ConstructionandCharacterizationofaFulllengthcDNALibraryandIdentificationof GenesInvolvedinSalinityStressinWild Eggplant(SolanumtorvumSwartz)

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ConstructionandCharacterizationofaFulllengthcDNALibraryandIdentificationof
GenesInvolvedinSalinityStressinWild
Eggplant(SolanumtorvumSwartz)
DatasetinHorticulture,EnvironmentandBiotechnology·February2012
ImpactFactor:0.73·DOI:10.1007/s13580-012-0089-0

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AnhuiAgriculturalUniversity(AHAU)
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Hort. Environ. Biotechnol. 53(2):158-166. 2012.
DOI 10.1007/s13580-012-0089-0

Research Report

Construction and Characterization of a Full-length cDNA Library and
Identification of Genes Involved in Salinity Stress in Wild Eggplant
(Solanum torvum Swartz)
1,2ಳ

Gang Chen
1

2ಳ

1

1,2*

2

2

, Hua Wang , Jun-Yi Gai , Yue-Lin Zhu , Li-Fei Yang , Qian-Qian Liu ,
2
2
Gong-Chen Zhang , and Guo-Hu Chen

National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China
2
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*Corresponding author:
These authors contributed equally to this work.

ಳ

Received October 9, 2011 / Revised February 10, 2012 / Accepted February 15, 2012
GKorean Society for Horticultural Science and Springer 2012

Abstract. The objectives of this paper were to construct a full-length cDNA library and to isolate genes that confer salt
tolerance from the leaves of salinity-tolerant wild eggplant variety, ‘Torvum Vigor’ (Solanum torvum Swartz). A full-length
cDNA library from the leaves was successfully constructed by a switching mechanism at 5’-end of RNA transcript
(SMART) approach and a long-distance PCR (LD-PCR) technique. The titer of the primary cDNA library was 3.6 ×
106 cfumL-1 and that of the amplified library was 1.2 × 1010 cfumL-1. Gel electrophoresis results showed that most
of the cDNA inserts ranged from 0.40 to 2.5 kb, with a recombination rate of 99%. A total of 427 randomly selected
positive clones were sequenced. After removing the unsuccessful reads, 364 datasets were obtained and have been
submitted to the NCBI Nucleotide Sequence Database under GenBank accession numbers JK265131-JK265494. Among
the 364 submitted sequences, 74.45% of them contained full-length coding regions. BLASTX analysis revealed that
62.36% of the ‘Torvum Vigor’ expressed sequence tags (ESTs) possessed homology to known or putative proteins of
other organisms. Seven genes that might be responsible for the encoding of known proteins in other organisms were
identified to confer salt tolerance. This evidence demonstrated that the cDNA library constructed was a full-length
library of high quality. It could be a useful resource for further research in the cloning of stress-related genes, which
could be utilized in the genetic improvement of vegetable crops using transgenic technology.
Additional key words: ESTs, gene cloning, salt-tolerance gene, stress-related gene, switching mechanism at 5’-end
of RNA transcript (SMART)

Introduction
The genetic resources of wild plant species have been
assessed for resistance against the most serious biotic and
abiotic stresses (Blestsos et al., 1998). Attempts at crossing
vegetable crops with their wild relatives resulted in limited
successes due to hybrid incompatibilities. However, the
application of plant biotechnology, particularly the exploitation

of genetic transformation for gene transfer, has offered great
opportunities to overcome the reproductive isolation between
different plant species (Collonnier et al., 2001). With regard
to genetic engineering, the availability of stress-tolerance
genes for vegetable crop improvement is one of the key

factors for a successful gene transfer. Genetic transformation
mediated by Agrobacterium tumefaciens has been performed
to facilitate the introduction of agronomically important traits
from wild relatives into cultivated vegetable crops (Sihachakr
et al., 1994). Among the wild relatives of eggplant, Solanum
torvum has been a useful source of tolerance to both biotic
and abiotic stresses and also as a valuable genetic resource
for rootstocks (Daunay et al., 1991; Wei et al., 2009).
ESTs are inexpensive and important gene-cloning tools
(Ohlrogge and Benning, 2000; Yang et al., 2009). Largescale cDNA sequencing and EST analyses have been rapid
and efficient ways to identify novel cDNAs that provide a
basis to investigate the genetic components essential to various

Electronic supplementary material: The online version of this article (doi:10.1007/s13580-012-0089-0) contains supplementary material, which
is available to authorized users.


