- Methodology article
- Open Access
Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey
- Chen-Tran Hsu1,
- De-Chih Liao1,
- Fu-Hui Wu1,
- Nien-Tze Liu1,
- Shu-Chen Shen2,
- Shu-Jen Chou3,
- Shu-Yun Tung4,
- Chang-Hsien Yang5,
- Ming-Tsair Chan†1, 6Email author and
- Choun-Sea Lin†1Email author
© Hsu et al; licensee BioMed Central Ltd. 2011
- Received: 28 November 2010
- Accepted: 7 April 2011
- Published: 7 April 2011
Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied.
Several molecular biology tools were used to isolate flower-specific gene promoters from Oncidium 'Gower Ramsey' (Onc. GR). A cDNA library of reproductive tissues was used to construct a microarray in order to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues, and the subcellular locations of the corresponding proteins were identified using lip transient transformation with fluorescent protein-fusion constructs. BAC clones of the 5 genes, together with 7 previously published flower- and reproductive growth-specific genes in Onc. GR, were identified for cloning of their promoter regions. Interestingly, 3 of the 5 novel flower-abundant genes were putative trypsin inhibitor (TI) genes (OnTI1, OnTI2 and OnTI3), which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable A. thaliana transformation analyses.
By combining cDNA microarray, BAC library, and bombardment assay techniques, we successfully identified flower-directed orchid genes and promoters.
- Bacterial Artificial Chromosome
- Bacterial Artificial Chromosome Clone
- Bacterial Artificial Chromosome Library
- Transient Transformation
- Tandem Repeat Sequence
The Orchidaceae family comprises an estimated 35,000 species and is one of the largest families of flowering plants. The Oncidiinae subtribe consists of ~70 closely related genera and >1400 species, of which Oncidium is the largest genus [1, 2]. Like other orchids, Oncidiinae can be easily crossed intergenerically, or across species, to produce flowers with unique colors, fragrances and shapes. Oncidium has become a commercially important flower in the orchid industry. Oncidium 'Gower Ramsey' (Onc. GR) is one of the most important Oncidium cut-flower varieties; it is an interspecific hybrid derived from Onc. flexuosum, Onc. sphacelatum and Onc. varicosum. Onc. GR is a yellow flower variety that can flower year-round. The length of inflorescence is ~1 m, with hundreds of ca. 4 cm flowers.
Functional genomic studies of orchids remain a challenge owing to large genome size, low transformation efficiency and long life cycles . However, gene transformation of Onc. GR has been established , offering an alternative strategy for Oncidium breeding and making it a priority to investigate and obtain Oncidium promoters. To date, several strategies have been used to investigate orchids at the genomic level. Sequence homology searches have identified homologous genes in Oncidium [5–11], and expressed sequence tag (EST) databases have been used for gene cloning [12–18]. Because model plants, such as rice and A. thaliana, do not contain all plant genes, and because some genes related to the unique morphological and physiological characteristics of Oncidium, such as the flower and pseudobulbs cannot be identified using sequence homology, an Oncidium-specific cDNA library of pseudobulbs and flowers has been established that contains a large amount of genetic information [12–18]. However, gene expression patterns cannot be predicted by nucleic acid sequences. Furthermore, several of the non-model plant EST sequences are not full-length sequences.
To clone full-length genes and promoters, further processing is necessary, such as rapid amplification of complementary DNA ends (RACE) for full-length cDNA, or genomic walking for promoter studies [8, 15, 16]. These techniques are difficult to apply to Onc. GR because its genome is complex and has not been sequenced. Bacterial artificial chromosome (BAC) libraries are an alternative tool for full-length gene and promoter cloning. To obtain such libraries, genomic DNA is cut into pieces of ~100 kb, cloned into a vector and stored in bacteria, making it is easier to obtain the promoter and the full length of the target gene without interference from homologs in the genome. Various strategies can then be used to identify the clones that contain target genes [19–22], and the identified clones can be sequenced directly to obtain the full-length gene sequence.
In this report, a cDNA microarray, a BAC library and a bombardment assay were combined to establish a novel platform that was used to identify and clone the Onc. GR genes and promoters abundantly expressed in Onc. GR flowers. This approach, combining multiple tools provides a fast, easy to use and convenient strategy for obtaining useful genetic information about Oncidium.
