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Promoter of chrysanthemum actin confers high-level constitutive gene expression in Arabidopsis and chrysanthemum
Scientia Horticulturae 211 (2016) 8–18
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Promoter of chrysanthemum actin confers high-level constitutive gene expression in Arabidopsis and chrysanthemum Joon Ki Hong a,1 , Eun Jung Suh a,1 , Soo-Jin Kwon b , Seung Bum Lee a , Jin A. Kim a , Soo In Lee a , Yeon-Hee Lee a,∗ a Agricultural Biotechnology Department, National Institute of Agricultural Sciences, Rural Development Administration, 370 Nongsaengmyeong-ro, Jeonju-si, Jeollabuk-do, Republic of Korea b R&D Coordination Division, Rural Development Administration, 300, Nongsaengmyeong-ro, Jeonju-si, Jeollabuk-do, Republic of Korea
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Article history: Received 13 May 2016 Received in revised form 2 August 2016 Accepted 7 August 2016 Keywords: Actin Chrysanthemum GUS Histochemical analysis Promoter Transgenic plant
a b s t r a c t A promoter that confers high-level constitutive expression of transgenes in crop plants, designated the CmActin promoter, was isolated from chrysanthemum (Chrysanthemum morifolium Ramat. ‘White Wing’). We characterized Arabidopsis and chrysanthemum transformants bearing a CmActin promoter-glucuronidase (GUS) reporter construct. Analysis of GUS expression demonstrated that the promoter directed reporter gene expression during all developmental stages and tissues of transgenic Arabidopsis except in seeds. Higher expression of GUS was observed in organs and tissues of transgenic chrysanthemum compared to those of 35S promoter-GUS transgenic plants. In addition, BrSRS expression driven by the CmActin promoter induced dwarf and compact plants with narrow upwardly curled leaves and short petioles. Transgene expression driven by the CmActin promoter was very similar to that of the 35S promoter in Arabidopsis and was significantly stronger than that of the 35S promoter in chrysanthemum, suggesting that the CmActin promoter can induce constitutive gene expression in various plants and that its function is presumably conserved in different species. The CmActin promoter may provide a practical choice for high-level constitutive expression of target genes and could be a useful tool in floriculture plant genetic engineering. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A promoter initiates and regulates transcription, which is the most important step in gene expression (Xiao et al., 2005). A promoter that strongly and constitutively expresses a foreign gene is required to generate useful transgenic plants with desirable phenotypes to genetically engineer plants (Shirasawa-Seo et al., 2002). A number of promoters confer constitutive expression of foreign genes in transgenic plants regardless of developmental and environmental cues. Among them, the 35S promoter from cauliflower mosaic virus has been the most widely used in various configurations as a strong and constitutive promoter to introduce foreign genes into various plant species (Benfey and Chua, 1990; Samac et al., 2004; Shirasawa-Seo et al., 2002). The foreign gene under the
∗ Corresponding author. E-mail addresses: [email protected] (J.K. Hong), [email protected] (E.J. Suh), [email protected] (S.-J. Kwon), [email protected] (S.B. Lee), [email protected] (J.A. Kim), [email protected] (S.I. Lee), [email protected] (Y.-H. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2016.08.006 0304-4238/© 2016 Elsevier B.V. All rights reserved.
35S promoter is expressed in all tissues and organs during plant growth and development. The 35S promoter is a useful tool that functions as a promoter element for constitutive high-level expression of foreign genes in plants. Several studies have shown that reporter genes driven by the 35S promoter display unequal tissue and developmental expression patterns, and that transgene expression from the 35S promoter in other plants causes loss of the transgenic phenotypic characteristics (Chen et al., 2013; Noda et al., 2013). In addition, it is less effective for monocots, such as cereals (Morita et al., 2012). The 35S promoter gives rise to the gene-silencing phenomenon, which inactivates genes at the post-transcriptional level (Chen et al., 2013; Dong and von Arnim, 2003). Thus, many constitutive promoters isolated from various plants have an activity similar to that of the 35S promoter in transgenic plants, including Gmubi from soybean (Hernandez-Garcia et al., 2009), tCUP from tobacco (Foster et al., 1999), RUBQ1 and OsAct2 from rice (He et al., 2009; Wang and Oard, 2003), ubi4 and ubi9 from sugarcane (Wei et al., 2003), ubi7 from potato (Garbarino et al., 1995), and ZmUbi1 from maize (Cornejo et al., 1993). These promoters are commonly used to drive ectopic gene expression and generate transgenic plants (Chen et al., 2013).