Hort. Environ. Biotechnol. 53(2):158-166. 2012.

physiological functions (Uno et al., 2008). In particular,
full-length cDNAs can be utilized as additional useful information on gene discovery and the subsequent functional
analysis (Wiemann et al., 2003), and full-length cDNA
libraries can be used in the large-scale discovery of genes.
Using the switching mechanism at 5’-end of RNA transcript

(SMART) technology for the enrichment of full-length cDNAs
is very straightforward, and the percentage of full-length
clones in the library is much higher in comparison to the
conventional libraries (Zhou et al., 2011). However, little
has been reported about the genes encoding agronomically
important traits in Solanum torvum.
In this study, we aimed to construct a full-length cDNA
library, to conduct EST analyses, and to isolate genes
conferring salinity tolerance from the leaves of ‘Torvum
Vigor’ to lay solid foundations for the further utilization of
the gene resources from Solanum torvum for the improvement of vegetable crops by genetic transformation.

Materials and Methods
3ODQW 0DWHULDO DQG &XOWXUH &RQGLWLRQV
The experiment was conducted in the insect-preventing
net-house of the Nanjing Agricultural University from June
to August in 2010. The salinity-tolerant wild eggplant variety
‘Torvum Vigor’ (Solanum torvum Swartz) (purchased from
Takii & Co., Japan) was used as the experimental material.
On June 8, the seeds were surface sterilized with sodium
hypochlorite containing 5% active chloride for 5 min, soaked
for 10 h in distilled water after being washed 5 times, then
germinated at 32/25G(day/night) for 7 d on moist filter
paper in Petri dishes (11 cm in diameter), as described by
Wei et al. (2009). On June 15, the uniformly germinated
seeds were sown in 45 plastic pots (60 cm [height] × 45 cm
[upper diameter] × 45 cm [lower diameter]) filled with a 1:1
mixture of peat and vermiculite, as described by Chen et al.
(2011), at a rate of 1 seed per pot. The plants were grown
under natural light, and the midday photosynthetic photon

-2
-1
flux density was between 550 and 950 ȝmolm sec (for
June-August, 2010). The average day/night temperature was
33/20, and the relative aerial humidity was between 60
and 80%. To minimize the individual variability, the experiment was a completely randomized design with three replications, providing 15 plants per replication. On June 26,
after the emergence of both cotyledons, each pot was
irrigated with 1 L of half-strength Enshi standard nutrient
solution (ESNS) (Zhu and Ito, 2000) every two days. When
the seedlings had reached the three-true-leaf stage on July
14, they were subjected to salinity stress with NaCl (100
-1
mmolL ) dissolved into the nutrient solution at 3-day
intervals for 3 times. After the salinity stress sampling of

159

leaves was conducted at 3-day intervals for 12 times; the
leaf samples (0.5 g) were collected from 6 randomly selected
plants per replication. All of these leaf samples were mixed,
frozen immediately in liquid nitrogen, and stored at -80
until use.
&RQVWUXFWLRQ RI WKH )XOOOHQJWK F'1$ /LEUDU\ DQG
4XDOLW\ $QDO\VLV
Total RNA was extracted from ‘Torvum Vigor’ sample of
mixed leaves using the TRIzol Reagent Kit (Invitrogen,
USA) according to the manufacturer’s instructions. The quality
of total RNA was verified by the demonstration of intact
28S and 18S rRNA by agarose gel electrophoresis and an
absorbance ratio (A260/A280) of 1.8 to 2.0 (Chotwiwatthanakun

