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 Fulllength 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 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 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 2ಳ 1,2* 2 , Hua Wang , Jun-Yi Gai , Yue-Lin Zhu , Li-Fei Yang , Qian-Qian Liu , 2 Gong-Chen Zhang , and Guo-Hu Chen National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China *Corresponding author: ylzhu@njau.edu.cn 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 min, soaked for 10 h in distilled water after being washed times, then germinated at 32/25G(day/night) for 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 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 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 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 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 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 min, followed by amplification for 28 cycles with a program of 94Gfor 30 s, 55Gfor 30 s, and 72Gfor A final extension was carried out at 72G for 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 $QQRWDWLRQ The raw sequence data were edited using a phred/phrap/ cross_match package (Ewing and Green, 1998) From the DNA sequencer, the base-calling of the trace files were performed by phred, the vector and adapter sequences were trimmed by cross_match, and the data with high-quality values (QV • 20) were isolated BLASTn was used to compare the primarily edited ESTs with known sequences deposited in GenBank (E-value ” 1E-5) BLASTX analysis was applied to search sequence similarities of the primarily edited ESTs against the known or putative protein sequences in the non-redundant database of the National Center for Biotechnology Information (NCBInr) (E-value ” 2E-5) databases A ‘possible full-length cDNA’ was defined as when the 5’-deduced amino acid sequence (N-terminus) matched the residues between positions and 10 following the initiation methionine The ‘non-full-length cDNA’ was that matching the amino acids extending beyond from the 11th amino acid following the initiation methionine (Qi et al., 2008) After searching the NCBI database, the bestmatched ESTs were selected to calculate the percentage of full-length cDNA inserts (Barrett et al., 2005) Results (YDOXDWLRQ RI WKH )XOOOHQJWK F'1$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 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 Agarose gel (1%) electrophoretogram of total RNA extracted from the leaves of ‘Torvum Vigor’ M, pHY DNA size marker (Takara) Fig 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\ 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 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 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 (BADH2) (1-the number of ESTs matched in GenBank, FJ228482-the accession number of genes of Fig 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 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 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    -.  1DFHW\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); stressassociated protein (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 -1 be above × 10 cfumL In the present study, the titer of -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 (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 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.) 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