Characterization of an A-type cyclin-dependent kinase gene from ...

Biologia 67/2: 360—368, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0016-y

Characterization of an A-type cyclin-dependent kinase gene from Dendrobium candidum Gang Zhang1,2, Chao Song1, Ming-Ming Zhao1, Biao Li1 & Shun-Xing Guo1* 1

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, People’s Republic of China; e-mail: [email protected] 2 College of Pharmacy and Shaanxi Provincial Key Laboratory for Chinese Medicine Basis & New Drugs Research, Shaanxi University of Chinese Medicine, Xi’an, Shaanxi 712046, People’s Republic of China

Abstract: Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and postembryonic development of organisms. To better understand the molecular mechanisms of CDKs involved in embryogenesis regulation in the endangered medicinal plant Dendrobium candidum Wall. ex Lindl., a 1229-bp full-length cDNA of an A-type CDK gene, Denca;CDKA;1, was identified using 3’ rapid amplification of cDNA end (RACE) PCR. Denca;CDKA;1 was predicted to encode a 294 amino acid residue-long protein of 33.76 kDa with an isoelectric point of 7.72. The deduced Denca;CDKA;1 protein contained a conserved serine/threonine-protein kinase domain (S-TKc) and a canonical cyclinbinding “PSTAIRE” motif. Multiple sequence alignment indicated that members of CDKA family from various plants exhibited a high degree of sequence identity ranging from 82% to 93%. A neighbor-joining phylogenetic tree showed that Denca;CDKA;1 was clustered into the plant group and was distant from the animal and fungal groups. The modeled three-dimensional structure of Denca;CDKA;1 exhibited the similar functional structure of a fold consisting of β-sheets and α-helices joined by discontinuous random coils forming two relatively independent lobes. Quantitative real-time PCR analysis revealed that Denca;CDKA;1 transcripts were the most abundant in protocorm-like bodies with 4.76 fold, followed by that in roots (4.19 fold), seeds (2.57 fold), and stems (1.57 fold). This study characterized the novel Denca;CDKA;1 gene from D. candidum for the first time and the results will be useful for further functional determination of the gene. Key words: cyclin-dependent kinase; Dendrobium candidum; cell cycle; cyclin-binding motif; gene expression; protocormlike body. Abbreviations: CAK, CDK-activating kinase; cdc, cell division cycle; CDK, cyclin-dependent kinase; CKL, cyclin dependent kinase like; Ct, cycle threshold; CTAB, cetyltrimethyl ammonium bromide; GSP, gene specific primer; ICK, inhibitor of CDK; M-MLV RT, moloney leukemia virus reverse transcriptase; ORF, open reading frame; PDB, protein data bank; PLB, protocorm-like body; qRT-PCR, quantitative real-time polymerase chain reaction; RACE, rapid amplification of cDNA ends; S-TKc, serine/threonine-protein kinase domain; UTR, un-translated region.

Introduction Cell cycle in eukaryotes is tightly governed by a family of serine/threonine protein kinases known as cyclindependent kinases (CDKs). The CDKs are initially activated by forming complex in association with corresponding regulatory cyclin subunits. For fine tune of the cell cycle initiation and progression, the activities of CDKs are regulated at multiple levels by several mechanisms, including phosphorylation, dephosphorylation, and binding of interacting proteins, like CDKactivating kinases (CAKs) or inhibitors of CDK (ICKs) (Francis 2007). The CDK molecular machinery of cell cycle regulation has been well adapted to intrinsic developmental and external environmental changes in diverse eukaryotic species. The cell division cycle 2 (Cdc2) in Schizosaccha-

romyces pombe and Cdc28 in Saccharomyces cerevisiae encode the prototypical CDK, implicating in yeast cell cycle regulation (Hartwell et al. 1974; Hindley & Phear 1984). Considerable efforts have been performed on the identification of CDK genes in plants, ultimately leading to clearly elucidation of CDK pathways in cell cycle control. Up to now, a total of 152 CDK genes have been identified from 41 plant species and are classified into eight classes, CDKA to CDKG and CKL (cyclin dependent kinase like) based on types of cyclin-binding motifs (Vandepoele et al. 2002; Tank & Thaker 2011). CDKA, an orthologue of yeast Cdc2/Cdc28, has a conserved PSTAIRE motif and regulates the G1/S and G2/M phase progression (Gutierrez 2005). In contrast, the expression of CDKB, containing the altered PPTALRE or PPTTLRE motif, is restricted in the late S/M phase (Gutierrez 2005). Plant C- and E-type CDKs with other