+
et al., 2008). The poly A mRNA was purified from the total
RNA using the PolyTract mRNA Isolation Kit (Promega,
USA), and the full-length cDNA library of the ‘Torvum
Vigor’ leaves was constructed using the SMART cDNA
Library Construction Kit (Clontech, USA). The reverse transcription step was performed using PowerScript reverse
transcriptase with the 5’-SMART oligonucleotide primer
and the CDS III/3’ PCR primer provided in the kit. The
double-stranded cDNA (ds-cDNA) was obtained by LDPCR with the 5’ PCR primer and the CDS III/3’ PCR primer
using the Advantage 2 PCR Kit (Clontech, USA) (Qi et al.,
2008). The ds-cDNA was digested with SfiI and fractionated
by size on a CHROMA SPIN+TE-1000 column (Clontech,
USA) before subcloning into a dephosphorylated pBluescript
II SK (+) vector (Stratagene, USA). The recombinant plasmids
were transformed into Escherichia coli DH10B competent
cells by electroporation (Siguret et al., 1994). The inserted
fragment sizes of the positive recombinants were analyzed
by PCR amplification using the vector-specific T3 and T7
primers (Qi et al., 2008). The PCR program was as follows:
denaturation at 95Gfor 5 min, followed by amplification
for 28 cycles with a program of 94Gfor 30 s, 55Gfor 30 s,
and 72Gfor 2 min. A final extension was carried out at
72G for 5 min. The PCR products were visualized by
electrophoresis through 1% agarose gels.
6DPSOH 3UHSDUDWLRQ IRU (67 6HTXHQFLQJ
The positive clones were selected randomly from the
library and were freshly grown overnight at 37Gon LuriaBertani (LB) -ampicillin medium containing isopropyl-ȕ-Dthiogalactopyranoside (IPTG) and 5-bromo-4- chloro-3-idolylȕ-D-galactoside (X-Gal) for colony selection (Ruszczyk et
al., 2008). Plasmids of each positive clone were extracted
TM
using the PureLink Plasmid DNA Purification Kit (Invitrogen,

USA); cDNA inserts were sequenced from the 5’-end using
M13 reverse primer (5’-CAGGAAACAGCTATGACC-3’)
TM
and the ABI Prism BigDye Terminator Cycle Sequencing


160 Gang Chen, Hua Wang, Jun-Yi Gai, Yue-Lin Zhu, Li-Fei Yang, Qian-Qian Liu, Gong-Chen Zhang, and Guo-Hu Chen

Kit (Applied Biosystems, USA) with the MegaBACE 1000
DNA sequencer (Pharmacia, USA). The sequencing was performed by the Beijing Genomics Institute, Beijing, China.
6HTXHQFH 5DZ 'DWD 7ULPPLQJ DQG )XQFWLRQDO
$QQRWDWLRQ
The raw sequence data were edited using a phred/phrap/
cross_match package (Ewing and Green, 1998). From the
DNA sequencer, the base-calling of the trace files were
performed by phred, the vector and adapter sequences were
trimmed by cross_match, and the data with high-quality
values (QV • 20) were isolated.
BLASTn was used to compare the primarily edited ESTs
with known sequences deposited in GenBank (E-value ”
1E-5). BLASTX analysis was applied to search sequence
similarities of the primarily edited ESTs against the known
or putative protein sequences in the non-redundant database
of the National Center for Biotechnology Information (NCBInr)
(E-value ” 2E-5).

databases. A ‘possible full-length cDNA’ was defined as
when the 5’-deduced amino acid sequence (N-terminus)
matched the residues between positions 2 and 10 following
the initiation methionine. The ‘non-full-length cDNA’ was

that matching the amino acids extending beyond from the
11th amino acid following the initiation methionine (Qi et
al., 2008). After searching the NCBI database, the bestmatched ESTs were selected to calculate the percentage of
full-length cDNA inserts (Barrett et al., 2005).

