erase), GE653491 (Heat shock N-terminal domain protein),
GE654013 (Zeaxanthin epoxidase), GE653702 (RUB1 conjugating
enzyme), GE653741 (cysteine proteinase), GE653602 (Gamma-
tocopherol methyltransferase), GE653710 (Histone mono-ubiquitina-
tion 2) and GE653455 (Lipid transfer protein) in leaves of control
(well watered, the right bar) and drought stressed (the left bar) plants
through real-time PCR. Solid bars represent FAM (carboxyfluores-
cein) signals during the reaction
Mol Biol Rep (2012) 39:1011–1018
Similar approach was used by a number of researchers to
validate the stress responding genes [23, 24].
Conclusion
As biotic and abiotic stresses are the emerging major
tribulations for the global agricultural practices. So it is
need of time to produce stress tolerant crops which have
the ability to give better yields under biotic and abiotic
stresses. The present study resulted; identification of 39
biotic and abiotic stresses resistant ESTs in Asiatic desi
cotton (G. arboreum). These findings will help to develop
stress resistant crop varieties for better yield and growth
performance under stresses.
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