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Dendrobium candidum gene for cyclin-dependent kinase A-type variant cyclin-binding sequences regulate RNA polymerase II via a phosphorylation cascade (Barroco et al. 2003) and their involvement in cell cycle is unclear yet. Classes CDKD and CDKF are two distinct CAKs that function by phosphorylating CDKAs (Umeda et al. 2005; Takatsuka et al. 2009). CDKG with PLTSLRE motif plays a role in cell differentiation process (Menges et al. 2005). Additionally, 15 CKLgenes were discovered by microarray analysis (Menges et al. 2005) and their functions remain to be investigated. Class CDKA is the only one that could complement the yeast cdc2/cdc28 mutant phenotype and is the most well characterized fundamental CDK in plants. It has been widely reported that CDKA has a crucial function in early developmental gametogenesis and embryogenesis processes (Hemerly et al. 1995, 2000; Iwakawa et al. 2006; Nowack et al. 2006; Dissmeyer et al. 2007). In Arabidopsis, over-expression of a dominant negative form of CDKA;1 showed severe defects during embryogenesis (Hemerly et al. 2000). Knockout of CDKA;1 resulted in embryo lethality, and the cdka;1 null mutants were defective in pollen development, exhibiting a gametophyte lethal phenotype (Iwakawa et al. 2006; Nowack et al. 2006). Increasing evidence has revealed that CDKA;1 also plays an important role in post-embryonic development. Transgenic tobacco over-expressed with a dominant negative form of Arath;CDKA;1 displayed reduction of cell division rate, and thus yielded smaller plants (Hemerly et al. 1995). A dwarf phenotype with fewer and larger leaf cells could be observed in Arabidopsis mutants with the weak cdka;1 allele (Dissmeyer et al. 2007). However, plants exhibited normal morphogenesis with normal developmental timing in both cases. These results suggest the biological importance of CDKA;1 for a wide range of developmental processes during the plant life cycle. Dendrobium candidum Wall. ex Lindl. (Orchidaceae) is one of most valuable medicinal plants widely used in traditional Chinese medicine for maintaining tonicity of the stomach and promoting body fluid production (Li et al. 2009). Phytochemical research has demonstrated various active constituents from the herb, such as alkaloids, fluorenones, sesquiterpenoids, bibenzyls, phenanthrenes, and polysaccharides, which exhibit diverse pharmacological effects, including antioxidant, immune stimulating, anti-tumor, and anti-mutagenic activities (Li et al. 2009; Luo et al. 2010; Xiao et al. 2011). Given these characters of D. candidum, there is an urgent need for its functional genomics research from multiple aspects, like plant growth and development regulation, adaptation to abiotic and biotic stresses, etc. Few reports on genetic transformation and molecular plant physiology of Dendrobium species have just emerged with limited achievements (Men et al. 2003; Chen et al. 2005; Suwanaketchanatit et al. 2007). In attempt to elucidate the molecular mechanism of plant growth regulation, we constructed a suppression subtractive hybridization (SSH) cDNA library enriched for genes specifically with induced expression levels in D. candidum roots infected by a compatible my-

