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Isolation and molecular characterization of an ethylene response
factor NtERF1-1 in Nicotiana tabacum cv. Xanthi
JUN-SHAN GAO , LI HU , PENG XIE , YAN MENG , YONG-PING CAI and YI LIN*
School of Life Sciences, Anhui Agricultural University, 230036 Hefei, Anhui, China
*Corresponding author (Fax, +86 551 65786967; Email, [email protected])
Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and
biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from
Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1
encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence
analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino
acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and
real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In
addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response
inducted by the interaction between the N gene and TMV.
[Gao J-S, Hu L, Xie P, Meng Y, Cai Y-P and Lin Y 2014 Isolation and molecular characterization of an ethylene response factor NtERF1-1 in
Nicotiana tabacum cv. Xanthi. J. Biosci. 39 887–897] DOI 10.1007/s12038-014-9473-5
1.
Introduction
Plants suffer from many diseases caused by bacteria, fungi,
viruses and other organisms. Yet, plants have employed a
variety of strategies to protect themselves against invading
pathogens (Burch-Smith et al. 2007). N gene is a member of
the toll interleukin-1 receptor/nucleotide-binding domain /
leucin-rich repeats (TIR-NB-LRR) class of plant resistance
genes, which confers resistance to Tobacco mosaic virus
(TMV) (Whitham et al. 1994; Bent and Mackey, 2007). It
encodes two alternatively spliced mRNAs, which are all
required for N function (Dinesh-Kumar et al. 1995, 2000).
When Nicotiana tabcum cv. Xanthi NN carrying the N gene
is infected by TMV, an interaction between the N gene
product and p50, a helicase domain of TMV replicase gene,
causes a hypersensitive response (HR) at the site of infection
(Yang et al. 2006; Niemeyer et al. 2013), which results in
programmed cell death and limits the spread of TMV, also
leading to acquiring systemic resistance (Martin et al. 2003;
Gao et al. 2010). The phenomenon is explained by the genefor-gene hypothesis (Flor 1971).
Keywords.
Apetala2/Ethylene Response Factor (AP2/ERF) is a large
family of transcription factors that plays significant roles in
response to biotic and abiotic stresses by recognizing the cisacting element AGCCGCC, which is present in the promoter
region of pathogenesis-related genes and is known as the
GCC box (Sakuma et al. 2002; Zhang et al. 2008). To date,
many ERF genes are cloned from various plants, such as
Arabidopsis AtERF1-5 (Fujimoto et al. 2000), tomato
LeERF1-4 (Tournier et al. 2003) and soybean GmERF3
(Zhang et al. 2008). In addition, AP2/ERF can induce the
expression of pathogenesis-related genes and enhance disease resistance in plants. A large number of evidences demonstrate that AP2/ERF is involved in biotic stresses, and
participates in the signal transduction pathway related to
disease resistance. For example, the overexpression of
TiERF1 enhances the resistance to sharp eyespot in transgenic wheat (Liang et al. 2008). GbERF enhances the resistance to Pseudomonas syringae in transgenic tobacco (Qin
et al. 2006). BrERF11, isolated from a Chinese cabbage,
improves resistance to Ralstonia solanacearum in transgenic
tobacco (Lai et al. 2013). Transient expressions of the two
AP2/ERF; Nicotiana tabacum; NtERF1-1; RT-PCR; TMV
http://www.ias.ac.in/jbiosci
Published online: 20 October 2014
J. Biosci. 39(5), December 2014, 887–897, * Indian Academy of Sciences
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Jun-Shan Gao et al.
IbERF(1/2) suggest that they play a role in the signal transduction pathway as a transcriptional activator causing defence response to stress (Kim et al. 2012). The
overexpression of OsERF922 enhances susceptibility to
Magnaporthe oryzae, and knockdown of OsERF922 by
means of RNAi increases resistance against M. oryzae (Liu
et al. 2012). Pp-ERFs, ERF transcriptional activators in
peach, are seen in response to Xanthomonas campestris pv.
(Sherif et al. 2012). MdERF3/4/5/6 are induced by Botrytis
cinerea infection in Malus domestica (Akagi et al. 2012).