Using cDNA microarray to identify genes highly expressed in flowers
A cDNA microarray was used to identify genes that are abundantly expressed in flowers. PCR products of 1065 clones from the cDNA library of Onc. GR were spotted on to slides to establish a flower-derived microarray. A total of 77 clones were upregulated by >3-fold and 42 clones were downregulated >3-fold relative to the leaves (data not shown).
Sequencing revealed that several clones were repeated. Among the 77 clones corresponding to genes highly expressed in flowers, 57 were unique genes. Among the clones corresponding to genes highly expressed in leaves, 3 were related to photosynthesis/chloroplasts (chloroplast chlorophyll a/b-binding protein, NADH dehydrogenase, and photosystem II 10 kDa protein) as expected; photosynthesis-related genes were highly expressed in leaves.
Onc. Gower Ramsey genes that are abundantly expressed (> 7.5×) or repressed (< 0.06×) in flower tissues
Cytosolic malate dehydrogenase
3-phosphoinositide-dependent protein kinase
NADH dehydrogenase subunit
Chlorophyll a/b-binding protein
40S ribosomal protein
Promoter cloning using a BAC library
Primers used for RT-PCR and BAC screening
ACA TTC AGA AGG AGT CAA CCC
Identifying protein sub-cellular localization using fusion with fluorescent proteins
For the Oncidium genes investigated, no difference in the fluorescence patterns was observed when proteins were expressed as N- or C-terminal fusions with a fluorescent protein (Figure 4G and 4H, OnTI1). The 3 OnTI proteins were seen as aggregated particles in the cells (Figure 4G-J). The subcellular locations of these proteins differed from endomembrane markers, such as mitochondria (Figure 4H). For YFP-OnExpasin, fluorescent signals were evident in the intercellular space and at the cell wall (Figure 4K), and for OnDRRP fluorescent signals appeared as a network system throughout the cell (Figure 4L).
Use of multiple tools to identify promoters
Identification of Oncidiumreproductive-specific expression of genes using cDNA microarray
The aim of this study was to establish a successful combination of integrated tools to obtain genetic information about the commercially important cut flower Onc. GR. A combination of a cDNA library, a microarray, a BAC library and transient transformation was effective. However, the microarray and cDNA library that was used had several limitations: (1) In gene families that have conserved regions and share sequence identity, binding occurs that can limit the specificity of the data. For example, we found that gastrodianin, aquaporin and cytosolic malate dehydrogenase gave false positives. (2) The clone number was limited. There were only 1065 clones in the microarray, which cover only a fraction of the Oncidium genome. The estimated genome size is 1C = 2.84 pg, http://data.kew.org/cvalues/CvalServlet?querytype=1. The estimated coverage of the Onc. GR BAC library is thus 1.28 fold, thereby limiting its possible uses. (3) Only a few genes that are highly expressed in leaves were identified because the microarray was composed from a flower cDNA library. To widen the use of this array, more sequence information needs to be integrated. For example, further libraries must be derived from different tissues and treatments. Sequences from next generation sequencing are an alternative resource for obtaining this data. In comparison to the traditionally employed method (i.e. construction of an EST library, storage and sequencing of each clone using Sanger sequencing technology), using high-throughput approaches allows several thousand ESTs to be obtained cost-effectively from different tissues with less space and effort. Specific gene sequences can then be printed and a microarray yielding more detailed data can be useful for a variety of applications.
BAC library construction is a useful tool for cloning promoters
Tandem repeats in the promoter and gene sequences used in this report.
Two strategies are used for BAC library screening: hybridization and PCR screening. As the gene sequences of the target genes were known in this study, the PCR screening strategy could be adopted. Recent improvements in PCR technology and protocols have made BAC screening more efficient and several genes have been successfully cloned using PCR to screen BAC libraries [19–22]. We thus used this strategy to obtain BAC clones containing genes of interest in the Onc. GR library.
