Botanical Studies (2010) 51: 7-16.
molecular biology
Construction of a full-length enriched cDNA library and analysis of 3111 ESTs from roots of Bupleurum chinense DC.
Chun SUI, Jian-He WEI*, Shi-Lin CHEN, Huai-Qiong CHEN, L.M. DONG, and Cheng-Min YANG
Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, P. R. China
(Received June 24, 2008; Accepted June 18, 2009)
ABSTRACT. Radix Bupleuri (Chaihu), sourced from the dried roots of Bupleurum species, is a traditional Chinese medicine with anti-inflammatory, anti-pyretic, and anti-hepatotoxic efficacy. It is widely used in China, Japan, Korea, and other countries in south Asia. A full-length enriched cDNA library derived from the roots of B. chinense DC. was constructed for the first time by the SMART technique in this study to initiate the functional genomic research of this important medicinal plant. The titre of the library was 1.1><106. From the library, randomly selected 3902 clones were 5' single-pass sequenced, among which 3111 high quality ESTs were generated and 1650 uniESTs were identified with 377 contigs and 1273 singleton ESTs. The estimated average cDNA insert size was 1.1 kb, and the fullness ratio was ca. 51.5%. BlastX analysis of all uniESTs resulted in 949 (57.5%) homology to previously identified genes, 680 (41.2%) matched to unknown, unnamed, or hypothetical protein genes, and 21 clones had no hit. Gene ontology (GO) annotation of uniESTs showed that approximately 1002, 957, and 861 were assigned molecular function, biological process, and cellular component GO terms, respectively. KEGG pathway analysis indicated that 307 uniESTs may be involved in 31 metabolic pathways, in which at least five uniESTs were contained, by comparing each with Arabidopsis metabolic pathways. Through SSR searching within 1650 uniESTs, 86 potentially useful SSR loci were identified in 82 uniESTs. The library and EST data provide a platform to study the molecular mechanisms of various physiological phenomena of Bupleurum. The set of SSR loci would potentially be useful molecular markers for the germplasm identification, genetic diversity analysis, and gene mapping of Bupleurum.
Keywords: Bupleurum chinense DC.; Full-length enriched cDNA library; ESTs; Switching mechanism at 5' end of RNA transcript (SMART); SSR.
introduction
Radix Bupleuri (Chaihu), sourced from the dried roots of Bupleurum species (Umbelliferae family), is an impor­tant Traditional Chinese Medicine (TCM) which has anti­inflammatory, anti-pyretic, and anti-hepatotoxic properties (Yen et al., 2005; Pan, 2006). It had been used in ancient China for about 2, 000 years when it was recorded in "Shen Nongs Materia Medica," the first monographic work of its kind in China. According to the "Pharmacopoeia of the People s Republic of China," B. chinense DC. is one of the two official Radix Bupleuri source species (the other being B. scorzonerifolium Willd.) widely distributed in China as a wild and cultivated species. It has also been introduced
to other countries. Crude drug, decoction pieces, and ex­tracts of Radix Bupleuri are annually exported from main­land China to Japan, Korea, and Southeast Asian countries. However, B. falcatum L., restricted to Japan and Korea, and B. kaoi Liu, C. Y Chao & Chuang, endemic to Taiwan, have also been pharmacologically studied and used as the source species for Radix Bupleuri in their native countries, respectively. Most research about Bupleurum has focused on its classification (Urgamal et al., 2007), authentication (Yang et al., 2007), cultivation and breeding (Wei et al., 2003), physiology (Aoyagi et al., 2001), identification of medicinal compounds (Liu et al., 2002), and pharmacol­ogy (Pan, 2006). Pharmacologically active components like saikosaponins, volatile oils, and polysaccharides have been found in Radix Bupleuri. Saikosaponins, a class of triterpenoid saponin, make up its major active component, and these are used as a quality control standard for me­dicinal Bupleurum. Little molecular information about the
*Corresponding author: E-mail: wjianh@263.net; jhwei@implad.ac.cn； Tel: +86-10-6281-8841; Fax: +86-10-6281-8841.
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Botanical Studies, Vol. 51, 2010
secondary metabolism of these species is available. A few laboratories have recently launched preliminary studies to research the biosynthetic pathways of saikosaponins (Kim et al., 2006; Chen et al., 2007).