TaPIEP1, a pathogen-induced ERF gene, confers resistance
to Bipolaris sorokiniana in wheat (Dong et al. 2010). The
expression of NtERF5 increases tolerance to TMV (Fischer
and Dröge-Laser, 2004). The overexpression of OPBP1 exhibits distinct enhancing of resistance to Magnaporthe oryzae
and Rhizoctonia solani in rice (Chen and Guo, 2008). The
expression of NtERF3 is up-regulated by TMV infection in
N. tabacum cv. Samsun NN harbouring the resistance N gene,
and enhances resistance to TMV (Ogata et al. 2012). EREB1
plays a vital role in the signal transduction pathways of biotic
stress and the overexpression of EREB1 has been suggested to
serve as a viable approach to enhance disease resistance in
cotton (Meng et al. 2010). ORA59, cloned from Arabidopsis
thaliana, strengthens plant resistance to pathogens (Pré et al.
2008). There is evidence supporting that the overexpression of
TSRF1 results in changes of the response in tobacco to pathogen (Zhou et al. 2008).
In this study, a member of AP2/ERF family, NtERF1-1,
was isolated from N. tabacum cv. Xanthi NN, which may
play a key role in the signal transduction pathway of HR
induction based on its bioinformatics and expression analyses. We suggest that NtERF1-1 could bind specifically to the
GCC-box, and induce pathogenesis-related (PR) genes to
express, and enhance resistance to TMV.
2.
2.1
Materials and methods
Plant materials and growth conditions
Tobacco (N. tabacum cv. Xanthi NN) plants, which possess the
N gene for resistance to TMV, provided by Tobacco Research
Institute of Chinese Academy of Agricultural Sciences, were
grown in a virus-free growth chamber at 25°C with a 16/8 h
light/dark cycle. Water with nutrient solution was given twice a
week. Seven-week-old plants were used in this study.
2.2
TMV inoculation
A wild-type TMV-OM virus was inocluated on TMVsensitive tobacco plants (N. tabacum cv. Xanthi NN), and
the infected leaves were stored at –80°C. Before inoculation,
TMV was prepared by grinding infected tobacco leaves in
J. Biosci. 39(5), December 2014
10 mM phosphate buffer (pH7.0) and purified by ultracentrifugation method (Konagaya et al. 2004). The precipitate
was suspended in phosphate buffer and diluted to a concentration of 5 ng/μL. TMV inoculation was performed according to the method reported previously (Gao et al. 2007). The
inoculated plants were maintained in a growth chamber at
25°C under 16/8 h photoperiod. HR symptoms were carefully monitored. The inoculated tobacco leaves were collected at the different time after infection and stored at −80°C for
use.
2.3
Isolation of NtERF1-1 full-length cDNA
Total RNA was extracted from these leaves inoculated with
TMV at 48 hours post inoculation (hpi) using RNA extraction reagent kit according to the manufacturer’s protocols
(Tiangen Biotech, Beijing). The concentration and quality of
extracted RNA were evaluated by 1.0% agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo,
USA), respectively. Reverse transcription polymerase chain
reaction (RT-PCR) was performed by the RT-PCR kit
(Promega, USA). The first strand cDNA was synthesized
from 3 ng total RNA according to the manufacturer’s recommendations. Internal cDNA was amplified with ERF-F
(5′-ATCCAGGGAAAAAGAGCCGTG-3′) and ERF-R (5′CAACGCGAGCGAATGATTTCT-3′), which were designed according to the consensus nucleotide sequences of
ERF genes from Arabidopsis, soybean, tomato and other
tobacco species. The 3′ and 5′ distal cDNAs were obtained
by rapid amplification of cDNA ends (RACE) method using
the SMART RACE cDNA amplification kit (Clontech,
USA). 3′-RACE products of NtERF1-1 were generated by
gene-specific forward primers ERF3′-1 (5′-ATCTCAGCCT
CGGTGGATCCCGTAGTT-3′) and ERF3′-2 (5′-CTGGAT
GTATTACGCCGGTGAATC-3′). 5′-RACE products of
NtERF1-1 were generated by gene-specific reverse primers
ERF5′-1 (5′-CTCAACGGTACTGCTTTGGCTCGGACT3′) and ERF5′-2 (5′-CGTATCAAAAGTGCCAAGCCAA
AC-3′). All these amplification reactions were carried out
in a programmable thermal controller (Whatman Biometra,
CA). The obtained PCR products were electrophoresed on a
1.2% agarose gel and were purified to insert into pEASY-T1
vector (Trans, Beijing). For sequence determination, at least
3–5 independent positive clones were selected and sequenced (Sangon, Shanghai). The overlapping sequences
were assembled by DNAstar software to obtain the fulllength cDNA sequence of NtERF1-1.
