Construction and analysis of a cDNA library from yellow-fruit ginseng ( Panax ginseng C . A . Meyer . ) leaf tissue

The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ES672876-ES673253). ESTs were annotated, analyzed by BlastX and functional classified based on gene ontology, the results showed that 221 ESTs showed significant similarities to gene sequences in Nr database and were known genes, 21 ESTs were non-significance and unknown function genes, and 136 ESTs were considered novel genes. Most of the ESTs appeared to be related to physiological and cellular processes.


INTRODUCTION
Ginseng (Panax ginseng C. A. Meyer), a perennial herb from the Araliaceae family, is one of the most commonly utilized medicinal plants.Ginseng is considered to be one of the most potent medicinal plants that have been used to bolster immunity, provide nutrition, ameliorate fatigue and enhance resistance to stress, disease and exhausttion.Ginsenosides, which are triterpene glycosides (saponins), are believed to be the main active compounds in ginseng tissues (Choi et al., 2005;Kim et al., 2006).More than 30 different ginsenosides have been isolated from ginseng plants (Sun et al., 2001;Zheng et al., 2001).Despite the considerable commercial interest in ginsenosides, little is known about the genes and biochemical pathways of ginsenoside biosynthesis.'Jilin yellow-fruit ginseng' is identified as a homozygous recessive mutant from ordinary panax ginseng and contains high levels of ginsenosides, rich proteins, amino acids and other nutrients (Zhao et al., 1998).
cDNA library is one of the basic means to study functional genomics.Expressed sequence tags (ESTs) are partial sequences of randomly selected complementary DNA (cDNA) clones; automated sequencing techniques make it possible to generate large numbers of EST at one time.Expressed sequence tags (EST) analysis is an effective method to discover novel genes and investigate gene expression in different organs and tissues (Wang et al., 2006).The generation and analysis of expressed sequence tags provides useful information on development, metabolism and signaling in various organisms.Expressed sequence tags have applications in the discovery of new genes, mapping of the genome and identification of coding regions in genomic sequences (Miyahara et al., 2000).
In the study, we constructed a cDNA library of yellowfruit ginseng leaf tissue and obtained 378 high quality EST sequences.Through EST analysis and gene ontology, this will help us to understand the gene expression pattern, discover novel genes and study the biochemical pathways of ginsenoside biosynthesis.

Plant materials
Actively growing 2 year old yellow-fruit ginseng (Panax ginseng C. A. Meyer) leaves were obtained from ZuoJia, Jilin province, China, on June 20, 2006.The harvested leaves were immediately frozen in liquid nitrogen and then stored at -70°C until RNA isolation.

Isolation and quantification of RNA
The total RNA was isolated from ginseng leaf tissue using SDS methods, as described by Yang et al. (2008).Total RNA was quantified by measuring the optical density of a dilute RNA solution.The integrity of the RNA was analyzed using 1.1% agarose/EtBr gel electrophoresis.The purity of the RNA was checked by the ratio of OD260/OD280.

cDNA synthesis and library construction
In accordance with the creator tm SMART TM cDNA library construction kit user manual provided by the manufacturers (Clontech), total RNA (1.0 µg) as starting material was reverse transcribed to synthesize first-strand cDNA, and double-strand cDNA was synthesis by LD-PCR.5 µg of the double-stranded cDNA were taken for analysis by electrophoresis on a 1.1% agarose/EtBr gel.Then the amplified double-strand cDNA was digested with proteinase K and Sfi I.After digestion, cDNA size fractionation was performed using chroma spin-400 columns to collect cDNA large than 400 bp and checked the profile of fractions on a 1.1% agarose/EtBr gel.The cDNA was ligated to the Sfi I-digested dephosphorylated pDNR-LIB vector provided with the kit and electroporated into DH5α Escherichia coli bacteria to develop the cDNA library of ginseng leaf tissue.To make a large, stable quantity of a high-titer stock of the library, we amplified the primary cDNA library.

