Construction and Characterization of a Fulllength cDNA Library and Identification of Genes Involved in Salinity Stress in Wild Eggplant (Solanum torvum Swartz)

Abstract.The objectives of this paper were to construct a fulllength cDNA library and to isolate genes that confer salt tolerance from the leaves of salinitytolerant wild eggplant variety, ‘Torvum Vigor’ (Solanum torvum Swartz). A fulllength cDNA library from the leaves was successfully constructed by a switching mechanism at 5’end of RNA transcript (SMART) approach and a longdistance PCR (LDPCR) technique. The titer of the primary cDNA library was 3.6 × 10 6 cfumL 1 and that of the amplified library was 1.2 × 10 10 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 JK265131JK265494. Among the 364 submitted sequences, 74.45% of them contained fulllength 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 fulllength library of high quality. It could be a useful resource for further research in the cloning of stressrelated genes, which could be utilized in the genetic improvement of vegetable crops using transgenic technology.

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257645012

Construction and Characterization of a Full-length cDNA Library and Identification of

Genes Involved in Salinity Stress in Wild

Eggplant (Solanum torvum Swartz)

Dataset in Horticulture, Environment and Biotechnology · February 2012

Impact Factor: 0.73 · DOI: 10.1007/s13580-012-0089-0

CITATIONS

2

READS

46

8 authors, including:

Guo-Hu Chen

Anhui Agricultural University (AHAU)

6 PUBLICATIONS 13 CITATIONS

SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate,

letting you access and read them immediately.

Available from: Guo-Hu Chen Retrieved on: 23 May 2016

Construction and Characterization of a Full-length cDNA Library and Identification of Genes Involved in Salinity Stress in Wild Eggplant

( Solanum torvum Swartz)

Gang Chen 1,2ಳ, Hua Wang 2ಳ, Jun-Yi Gai 1 , Yue-Lin Zhu 1,2* , Li-Fei Yang 2 , Qian-Qian Liu 2 ,

1

National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China

2

College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

*Corresponding author: ylzhu@njau.edu.cn

ಳ

These authors contributed equally to this work.

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)

Research Report

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.

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) Large- scale 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

Hort Environ Biotechnol 53(2):158-166 2012. 159

physiological functions (Uno et al., 2008) In particular,

full-length cDNAs can be utilized as additional useful

infor-mation 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

improve-ment 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

flux density was between 550 and 950 ȝmolm-2sec-1 (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

experi-ment was a completely randomized design with three

repli-cations, 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

mmolL-1) dissolved into the nutrient solution at 3-day

intervals for 3 times After the salinity stress sampling of

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 tran-scription 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 LD- PCR 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 Luria- Bertani (LB) -ampicillin medium containing isopropyl-ȕ-D- thiogalactopyranoside (IPTG) and 5-bromo-4- chloro-3-idolyl- ȕ-D-galactoside (X-Gal) for colony selection (Ruszczyk et al., 2008) Plasmids of each positive clone were extracted using the PureLinkTM Plasmid DNA Purification Kit (Invitrogen, USA); cDNA inserts were sequenced from the 5’-end using M13 reverse primer (5’-CAGGAAACAGCTATGACC-3’) and the ABI Prism BigDyeTM Terminator Cycle Sequencing

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).

Kit (Applied Biosystems, USA) with the MegaBACE 1000

DNA sequencer (Pharmacia, USA) The sequencing was

per-formed 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)

(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

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 best- matched ESTs were selected to calculate the percentage of full-length cDNA inserts (Barrett et al., 2005)

Results

([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 RNA; the concentration of total RNA was 1.365 µgµL-1, 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

Hort Environ Biotechnol 53(2):158-166 2012. 161

Fig 3 The amplification of double-stranded cDNA by LD-PCR

as visualized by agarose gel (1%) electrophoresis M, pHY DNA

size marker (Takara).

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)

range of 0.4 to 2.5 kb, indicating that ds-cDNA was

successfully synthesized by the LD-PCR technique (Fig 3)

&KDUDFWHULVWLFV RI WKH )XOOOHQJWK F'1$ /LEUDU\

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, 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

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

(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

(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 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.