2.4
Sequence and protein structure analysis
The physical and chemical properties of NtERF1-1 and
deduced amino acid sequence were analysed by DNAstar
NtERF1-1 from Nicotiana tabacum cv. Xanthi
and DNAMAN 5.0 software, respectively. Open reading
frame (ORF) of NtERF1-1 was checked for use by the NCBI
ORF Finder online (http://www.ncbi.nlm.nih.gov/gorf/
gorf.html). Comparisons of nucleotide and amino acid sequence homologies were made by BLAST and DNAMAN
5.0 software. The phylogenetic tree of NtERF1-1 and other
genes was constructed using MEGA 4.0 software. Secondary
structure, signal peptide and transmembrane domain of the
NtERF1-1 protein were analyaed by PredictProtein online,
SignalP 3.0 Server and TMHMM Server v.2.0, respectively.
Meanwhile, the conserved structural domain and posttranslational modification site of the NtERF1-1 protein were
calculated with DNAMAN 5.0 and PredictProtein online,
respectively.
2.5
Semi-quantitative RT-PCR and real-time quantitative
PCR
To detect the expression profile of NtERF1-1, total RNA was
isolated from N. tabacum cv. Xanthi NN leaves of 0, 12, 24,
36, 48, 60 and 72 hpi with TMV using RNA extraction
reagent kit (Tiangen Biotech., Beijing). The first strand
cDNA was synthesized from 1 μg total RNA using the first
strand cDNA synthesis kit (Takara, Dalian) according to the
manufacturer’s instructions. For semi-quantitative RT-PCR,
gene-specific primers were designed from the flanking sequence of NtERF1-1 cDNA as follows: 5′-TTGTAGTCCG
AGCCAAAGCAGTA-3′ (forward) and 5′-TATGAAGA
AATCACTCGCTCTCGTT-3′ (reverse). The tobacco Actin
gene (Accession number EU938079.1) was used as an internal control, which was amplified with the forward primer
(5′-TCGTCTGTGATAATGGGACTGGA-3′) and the reverse primer (5′-AAAGGGATAGAACGGCTTGAATG3′). To further analyse the NtERF1-1 gene expression, realtime quantitative RT-PCR was performed using the following specific primer pairs: 5′-TATTTAGCAACGGTGTGG
GTAGG-3′ (forward) and 5′-GAAGAAATCACTCGCTCT
CGTTG-3′ (reverse). Likewise, the Actin gene was used as
an internal control, which was amplified using the primer
pairs: 5′-AGGGTTTGCTGGAGATGATGCT-3′ (forward)
and 5′-GGTGCTTCAGTGAGTAGTACAGGG-3′
(reverse).
3.
3.1
Results
HR induction by TMV infection
To investigate the time and symptom of HR induction, we
inoculated N. tabacum cv. Xanthi NN plants with TMV. The
inoculated plants were incubated at 25°C for 3 days and the
induction of HR was observed at different time interval after
inoculation. The results showed that a local lesion began to
889
exhibit at around 36 hpi, but necrotic lesions were induced
clearly at 48 hpi. As time post-inoculation went on, the size
and quantity of lesions increased gradually. Until 72 hpi, the
HR spread to all inoculated leaves (figure 1).