Three trypsin inhibitor genes, OnTI1, OnTI2 and OnTI3, which are highly expressed in flowers, are tandemly duplicated
Three tandemly duplicated genes, OnTI1, OnTI2 and OnTI that are highly expressed in flowers were identified. Gene duplications that encode similar gene functions are a common phenomenon in plants and are thought to have contributed to the origin of evolutionary 'novelties' . For example, it has been proposed that in the early evolution of orchids, two rounds of DEFICENS-like MADS-box gene duplications generated the genes that were probably recruited to distinguish the different types of orchid perianth organs . Information about tandem duplicates can be useful in investigations pertaining to gene duplication. For example, the cinnamyl alcohol dehydrogenase gene , the R2R3-MYB family of transcription factors genes  and NAC domain transcription factors genes  are tandemly duplicated in Populus trichocarpa. These genes have been duplicated from the same ancestral gene, allowing the expression pattern of these genes to be correlated. An investigation of the gene locations of the NAC domain transcription factors in Populus trichocarpa showed that 6 pairs of NACs are present as tandem duplicates, represented in tandem clusters of 2 or 3 genes each. In the tandemly duplicated clusters with 3 genes, the expression patterns of 2 of the genes were found almost identical. However, in the tandemly duplicated clusters with 2 genes, the gene expression levels differed significantly . In the current study, the expression patterns of OnTI genes were similar. On the basis of sequence homology, we discovered 4 conserved regions upstream of OnTI3 similar to OnTI2 (region 1) and OnTI1 (regions 2-4). We tentatively suggest that these OnTIs may be derived from the same ancestral gene.
Several di- or tri-nucleotide tandem repeats were evident in the flower-related genes (Table 3). Because information on Oncidium is limited, the biological significance of tandem repeats in these genes remains unclear. The end sequencing of this BAC library may provide suitable information for identifying the relationship between flower-related genes and tandem repeat sequences.
Transient transformation is a suitable tool for determining the subcellular localization of protein
The subcellular location of a protein is related to its function. For example, photosynthesis-related proteins are located in chloroplasts. Therefore, experiments aimed at determining the specific localization of proteins can provide information on biological processes . Computational prediction is one method used to investigate the subcellular localization of a protein . However, as yet, no suitable reference database exists for Oncidium. Experimentally, the subcellular localization of a protein can be studied by imaging it after fusion with a fluorescent protein [30, 31]. However, no suitable protocol for investigating subcellular localization has so far been established for orchids. In this report, a transient transformation system for the orchid lip using markers derived from different species as fluorescent markers was established to study subcellular localization of proteins.
Trypsin inhibitors can be used to reduce trypsin activity, which can play an active role against pests and diseases . The expression of trypsin inhibitor genes can also be induced by water stress  and stress-related plant growth regulators [34, 35]. Constitutive expression of a trypsin inhibitor can improve plant tolerance to abiotic stress [34, 35]. Trypsin inhibitors are present in all protein bodies, and to a lesser extent in the nucleus and intercellular space [36, 37]. Here, we found that OnTI proteins can form particles similar to protein bodies, but they were not in the nucleus or intercellular space.
Expansins are a superfamily of proteins crucial in loosening the cell wall. The expansins consist of 2 domains, the glycoside hydrolase family 45 (GH45) catalytic domain and group-2 grass pollen allergens. Experimental evidence indicates that expansins can induce slippage of cellulose microfibrils in the cell wall which becomes loosened . The expansin was located in the cell wall and in the intercell wall spaces [39, 40]. The fluorescent signal for OnExpansin was located around the cell wall; according to the results obtained using RT-PCR, OnExpansin was highly expressed in the lips and during lip expansion. Therefore, this gene may be correlated with Onc. GR lip development.
In summary, the localizations of the proteins we investigated are correlated with their predicted functions, but the roles of these genes during Oncidium flower development are unknown as their overexpression in A. thaliana flowers did not result in any significant change in terms of flowering time or morphology.
Useful genetic information can be mined using this integrated platform
Promoters of Oncidium were successfully cloned using a combination of a cDNA library, microarray, BAC library and transient transformation. Transformation of Oncidium is time-consuming and requires considerable human resources. Use of a transient expression system reduced the time required to obtain preliminary information to ~1 week. This approach is thus more time-efficient than genomic walking and stable transformation methods, and allows investigators to estimate experimental priorities.