Results

(YDOXDWLRQ RI WKH )XOOOHQJWK F'1$V
Estimation of the full-length cDNAs was based on the
similarity alignment results acquired using BLASTX analysis
of the 5’-end EST sequences. A cDNA was tentatively
scored as ‘full-length’ if the 5’-deduced amino acid sequence
(N-terminus) of this cDNA matched the initiation methionine
of a ‘complete protein sequence’ in the NCBInr protein

([WUDFWLRQ RI 7RWDO 51$
Agarose gel (1%) electrophoresis of total RNA from the
sample of mixed ‘Torvum Vigor’ leaves is shown in Fig. 1.
The total RNA extracted with the TRIzol reagent showed
two clear bands corresponding to ribosomal 28S and 18S
-1
RNA; the concentration of total RNA was 1.365 µgµL ,
and the ratio of A260/A280 of total RNA was 1.954. These
data showed that high-quality total RNA was successfully
isolated from ‘Torvum Vigor’ mixed-sample leaves. Agarose
+
gel electrophoresis (1%) of the poly A mRNA showed a
dispersion band (Fig. 2). These results indicated that the
isolated mRNA could be used to synthesize cDNA. The
double-stranded cDNA produced by LD-PCR was analyzed

by 1% agarose gel electrophoresis, and the results showed
that the majority of the ds-cDNA was concentrated in the

Fig. 1. Agarose gel (1%) electrophoretogram of total RNA extracted
from the leaves of ‘Torvum Vigor’. M, pHY DNA size marker
(Takara).

Fig. 2. Agarose gel (1%) electrophoretogram of the purified mRNA
from the total RNA in the leaves of ‘Torvum Vigor’. M, pHY DNA
size marker (Takara).


Hort. Environ. Biotechnol. 53(2):158-166. 2012.

161

range of 0.4 to 2.5 kb, indicating that ds-cDNA was
successfully synthesized by the LD-PCR technique (Fig. 3).

could be a critical resource when used for isolating and
identifying full-length expressed genes.

&KDUDFWHULVWLFV RI WKH )XOOOHQJWK F'1$ /LEUDU\
6
The titer of the primary cDNA library was 3.6 × 10 cfu
-1
10
mL and that of the amplified library was 1.2 × 10 cfu
-1
mL , with a recombination rate of 99%. Sixteen positive

clones were randomly picked from the cDNA library, and
the inserted cDNA fragments were confirmed by PCR
amplification, which revealed that most of the cDNA inserts
ranged from 0.4 to 2.5 kb (Fig. 4). These data showed that
the cDNA library of ‘Torvum Vigor’ had large inserted
fragments, a high titer and a high recombination rate, which

(67 6HTXHQFLQJ $QDO\VLV
Fig. 5 shows the PCR results of portions of the positive
clones from the constructed library after the blue/white
selection; a total of 427 positive clones were sequenced.
After removing the vector sequence and terminal sequences
of low reliability, a total of 364 high-quality sequence data
were obtained. The average readable sequences after vector
removal and quality trimming were 520 bp.
The obtained 364 ESTs were subjected to a BLASTn
search against Arabidopsis thaliana and two species within
Solanaceae. The homology search against Arabidopsis thaliana
revealed that 11.81% (43/364) ESTs had significant homology
(E-value ” 1E-5). When the sequence alignment was restricted
to Solanaceae, that is, Solanum lycopersicum and Solanum
tuberosum, 38.74% (141/364), and 5.22% (19/364) of the
‘Torvum Vigor’ ESTs sequences showed a match to ESTs
from each of the species, respectively.

Fig. 3. The amplification of double-stranded cDNA by LD-PCR
as visualized by agarose gel (1%) electrophoresis. M, pHY DNA
size marker (Takara).

(67V ,QYROYHG LQ 6DOLQLW\ 6WUHVV

A BLASTX analysis revealed that 62.36% (227/364) of
the ESTs could be functionally classified with the known or
putative proteins in the NCBInr databases, whereas 37.64%
(137/364) ESTs indicated low or no significant similarity to
any of the proteins in the published databases.
The ‘Torvum Vigor’ ESTs possessed homology to known
or putative proteins of other organisms, which helped to
reveal the functional identities of these ‘Torvum Vigor’ ESTs.
Seven genes that contained the initiation codon (ATG) of
the open reading frame (ORF) were functionally assigned as
proteins involved in salinity stress, including: betaine aldehyde
dehydrogenase 2 (BADH2) (1-the number of ESTs matched
in GenBank, FJ228482-the accession number of genes of

Fig. 4. The PCR products of cDNAs cloned randomly from the full-length cDNA library. Lanes 1-16, cDNA fragments amplified from
randomly selected positive clones with the vector-specific T3 and T7 primers by PCR. M, pHY DNA size marker (Takara).