3.2
Isolation and characterization of the NtERF1-1 gene
A NtERF1-1 gene, belonging to AP2/ERF transcription factors, was cloned from N. tabacum cv. Xanthi NN by RTPCR. To determine the full-length coding sequences of the
NtERF1-1 cDNA, the 5′ and 3′ distal cDNAs were amplified
by RACE from the total RNA extracted from N. tabacum cv.
Xanthi NN, and assembled with the overlapping internal
cDNA sequences. The reconstructed, full-length coding sequences of NtERF1-1 cDNA was of 1091 base pairs (bp),
containing 114 nucleotides (nt) 5′-untranslated regions
(UTR) and 287 nt 3′-UTR and 690 bp ORF, which encoded
229 amino acid residues (Genbank accession number
KC111203). The deduced NtERF1-1 protein had an estimated molecular mass of 24.58 kDa and contained an AP2
domain (figure 2). The results showed that the NtERF1-1
gene was a member of AP2/ERF transcription factor family.
Also, a prediction of the NtERF1-1 protein structure demonstrated that mean isoelectric point and hydrophobic property were −0.474 and pH 9.9, respectively.
The amino acid sequences of the full-length NtERF1-1
protein and AP2/ERF transcription factors from diverse
plants were shown in figure 3. The amino acid sequence
analyses revealed that the full-length NtERF1-1 protein had
95% identity to the NtERF6b protein from N. tabacum cv.
Samsun NN (Ogata et al. 2012), and 95% identity to the
NbCD1 protein from N. benthamiana (Nasir et al. 2005), but
only 63%, 71% identities to the Arabidopsis ERF4 protein
and the Populus ERF46 protein, respectively (Yang et al.
2005; Tuskan et al. 2006). Phylogenetic tree was constructed
using the BLAST program (figure 4). The results showed
that the NtERF1-1 protein was closely related to NtERF6b
and NbCD1 proteins, which were from the same genera
Nicotiana. This analysis indicated that NtERF1-1 was the
closest to NtERF6b and might play a role similar to
NtERF6b.
3.3
Structural features of NtERF1-1 protein
To characterize the NtERF1-1 protein, we used
PredictProtein software, which was an online analysis database tool (http://www.predictprotein.org/), to calculate the
secondary structure of NtERF1-1. From figure 5, we conclude that the minimum consecutive exposed residues were
10 amino acids. The structure included 12% cutoff, and
predicted that the nucleolus organization region was 70J. Biosci. 39(5), December 2014
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Jun-Shan Gao et al.
Figure 1. HR induced by TMV infection in N. tabacum cv. Xanthi NN. (A–G) Symptoms of HR at 0, 12, 24, 36, 48, 60 and 72 hpi (hours
post inoculation), respectively. Arrows indicate the appearance of lesion.
229. In the secondary structure of NtERF1-1, Helix, Strand
and Lop were 7.0%, 8.3% and 84.7%, respectively.
By the SignalP 3.0 Server online (http://www.cbs.dtu.dk/
services/SingalP/), we calculated whether the NtERF1-1
protein had a signal peptide. The results indicated that C
score of the 30th amino acid (E) was 0.427, and the S score
of the 9th amino acid (L) was 0.950 for the highest mark.
Combining with the systems of C score and S score derived
from the optimal cutting sites were the 29th and 30th amino
acids, respectively. Simultaneously, the maximum of Y
score was 0.586. These results showed that the NtERF1-1
protein contained a signal peptide. NtERF1-1 could be synthesized in cytoplasm, and subsequently transported to a
corresponding site (nucleus) to specifically combine to the
corresponding cis-element, in order to regulate the target
gene expression.
To detect whether the NtERF1-1 protein contained a
transmembrane domain, we used TMHMM Server v.2.0
online (http://www.cbs.dtu.dk/services/TMHMM/). According to figure 6, we could speculate that there was not a
transmembrane domain involved in the NtERF1-1 protein.
3.4
Expression analysis of NtERF1-1 gene in Xanthi NN
To determine the expression level of NtERF1-1 gene in
Xanthi NN inoculated with TMV, we carried out semiJ. Biosci. 39(5), December 2014
quantitative RT-PCR and real-time quantitative PCR using
specific primers for the respective mRNAs. DNA fragments
of the expected sizes were amplified only after the RT
reaction from total RNA of Xanthi NN leaves at different
time points post TMV inoculation. The results indicated that
the expression levels of NtERF1-1 were higher at both 48 hpi
and 60 hpi than other time points. Subsequently, the expression level of NtERF1-1 decreased a little (figure 7A–B).