A full-length enriched cDNA library, with higher level full-length cDNA sequences (usually 30-70% of the total sequenced clones) than that of EST or cDNA common library, has been constructed and annotated largely from model organisms like Arabidopsis, rice, maize, Drosoph-ila, and mice to analyze the functions of genes (Jia et al., 2006). As to medicinal plants, EST or common cDNA li­braries were constructed from plant species such as Panax ginseng (Jung et al., 2003; Kim et al., 2006) and Crocus sativus (D'Agostino et al., 2007). For the Umbelliferae family, the first cDNA library was constructed using carrot somatic embryos (Lin et al., 1996). Subsequently, seven cDNA or EST libraries have been generated according to dbEST (Vilaine et al., 2003; Kwon et al., 2004; Divol et al., 2005; Park and Park, 2006). Most of these cDNA libraries were subtractive or differentially displayed using materials with special treatment, and the numbers of se-quenced ESTs or cDNAs from these cDNA libraries were small. For plants in the genus Bupleurum, only one sub-tractive EST library was constructed using the adventitious roots of B. kaoi (Chen et al., 2007).
In this study, we constructed a full-length enriched cDNA library of B. chinense root, sequenced and bioinformatically analyzed more than three thousand ESTs. The cDNA library and these sequence data will help to isolate some metabolic functional genes and also will increase useful bioinformatics references for research on other umbelliferous plants. We also identified some interesting potential SSR markers detected in our ESTs. This is the first report to analyze a full-length enriched cDNA library using medicinal roots of traditionally cultivated Bupleurum.
materials and methods
Plant materials
Bupleurum chinense cv. Zhongchai No. 1, which is a mass-selected cultivar of B. chinense, was used to construct a full-length enriched cDNA library. The roots of one-year old plants were harvested when flowering, frozen immediately in liquid nitrogen, and then stored at -80°C until RNA was extracted.
RNA extraction and full-length enriched cDNA library construction
Total RNA was extracted using TRIzol reagent (GIBCO BRL) according to the manufacturer's guideline. From approximately 6 g of frozen tissue, 728 fig total RNA was obtained. About 8.84 fig mRNA was then isolated using an Oligotex mRNA Kit (QIAGEN). The full-length cDNA library was constructed from approximately 3 fg mRNA by the SMART technique (Wellenreuther et al., 2004). After first-strand cDNA synthesis, long distance
PCR (LD-PCR) and proteinase K digestion, PCR products
were digested with restriction enzyme SfiI to generate directional cloning ends. The 5^I-digested double-strand cDNA was then size fractioned by a CHROMA SPIN-400 Column, and six fractions of cDNA fragments (ranging from 500 bp to 4 kb) were pooled. Fractioned cDNA was cloned into 5^I-digested reconstructive pBluescript II SK vector (sequences between EcoRl and NotI sites replaced by Sfil A and Sfil B adaptors for directional insertion) and transformed into Escherichia coli DH-5a competent cells.
Evaluation of full-length enriched cDNA library
and sequence analysis of ESTs
One microlitre ligation products were transformed to evaluate the recombination rate and capacity of a full-length enriched cDNA library. Plasmid DNA from a total of 136 clones was extracted, SfiI-digested, and analyzed on 1% agarose gel. Fragment size was estimated using AlphaImager 2200. Three thousand, nine hundred and two clones were randomly selected and 5' single-pass sequencing was performed using an ABI 3730 Sequencer. The Phred/Phrap/Consed software package was used to call bases, remove vector sequences and low quality readings, and assemble uniESTs. Inserts with lengths >=450 bp and N<5 were defined as valid sequences. All sequences were compared to genes in the non-redundant protein database (nr) and nucleic acid databases (nt) of NCBI using BlastX and BlastN searches. Sequences with identity>90% over 100 bp were clustered as single uniESTs. To evaluate the fullness ratio of the library, clones corresponding to the identified genes were aligned to determine whether they contained a 5' UTR and a putative ATG translation initiation codon. When a sequence contained a putative translation initiation codon, it was defined as a full-length cDNA. Assembled uniESTs were annotated via a GO (Gene Ontology) term using a GoPipe standalone package (Chen et al., 2005) and further compared with the Arabidopsis metabolism pathway defined by KEGG (http ://www.kegg.com) after BlastX of uniESTs and Arabidopsis protein sequences with a threshold E-value 1E-10 and an overall identity of 50%. Additionally, uniESTs assembled in the library were compared with all nucleotide squences of Bupleurum obtained from GenBank via a BlastN algorithm.