162 Gang Chen, Hua Wang, Jun-Yi Gai, Yue-Lin Zhu, Li-Fei Yang, Qian-Qian Liu, Gong-Chen Zhang, and Guo-Hu Chen

Fig. 5. The PCR results of portions of the positive cDNA clones from the constructed full-length cDNA library after the blue/white selection.
Lanes 1-48, cDNA fragments amplified by PCR from positive clones. M, DL2000 DNA size marker (Takara).
Table 1. BLASTX analysis of seven isolated genes with open reading frames from the constructed full-length cDNA library involved
in salinity stress.
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known function in GenBank); N-acetyl-glutamate synthase
(NAGS) (1, FJ543466); 14-3-3 protein (1, GQ422966); stressassociated protein 5 (SAP5) (1, FJ442191); cyclophilin-like
(1, DQ235183); histone H1E (1, EF522839); and glycine
rich protein-like (1, DQ241844) (Table 1). Other 264 genes
with ORFs were listed in the Supplemental Table.
(VWLPDWLRQ RI WKH )XOOOHQJWK F'1$V
The full-length cDNAs in the library were estimated

based on our BLASTX analysis. The total full-length cDNA
sequences accounted for 74.45% (271/364) of all of the 364

submitted sequences (ESTs significantly matched with an
-10
E-value < 10 ), and the cDNA sequences without full-length
coding regions occurred at a low frequency (25.55%). These
results indicated that the SMART cDNA synthesis methods
produced a cDNA library enriched in full-length cDNA
clones, thus providing a foundation for further functional
analysis of stress-related genes.

Discussion
Plant breeders are always interested in obtaining new


Hort. Environ. Biotechnol. 53(2):158-166. 2012.

germplasm with increased yield, improved quality, and resistance to diseases and pests through interspecific hybridization
using wild species as one of the parental materials (Chandra
et al., 2004). Nevertheless, cross incompatibilities exist in
interspecific crosses between cultivated plants and their wild
relatives. One of the effective alternatives to overcome such
reproductive isolation is the application of genetic transformation either via Agrobacterium-mediated methods or
particle bombardment to facilitate the transfer of stress
-tolerance genes from wild relatives into cultivated vegetable
crops (Kashyap et al., 2003). The availability of stresstolerance genes for vegetable crop improvement is one of
the key factors for the success of gene transfer. Solanum
torvum, a wild relative of eggplant, has been shown to
possess resistance to nearly all known diseases and pests of

cultivated eggplant (Gousset et al., 2005). Therefore, attempts
have been made in the present study to construct a ‘Torvum
Vigor’ (Solanum torvum Swartz) full-length cDNA library
with the SMART cDNA synthesis method combined with
LD-PCR. Construction of a full-length cDNA library is very
useful for the isolation and functional analysis of target
genes (Yamagishi et al., 2011), and the following two factors
have been shown to play an important role in the construction
of a full-length cDNA library. One, high-quality mRNA is
critical to the creation of full-length cDNA; in our study, the
mRNA quality was extremely high (Fig. 2). The other factor
involves a wide representativeness of the library: the titer of
a cDNA library could be used as an evaluation criterion of
the representativeness of the library (Yang et al., 2009). In
general, it has been suggested that the titer of cDNA library
6
-1
be above 1 × 10 cfumL . In the present study, the titer of
6
-1
the primary cDNA library was 3.6 × 10 cfumL and that
10
-1
of the amplified library was 1.2 × 10 cfumL . Furthermore,
insert detection by PCR amplification revealed that the
majority of positive clones in the library contained fragments
of large size. These results indicated that the constructed
cDNA library was a full-length library of high quality,
which could serve as an important resource for the isolation
of stress-related genes to be utilized in the genetic improvement of vegetable crops using genetic engineering.