4.
Discussion
AP2/ERF genes, which possess GCC-box binding activity,
were first isolated from tobacco (Ohme-Takagi and Shinshi,
1995), and increasing evidence indicates that they play an
important role for transcription activators/repressors in modulating plants defence response (Sohn et al. 2006; Hu et al.
2004; Fujimoto et al. 2000). In this study, a tobacco transcriptional activator NtERF1-1 was cloned from N. tabacum
cv. Xanthi NN, which encoded 229 amino acid residues. The
deduced NtERF1-1 protein had an estimated molecular mass
of 24.58 kDa and contained an AP2 domain of 59 amino
acids. However, NtERF1-1 did not contain a conserved (L/F)
DLN (L/F) XP sequence at their C-terminal, also called
ERF-associated amphiphilic repression (EAR) motif (Ohta
et al. 2001). According to the numbers and similarities of the
DNA-binding domains, the AP2/ERF transcription factors
NtERF1-1 from Nicotiana tabacum cv. Xanthi
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Figure 2. Nucleotide and deduced amino acid sequences of a full-length NtERF1-1 cDNA in N. tabacum cv. Xanthi NN. A conservative
AP2 motif is marked with box. The initiation codon and termination codon are marked using double underline.
are classified into four subfamilies: AP2 (APETALA2),
RAV (related to ABI3/VP1), DREB (dehydration-
responsive element binding protein) and ERF (ethyleneresponsive factor) (Sakuma et al. 2002). The cloned
J. Biosci. 39(5), December 2014
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Jun-Shan Gao et al.
NtERF1-1 from Nicotiana tabacum cv. Xanthi
893
Figure 4. Phylogenetic tree of NtERF1-1 with other ERF proteins. AtERF3 (accession no. NP_175479), AtERF9 (accession no.
NP_199234), NtERF5 (accession no. AAU81956), NtERF111 (accession no. BAL68166), SlTSRE1 (accession no. NP_001234818),
SlTERF1 (accession no. NP_001234841), TaERFL1c (accession no. ABC65860), PtERF44 (accession no. XP_002329798), GmERF7
(accession no. AEQ55266), CaERFLP1 (accession no. AAS20427).
NtERF1-1 protein contained a conserved alanine at 14th and
aspartic acid at 19th in the AP2/ERF domain, and suggested
that NtERF1-1 belonged to a member of the ERF subfamily.
Meanwhile, the conserved sites of AP2 domain suggest this
kind of transcription factors binding specifically to different
cis-acting elements (Allen et al. 1998).
Up to now, a large number of ERF transcription factors
have been described in different plant species and presented
a diversity of regulatory functions. For instance, GmERF4
increases resistance not only to biotic stresses (TMV) but
also to abiotic stresses (cold, drought, salt) (Zhang et al.
2010). HARDY can improve plant tolerance to salt and
drought in Arabidopsis thaliana (Abogadallah et al. 2011).
JERF3 enhances tolerance of tobacco seedlings to salt,
drought and freezing (Wu et al. 2008).
Previous reports have demonstrated that ERFs play important roles in activating PR genes and triggering defence
responses following pathogen attack (Sakuma et al. 2002).
Specially, when tobacco harbouring the N gene for resistance
is infected with TMV, ERFs up-regulate some defence
genes, and induce HR at the site of virus infection to limit
the spread of the pathogen (Gilchrist 1998; Heath 2000). The
pathogen gets confined around the necrotic lesions, and
therefore systemic infection is prevented to induce a state
of pathogen-nonspecific resistance throughout the plant.
These events activate a signal transduction pathway that
results in the induction of kinase cascades (Kunkel and
Brooks, 2002). Consequently, the plant develops a broadspectrum resistance against many pathogens, a phenomenon
known as systemic acquired resistance (SAR) (Sticher et al.