SSR searching
All uniESTs obtained in sequence assembly were analyzed for SSR using SSRHunter 1.3 (Li and Wan, 2005). To ensure the accuracy, only SSR loci within a 600 bp range of uniEST sequences were counted and for later primer design, at least 50 bp nucleotides were present before and after SSR loci. If two or more SSRs were within one uniEST, and the distance between them was less than 50 bp, they were identified as one compound SSR locus. Dinucleotides that were equal or repeated more than 9 times and tri-nucleotides repeated over 5 times were selected.
SUI et al. ― cDNA library construction and ESTs analysis of B. chinense
9
results
Overall features of B. chinense root full-length enriched cDNA library
One microlitre of transformed ligation mixture yielded 1113 recombinant clones, and the titre of the full-length enriched cDNA library was about 1.1x106 clones. To evaluate the size and distribution of insert cDNA clones, a total of 136 clones were randomly selected and digested by Sfil, allowing us to obtain average cDNA insert sizes and the cDNA length distribution profiles. Most insert lengths ranged from 0.5 to 2 kb. The estimated average insert size was 1.1 kb. The size distribution of insert DNA clones is shown in Figure 1.
A total of 3902 recombinant clones were randomly selected and sequenced from the 5' end. After eliminating low-quality sequences and contaminated clones, 3111 valid sequences in all were obtained. Clustering and assembly of these ESTs resulted in a total of 1650 uniESTs with 377 contigs and 1273 singleton ESTs. BlastX and BlastN analysis showed that of 1650 uniESTs, 949 (57.5%) were homologous to previously identified genes, 680 (41.2%) matched to unknown, unnamed, or hypothetical protein genes, and 21 clones had no hit. Clones corresponding to the identified genes were tested to determine whether they contained a 5' UTR and a putative ATG translation initiation codon. Among these sequences matched to a known gene, 489 (ca. 51.5%) clones were predicted to contain a putative ATG translation initiation codon, and among them 159 single sequences were deposited in the dbEST division of GenBank (Accession numbers: FG341847-FG342005). General features of the library and sequencing statistics are listed in Table 1.
Gene Ontology annotation and metabolism pathway analysis
All uniESTs were assigned the Gene Ontology (GO) terms using the sequence comparison results of BlastX. Of the uniESTs, 1002 (60.7%), 957 (58.0%), and 861 (52.2%) were assigned molecular function, biological process, and cellular component GO terms, respectively. One uniEST did not exclusively belong to a single GO term. Figure 2 illustrates the GO term distribution of all uniESTs. The first two largest categories in three GO terms were binding and catalytic activity, physiological process and cellular process, cell and intracellular. These results which relate to the storage function of the root were consistent with similar studies on P. ginseng root (Jung et al., 2003). Comparison with the KEGG Arabidopsis pathway showed that 307 uniESTs represented 31 metabolic pathways on the prerequisite that each pathway contained at least 5 uniESTs (Table 2). The full-length enriched cDNA library we constructed is thus useful in identifying functional genes of B. chinense. BlastN analysis of our uniESTs with Bupleurum nucleotide sequences obtained from GenBank indicated that only three were homologous and the hits were AM409304 (cinnamic acid 4-hydroxylase),
Figure 1. Size distribution of cDNAs in the library. Fragment sizes were determined by sfil digestion of 136 random selected clones.
Table 1. General features of root of B. chinense full-length enriched cDNA library and sequencing statistics.
Titre (pfu)
Average cDNA insert size Total clones sequenced Sequences passed qualiy check Clustered (contigs) Unassembled (singletons) Unigenes (contigs+ singletons) Observed redundancy"1 No hit to nr (BLASTX)
1.1x106 1.1 kb 3902 3111 (79.7%) 377 1273 1650 88.5%
22
EST matches with E-value in BLASTX > 1><10—14 366 (11.8%) EST matches with E-value in BLASTX < 1><10—14 2724 (88.2%)
aOberved redundancy: (EST# after quality check-Unigene #)/ Unigene # (Lindqvist et al., 2006).
AM409290 (short-chain dehydrogenase/reductase), and AM409292 (omega-6 fatty acid desaturase). Therefore, nearly all uniESTs that we sequenced may represent novel transcripts from Bupleurum.
To visualize the transcript abundance of Bupleurum root, the contigs assembled from the library were analyzed. Of these, the sixteen most abundant transcripts (EST number equal to or larger than 18) observed in this library are listed in Table 3. The contig with the largest number of ESTs was identified to be dehydrin protein. Contigs with the second and third largest ESTs were a pathogenesis-related protein-like protein 1 and a putative
10
Botanical Studies, Vol. 51, 2010
stress-responsive protein, respectively. Moreover, two proteins belonging to the LEA family and another stress-related protein ranked among the sixteen contigs with the most ESTs.