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corrhizal fungus (unpublished data). A 594–bp cDNA (Dc594) encoded amino acid sequence was found to be highly homologous (95%) to N-terminal 184 amino acid residues of the Chenopodium rubrum CDKA (GenBank accession no. CAA71242). We therefore hypothesized in this paper that CDKA should be important in regulating cell cycle and embryonic growth in D. candidum. Here, we report the identification of an A-type CDK gene with a full-length cDNA from D. candidum, designated as Denca;CDKA;1. The molecular characteristics of Denca;CDKA;1 were analyzed, and its tissue specific expression profiles were also examined by quantitative real-time PCR (qRT-PCR). Our research will be useful for further functional characterization of CDKA;1 involving in cell cycle, growth and development regulation in D. candidum. Material and methods Plant materials and samplings The aseptic seedlings and protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl. (Orchidaceae), kept in National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, China, were used in this study. The seedlings were grown in artificial media for two months as described previously by Song & Guo (2001). The PLBs at the stage 3 (Stewart et al. 2003) were maintained in hormone-free 1/2 Murashige-Skoog medium consisting of 1/2-strength macro and micro elements and all of the vitamins described by Murashige & Skoog (1962), 20 g/L sugar, and 6.5 g/L agar (Men et al. 2003). Capsules of D. candidum were collected from Xishuanbanna, Yunnan, China (November, 2010) and germinating seeds at the stage 1–2 (Stewart et al. 2003) were obtained according to the designated procedures (Wang et al. 2011). All plant materials were kept in a high-humidity (75 ± 5%) chamber at 25±2 ◦C under a 16-h photoperiod. Intact root, leaf, stem, PLB, and seed tissues were prepared, immediately frozen in liquid nitrogen and stored at −80 ◦C prior to total RNA extraction. Total RNA isolation and quality control Total RNA was extracted according to the cetyl trimethyl ammonium bromide (CTAB) – LiCl protocol (Iandolino et al. 2004). Genomic DNA was removed withRQ1 RNase-free DNase (Promega, Madison, WI, USA). The quality and integrity of the total RNA were determined using a formamide denaturing gel along with an RNA ladder (Invitrogen, Carlsbad, CA, USA) for comparison, and quantity was examined using a NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 3’ RACE-PCR and sequence analyses To obtain the full length cDNA of CDKA;1 from D. candidum, a forward gene specific primer (GSP) 5’-CTATCAG ATTCTCCGTGGCATTGCTTACTG-3’ was designed with the Primer 3 (Rozen et al. 2000) and synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). 3’ rapid amplification of cDNA end (3’ RACE) PCR was employed with the SMARTT M RACE cDNA Amplification Kit (Clontech, USA). A mixture of total RNA was utilized for 3’ ready cDNA synthesis. The 3’ RACE-PCR was performed in a MJ Research PTC-200 thermocycler (MJ Research, Alameda, CA, USA) using the following parameters: 94 ◦C for 3 min;

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Fig. 1. The 3’ RACE-PCR products detected by a 1.8% agarose gel. Lane 1 represents the amplification products. Lane 2 shows the standard DNA marker of DL2000. Kb indicates the size for various DNA fragments of DL2000.

32 cycles of 94 ◦C for 1 min, 68 ◦C for 90 s; and a final extension at 68 ◦C for 7 min. PCR products were loaded on 1.8% agarose gel with ethidium bromide staining (Sigma-Aldrich, St. Louis, MO, USA). The target band was excised from gel and cloned into the pGEM-T vector (Promega, Madison, WI, USA) followed by transformation into Escherichia coli JM109 competent cells (TaKaRa, Dalian, China). Recombinant clones were sequenced on an ABI PRISM 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). Analyses of cDNA sequence were carried out using a series of online tools with basic local alignment search tool (BLAST) ( http://www.ncbi.nlm.nih.gov/blast/), open reading frame (ORF) Finder (http://www.ncbi.nlh.nih.gov/ gorf/gorf.html), VecScreen (http://www.ncbi.nlm.nih.gov/ VecScreen/VecScreen.html), and CAP3 Sequence Assembly Program (Huang & Madan 1999). The deduced Denca; CDKA;1 protein was characterized with ExPASy Proteomic tools (http://www.expasy.ch/tools/). Homologybased structural modeling was performed by Swiss-Model (Arnold et al. 2006). Vector Alignment Search Tool (VAST) ( http://www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb. shtml) was used for domain detection followed by the display of three-dimensional structure with Cn3D 4.3 (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d. shtml). Multiple sequence alignment was conducted using MegAlign under the Lasergene software package of DNAstar 6.0 (Madison, WI, USA). A neighbor-joining phylogenetic tree was generated by MEGA 4.0 (Tamura et al. 2007) with 1000 repeats. Quantitative RT-PCR analyses Reverse transcription was performed using the Moloney Leukemia Virus Reverse Transcriptase (M-MLV RT) (Promega, Madison, WI, USA) following the manufacturer’s instructions. Tissue specific expression patterns of Denca; CDKA;1 were determined using qRT-PCR analyses. A D.