1997; Durrant and Dong, 2004).
In this study, we isolated a AP2/ERF transcription factor,
NtERF1-1, from N. tabacum cv. Xanthi NN. The experimental results indicated that the NtERF1-1 gene expression was
observed at 36 hpi and was markedly up-regulated at around
48–60 hpi. Subsequently, the gene expression was slightly
down-regulated at 72 hpi. The results were in accordance
with other such reports (Otsuki et al. 1972; Mittler et al.
ƒFigure 3.
Comparison of amino acid sequences among NtERF1-1, NtERF6b (accession no. BAJ72666), NbCD1 (accession no. BAD99476),
AtERF4 (accession no. NP_188139), AtERF12 (accession no. NP_174158), LeERF2 (accession no. AAP32202), PtERF46 (accession no.
XP_002327909), SlERF3 (accession no. AA034705), StERF3 (accession no. ABK96798), TaERFL1a (accession no. ABC65858). Amino acid
identity of 100% is marked with a black background, amino acid identity of higher than 50% is marked with a dark grey background, and amino
acid identity of more than 33% is marked with a light grey background. The alanine and aspartic acid residues at positions of 14 and 19 in the
AP2/ERF domain are marked with open dots. The nuclear localization signal sequences are marked with solid dots.
J. Biosci. 39(5), December 2014
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Jun-Shan Gao et al.
Figure 5. Predicted signal peptide of the NtERF1-1 protein. C score: score for original cleavage site; S score: score for the signal peptide;
Y score: score for general cleavage sit.
1996). In addition, from figure 1, we found clearly that a
local lesion appeared at approximately 48 h after TMV
inoculation. Based on the PredictProtein database online,
we concluded that the NtERF1-1 protein could locate in
cytoplasmic matrix or organelle matrix or nucleus, which
was similar to some AP2/ERF proteins (Solano et al. 1998;
Yang et al. 2005). We had also analysed the activation site
and found that it contained protein kinase C phosphorylation
site, casein kinase II phosphorylation site, N-myristoylation
site, amidation site, serine proteases, histidine active site, and
Figure 6. Predicted transmembrane domain for deduced amino acid of the NtERF1-1 protein.
J. Biosci. 39(5), December 2014
NtERF1-1 from Nicotiana tabacum cv. Xanthi
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Figure 7. Expression pattern of NtERF1-1 gene in Xanthi NN inoculated with TMV. (A) Semi-quantitative PCR analysis of the NtERF1-1
gene expression. 0, 12, 24, 36, 48, 60 and 72 hpi represent the different timepoint after TMV inoculation. A house-keeping gene Actin was
used as an internal control to normalize the template quantity. (B) Real-time quantitative PCR analysis of the NtERF1-1 gene expression.
0h, 12h, 24h, 36h, 48h, 60h and 72h represent the different time points after TMV inoculation. Each independent experiment was performed
three times.
so on. These phosphorylation sites represented that the
NtERF1-1 protein was involved in a variety of signal transduction pathway. Because the protein kinases and protein
phosphatases could make protein phosphorylation or dephosphorylation in the process of signal transduction, and
thus regulated the target gene expression. On the basis of
these, we speculated that the NtERF1-1 gene was involved in
HR induction and may play an important role in HR signal
transduction pathway. In future, we will provide enough
evidence to prove the functional role of the NtERF1-1 gene
during HR induction or under abiotic stresses for developing
a new strategy to increase resistance to TMV and offering a
fundamental basis for crop genetic breeding.
Acknowledgements
This work was supported by the Natural Science Fund of
Anhui Province (no. 11040606M67 to J-S G) and PhD
Program Foundation of Ministry of Education of China
(no. 20123418120001 to J-S. G.). It was also supported by
the National Natural Science Fund of China (no. 31172270
to Y. M.) and the Scientific Research Foundation for the
Excellent Talents of Anhui Agricultural University (no.
yj2009-25 to J-S G).
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MS received 11 April 2014; accepted 07 August 2014
Corresponding editor: MAN MOHAN JOHRI
J. Biosci. 39(5), December 2014