SSR discovery
A total of 86 potentially useful SSR loci were identified in 82 uniESTs (four uniESTs have two loci each), comprising ca. 4.97% and 2.64% of the total uniESTs and total EST sequences, respectively. Trinucleotide repeats were the most abundant (48.8%), followed by di- (46.5%) and hexanucleotide repeats (1.2%). Also there were two loci with di- and trinucleotide compound repeats and one locus with di- and pentanucleotide compound repeats (Table 4).
discussion
Radix Bupleuri is an important and commonly used TCM in mainland China and Taiwan, Japan, Korea, and Southeast Asian countries, but molecular biology about
the source species is poorly researched. Chen et al. (2007) reported the construction of PCR-select cDNA subtrac­tion libraries and transcriptional changes in MeJA-induced adventitious roots of B. kaoi. They obtained a total of 834 ESTs representing 532 uniESTs. Kim et al. (2006) cloned core sequences of five isoprenoid pathway genes by homology-based RT-PCR and determined the correlation of transcripts of these genes with saikosaponin accumula­tion in B. falcatum. In our study, a set of 3111 ESTs rep­resenting 1650 uniESTs acquired from B. chinense would expand the genic sequence pool of the genus Bupleurum. Approximately 51.5% full-length cDNAs of those se­quences which match to a known gene would provide a robust approach to identifying different functional genes in agriculture and pharmacology. ln addition, compared to other crops, umbelliferous crops receive little research attention, especially in molecular biology (Rubatzky et al., 1999). Before we submitted our ESTs (May of 2008), only 8226 nucleotide sequences (5863 Nucleotides + 2363 ESTs) were registered, and only five genera~Apium (2224 ESTs), Daucus (640 Nucleotides + 38 ESTs), Eryn-gium (477 Nucleotides), Cymopterus (233 Nucleotides), and Petroselinum (214 Nucleotides)―had more than 200 nucleotide sequences according to GenBank. Therefore the sequence information we obtained from B. chinense could also provide a useful basis for researching other impor­tant medicinal plants and vegetables in the Umbelliferae family, e.g. Angelica sinensis (Oliv.) Diels, A. dahurica Maxim., Saposhmikovia divaricata (Turcz.) Schischk., Pimpinella anisum L., Carum carvi L., Daucus carota L. and Apium graveolens L.
Of sixteen contigs with the most abundant ESTs, nine had a (putative) relationship with stress responses, including biotic stresses (contig 25, 110) (Mosolov and Valueva, 2005) and abiotic stresses like drought, cold, or salt (contig 23, 115, 50, 41, 310) (Chang and Zhu, 2002; Lopez et al., 2004; Yakubov et al., 2005; Goyal et al., 2005). This may suggest that the natural drought environment experienced in November in Beijing induced the anti-stress responses that Bupleurum plants exhibit when flowering. Contig 45 with the fourth most abundant EST numbers showed a high homology with Broad bean wilt virus 2 (BBWV2) or Patchouli mild mosaic virus (PatMMV), which both belong to the genus Fabavirus and share a high level of similarity (Qi et al., 2000). BlastX and BlastN implied that the nucleoside and amino
acid identities were 92-95% and 89-100% (different
from each EST clone), respectively. The homology length was 510-797 bp and 142-265 amino acids, and the homology position was the RNA-dependent RNA polymerase (RdRp) encoded fragment of the virus RNA1. In addition, another contig (4 ESTs) and one singleton, which had homology with a different fragment of RNA1 of BBWV2 or PatMMV, were in the library. From these data, we concluded primarily that Bupleurum plants used for constructing the cDNA library were infected by a virus strain or isolate belonging to the genus Fabavirus in the family Comoviridae. According to Plant Virus Online
Figure 2. Gene Ontology annotation of 1650 uniESTs. A, B and C indicate molecular function, biological process, and cellular component, respectively.
SUI et al. ― cDNA library construction and ESTs analysis of B. chinense 11
Table 2. A list of 31 pathways bearing at least five B. chinense homologous uniESTs by BlastX with protein sequences of Arabidopsis.
KEGG identifier
Pathway
Unigene Nos.