There are 4 conserved regions in the promoter regions of OnTI genes. The OnTI1 promoter study demonstrated that box 1, box 3 and box 4 were not related to flower expression. The OnTI2 promoter, which does not have these regions, can be expressed in flowers. The most important region controlling the repression in leaves is situated between box 2 and the repeat region. There is a potential Agamous binding site in this region and there is a similar region in the OnTI2 promoter region (TAATGTTACGAAATAAAATATCACTCCTGAATATA). Unlike the repression of OnTI2 in leaves, the most important region for flower expression in OnExpansin is located between -113 to -334 bp. It is expected that the regulation of OnExpansin expression is different from that of OnTI2. Interestingly, 2 potential TF-binding domains (an Agamous and an AtHB9 binding site) are flower or development related. The relevance of the Agamous binding site for gene repression in leaves and flower expression, however, requires further investigation.
The promoter regions of OnTI, OnExpansin and Oncidium MADS genes contain nucleotide tandem repeat sequences (Table 3). However, promoter studies demonstrated that the tandem repeats in OnTI1 and OnExpansin promoters are not related to gene expression. According to our data, the promoter region controlling flower/leaf expression is within 1 kb of the promoter. Analysis of other gene promoters (OnTI1 and OnDRRP) produced similar results (data not shown).
The clones which contain ~ 1 kb promoter regions fused with GUS were transformed into A. thaliana. Although GUS staining was more prominent in flowers, there were some unexpected results. In OnExpansin, GUS staining was evident in the leaves despite the RT-PCR results demonstrating that OnExpansin is predominantly expressed in the lips of Oncidium. In A. thaliana, GUS was weakly expressed in petals, but highly expressed in anthers and styles (Figure 6). The OnTI genes were predominantly expressed in the Oncidium lip and callus. However, there was no GUS staining in the petals of the A. thaliana transformants. These results may be due to the absence of a transcription factor that can recognize the Oncidium binding site, highlighting the necessity of identifying species-specific promoters. The promoters we found were only 1 kb in size. The region that controls the specific organ of interest may not have been included, producing unexpected results in stable A. thaliana transformation.
A cDNA library, a microarray, a BAC library and transient transformation were combined to identify gene promoters highly expressed in the flowers of Oncidium Gower Ramsey, a commercially important cut flower. Classical approaches of identifying orchid genes and promoters - in particular the genome walking method - cannot easily be performed when regions of high DNA sequence homology tandem repeats and tandemly duplicated genes are present. Gene sequences of interest were identified successfully using BAC sequencing. Using lip transient transformation, GUS reporter gene fusion constructs with various lengths of promoters were introduced into the lip to determine promoter activity. Furthermore, the subcellular localization of proteins encoded in these genes was also determined in this system. With this combination of approaches, 5 novel Oncidium gene promoters of genes abundantly expressed in flowers were cloned and confirmed. These promoters can be used to express genes in floral organs and change the flower phenotype without affecting the vegetative tissues.
Flowering Onc. GR (a tetraploid interspecific hybrid) were obtained from a local grower (Yung Hsin Orchid Nursery, Taichung, Taiwan). The orchids were maintained in the greenhouse at Academia Sinica, Taipei, Taiwan. A voucher specimen was deposited at the National Museum of Natural Science, Taichung, Taiwan.
Onc. Gower Ramsey flower cDNA library construction
Onc. GR flowers were used as the materials for cDNA library construction. Total RNA and poly(A)+ mRNA were isolated using Trizol reagent (Invitrogen, Carlsbed, CA, USA) and the Oligotex Midi mRNA kit (Qiagen, Venlo, The Netherlands), respectively, according to the manufacturer's instructions. The cDNA library was constructed using the Long Distance PCR SMART cDNA Library Construction kit (Clontech, Mountain View, CA, USA) following the manufacturer's instructions. The cDNAs were cloned into the pDNR-LIB vector (Clontech). Colonies were picked up, collected in 96-well plates, and stored at -80°C.
Microarray preparation followed the procedure described by Wu et al.  for the preparation of a bamboo microarray. A total of 1065 cDNAs [GenBank: HS521830-HS522791; HS524614-HS524707] derived from the Onc. GR flower cDNA library were amplified using PCR, incorporating the T3 and M13 reverse universal primers. The PCR products were purified using the MultiScreen PCR96 Filter Plate (Millipore Corp., Bedford, MA, USA) and eluted with 100 μl of 0.1× TE buffer (1 mM Tris and 0.1 mM EDTA, pH 8.0). Purified PCR products were printed on GAPS II-coated slides (Corning, New York, NY) using the OmniGrid 100 microarray (Genomic Solutions, Ann Arbor, MI, USA), and arranged into two 1.8 × 1.8-cm arrays (spot size: 100 μm). After printing, the slides were left to dry overnight. These DNAs were cross-linked to the slide by baking the array for 2 h at 80°C.
