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© 2013 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com

Introduction

Abiotic stress, such as drought, salinity, and ex- treme temperature, is one of the primary causes of crop loss worldwide, reducing the average pro- duction of major crop plants. Salinity is a serious worldwide problem, with more than 800 million hectares of land affected throughout the world, which accounts for 6% of world’s total land area (Munns and Tester, 2008). In China, more than 90 million hectares are exposed to salinity or se- condary salinity. Xinjiang occupies one-sixth of the area of China, but more than one-third of the land is salinized. Therefore, developing salt-tole- rant varieties of crops is an important breeding goal in Xinjiang. Owing to the extreme ecological environment, salt-tolerant organisms are abun- dant in Xinjiang, with abundant gene resources.

An important research project is to mine salt- resistance genes from such plants which can be used in future crop improvement.

Olimarabidopsis pumila (Stephan) Al-Shehbaz, O’Kane & R. A. Price [synonym: Arabidopsis pumila (Stephan) N. Busch] habitates semi-arid and semi-salinized areas in Xinjiang. O. pumila is a close relative of Arabidopsis thaliana, but it is more tolerant to salt stress than A. thaliana (Hoffmann et al., 2010; Roy et al., 2010). Although the size of the 1C DNA (173 Mbp) of O. pumila is comparable to that of A. thaliana (167 Mbp), its genome size is about twice as large as that of A. thaliana (Hoffmann et al., 2010). O. pumila has 2n = 32 chromosomes, but it has the lowest mean DNA content per chromosome of the four Bras- sicaceae species A. thaliana, Arabis auriculata, and Arabis montbretiana. The estimated time of divergence of A. thaliana and O. pumila is 10 – 14 million years (Mya) (Clauss and Koch, 2006; Roy et al., 2010).

Although much progress has been made in the discovery of salt tolerance genes and mechanisms

Olimarabidopsis pumila and Preliminary Analysis of Expressed Sequence Tags

Yun-Xia Zhaoa, Yan-Ling Weia, Ping Zhaob, Cheng-Bin Xiangb, Fang Xua, Chao Lia, and Xian-Zhong Huanga,*

a Key Laboratory of Agrobiotechnology, College of Life Sciences, Shihezi University, Shihezi 832003, P. R. China. Fax: +86 993 2057216. E-mail: xianzhongh106@163.com

b School of Life Sciences, University of Science and Technology of China, Hefei 230027, P. R. China

* Author for correspondence and reprint requests

Z. Naturforsch. 68 c, 499 – 508 (2013); received March 15/October 23, 2013

Olimarabidopsis pumila is a close relative of the model plant Arabidopsis thaliana but, unlike A. thaliana, it is a salt-tolerant ephemeral plant that is widely distributed in semi-arid and semi-salinized regions of the Xinjiang region of China, thus providing an ideal candidate plant system for salt tolerance gene mining. A good-quality cDNA library was constructed using cap antibody to enrich full-length cDNA with the gateway technology allowing library construction without traditional methods of cloning by use of restriction enzymes. A prelimi- nary analysis of expressed sequence tags (ESTs) was carried out. The titers of the primary and the normalized cDNA library were 1.6 · 106 cfu/mL and 6.7 · 106 cfu/mL, respectively. A total of 1093 clones were randomly selected from the normalized library for EST sequenc- ing. By sequence analysis, 894 high-quality ESTs were generated and assembled into 736 unique sequences consisting of 72 contigs and 664 singletons. The resulting unigenes were categorized according to the gene ontology (GO) hierarchy. The potential roles of gene products associated with stress-related ESTs are discussed. The 736 unigenes were similar to A. thaliana, A. lyrata, or Thellungiella salsuginea. This research provides an overview of the mRNA expression profi le and fi rst-hand information of gene sequence expressed in young leaves of O. pumila.

Key words: Olimarabidopsis, Comparative Genomics, Gene Expression

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of salt tolerance, salt tolerance in plants is still a very complex problem. Salt stress signal transduc- tion still needs to be explored further.

Expressed sequence tags (ESTs) are short (usually about 300 – 500 bp), single-pass sequence reads from mRNA (cDNA), which are used in the identifi cation of gene transcripts, in gene disco very, and in gene sequence determination (Adams et al., 1991; Boguski et al., 1993; Alba et al., 2004). ESTs have proven to be an effi cient, rapid means to identify novel genes involved in tolerance to environmental stress (Jha et al., 2009; Nishiuchi et al., 2010). Large-scale cDNA sequencing and EST analyses have been suc- cessfully used to identify stress tolerance genes in a large number of plants, such as Suaeda salsa (Zhang et al., 2001), Avicennia marina (Mehta et al., 2005), Thellungiella halophila (Du et al., 2008;

Taji et al., 2008), Salicornia brachiata (Jha et al., 2009), and Suaeda asparagoides (Ayarpadikannan et al., 2012).