KEGG
identifier
Pathway
Unigene Nos.
ath03010
Ribosome
58
ath03060
Protein export
7
ath00010
Glycolysis
17
ath00051
Fructose and mannose metabolism
6
ath00190
Oxidative phosphorylation
17
ath00071
Fatty acid metabolism
6
ath04120
Ubiquitin mediated proteolysis
17
ath00100
Biosynthesis of steroids
6
ath00710
Carbon fixation
13
ath00271
Methionine metabolism
6
ath03050
Proteasome
12
ath00450
Selenoamino acid metabolism
6
ath00230
Purine metabolism
11
ath00632
Benzoate degradation via CoA ligation
6
ath01040
biosynthesis of unsaturated fatty acids
11
ath00220
Urea cycle and metabolism of amino groups
5
ath00350
Tyrosine metabolism
10
ath00251
Glutamate metabolism
5
ath00620
Pyruvate metabolism
10
ath00360
Phenylalanine metabolism
5
ath00680
Methane metabolism
10
ath00480
Glutathione metabolism
5
ath00500
Starch and sucrose metabolism
9
ath00562
lnositol phosphate metabolism
5
ath04070
Phosphatidylinositol signaling system
8
ath00940
Phenylpropanoid biosynthesis
5
ath00020
Citrate cycle (TCA cycle)
7
ath00960
Alkaloid biosynthesis ll
5
ath00030
Pentose phosphate pathway
7
ath04130
SNARE interactions in vesicular transport
5
ath00380
Tryptophan metabolism
7
Table 3. BlastX results of 16 contigs with most redundant ESTs in the B. chinense full-length enriched cDNA library.
Contig
No. of
Percentage
NCBI BlastX
ESTs
of total
Accession No.
Species
Gene name
e-value
contig23
147
4.73%
BAD86644
Daucus carota
Dehydrin protein
8e-63
contig25
119
3.83%
BAD04841
Daucus carota
Pathogenesis-related protein-like protein 1
1e-116
contig13
65
2.09%
AAT01418
Tamarix androssowii
Putative stress-responsive protein
3e-93
contig45
48
1.54%
BAB83045
Broad bean wilt virus 2
210 kDa protein precursor
1e-144
contig110
45
1.45%
AAU81597
Petunia x hybrida
Cysteine proteinase inhibitor
1e-140
contig115
30
0.96%
CAA33406
Brassica napus
Late embryogenesis abundant protein 76
1e-119
contig182
24
0.77%
CAO65275
Medicago truncatula
Eukaryotic/archaeal ribosomal protein S3
9e-26
contig310
22
0.71%
AAC62510
Pimpinella brachycarpa
Metallothionein-1-like protein
0.0
contig6
20
0.64%
AAK83601
Arabidopsis thaliana
Glyceraldehyde-3-phosphate dehydrogenase
1e-119
contig50
20
0.64%
AAA61564
Gossypium hirsutum
Desiccation protectant protein Lea14 homolog
1e-114
contig53
20
0.64%
EAY98759
Oryza sativa
Hypothetical protein OsI—019992
1e-126
contig109
20
0.64%
AAD51854
Vitis vinifera
Stress related protein
8e-76
contig41
19
0.61%
ABB29477
Panax ginseng
Tonoplast intrinsic protein
1e-150
contig158
19
0.61%
AAR20771
Arabidopsis thaliana
GRAM domain-containing protein / ABA-responsive protein-related
1e-135
contig10
18
0.58%
AAY87906
Sesamum indicum
Caleosin B
1e-88
contig97
18
0.58%
ABL67651
Citrus cv. Shiranuhi
Putative auxin-repressed/dormancy-associated protein
1e-73
Total
654
21.02%
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Botanical Studies, Vol. 51, 2010
Table 4. A list of 86 SSR loci identified in 82 uniESTs with repeat motifs and BlastX results shown.