candidium elongation factor, DcEF1α, was used as the internal control. The primer sequences were: CDKA;1-FP 5’-TTCTCCGTGGCATTGCTTACTG-3’, CDKA;1-RP 5’TCCAAGGAGGATTTCTGGTGCT-3’; and DcEF1α-FP: 5’-TCAGGCTGACTGTGCTGTCCT-3’, DcEF1α-RP: 5’GTGGTGGCGTCCATCTTGTT-3’. The qRT-PCRs were performed with the SYBR Premix Ex TaqTM (TaKaRa, Dalian, China) on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Templates were the 40× diluted cDNAs from each 2 µg RNA sample. The qRT-PCR reactions were set up to a total volume of 25 µL containing 12.5 µL 2× SYBR Premix Ex TaqTM Master Mix, 0.5 µL each primer (10 µM), 0.5 µL ROX Reference Dye, 2 µL cDNA template, and 9 µL double distilled water. The thermal conditions were: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C 15 s, 60 ◦C 1 min. All reactions, including non-template controls, were carried out three-times with three independent biological replicates in each time. All qRT-PCR products were subjected to melting curve analysis and verified by gel electrophoresis. Cycle threshold (Ct) values were generated from the ABI PRISM 7500 Software Tool (Applied Biosystems, Foster City, CA, USA). The relative expression ratios were calculated using the comparative ∆∆CT method of relative gene quantification (Pfaffl 2001). A probability (P) value equal or less than 0.05 was used to determine the significance of difference between samples or its relative quantity of RNA was at least two-fold higher or lower than in the control sample.

Results Cloning of a full-length Denca;CDKA;1 cDNA Based on Dc594, the forward GSP was designed and used for 3’ RACE. With 3’ ready cDNA as the template, 3’ RACE-PCR was conducted and yielded a specific band (Fig. 1). The amplicon was retrieved and cloned. Sequencing analysis revealed an 872-bp cDNA with a poly-A tail, after trimming vector and adaptor using VecScreen. Being assembled with Dc594 using CAP3, a 1229-bp nucleotide sequence was obtained and an ORF (35-919) was identified flanked by 34 bp and 285 bp in the 5’ and 3’ un-translated regions (UTR), respectively (Fig. 2A). BLASTX analysis demonstrated that the ORF exhibited 92% identities with Pinco;CDKA;1 (GenBank accession no. CAA56815), and contained the canonical cyclin-binding “PSTAIRE” motif, unique to the plant A-type CDKs. Moreover, to verify the accuracy of the identified sequence, RT-PCR with primers spanning ORF was applied followed by sequence analysis, which successfully passed validation (data not shown). The putative initiation codon in the context of TGAATGG did not comply with the Kozak consensus initiator A/GNNATGG (Kozak 1987). Nevertheless, we designated the 1229-bp cDNA as Denca;CDKA;1 (Dendrobium candidum CYCLIN DEPENDENT KINASE) and deposited in GenBank (Benson et al. 2012) under the accession of HQ904083, in accordance with the conventional plant CDK nomenclature (Joubès et al. 2000). Sequence analyses of the deduced Denca;CDKA;1 protein The ORF of Denca;CDKA;1 was deduced to encode


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Fig. 2. (A) The full length cDNA of Denca;CDKA;1 and the deduced amino acid sequence of the coding region. Triple nucleotide bases boxed are start and stop codons, respectively; Shadowed residues indicate the cylcin-binding motif. Numbers show different positions of cDNA and coding amino acids. (B) Conserved domain analysis of Denca;CDKA;1 using Interproscan. The S-TKc (4-287) in red shows the serine/threonine-protein kinase domain.

a 294–amino acid residue protein with the molecular weight of 33.76 kDa and an isoelectronic point (pI) of 7.72 calculated using Compute pI/MW. ScanProsite and InterProScan tools analyses of the deduced Denca;CDKA;1 protein identified a serine/threonineprotein kinase domain (S-TKc, residues 4-287) (Fig. 2B) that was characteristic of the eukaryotic serine/threonine protein kinases. The protein kinases ATP-binding region (10–33), serine/threonine protein kinases activesite signature (123–135), T-loop region (147–172), and SUC/CKS-binding motif (207–244) were all included in the S-TKc domain (Fig. 3). Moreover, PROSITE Scan analysis revealed different types of motifs, including an N-glycosylation site (27–30), a cAMP- and cGMP-dependent protein kinase phosphorylation site (274–277), a tyrosine kinase phosphorylation site (9– 15), and a leucine zipper pattern (249–270), four protein kinase C phosphorylation sites (72–74, 120–122, 273–275, 277–279), four N-myristoylation sites (43– 48, 114–119, 148–153, 191–196), and five casein kinase II phosphorylation sites (93–96, 183–186, 208– 211, 222–225, 233–236). In addition, PSORT suggested that Denca;CDKA;1 would principally locate in cytoskeleton with a high probability of 86%. No signal peptide or trans-membrane region was observed using SignalP 3.0 or TMHMM analyses, respectively.