In this study, we made use of the gateway tech- nology to construct a high-quality normalized and full-length cDNA library from young leaves of O.

pumila plants exposed to 500 mM NaCl stress, and performed a preliminary functional analysis of ESTs in young shoots of O. pumila.

Material and Methods

Plant material and salt stress treatment

O. pumila seeds were collected from the natural semi-salinized land surrounding the Mashroom Lake in Shihezi that is located in the north of Xinjiang province of China. Seeds were surface- sterilized and placed in square Petri dishes with 0.5 × Murashige and Skoog (MS) medium (pH 5.7), with 1% (w/v) sucrose and 0.1% (w/v) agar, at 4 °C for 5 d in the dark to synchronize germi- nation. The plates were incubated in a growth chamber under a 14-h light/10-h dark cycle at 22 °C. After 7 d, the seedlings were transplanted into soil and kept in a growth room with a 14-h photoperiod. When plants were 4 weeks old, the soil was watered with 0.5 × MS nutrient solution supplemented with 500 mM NaCl and the plants were grown for 14 h as described previously (Ni et al., 2007) before being sampled for RNA isola- tion.

RNA extraction and construction of the primary cDNA library

Total RNA was isolated from the leaves using the plant RNA Mini-Prep kit (Qiagen, Hilden, Germany). The mRNA was isolated from total RNA with the FastTrack MAG mRNA isolation kit (Invitrogen, Carlsbad, CA, USA). With the materials, a full-length cDNA library was prepared using the Superscript full-length library construc- tion kit (Invitrogen) according to the manufac- turer’s protocol. First-strand cDNA was synthe- sized using Superscript III reverse transcriptase with the 3' primer attB2-(d)T22 VN: 5'-biotinyl- GGGGACAACTTTGTACAAGAAAGTTGGG (T)22VN-3', where N = A, C, G, or T; V = A, G, or C. The underlined sequences are attB2 sequences.

The 3' primer is biotinylated to block blunt-end ligation of the 5' primer adapter to the 5' end of the fi rst-strand cDNA during the adapter liga- tion step. Truncated cDNA/RNA was digested by RNase I treatment of incomplete RNA: DNA hybrids. Full-length fi rst-strand cDNA was mag- netically captured by Cap-antibody beads and then eluted by wash buffer (Invitrogen). The 5' prime adapter with the 5' attB1 primer sequence 5'-ACAACTTTGTACAAAAAAGTTGG-3' was ligated to the 3' end of the fi rst-strand cDNA.

The second-strand cDNA was synthesized using the 5' primer extension method. The 100-μL reaction mixture containing 10 μL 10× high-fi del- ity PCR buffer, 4 μL dNTP mix (10 mM of each dNTP), 79 μL fi rst-strand cDNA (about 1 μg) with the 5' primer adaptor, 1 μL 5' primer (100 ng/μL), and 1 μL Platinum® Taq DNA polymerase high fi delity (Invitrogen) was incubated at 68 °C for 20 min, then at 72 °C for 20 min. Double-strand cDNA was size-fractionated by Sephacryl® S-500 HR column chromatography (Invitrogen) to gen- erate a cDNA library with an average cDNA in- sert size of approximately 1,000 bp.

To construct a gateway entry cDNA library, the size-fractionated cDNA was ligated into the plasmid vector pDONR222 (Invitrogen) using BP Clonase (Invitrogen). The recombination re- action was allowed to proceed at 25 °C for 16 h, and terminated by mixing with 2 μL proteinase K (Invitrogen) and incubating at 37 °C for 10 min.

The resultant mixture was used to transform the ElectroMAX™ DH10B™ T1 phage-resistant cells (Invitrogen). Stocks of electro-transformed

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Escherichia coli cells were stored at –80 °C in glycerol.

Construction of the normalized cDNA library Genomic DNA of O. pumila was isolated using the cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). Genomic DNA (120 μg) was digested with EcoR I (TaKaRa, Dalian, China). After purifi cation and recovery, the DNA fragments were fi lled in using the Kle- now fragment (Invitrogen). The treated geno- mic DNA was mixed with Dynal magnetic beads (Dynabeads® M-280 Streptavidin) (Invitrogen) to form the affi nity system.