*HQ%DQN

DFFHVVLRQ QR

6L]H

ES )XQFWLRQDO DQQRWDWLRQ 0DWFKLQJ VSHFLHV

4XHU\

FRYHUDJH  (YDOXH

,GHQWLW\  -.  %HWDLQH DOGHK\GH GHK\GURJHQDVH  6RODQXP O\FRSHUVLFXP    -.  1 DFHW\OJOXWDPDWH V\QWKDVH 6RODQXP O\FRSHUVLFXP    -.    SURWHLQ 6RODQXP FKDFRHQVH  (  -.   6WUHVVDVVRFLDWHG SURWHLQ  6RODQXP O\FRSHUVLFXP  (  -.   &\FORSKLOLQOLNH 6RODQXP WXEHURVXP    -.   +LVWRQH +( 1LFRWLDQD WDEDFXP  (  -.  *O\FLQH ULFK SURWHLQOLNH 6RODQXP WXEHURVXP  ( 

known function in GenBank); N-acetyl-glutamate synthase

(NAGS) (1, FJ543466); 14-3-3 protein (1, GQ422966); stress-

associated 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 E-value < 10-10), 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. 163

germplasm with increased yield, improved quality, and

resist-ance 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

trans-formation 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 stress-

tolerance 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

be above 1 × 106 cfumL-1 In the present study, 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 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

improve-ment 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

develop-mental 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

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 deter-mined 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-acetyl- glutamate 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

et al., 1992) JK265448 is predicted to encode the stress-

associated 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

intro-duction 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

predominant enhancement of salt tolerance (400 mmolL-1

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

elevation of salt tolerance (120 mmolL-1 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

Literature Cited

Aitken, A., D.B Collinge, B.P.H van Heusden, T Isobe, P.H Roseboom,

G Rosenfeld, and J Soll 1992 14-3-3 proteins: A highly conserved, widespread family of eukaryotic proteins Trends Biochem Sci 17:498-501.

Altschul, S.F., T.L Madden, A.A Schäffer, J.-H Zhang, Z Zhang,

W Miller, and D.J Lipman 1997 Gapped BLAST and PSI-BLAST:

A new generation of protein database search programs Nucleic Acids Res 25:3389-3402.

Barrett, T., T.O Suzek, D.B Troup, and S.E Wilhite 2005 NCBI GEO: Mining millions of expression profiles-database and tools Nucleic Acids Res 33:562-566.

Baunsgaard, L., A.T Fuglsang, T Jahn, H.A.A.J Korthout, A.H de Boer, and M.G Palmgren 1998 The 14-3-3 proteins associate with the plant plasma membrane H+-ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system Plant J 13:661-671.

Bletsos, F.A., D.G Roupakias, M.L Tsaktsira, A.B Scaltsoyjannes, and C.C Thanassoulopoulos 1998 Interspecific hybrids between

three eggplant (Solanum melongena L.) cultivars and two wild species (Solanum torvum Sw and Solanum sisymbriifolium Lam.) Plant

Breeding 117:159-164.

Chandra A., M.L Gupta, I Ahuja, G Kaur, and S.S Banga 2004

Intergeneric hybridization between Erucastrum cardaminoides and two diploid crop Brassica species Theor Appl Genet 108:1620-

1626

Chen, A.-P., G.-L Wang, Z.-L Qu, C.-X Lu, N Liu, F Wang, and

G.-X Xia 2007 Ectopic expression of ThCYP1, a stress-responsive

Hort Environ Biotechnol 53(2):158-166 2012. 165

cyclophilin gene from Thellungiella halophila, confers salt tolerance

in fission yeast and tobacco cells Plant Cell Rep 26:237-245.

Chen, L., Q.-Q Liu, J.-Y Gai, Y.-L Zhu, L.-F Yang, and C Wang

2011 Effects of nitrogen forms on the growth and polyamine

contents in developing seeds of vegetable soybean J Plant Nutr

34:504-521.

Chotwiwatthanakun, C., J Ngopon, S Unajak, and S Jitrapakdee

2008 The ribophorin I from Penaeus monodon shrimp: cDNA

cloning, expression and phylogenetic analysis Comp Biochem

Physiol 150:331-337.

Collonnier, C., I Fock, V Kashyap, G.L Rotino, M.C Daunay, Y

Lian, I.K Mariska, M.V Rajam, A Servaes, G Ducreux, and D

Sihachakr 2001 Applications of biotechnology in eggplant Plant

Cell Tiss Org Cult 65:91-107.

Daunay, M.C., R.N Lester, and H Laterrot 1991 The use of wild

species for the genetic improvement of brinjal eggplant (Solanum

melongena) and tomato (Lycopersicon esculentum), p 389-413

In: J.H Hawkes, R.N Lester, M Nee, and N Estrada (eds.)

Solanaceae III: Taxonomy, Chemistry, Evolution, Vol 27 Royal

Botanic Gardens Kew and Linnean Society, London, UK.