EST sequencing for full-length cDNA has proven to be a
rapid and effective strategy for examining the expression
patterns of genes in a specific tissue or at a specific developmental stage (Gueguen et al., 2003). Full-length cDNAs are
also useful tools for the analysis of expression profiles
because the cDNA population in each full-length cDNA
library should closely represent the transcripts of the used
materials: the number of ESTs matching a particular gene
should reflect the abundance of their corresponding cDNAs
in the non-normalized library (Ewing et al., 1999). After
excluding the ambiguous and incomplete sequences, a total
of 364 EST sequences were obtained, which provides the

163

first nucleotide sequence data for ‘Torvum Vigor’ (Solanum
torvum Swartz). As a molecular basis of information on
whole genomes, the accumulation of EST sequences is a
promising strategy for studies in plant molecular biology
(Rudd, 2003). The ‘Torvum Vigor’ ESTs sequenced in this
study could be a potential resource for comprehensive genomic
studies and also for expanding the scope of comparative
biology in Solanum species. Therefore, the accumulation of
EST information would facilitate molecular biology in ‘Torvum
Vigor’.
BLASTX also assigned putative functions to the determined EST sequences (Altschul et al., 1997), revealing that
74.45% of the submitted sequences contained full-length
coding regions. Moreover, 62.36% of the ESTs could be
functionally predicted based on known or putative proteins.
Seven genes (JK265299, JK265455, JK265378, JK265448,
JK265400, JK265205, and JK265278), which might encode

known proteins in other organisms, were identified to confer
salt tolerance (Table 1). JK265299 (the betaine aldehyde
dehydrogenase gene, BADH2) and JK265455 (the N-acetylglutamate synthase gene, NAGS) are the two key genes that
regulate the biosynthesis of glycine betaine and ornithine in
plants (McCue and Hanson, 1992; Slocum et al., 2005).
Moreover, both BADH2 and NAGS have been reported to
confer salt tolerance (Hibino et al., 2001; Kalamaki et al.,
2009; McCue and Hanson, 1992). The cellular responses of
salt-tolerant plants to salinity stress include the synthesis
and accumulation of a class of osmoregulation substances
known as compatible solutes. Betaine, as one of these
osmolytes, plays an important role in osmoregulation in
most higher plants, and betaine has been found to accumulate
in many plant species in response to salinity (Rhodes and
Hanson, 1993). In higher plants, glycine betaine is synthesized
by a two-step oxidation of choline. In the first step, choline
monooxygenase (CMO) catalyzes choline to betaine aldehyde;
the second step is mediated by betaine aldehyde dehydrogenase
(BADH, EC 1.2.1.8), which catalyzes the conversion of
betaine aldehyde to glycine betaine (Rhodes and Hanson,
1993; Weretilnyk and Hanson, 1989). Compatible solutes or
osmoprotectants act as non-toxic solutes for cytoplasmic
osmoregulation and can also partly reverse the damaging
effects of salts on proteins and membranes (Yancey et al.,
1994). In plants, NAGS has been shown to serve as the
regulation point of arginine biosynthesis (Slocum et al.,
2005), resulting in the increasing of ornithine levels and
elevating the salt tolerance (Kalamaki et al., 2009). JK
265378 encodes a 14-3-3 protein; these proteins appear to
play important roles in regulating nitrate reductase enzymes

+
(Moorhead et al., 1996) and the plasma membrane H ATPase (Baunsgaard et al., 1998). The 14-3-3 proteins also
participate in protein transport into the mitochondria and the
transcriptional regulation of some stress-related genes (Aitken