The isolation of the plasmids containing the primary cDNA library was performed as pre- viously described by Xiang et al. (1994). The mix- ture of the plasmids was added to the above af- fi nity system for saturation hybridization (Reqan et al., 2000), and then eluted with wash buffer (In- vitrogen). The eluted plasmids were transformed into the ElectroMAX™ DH10B™ T1 phage- resistant cells. After recovery, cells were plated on Luria-Bertani (LB) agar medium containing 50 μg/mL kanamycin. A fraction of the bacterial colonies was used for plasmid preparation and li- brary checking, whereas the remaining bacterial colonies were washed off the agar plates with li- quid LB medium containing 50 μg/mL kanamycin and completely resuspended. The suspension was mixed with an equal volume of 80% glycerol, and aliquots were stored as normalized cDNA library stocks at –80 °C.

Titration of the cDNA library

Ten μL electro-transformed E. coli cells carrying the primary and normalized library, respectively, were diluted 1000-fold, and 50 μL were spread on an LB plate containing 50 μg/mL kanamycin. The plates were incubated at 37 °C overnight to deter- mine the colony forming units (cfu). The number of clones was counted to calculate the library ti- ter according to the formula: cfu/mL = number of plaques · dilution factor · 103 μL. The size of the inserted fragments was determined by polymer- ase chain reaction (PCR) in 40 to 60 randomly selected clones as described by Ni et al. (2007).

EST sequencing, editing, and assembly

Clones of the normalized cDNA library were randomly selected from LB agar plates sup- plemented with 50 μg/mL kanamycin. After manual picking, clones were grown overnight in standard LB/kanamycin medium, and plas- mids were isolated by the alkaline lysis method (Birnboim and Doly, 1979). In addition, selected clones were stored at –80 °C as glycerol stocks.

Sequencing was carried out from the 5' end of the cDNA inserts with the M13 forward primer (5'-GTAAAACGACGGCCAG-3') using an ABI PRISM 3730xl automated DNA sequencer (Ap- plied Biosystems, Grand Island, NY, USA) at the Sequencing Center of the Beijing Genomics Insti- tute, Beijing, China.

The trimming process of all sequences, which included the base calling, the removal of the low-quality sequences, sequences from the vec- tors, and poly (A) tails, was conducted essentially as described by Lai et al. (2011). After cleaning, sequences shorter than 100 nucleotides were dis- carded. High-quality ESTs were aligned and as- sembled into contigs using Codon Code Aligner software (http://www.codoncode.com), when the criterion of a minimum identity of 95% over 40 bp was met. When an EST could not be as- sembled with others in a contig, it remained as a ‘‘singleton’’. The contigs and singletons should thus correspond to sequences of unique genes (unigenes).

Functional annotation of unigenes

All unique sequences were searched for puta- tive open reading frames (ORF) with the pro- gram gorf (http://www.ncbi.nlm.nih.gov/gorf/

gorf.html), and the largest ORF sequences were used for functional analysis. Sequence similar- ity searches were performed using the BLAST program (http://ncbi.nlm.nih.gov/blast/) and the sequences then compared to those in a variety of databases including NCBI nt (non-redundant nucleotide database), NCBI nr (non-redundant protein database) (http://www.ncbi.nlm.nih.gov), and Swiss-Prot (http://www.expasy.org/sprot/) which contains all nucleotide or protein sequenc- es submitted to the public databases, with a cut- off value of E < 1e-5. To assign gene ontology (GO) terms, the unique sequences were function- ally categorized using the GO annotation tool in TAIR (http://www.arabidopsis.org/tools/bulk/go/).

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The distribution of GO terms in the main onto- logy categories of cellular components, molecular functions, and biological processes was examined.

Results

Total RNA isolation and double-strand cDNA synthesis

To construct the cDNA library, total RNA was isolated from O. pumila leaves stressed by 500 mM

NaCl for 14 h. The A260/A280 ratio of isolated RNA was found to be 2.0 confi rming good quality of the isolated RNA. Agarose gel electrophoresis of the total RNA revealed distinct bands of 28S and 18S rRNA, respectively, the concentration of 28S rRNA being about twice that of 18S rRNA.

Double-strand cDNA was analysed on an 1.0%

agarose gel. The ds cDNA appeared as a 0.5- to 5-kb smear on the gel, which confi rmed the suc- cessful synthesis of ds cDNA.