Didierjean, L., P Frendo, and G Burkard 1992 Stress responses

in maize: Sequence analysis of cDNAs encoding glycine-rich

proteins Plant Mol Biol 18:847-849.

Ewing, B and P Green 1998 Base-calling of automated sequencer

traces using phred II Error probabilities Genome Res 8:186-

194.

Ewing, R.M., A.B Kahla, O Poirot, F Lopez, S Audic, and J.M

Claverie 1999 Large-scale statistical analyses of rice ESTs reveal

correlated patterns of gene expression Genome Res 9:950-959.

Gousseta, C., C Collonniera, K Mulyab, I.K Mariskab, G.L Rotinoc,

P Bessed, A Servaesa, and D Sihachakr 2005 Solanum torvum,

as a useful source of resistance against bacterial and fungal diseases

for improvement of eggplant (S melongena L.) Plant Sci 168:319-

327.

Gueguen, Y., J.P Cadoret, D Flament, C Barreau-Roumiguière, A.L

Girardot, J Garnier, A Hoareau, E Bachère, and J.M Escoubas

2003 Immune gene discovery by expressed sequence tags generated

from hemocytes of the bacteria-challenged oyster, Crassostrea

gigas Gene 303:139-145.

Guo, B.-H., Y.-M Zhang, H.-J Li, L.-Q Du, Y.-X Li, J.-S Zhang,

S.-Y Chen, and Z.-Q Zhu 2000 Transformation of wheat with

a gene encoding for the betaine aldehyde dehydrogenase (BADH)

Acta Bot Sin 42:279-283.

Hartl, F.U 1996 Molecular chaperones in cellular protein folding

Nature 381:571-579.

Hibino, T., Y.-L Meng, Y Kawamitsu, N Uehara, N Matsuda, Y

Tanaka, H Ishikawa, S Baba, T Takabe, K Wada, T Ishii, and

T Takabe 2001 Molecular cloning and functional characterization

of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating

mangrove Avicennia marina (Forsk.) Vierh Plant Mol Biol

45:353-363.

Jia, G.-X., Z.-Q Zhu, F.-Q Chang, and Y.-X Li 2002 Transformation

of tomato with the BADH gene from Atriplex improves salt

tolerance Plant Cell Rep 21:141-146.

Kalamaki, M.S., D Alexandrou, D Lazari, G Merkouropoulos, V

Fotopoulos, I Pateraki, A Aggelis, A Carrillo-L ȩpez, M.J Rubio-

Cabetas, and A.K Kanellis 2009 Over-expression of a tomato

N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis

thaliana results in high ornithine levels and increased tolerance

in salt and drought stresses J Exp Bot 60:1859-1871.

Kashyap, V.S.V KumarC CollonnierF Fusari, R Haicour, G.L

Rotino, D Sihachakr, and M.V Rajam 2003 Biotechnology of

eggplant Sci Hortic 97:1-25.

Kishitani, S., T Takanami, M Suzuki, M Oikawa, S Yokoi, M Ishitani,

A.M.A Nakase, T Takabe, and T Takabe 2000 Compatibility

of glycinebetaine in rice plants: Evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley Plant Cell Environ 23:107-114.

Kumar, S., A Dhingra, and H Daniell 2004 Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance Plant Physiol 136:2843- 2854.

Mayer, M.P and B Bukau 1998 Hsp70 chaperone systems: Diversity

of cellular functions and mechanism of action Biol Chem 379: 261-268.

McCue, K.F and A.D Hanson 1992 Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression Plant Mol Biol 18:1-11.

Moorhead, G., P Douglas, N Morrice, M Scarabel, A Aitken, and

C MacKintosh 1996 Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin Curr Biol 6:1104-1113.

Mousavi, A and Y Hotta 2005 Glycine-rich proteins: A class of novel proteins Appl Biochem Biotech 120:169-174

Ohlrogge, J and C Benning 2000 Unravelling plant metabolism

by EST analysis Curr Opin Plant Biol 3:224-228.

Qi, J.-L., W.-J Zhang, S.-H Liu, H Wang, D.-Y Sun, G.-H Xu, M.-W Shi, Z Liu, M.-S Zhang, H.-M Zhang, and Y.-H Yang

2008 Expression analysis of light-regulated genes isolated from

a full-length-enriched cDNA library of Onosma paniculatum cell

cultures J Plant Physiol 165:1474-1482.

Rhodes, D and A.D Hanson 1993 Quarternary ammonium and tertiary sulfonium compounds in higher plants Annu Rev Plant Physiol Plant Mol Biol 44:357-384.