Multiple sequence alignment and phylogenetic analyses The deduced amino acid sequence of Denca;CDKA;1 was compared with 32 CDKAs from a number of plants using the MegAlign program under DNAstar 6.0 (Fig. 3). The overall sequence alignment indicated that the CDKAs were highly conserved among these plant species with a high degree of identities (82– 93%). For example, Denca;CDKA;1 shared the highest identity (93%) with Picab;CDKA;1 (GenBank accession no. CAA544746) and the lowest (82%) with Antma;CDKA;1 (GenBank accession no. CAA66234). For phylogenetic analysis, seven homologous CDKs from several representative fungi and animals were included in the comparison with the plant CDKAs mentioned above to generate a comprehensive neighborjoining tree using MEGA 4.0 (Fig. 4). The significance level of the tree was examined by bootstrap testing with 1000 repeats and the cut-off value of 50% was accepted as indicative of a well-supported branch. The results revealed three distinct categories of animal, fungus and plant for the CDKs analyzed. Within the plant group, except CDKAs all shared the same ancestor of a subgroup consisting of the Brassica and Arabidopsis CDKAs (GenBank accession no. AAA92823, XP 002875923, and NP 566911), the other CDKAs in different monocots and dicots including Denca;CDKA;1 could not be further divided specifically, though an-


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Fig. 3. Alignment of the amino acid sequences of Denca;CDKA;1 with 32 homologous CDKAs from various plants using MegAlign under the Lasergene software package of DNAstar 6.0. The proteins included in the comparison were shown with the abbreviations of plant species followed by their GenBank accession numbers with a dash “-” as follows: Ac-BAA21673, Allium cepa; Am-CAA66233, Antirrhinum majus; At-NP 566911, Arabidopsis thaliana; Bn-AAA92823, Brassica napus; Cn-ACX54361, Cocos nucifera; Cr-CAA71242, Chenopodium rubrum; Cs-BAE80323, Camellia sinensis; Dc-CAD43850, Daucus carota; Dc-HQ904083, Dendrobium candidum; GhABV64386, Gossypium hirsutum; Gm-ABW23441, Glycine max; Ht-AAL47481, Helianthus tuberosus; Hv-BAK01471, Hordeum vulgare; Nt-AAB02567 and Nt-AF289467, Nicotiana tabacum; Os-ABF93569 and Os-CAA42922, Oryza sativa; Pa-CAA54746, Picea abies; Pc-AAC41680, Petroselinum crispum; Pc-CAA56815, Pinus contorta; Pd-ABD14373, Prunus dulcis; Ph-CAA73997, Petunia hybrida; Ps-BAA33152, Pisum sativum; Sb-BAE06268 and Sb-BAE06269, Scutellaria baicalensis; Sb-CAZ96037, Sorghum bicolor; Sl-CAA76701, Solanum lycopersicum; Sr-CAA99991, Sesbania rostrata; Ta-CAA54746, Triticum aestivum; Va-AAA34241, Vigna aconitifolia; and Zm-AAA33479, Zea mays. AAK16652 and XP 002875923 represent the CDKA;1 of Populus tremula×tremuloides and Arabidopsis lyrata, respectively. The S TKc is marked with thick dot line above the sequence. The different functional regions are indicated by black lines below the corresponding sequences. The three conserved phosphorylation sites of CDKAs are indicated by asterisks. The black boxes indicate identical residues, and hyphens indicate gaps introduced to optimize the alignment.

other subgroup were clearly made up of CDKAs from four closely related monocots – rice, wheat, maize, and barley. In addition, two CDKAs of Picea abies and Pinus contorta from Gymnospermae were clustered to-

gether. Taken fungus as the root, plant CDKAs seemed to have evolved before fungi and animals were differentiated. After plants were separated, the genes mutated in different plants detected in this study.


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Fig. 4. A neighbor-joining phylogenetic tree of 33 CDKA;1s from diverse plant species and 7 homologues from fungi and animals using MEGA 4.0. The whole protein sequences were used in the comparison. The fungal CDKs were rooted in the tree. The numbers at branches represent bootstrap values (≥50%) based on 1000 replications. Branches are labeled by species names with the GenBank or UniProt database accession numbers, indicating the corresponding genes. Denca;CDKA;1 is represented by Dendrobium candidumHQ904083 emphasized in bold. Asterisks indicate genes of monocots and number signs indicate those from Gymnospermae.

Fig. 5. The three-dimensional structure of Denca;CDKA;1 obtained using the homology-based modeling with Arath;CDKA;1 (PDBcode: 1jsu) as the template. The N- and C-terminal lobes are signified by pink and blue, respectively. The conserved “PSTAIRE” motif for binding cyclin is shown in yellow. The β-strands and α-helices are illustrated as golden arrows and green sticks, respectively. The Asp127 in the active site (123–235) is shown as a red asterisk.