Construction and analysis of the full-length cDNA library

A primary cDNA library was constructed with a titer of 1.6 · 106 cfu/mL. The total library capac- ity was 4.8 · 106 cfu. The quality of the library was assayed by PCR amplifi cation of 96 colonies ran- domly selected. The recombination rate was more than 95%. The size of the inserts ranged from 500 to 3000 bp, with an average size of about 1000 bp.

The titer of the normalized cDNA library was 6.0 · 106 cfu/mL, and the capacity of the total li- brary was 6.7 · 106 cfu. PCR amplifi cation of ran- domly picked clones confi rmed that the average insert size was again about 1000 bp (Fig. 1). The recombination rate was more than 95%. Thus the normalized and full-length cDNA library, respec- tively, had high titers, high recombination rates, and large inserts.

Generation, assembly, and analysis of O. pumila ESTs

From the normalized cDNA library, a total of 1093 cDNA clones were randomly picked and sequenced from the 5' terminal using the primer M13-F. All raw EST sequences were trimmed of vector sequences and the poly (A) tails. The Fig. 1. Agarose gel assays for cDNA inserts in the normalized cDNA library. Thirty-two randomly selected clones from the normalized cDNA library of O. pumila were analysed by PCR using the primers M13-F and M13-R. M, 1-kb Plus DNA ladder.

Table I. Summary of the ESTs from cDNA clones ob- tained from Olimarabidopsis pumila.

Feature Value

Total ESTs 1093

High-quality ESTs 894

Contigs 72

ESTs in contigs 230

Singletons 664

Unique sequences 736

Redundancy (%) 17.86

Average length of unigene

sequences (bp) 831.5

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low-quality sequences below the minimum length (100 bp) were discarded. This resulted in 894 high- quality ESTs with an average length of 820 bp.

These EST resources are suitable for salt toler- ance gene discovery and as molecular markers.

The 894 high-quality ESTs were assembled into 736 unigenes, including 72 (9.8%) contigs and 664 (90.2%) singletons (Table I). The average length of the unigene sequences was 831.5 bp, ranging from 216 bp to 1424 bp (Table I). Fig. 2 compares the distribution of the sequence lengths before and after sequence assembly. The distribution of EST frequencies after clustering is shown in Fig. 3.

Of the 72 contigs, 46 (63.9%) contained 2 ESTs, 10 (13.9%) contained 3 ESTs, 7 (9.7%) contained 4 ESTs, and 6 (8.3%) contained 5 ESTs. There were few sequences (4.2%) representing more than six ESTs (Fig. 3), and there were only 16

contigs representing more than 4 ESTs (Table II), suggesting that the redundance in the normalized library was relatively low. Each of these clusters contained  5 ESTs, representing 12% of the to- tal number of ESTs obtained. On average, each contig was assembled from 3.2 sequences due to highly redundant ESTs, and the unigene average size was only 1.2 sequences. These data indicate the good quality of the normalized O. pumila cDNA library.

Functional annotation and categorization of unigenes

GO analysis has been widely used to classify gene functions (Ashburner et al., 2000). Three structured controlled vocabularies (ontologies) have been defi ned that describe gene products in terms of their associated biological processes, Fig. 2. Sequence length distribution of the ESTs before and after assembly.

Fig. 3. Frequency and distribution of O. pumila ESTs in the assembled contigs. Cluster size indicates the number of ESTs in 736 unique sequences including 664 singletons and 72 contigs.

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cellular components, and molecular functions in a species-independent manner (Berardini et al., 2004). All 736 unigenes were annotated, in- dicating that all of them had signifi cant matches against genes of A. thaliana, A. lyrata, and other plants, and were divided among the three GO categories (Fig. 4). On the whole, 652 genes were categorized under the “cellular component” cate- gory, 654 under “molecular function”, and 669 under “biological process”. The total number of GO mapping in each of the three ontologies may exceed the number of unigenes, because a given gene product may be assigned to more than one GO term.

Within the “cellular component” category, chloroplast (13.4%) and plastid (8.3%) genes are most enriched (Fig. 4A). Genes encoding pro- teins of membranes, including the plasma mem- brane (4.2%) and other membranes (9.6%), the nucleus (6.3%) and the cytosol (3.6%) are also signifi cantly enriched (Fig. 4A). Transcription fac- tors are crucial for regulating plant responses to biotic and abiotic stress (Singh et al., 2002). In the

"molecular function" category, most of the GO terms (40%) were included in "binding" proteins, which often act as transcription factors, such as nucleotide binding (8%), protein binding (6.7%), DNA and RNA binding (5.6%), nucleic acid bind-

ing (1.6%), and other binding (17.8%) (Fig. 4B).