Identification of the cDNA library
According to the library tittering protocol, the unamplified and amplified cDNA library were tittered.To identify the cDNA inserts of the recombinants and determined the percentage of recombinant clones, 16 plaques were randomly picked from plate.Then PCR was performed with M13 primers provided by the advantage 2 PCR kit.The PCR products checked on 1.2% TAE/agarose gel with DNA size markers.

Library sequencing and analysis
The cDNA library clones were plated into LB agar plate containing 30 µg/ml of chloramphenicol, white clones were picked in to 96 well plates randomly, plasmids DNA of each clone were prepared by standard alkaline lysis preparation protocol and then sequenced by Beijing genomics institute.Sequencing was performed in a Mega BACE1000 DNA capillary sequence machine.The raw expressed sequence tags (EST) were edited to remove vector and poor quality sequences.The remaining sequences were subjected to blast analysis against the non-redundant database on the GeneBank (http://www.ncbi.nlm.nih.gov/blast) for similarity.The confirmed sequences were submitted to the dbEST database of GeneBank.Identified genes were classified according to gene ontology.The network information of the gene ontology database is categorized into 3 groups: cellular component, molecular function and biological process.

RESULTS AND DISCUSSION
High-quality total RNA was isolated from the leaf tissue of ginseng.Electrophoresis of the total RNA on 1.1% agarose/EtBr showed distinct 28S and 18S rRNA bands and the ratio of intensities of 28S and 18S was about 2:1 (Figure 1), the ratio of OD 260 /OD 280 to the total RNA was 1.90.The total RNA isolated was integrated and suitable for constructing the cDNA library.The double-strand cDNA synthesized using LD-PCR was analyzed on a 1.1% agarose/EtBr gel and the product showed a smear from 0.2 to 2.5 kb (Figure 2).The double-strand cDNA fractionated using CHROMA SPIN-400 columns was  collected and pipette 3 µl of cDNA to run on a 1.1% agarose/EtBr gel at 150 V for 10 min.Electrophoresis results showed the cDNA in lanes 7 -11 was larger than 400 bp (Figure 3) and was ready to be ligated to the Sfi Idigested, dephosphorylated pDNR-LIB vector.
The titer of the un-amplified constructed cDNA libray was approximately 1.01 × 10 6 pfu/ml, the titer of the amplified cDNA library was 2.16 × 10 9 pfu/ml.16 independent clones were selected randomly from unamplified cDNA library and were amplified by PCR to check the percentage of recombinants and the insert size of the recombinants.The results showed the percentage of recom-binants was 93.75%, the cDNA inserts ranged from 0.3 to 2 kb (Figure 4), with an average size of 700 bp.The results indicated that the quality of cDNA library should be sufficient to identify the expressed genes in yellow-fruit ginseng.
A total of 400 randomly selected clones were sequenced from the library.Of these, 378 high quality sequences were obtained after deletion of the vector sequences and sequences with short base pairs.The obtained 378 EST sequences were submitted to GeneBank (accession No: ES672876-ES673253) and dbEST (ID: 46881386 46881763).Through BlastX analysis, 221ESTs showed significant similarities to gene sequences in Nr database and were known genes, 21 ESTs were non-significance and unknown function genes, and 136 ESTs, which have no matched were considered novel genes in P. giseng.The results of BLASTX search and annotation are shown in Table 1.The generated EST were categorized using gene ontology terms as shown in Table 2, which provide a structured vocabulary to describe a sequence according to its cellular component status, molecular function and biological process.Most EST appeared to be related to physiological and cellular processes.
In conclusion, we described the construction of a cDNA library from yellow-fruit ginseng leaf tissue, EST analysis and gene ontology.The results of analysis of 378 cDNA

Figure 1 .
Figure 1.Agarose gel electrophoresis of total RNA from yellow-fruit ginseng leaf.

Figure 4 .
Figure 4. 16 clones in the cDNA library were selected randomly to evaluate their insert sizes.Lanes 1-16: Products of inserts; lane M: DL2000 marker.

Table 1 .
List of BlastX searched and annotated expressed sequence tags from yellow-fruit ginseng leaf cDNA library.

Table 2 .
Gene ontology of expressed sequence tags from yellow-fruit ginseng leaf cDNA library.