164 Gang Chen, Hua Wang, Jun-Yi Gai, Yue-Lin Zhu, Li-Fei Yang, Qian-Qian Liu, Gong-Chen Zhang, and Guo-Hu Chen

et al., 1992). JK265448 is predicted to encode the stressassociated protein 5 (SAP5). A SAP-family gene encoding
an A20/AN1 zinc finger protein has been implicated in the
response to various environmental stresses (Solanke et al.,
2009). JK265400 encodes a cyclophilin-like protein. Cyclophilins
(Cyps) have been found in bacteria, fungi, insects, plants,
and mammals. These proteins belong to the cluster of
immunophilin proteins that possess peptidyl-prolyl cistrans
isomerase (PPIase) enzymatic activity, the rate-limiting step
in protein folding (Zhu et al., 2011). In plants, cyclophilin
genes are induced in response to biotic or abiotic stresses. It
has been reported that cyclophilin proteins in tobacco and
yeast play a very important role in increasing tolerance to
salt stress (Chen et al., 2007). JK265205 is predicted to
encode histone H1E, which is one type of molecular chaperone
that plays an important role in the mediation of the folding
of synthesized proteins and the refolding of denatured
proteins in the cell (Hartl, 1996). Chaperones have been
identified to aid in the translocation of newly synthesized
proteins and also to protect eukaryotic cells against the
effects of cellular stress (Mayer et al., 1998). JK265278 is
predicted to encode the glycine rich protein. Glycine-rich
proteins (GRPs) compose a large family of heterogenous

proteins characterized by a high content and repetitive
sequences of glycine residues based on (Gly-X)n motifs that
are usually found in ȕ-plated sheets with antiparallel strands
or form flexible coiled structures. Additionally, the expression
of GRPs seems to be regulated by external stimuli ranging
from pathogen infection to several forms of environmental
stress such as salinity (Mousavi and Hotta, 2005) and water
(Didierjean et al., 1992). We expect to carry out functional
analyses of these identified genes in future work.
Plant genetic transformation has provided plant breeders
with new opportunities for vegetable crop improvement.
Genetic engineering studies in vegetable crops need to be
further exploited for the introduction of stress-tolerance genes
encoding resistance to biotic and abiotic stresses. The introduction of BADH for elevated salinity tolerance has been
reported in several agronomical crops (Guo et al., 2000;
Kishitani et al., 2000; Wu et al., 2008). In addition, the
successful genetic transformation of carrot (Daucus carota
L. cv. Half long) for the overexpression of BADH via
particle bombardment has been reported to result in a
-1
predominant enhancement of salt tolerance (400 mmolL
NaCl) in the transgenic carrots (Kumar et al., 2004). Jia et
al. (2002) reported that Agrobacterium-mediated transformation
of AhBADH into a salt-sensitive tomato cultivar (Solanum
lycopersicum Mill. cv. Bailichun) resulted in a significant
-1
elevation of salt tolerance (120 mmolL NaCl) in the
transgenic tomatoes. These results demonstrated that BADH
is a potential gene resource in genetic engineering to increase
the salt tolerance of vegetable crops. It has been reported

that the engineering of the ornithine synthesis pathway by
the overexpression of a tomato N-acetyl-L-glutamate synthase
gene (SlNAGS1) in Arabidopsis thaliana induced an increase
in the ornithine levels and elevated the salt tolerance (Kalamaki
et al., 2009). Thus, a significant improvement in salinity
tolerance in vegetable crops can be achieved by engineering
a single gene. Because several salt tolerance genes were
isolated, genetic engineering appears to be a viable strategy
to enhance salinity tolerance in vegetables and other crops.
In conclusion, a full-length cDNA library from the leaves
of the salinity-tolerant wild eggplant variety, ‘Torvum Vigor’
(Solanum torvum Swartz), was constructed with the SMART
method. BLASTX analysis revealed that seven valuable
genes might encode known plant proteins were identified as
conferring salt tolerance. This cDNA library contributes to
the information for the ‘Torvum Vigor’ EST library and
also may facilitate the screening of full-length cDNAs. The
ESTs data identified in ‘Torvum Vigor’ may be useful in the
cloning of stress-related genes to increase the tolerance of
vegetable crops to biotic and abiotic stresses by genetic
transformation.
Acknowledgement: This work was financially supported
by National Key Specialized Project for Transgenic Researches
(2009ZX08004-011B) of China and A Project Funded by
the Priority Academic Program Development of Jiangsu
Higher Education Institutions.

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