Unigene ID
Repeat
NCBI BlastX
Species
Gene name
Bc-0002
(TA)6TT(TA)5
Petroselinum crispum
Common plant regulatory factor 7
Bc-0013
(AAG)8
Tamarix androssowii
Putative stress-responsive protein
Bc-0017
(TTC)5
Vitis vinifera
Hypothetical protein
Bc-0023
(TA)6(CA)5
Daucus carota
Dehydrin protein
Bc-0024
(AT)W
Glycine max
SCOF-1
Bc-0025
(TA)M
Daucus carota
Pathogenesis-related protein-like protein 1
Bc-0029
(TA)M
Ricinus communis
Cyclophilin
Bc-0085
(TCC)5
Vitis vinifera
CBF4 transcription factor
Bc-0113
(GAT)6
Solanum tuberosum
Histone deacetylase 2a-like
Bc-0115
(AT)。
Brassica napus
Late embryogenesis abundant protein 76 (LEA 76)
Bc-0119
(GT)5(AT)5
Arabidopsis thaliana
Unknown protein
Bc-0125
(TC)5(TA)6+(AT)„
Medicago truncatula
Hypothetical protein MtrDRAFT_AC174144g16v1
Bc-0137
(AT)W
No hit
Bc-0140
(AT)M
Arabidopsis thaliana
Late embryogenesis abundant protein-like
Bc-0150
(TG)n
Craterostigma plantagineum
Group 4 LEA protein
Bc-0199
(AT)M
Oryza sativa
Hypothetical protein OsI—031007
Bc-0211
(AAT)6
Capsicum annuum
WRKY-type transcription factor
Bc-0233
(TA)9+(AAC)7
Arabidopsis thaliana
Rcd1-like cell differentiation protein, putative
Bc-0254
(TTA)5
Lycopersicon esculentum
TAGL12 transcription factor
Bc-0259
(GT)„
Solanum tuberosum
Cinnamoyl CoA reductase
Bc-0274
(AT)9
Mesembryanthemum crystallinum
Major latex protein homolog
Bc-0285
(CA)6CT(CA)6
Vitis vinifera
Hypothetical protein
Bc-0309
(ACA)5
Ricinus communis
Cyclophilin
Bc-0310
(TTA)?
Pimpinella brachycarpa
Metallothionein-1-like protein
Bc-0341
(GCT)5
Arabidopsis thaliana
ARF GAP-like zinc finger-containing protein ZIGA3
Bc-0356
(ACT)5
Homo sapiens
hCG1999844
Bc-0413
(TA)9
Solanum tuberosum
snakin2
Bc-0418
(GGT)3+(TGG)5
Rattus norvegicus
PREDICTED: similar to keratin associated protein 10-7
Bc-0448
(AT)9
Arabidopsis thaliana
Phosphatidylethanolamine-binding family protein
Bc-0461
(AGA)5
Vitis vinifera
Hypothetical protein
Bc-0466
(CA)n
Vitis vinifera
Putative ripening-related protein
Bc-0473
(AT)9
Trichomonas vaginalis G3
Hypothetical protein TVAG—343280
Bc-0478
(TA)M
Vitis vinifera
Hypothetical protein
Bc-0507
(GGA)8
Lycopersicon esculentum
Mature anther-specific protein LAT61
Bc-0515
(CCG)5
Glycine max
Aspartate aminotransferase glyoxysomal isozyme AAT1 pre-
cursor
Bc-0557
(AT)9(TATAT)4
Oryza sativa
Os01g0633400 putative YZ1
Bc-0573
(CCG)5
Arabidopsis thaliana
RNA recognition motif (RRM)-containing protein
Bc-0603
(TC)9
Arabidopsis thaliana
UBC22 (ubiquitin-conjugating enzyme 18); ubiquitin-protein ligase
Bc-0635
(TAT)6
Ostreococcus tauri
Unnamed protein product
Bc-0659
(AGA)5
Glycine max
Histone deacetylase HDT1 (Histone deacetylase 2a)
Bc-0700
(GAA)2GAC(GAA)5 (AAG)2(GAA)4
Arabidopsis thaliana
Unknown protein
Bc-0776
(GCA)5
Drosophila melanogaster
Flap endonuclease 1 CG8648-PA
Bc-0794
(AT)U
Arabidopsis thaliana
Hypothetical protein
SUI et al. ― cDNA library construction and ESTs analysis of B. chinense
13
Table 4. (Continued)
Unigene ID
Repeat
Species
NCBI BlastX
Gene name
Bc-0821
(TCA)5(TC)8
Olea europaea
Cytochome b5
Bc-0824
(AAG)5
Solanum tuberosum
Eukaryotic translation initiation factor 2 beta subunit-like
Bc-0850
(AT)9
Lycopersicon esculentum
Lecithine cholesterol acyltransferase-like protein
Bc-0861
(AAG)5
Oryza sativa
Hypothetical protein
Bc-0861
(ATC)5
Oryza sativa
Hypothetical protein
Bc-0915
(AAT)u
Ipomoea batatas
ATP synthase delta' chain, mitochondrial precursor
Bc-0949
(GGA)6
Arabidopsis thaliana
Hypothetical protein
Bc-0960
(GAA)5
No hit
Bc-0973
(AC)9
Vitis vinifera
Hypothetical protein
Bc-0995
(TC)7(TA)s
Oryza sativa
Os10g0533900 unknown protein
Bc-1029
(AAG)6
Lycopersicon esculentum
Bax inhibitor
Bc-1045
(AT)9
Arabidopsis thaliana
Got1-like family protein
Bc-1063
(AT)9
Oryza sativa
Os02g0693200 DnaJ protein-like
Bc-1088
(AAG)5+(AAG)6
Arabidopsis thaliana
Transducin family protein / WD-40 repeat family protein
Bc-1120
(TTC)5
Arabidopsis thaliana
Unknown protein
Bc-1126
(TGT)5
Vitis vinifera
Hypothetical protein
Bc-1150
(TTC)5
Vitis vinifera
Unnamed protein product
Bc-1169
(GT)5+(AC)5
Vitis vinifera
Unnamed protein product
Bc-1186
(AC)W
Vitis vinifera
Unnamed protein product
Bc-1192
(TA)9
Homo sapiens
PREDICTED: hypothetical protein