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Fig. 6. The expression pattern of CDKA;1 in various D. candidum tissues by qRT-PCR analysis. Relative quantitation was calculated using the comparative ∆∆CT method (Pfaffl 2001). The raw qRT-PCR data of Denca;CDKA;1 transcripts were normalized to the expression levels of the internal control DcEF1α gene. Leaf and seed sample were taken as the calibrator (A and B), respectively. Mean and error bars are calculated from three independent replicates. The mean relative expression values are presented on the top of the columns.

Homology modeling of Denca;CDKA;1 protein Denca;CDKA;1 shared 86% identities to Arabidopsis thaliana CDKA;1 (GenBank accession no. NP 566911). Therefore, the three-dimensional structure of Arath; CDKA;1 (PDB code: 1jsu) was used as a unique template for homology modeling of Denca;CDKA;1 by Swiss-Model analysis (Fig. 5). The model exhibited a fold composed of a couple of β-strands and α-helices joined by discontinuous random coils forming the two relatively independent lobes. VAST analysis indicated that the small N-terminal lobe (13–82) had four βstrands and one C-helix, on which the conserved cyclinbinding “PSTAIRE” motif (45–51) was located. In contrast, the large C-terminal lobe (83–290) contained a spot of four β-strands and five α-helices. The residue Asp127 in the serine/threonine protein kinases activesite signature (123–135) was located deeply within the active-site cleft between the two lobes. Tissue-specific expression analysis of Denca;CDKA;1 Before proceeding with qRT-PCR analysis, regular PCR assay was employed to confirm primers’ specificity for the target and reference gene, which successfully passed validation by further sequencing analysis (data not shown). Next, to investigate the physiological role of CDKA;1, we isolated total RNAs from various tissues of D. candidum plants, including leaves, stems, roots, seeds, and PLBs, followed by reverse transcription on 2 µg RNA for each sample. The tissue specific expression profiles of CDKA;1 were then examined using qRT-PCR technique (Fig. 6). Overall, the results showed that CDKA;1 transcripts were the most abundant in PLBs with 4.76 fold over than in the leaves, followed by that in roots (4.19 fold). The expression level of Denca;CDKA;1 was relatively high in seeds with 2.57 fold, and low in stems (1.57 fold). Taken seed as the

calibrator, the amount ofCDKA;1 transcripts in PLBs accumulated to 1.86 fold (Fig. 6B). Discussion Characterization of diverse CDKA genes has been widely reported across a number of plant species. In this paper, we identified a novel A-type CDK gene, namely Denca;CDKA;1, from an endangered orchid medicinal plant species, D. candidum, for the first time. Denca;CDKA;1 is highly homologous (>90%) to a number of plant CDKAs, like CDKA;1 in Arabidopsis thaliana and tomato (Hemerly et al. 1995; Joubès et al. 1999), while relatively low similarity (roots>seeds>stems>leaves (Fig. 6A). It is likely that the transcriptional regulation of Denca;CDK;1 may be involved in development of organs by coordinating cell division and differentiation of different cell types. For orchids, plant regeneration from calli is generally through somatic embryogenesis of an intermediary PLB phase (Huan et al. 2004). In D. candidum, histological observation demonstrated that during embryogenic PLBs formation, embryogenic structures consisting of cells with densely stained cytoplasm and small vacuoles were developed from the inside and surface of calli (Zhao et al. 2008). No molecular evidence on its somatic embryogenesis has been published yet. In our study, Denca;CDKA;1 transcripts progressively increase during PLBs formation compared to the germinated seeds (Fig. 6B), which is in line with and supportive to the PLBs regeneration events reported by Zhao et al. (2008), indicating that embryogenic cells of PLBs are still competent to divide before differentiation of plantlets. This decrease of Denca;CDKA;1 expression in geminating seed embryo versus in embryogenic PLBs seems to coincide with a steady decrease in CDKAs expression during somatic embryo formation and maturation in Picea abies (Footitt et al. 2003) and Cocos nucifera (Montero-Cortés et al. 2010). Therefore, we propose that the Denca;CDKA;1 may play an essential role during embryogenic PLBs formation in D. candidum. Acknowledgements We thank Dr. Xianming Chen at Washington State University for critical review of the manuscript. This research was financially supported by the National Natural Science Foundation of China (No. 31070300, 31101608 and 31170314).

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Received June 27, 2011 Accepted November 12, 2011