Proteins having hydrolyase activity (11.3%) and transferase activity (10%) are dramatically en- riched compared to other terms (Fig. 4B). The un- known molecular function term (7.7%) is rather enriched (Fig. 4B).

The GO "biological process" is helpful for the functional classifi cation of analysed genes.

In the "biological process" category (Fig. 4C), a consider able number of ESTs were involved in response to abiotic and biotic stimuli (10%), response to stress (9.5%), and transport (5.5%), which is consistent with previous studies showing that plant adaptation to environmental stress in- volves the expression of stress-responsive genes and activation of various physiological and meta- bolic responses (Thomashow, 1999; Shinozaki et al., 2003). These three categories form the basis for mining stress-regulated genes.

Discussion

Construction and composition of a cDNA library of O. pumila

Construction and analysis of a cDNA library is still an essential technology in modern bio logy and functional genomics, as it provides much more detailed information on genomic mecha- Table II. Sixteen highly abundantly represented genes in the 894 high-quality ESTs (number of ESTs  4).

Contig no.

Contig length

Total of ESTs

Annotation (putative function) Identitya

(%) E valueb

1 1302 29 Chlorophyll a-b binding protein 1 (CAB1) 93 2e-129

2 866 13 Ribulose bisphosphate carboxylase small chain 3B 97 4e-89

3 1333 8 Ribulose bisphosphate carboxylase/oxygenase activase 78 3e-162

4 1254 5 Polyubiquitin 10 (UBQ10) 93 0

5 1120 5 Photosystem II protein psbY-2 (PSBY) 93 3e-59

6 961 5 Glutathione S-transferase 1 (GSTF6) 91 1e-120

7 913 5 Translationally controlled tumour protein-like protein (TCTP)

93 1e-106

8 821 5 Plastocyanin major isoform (DRT112) 83 1e-107

9 497 5 Metallothionein-like protein (AtMT-q) 73 1e-37

10 1424 4 S-Adenosylmethionine decarboxylase 1 beta chain (SAMDC)

98 3e-142

11 1216 4 Glyceraldehyde 3-phosphate dehydrogenase (GAPC2) 86 1e-180

12 1096 4 Catalase 3 (CAT3) 94 1e-178

13 1081 4 3-Ketoacyl-CoA thiolase 2 (PKT3) 80 1e-160

14 1007 4 Putative Atpm24.1 glutathione S-transferase 89 1e-142

15 996 4 Lhcb2 protein (Lhcb2.1) 93 1e-128

16 526 4 Metallothionein 3 (MT3) 62 2e-38

a With respect to the corresponding sequences of A. thaliana.

b The E value is the expect value which describes the random background noise. The lower the E value, or the closer it is to zero, the more "signifi cant" the match is.

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Fig. 4. Representation of GO mapping results for O. pumila unigenes: (A) GO term "cellular component"; (B) GO term "molecular function"; (C) GO term "biological process".

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nisms underlying diverse processes of the organ- ism. The key to the success of a cDNA library construction are the quality of cDNA and the ability to obtain high-quality cDNAs from limit- ed amounts of mRNA. Numerous improvements carried out in the past few years have been made in the construction of full-length cDNA librar- ies (Seki et al., 1998; Zhu et al., 2001; Kato et al., 2005; Ni et al., 2007). Nevertheless, the construc- tion of high-quality cDNA libraries is still a great challenge, and there is no single method that can resolve all the common problems associated with ligation-assisted conventional cDNA library con- struction. In the present study, we made use of cap antibody beads to capture and enrich the full- length cDNA, and constructed a normalized and full-length cDNA library by using site-specifi c recombination, avoiding that genes were cut by restriction enzymes. Using this approach, we con- structed an entry cDNA library of young leaves of O. pumila plants treated with 500 mM NaCl.

The titer of the cDNA library, the recombination rate, and the average insert size in the primary and normalized cDNA library are suitable for the needs of a standard cDNA library (Fig. 1).