Rudd, S 2003 Expressed sequence tags: alternative or complement

to whole genome sequences? Trends Plant Sci 8:321-329 Ruszczyk, A., M Joerink, C Guldenaar, T Hermsen, H.F.J Savelkoul, and G.F Wiegertjes 2008 cDNA expression library screening and identification of two novel antigens: ubiquitin and receptor for

activated C kinase (RACK) homologue, of the fish parasite Trypanosoma

carassii Fish Shellfish Immun 25:84-90.

Siguret, V., A.S Ribba, G Chérel, D Meyer, and G Piéru 1994 Effect of plasmid size on transformation efficiency by electroporation

of Escherichia coli DH5Į Biotechniques 16:422-426.

Sihachakr, D., M.C Daunay, and I Serraf 1994 Somatic hybridization

of eggplant (Solanum melongena L.) with its close and wild relatives

(A), p 255-278 In: Y.P.S Bajaj (ed.) Biotechnology in Agriculture and Forestry, Somatic hybridization in crop improvement (C), Vol

I Springer Verlag, Berlin, Germany.

Slocum, R.D 2005 Genes, enzymes and regulation of arginine biosynthesis in plants Plant Physiol Biochem 43:729-745 Solanke, A.U., M.K Sharma, A.K Tyagi, and A.K Sharma 2009 Characterization and phylogenetic analysis of environmental stress-

responsive SAP gene family encoding A20/AN1 zinc finger proteins

in tomato Mol Genet Genomics 282:153-164.

Uno, Y., Y Suzuki, H Wakaguri, Y Sakamoto, H Sano, N Osada,

K Hashimoto, S Sugano, and I Inoue 2008 Expressed sequence

tags from cynomolgus monkey (Macaca fascicularis) liver: A

systematic identification of drug-metabolizing enzymes FEBS Lett 582:351-358.

Wei, G.-P., L.-F Yang, Y.-L Zhu, and G Chen 2009 Changes in oxidative damage, antioxidant enzyme activities and polyamine contents in leaves of grafted and non-grafted eggplant seedlings under stress by excess of calcium nitrate Sci Hortic 120:443-451 Weretilnyk, E.A and A.D Hanson 1989 Betaine aldehyde dehydrogenase from spinach leaves: Purification, in vitro translation of the mRNA, and regulation by salinity Arch Biochem Biophys 271:56-63 Wiemann, S., A Mehrle, S Bechtel, R Wellenreuther, R Pepperkok, and A Poustka 2003 cDNAs for functional genomics and proteomics: the German Consortium C R Biologies 326:1003-1009.

Wu, W., Q Su, X.-Y Xia, Y Wang, Y.-S Luan, and L.-J An 2008

The Suaeda liaotungensis Kitag betaine aldehyde dehydrogenase

gene improves salt tolerance of transgenic maize mediated with

minimum linear length of DNA fragment Euphytica 159:17-25.

Yamagishi, J., H Wakaguri, S Sugano, S Kawano, K Fujisaki, C

Sugimoto, J Watanabe, Y Suzuki, I Kimata, and X.-N Xuan

2011 Construction and analysis of full-length cDNA library of

Cryptosporidium parvum Parasitol Int 60:199-202.

Yancey, P.H 1994 Compatible and counteracting solutes, p 81-109

In: K Strange (ed.) Cellular and molecular physiology of cell

volume regulation CRC Press, Boca Raton, USA.

Yang, L.-F., J.-Y Gai, Y.-L Zhu, G Chen, G.-P Wei, C Wang,

and Q.-Q Liu 2009 Construction and characterization of full-length

cDNA library and expressed sequence tags analysis in developing

seeds of vegetable soybean Hort Environ Biotechnol 50:51-56 Zhou, F., Y Wang, Y Guan, Y.-Y Xu, X.-D Gao, W.-T Wu, and B.-P Ye 2011 Construction and characterization of a cDNA library from shark regenerated hepatic tissue Fish Shellfish Immun 30: 1170-1177.

Zhu, C.-F., Y.-X Wang, Y.-B Li, K H Bhatti, Y.-C Tian, and J.-H Wu 2011 Overexpression of a cotton cyclophilin gene

(GhCyp1) in transgenic tobacco plants confers dual tolerance to salt stress and Pseudomonas syringae pv tabaci infection Plant

Physiol Biochem 49:1264-1271.

Zhu, Y and T Ito 2000 Effects of nutrient stress by split-root system

on the growth and K, Ca, and Mg contents at different stages

of hydroponically-grown tomato seedlings J Japan Soc Hortic Sci 69:677-683.