Total RNA from leaves and flowers (25 μg) was used for cDNA synthesis and labeling with either Cy3 or Cy5 dye, using the 3DNA Expression Array Detection kit for microarrays (Array 50, version 2, Genisphere, Hatfield, PA, USA). cDNA hybridization and washing procedures were performed according to the manufacturer's instructions. All experiments were carried out in 3 biological replicates (n = 3). Detailed information of the microarray has been deposited in the NCBI GEO database [GEO: GSE26504].
Semi-quantification using RT-PCR
Total RNA (5 μg) extracted from various tissues was subjected to RT-PCR. First-strand cDNAs were synthesized using M-MLV reverse transcriptase (RNase H Minus, Point mutant; Promega, Madison, WI, USA) and a poly(dT) primer. Each gene was amplified for 25 cycles using primers specific for each gene. Onc. GR ubiquitin was used as an internal control. The primers are given in Table 2.
BAC library construction
Young Onc. GR leaves (200 g) were collected for isolation of high molecular weight DNA according to Zhang et al. . The DNA was sheared randomly, and the fragments ligated into the pSMART-BAC vector (Lucigen, Middleton, WI, USA). The ligated DNA was transfected into E. coli strain 10G BAC-Optimized Electrocompetent cells (Lucigen).
Identification of BAC clones containing target genes using PCR
Sequences derived from the microarray experiments and the published flower-related genes were used to design primers. Primers that could amplify predicted genomic regions in the presence of Onc. GR genomic DNA (positive control, white box, Figure 8) were used for further screening. After superpool screening (149 reactions), plate screening (2-5 reactions) was performed, and row (16 reactions) and spot screening (24 reactions) were used to identify clones containing genes of interest.
BAC plasmid isolation and sequencing
BAC plasmids were isolated using the NucleoBond BAC 100 kit (NucleoSpin blood, Macherey-Nagel GmbH & Co KG, Germany) following the manufacturer's instructions, and sequenced using the Big Dye™ Terminator Cycle Sequencing Ready Reaction kit and an automated sequencer (Perkin-Elmer Applied Biosystems, CA, USA).
Bombardment assay was conducted as outlined below, modified from Chiou et al. . Purified recombinant plasmid DNA (2.5 μg) was isolated using the Midi Plus plasmid DNA extraction system (Viogene, Taipei, Taiwan) and coated onto gold particles (1 μm diameter) for bombardment transformation. Onc. GR flower lips and leaves were incubated on sucrose-free 1/2 MS  solid medium and bombarded using a pneumatic particle gun (Biolistic PDS-1000/He; Bio-Rad) set to the following conditions: 1350 psi helium pressure of projectile; 27 mm Hg partial vacuum; 9 cm target-distance. Bombarded lips were subsequently incubated on MS solid medium at 22°C overnight for further experiments.
Primers used for the construction of fluorescence protein fusion
Primers used in the promoter study.
AAA AAG CAG GCT AGG AAG GAC ACA CAA CTT
AGA AAG CTG GGT CAT TAG AGA GTA GGA GGT
To measure luciferase and GUS activities, 0.4 g of tissue was ground in a mortar after liquid nitrogen treatment. A volume of 1 ml of 1× CCLR Buffer (Promega, Madison, WI, USA) was added to the powder and incubated at room temperature for 5 min. The solution was centrifuged at 18,000 × g for 5 min and the supernatant collected for further measurements. Luciferase activity was determined using luciferase assay reagent (Promega). GUS-specific activities were determined using 2 mM of 4-methylumbelliferone glucoronide substrate .
Transcription binding sites and tandem repeats were analyzed using the Plant Promoter Analysis Navigator .
We thank Dr. Yu-Yun Chang for helpful discussions and Dr. Heiko Kuhn and Ms. Miranda Loney for editing the manuscript. This work was supported by the Development Program of Industrialization for Agricultural Biotechnology (to MTC and CSL), the Taiwan Seed Improvement and Propagation Station (to CSL) and Academia Sinica (to MTC and CSL) Taiwan.
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