Generation and analysis of ESTs

High throughout single-pass sequencing, gen- eration and analysis of ESTs have been proven to be a rapid and effi cient way of obtaining in- formation on gene expression patterns (Adams et al., 1991; Wu et al., 2002; Phukon et al., 2012). In the present study, a total of 1093 cDNA clones randomly selected from the normalized and full- length cDNA library of O. pumila were subjected to sequencing for generation of ESTs. A total of 894 high-qua lity ESTs were obtained from the cDNA library, putatively representing 736 uni- genes, including 72 contigs and 664 singletons.

We found that all ESTs had signifi cant homo- logy with genes from A. thaliana, A. lyrata, or Thellungiella salsuginea. As shown in Fig. 4, the unigenes were classifi ed into cellular components, molecular function, and biological process devel- oped by the Gene Ontology Consortium (Berar- dini et al., 2004). These genes covered a broad range of the GO functional categories.

A large proportion of genes were found to par- ticipate in metabolic processes (30.5%) including protein metabolism, DNA or RNA metabolism, and cell organization and biogenesis (Fig. 4C).

Our results showed that the terms of response to abiotic or biotic stimuli, responds to stress, and transport (Fig. 4C) were signifi cantly expressed.

At high salinity stress, plant can enhance salin- ity tolerance by Na+ exclusion and the control of Na+ transporters within the plant. Many studies concentrated on the Na+/H+ antiporter proteins in the plasma membrane and tonoplast which play essential roles in Na+ exclusion and compartmen- talization, for example SOS1 (Qiu et al., 2003), NHX1 (Pardo et al., 2006), and HKT1 (Haro et al., 2005). Previous studies showed that the Na+/ H+ antiporter (Apse et al., 1999; Gaxiola et al., 2001), H+-adenosine triphosphatase (H+-ATPase), and H+-inorganic pyrophosphatase (H+-PPase) (Maeshima, 2001) coordinately regulate Na+ con- centration. In a high salt stress environment, plants can survive by exclusion of excess Na+ from the cytoplasm and sequestration of Na+ from the cy- tosol to the vacuole towards the maintenance of ion homeostasis inside the cell. Detailed analysis of genes involved in responses to abiotic and bi- otic stimuli showed that genes encoding sodium/

hydrogen exchanger (NHX1) (JZ151532), vacu- ole H+-ATPase (JZ151828), plasma membrane H+-ATPase (JZ151872), late embryogenesis abundant (LEA) protein (JZ151694), NAC do- main transcription factor (JZ151841), aquaporin PIP2 – 3 (JZ152319), and other stress-induced proteins were found in these categories (Fig. 4C).

Of the 72 contigs, there are only nine contigs with more than 5 ESTs (Table II). The gene for which most ESTs (29) were obtained, was the gene encoding the chlorophyll a-b binding pro- tein (CAB1). The genes encoding ribulose bis- phosphate carboxylase small subunit 3B, ribulose bisphosphate carboxylase/oxygenase activase, and photosystem II protein psbY-2 (PSBY) were re- presented by 13, 8, and 5 ESTs, repectively (Table II). The representation of a high number of tran- scripts encoding the mentioned proteins involved in photosynthesis may indicate that the plants re- mained healthy even in the presence of NaCl (Jha et al., 2009). Genomic sequencing and compara- tive genomics indicated that many genes related to cation transport, abscisic acid signaling, and wax production possibly contributed to the suc- cess of Thellungiella salsuginea in stressful envi- ronments (Wu et al., 2012). There are many other genes putatively involved in stress responses of the O pumila genome the functions of which have not yet been analysed.

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This is the fi rst study of the transcriptome of O.

pumila, a close relative of A. thaliana and A. lyrata, based on a normalized cDNA library derived from young leaves. Through analysis of the ESTs and putative functional gene annotation, a large number of putative stress-regulated genes were identifi ed. Next, we will determine the expression profi les of these genes in response to salt stress.

The full length of these genes can be obtained easily, and their specifi c function in salt tolerance can be investigated by the transgene technology in model organisms, which will eventually provide new gene targets for improving crop resistance to abiotic stress by the genetic engineering technolo- gy. In addition, the resulting gateway entry cDNA library obtained can be transferred into various gateway destination vectors for gene expression

and functional analysis. The set of ESTs obtained will facilitate the comparable genomic based on unigenes within the different species of the Bras- sicaceae in the future.

Accession Numbers

All ESTs data are available on the internet (GenBank_Accn: JZ151519~JZ152412).

Acknowledgements

This work was supported by the National Natu- ral Science Foundation of China (31060149), the Program for Excellent Talents in Shihezi Universi- ty (RCZX200902), and the Natural Science Foun- dation of Shihezi University (2011ZRKXTD-06).

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