Bc-1192
(TG)6(TA)8
Homo sapiens
PREDICTED: hypothetical protein
Bc-1249
(TTC)6
Oryza sativa
Os03g0119000 ABR017Cp, putative, expressed
Bc-1269
(AT)。
Arachis hypogaea
Subtilisin-like protease
Bc-1275
(TA)M
Arabidopsis thaliana
ATRPABC24.3 ('Arabidopsis thaliana RNA polymerase I, II and III 24.3 kDa subunit'); DNA binding / DNA-directed RNA polymerase
Bc-1329
(TA)M
No hit
Bc-1335
(CA)11(TA)7
Vitis vinifera
Hypothetical protein
Bc-1377
(GT)10(GA)M
Arabidopsis thaliana
Subtilase family protein
Bc-1397
(TA)9
Vitis vinifera
Hypothetical protein
Bc-1419
(AGA)7
Oryza sativa
Hypothetical protein
Bc-1423
(AAC)5
Vitis vinifera
Hypothetical protein
Bc-1461
(GAA)5
No hit
Bc-1470
(CTA)7
Glycine max
Dehydration responsive element binding protein
Bc-1503
(TA)10
Cucumis sativus
3-ketoacyl-CoA thiolase; acetyl-CoA acyltransferase
Bc-1511
(AAGTAG)5
Vitis vinifera
Hypothetical protein
Bc-1511
(GCA)8A(ATG)5
Vitis vinifera
Hypothetical protein
Bc-1542
(TGA)6
Vitis vinifera
Hypothetical protein
Bc-1542
(AAG)5
Vitis vinifera
Hypothetical protein
Bc-1543
(GGT)5
Vitis vinifera
Hypothetical protein
Bc-1561
(CAA)5
Solanum tuberosum
Oligouridylate binding protein-like protein
Bc-1562
(AT)6+(AT)6
Prunus persica
Major latex-like protein
Bc-1563
(CTG)5
Medicago truncatula
Pumilio/Puf RNA-binding
Bc-1590
(GTC)5
Arabidopsis thaliana
Unknown protein
Bc-1630
(TG)9
Medicago truncatula
Galactose mutarotase-like
14
Botanical Studies, Vol. 51, 2010
(http://image.fs.uidaho.edu/vide/descr115.htm), one of the BBWV synonyms was Parsley virus 3, which infects parsley asymptomatically, but until now, no Fabavirus has been reported to infect Bupleurum plants. Virus inoculation, virion isolation, and complete sequence analysis will be needed to verify the virus infection of our experimental plant material.
Saikosaponins, the main pharmacologically active component of Bupleurum, are synthesized via the isoprenoid pathway by cyclization of 2, 3-oxidosqulene to produce oleanane (p-amyrin) or a dammarane triterpenoid skeleton, and the triterpenoid backbone is modified by P450 and glycosyltransferases (Haralampidis et al. , 2002; Chen et al., 2007). In the report of Chen et al. (2007), ESTs of a p-amyrm synthase gene (fi-AS), a UDP-glucosyltransferase gene, and two P450 genes were identified, and the transcripts of these genes were all upregulated in the adventitious roots of B. kaoi induced by MeJA. Of 3111 ESTs sequenced in our study, one 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase gene (HMGR), five glycosyltransferase genes (GT), and five cytochrome P450 genes (P450) were included. GT and P450 comprise superfamilies genes in plants. They are seldom cloned by the regular PCR-based method, and their function is difficult to determine. To screen the large-scale ESTs of induced or developmental EST or cDNA libraries and subsequently verify their function is an effective measure (Osbourn, 2003; Goossens et al., 2003). Whether these genes play a role in saikosaponin biosynthesis is of interest. Although previous investigation showed roots of flowering B. chinense plants had the highest content of saikosaponin (Yang et al., 2006), no more cDNAs involved in saikosaponin biosynthesis were identified in the sequenced cDNA pool. Consistent with that, the ginseng leaf cDNA library contained five ESTs of ginsenoside-synthesizing enzymes out of 2,896 ESTs (0.17%), and the ginseng root cDNA library featured two out of 3,808 ESTs (0.05%) (Kim et al., 2006). This phenomenon implies that the transcript abundances of genes involved in saikosaponin biosynthesis are naturally rather low or the plant developmental stage during which roots of plants have the highest content of saikosaponin may not be the time that saikosaponin is rapidly biosynthesized. Additionally, it is possible that the transcripts of some related genes degrade and regenerate rapidly. Some reports such as research by Kim et al. (2006) have shown that the gene expression responsible for the biosynthesis of secondary metabolic products is upregulated dramatically when metabolites are increased artificially. The behavior of plants when cultivated remains to be characterized.
SSR (also referred to as microsatellite), with remarkable attributes such as codominant inheritance and ease of detection, has become preferred over other molecular
markers like RAPD, lSSR, and AFLP. With the growing
amount of EST data, EST-SSRs have been identified by some plant EST or cDNA libraries (Lindqvist et al., 2006; Chen et al., 2006). Mining SSRs from EST data has proven to be a time and cost saving method (Ceresini et al., 2005).
During our SSR search in the B. chinense root full-length enriched cDNA library, only loci for which there were at least 50 bp for potential flanking primers before and after the repeat site were accounted for. Therefore, future work will need the design of primers for these 86 SSR loci. This could lead to the development of a set of SSR markers. These putative expressed SSR markers plus those genomic SSR markers we have developed for B. chinense (Sui et al., 2009) would be extremely useful for analyzing genetic diversity, germplasm identification, and genetic map construction for the Bupleurum genus plant.
Acknowledgements. This research was supported by the National Key Project of Scientific and Technical Supporting Programs funded by the Ministry of Science & Technology of China (No. 2006BAI09B01), by the Special Funds in Basic Scientific Research for Non-Profit Research Institutes financed by the Ministry of Finance, People's Republic of China (No. YZ-1-10), and by the Special Funds in Scientific and Technological Research financed by the State Administration of Traditional Chinese Medicine (No. 2004ZX06-3).
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Botanical Studies, Vol. 51, 2010
柴胡全長富集cDNA文庫構建及其3111個EST
序列測定分析
隋春魏建和陳士林陳懷瓊董樂萌楊成民
中國醫學科學院及北京協和醫學院藥用植物研究所
中藥柴胡，為柴胡屬植物的乾燥根，具有抗炎、返熱和保肝等功效。在中國、日本、韓國及南亞其
他國家和地區廣泛應用。本文利用SMART技術構建了藥用柴胡屬植物北柴胡(Bupleurum chinense DC.)
根的全長富集cDNA文庫，以啟動藥用柴胡功能基因組研究。文庫滴度為1.1X106 。從文庫中隨機選擇 了3902個克隆進行5'端單反應測序，獲得了3111個高品質EST序列，包括377個序列重疊群(contigs)
和1273個單一序列（singletons)共1650個獨立EST (uniESTs)。文庫插入片段平均長度約1.1 kb '全長
比率約51.5%。BlastX分析結果表明949 (57.5%)個獨立EST和已鑒定基因同源，680 (41.2%)個獨立
EST與GenBank中未知、未命名或推測的蛋白基因同源，21個獨立EST沒有找到同源基因。獨立EST
的GO (Gene Ontology)注釋顯示1002 、 957和861個獨立EST分別歸屬於細胞功能、生物過程和細胞
組分三大類。與KEGG中擬南芥代謝途徑進行了比較分析'結果表明'307個獨立EST可能參與31個
代謝途徑（每個代謝途徑至少包含5個獨立EST)。1650個獨立EST中82個含有總計86個微衛星位點
(SSR)。本研究首次構建的柴胡根全長富集cDNA文庫及測序分析的EST克隆，為研究這一中藥材的各
種生理現象的分子基礎提供了有效平臺。從文庫資料中挖掘的具有潛在標記價值的SSR位點將為柴胡
種質鑒定，遺傳多樣性分析及基因定位提供有效分子標記。
關鍵詞：北柴胡(Bupleurum chinense DC.)；全長富集cDNA文庫；表達序列標籤（ESTs) ； SMART (Switching mechanism at 5' end of RNA transcript)技術 ；SSR 0