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Chrysanthemum is a member of the Asteraceae family and among the most popular plants in the global floriculture industry (Noda et al., 2013). Various promoters have been used to introduce useful agronomic traits into chrysanthemums. Several efficient promoters have been developed for high-level transgene expression in chrysanthemums using the -glucuronidase (GUS) reporter gene, including Lhca3.St.1 from potato; UEP1, rbcS1, and cab from chrysanthemum; and elongation factor 1 (EF1) from tobacco (Shinoyama et al., 2012; Takatsu et al., 2000). However, introduced transgenes have occasionally been expressed at lower levels or silenced in chrysanthemum transformants (Shinoyama et al., 2012). In a previous study, induction of the heterogeneous 3 , 5 -hydroxylase gene under control of the Nicotiana tabacumm EF-1a promoter failed to yield delphinidin in transgenic chrysanthemum (Noda et al., 2013). Furthermore, GUS activity under control of the 35S promoter decreases in the next generation of nearly all transgenic plants (Takatsu et al., 2000). It appears that these constitutive promoters are not suitable for expressing transgenes in chrysanthemum, and that the expression of the transgenes and the resultant phenotypes may differ depending on the target species (Togami et al., 2006). Accordingly, novel plant sequences are needed that function as promoter elements suitable for the constitutive highlevel expression of target genes in chrysanthemum (Xiao et al., 2005). Here, we cloned and analyzed an actin gene promoter sequence from chrysanthemum (Chrysanthemum morifolium Ramat. ‘White Wing’) that functions as a promoter element for moderate constitutive expression. The CmActin gene promoter led to constitutive GUS reporter gene expression in tissues and organs during growth and development of transgenic Arabidopsis and chrysanthemum. In addition, constitutive expression of Brassica rapa SHI-related sequence SRS genes by the CmActin promoter induced identical phenotypic characteristics in transgenic Arabidopsis, such as dwarfism and upwardly curled leaves. These results suggest that the CmActin promoter is a useful and powerful constitutive promoter for baseline and practical studies in various plants including floral crops. 2. Materials and methods 2.1. Plant materials and growth conditions Chrysanthemum plants (Chrysanthemum morifolium Ramat. ‘White Wing’) were grown in a growth chamber under long-day conditions (16 h light/8 h dark photoperiod) at 25 ◦ C (Suh et al., 2015). Young leaves from 4-week-old plants with identical growth status were collected and prepared for cloning the CmActin promoter. Surface-sterilized Arabidopsis thaliana Columbia (Col-0) seeds were placed on MS (Murashige and Skoog) medium supplemented with vitamins, 1% sucrose, and 0.3% phytagel (Sigma, St. Louis, MO, USA) and stratified at 4 ◦ C for 3 days in the dark to induce synchronous germination. The stratified seeds were germinated under long-day conditions. The plants were transferred to horticultural substrate and grown at 23 ◦ C under long-day conditions for transformation. Samples were collected throughout the growth period. 2.2. Cloning and characterization of the CmActin promoter The chrysanthemum actin promoter was isolated using the genome-walking method according to the manufacturer’s instructions (Siebert et al., 1995; GenomeWalker Universal kit; Clontech, Palo Alto, CA, USA). Genomic DNA was isolated from chrysanthemum leaves using the cetyltrimethylammonium bromide (CTAB) method. Genomic DNA was digested with DraI, EcoRV, PvuII, and
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StuI blunt-end restriction enzymes and ligated to GenomeWalker adaptors. The desired genomic region was amplified via polymerase chain reaction (PCR) using the specific AP1 primer and actin-specific primer1 (GSP1; Supplementary Table 1). The PCR products were diluted 1:5000 and 1 L was used as template for the second PCR amplification (94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 2 min) in a 25 L reaction volume using nested GSP2 and AP2 primers. PCR reactions contained 1 × PCR buffer, 1.5 mM MgCl2 , 0.5 M each primer, 0.2 mM each dNTP, and 2.5 units Taq DNA polymerase (Takara, Shiga, Japan). The PCR amplicon was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced to confirm the fidelity of amplification. Exon-intron splice sites were analyzed and confirmed by comparing the Chrysanthemum lavandulifolium actin (ClActin) partial coding sequence (accession no. JN638568) and Chrysanthemum seticuspe actin (CsActin) coding sequence (accession no. AB679277) using GENSCAN software (http://genes.mit.edu/GENSCAN.html; Burge and Karlin, 1998) and GeneMark software (http://opal.biology.gatech.edu/GeneMark/; Lukashin and Borodovsky, 1998). The cis-acting elements of the promoter sequence were analyzed using plantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (http://www.dna.affrc.go.jp/PLACE/) databases (Higo et al., 1999; Rombauts et al., 1999). Signal peptide analysis was performed with the SignalP Version 4.1 (http://www.cbs.dtu.dk/services/SignalP/) program. The upstream sequence containing the first translation site (CmActin promoter, −1372 to +60) was retrieved from the isolated 1.8 kb CmActin gene fragment for analysis in transgenic plants. The CmActin gene promoter region was amplified by PCR from the isolated clone using the specific primer set (Supplementary Table 1). The CmActin promoter was digested with BamHI and SpeI and subcloned into the same restriction sites of the pCAMBIA1381 vector. The pCAMBIA2300 vector containing kanamycin-resistance gene (nptII) as a selectable marker was digested with XhoI to select transgenic chrysanthemum plants. Subsequently, the digested nptII fragment was subcloned into the CmActin promoter-GUS vector, which had previously been digested with the same enzymes. We also generated CmActin promoter-BrSRS constructs. The CmActin promoter was digested with PstI and BamHI and subcloned into the same restriction sites of the pCAMBIA1390 vector. The SpeI and EcoRI fragments of three BrSRS cDNAs were inserted into the sense orientations between the CmActin promoter and the nopaline synthase terminator. Correct insertion of the promoter region was confirmed by full vector sequencing. The constructs were transformed into Agrobacterium tumefaciens strains GV3101 and LBA4404 using the freeze-thaw shock method (Holsters et al., 1978). 2.3. Generation of transgenic Arabidopsis and chrysanthemum plants The binary vectors containing the CmActin promoter-GUS or CmActin promoter-BrSRS expression constructs were introduced stably into Arabidopsis plants using the Agrobacterium tumefaciensmediated spray method (Hong et al., 2012; Kim et al., 2007). Arabidopsis flowers were sprayed with A. tumefaciens GV3101 containing the binary vectors suspended in 5% sucrose and 0.05% Silwet-L77, the plants were incubated in a growth chamber at 23 ◦ C and 100% humidity for 1 day, and then they were grown in a growth chamber under a 16 h light/8 h dark photoperiod at 23 ◦ C. T1 seeds and progeny were germinated on MS medium containing 30 mg L−1 hygromycin or 50 mg L−1 kanamycin to select the transformants. T3 generation transgenic plants were selected for analysis. The chrysanthemum transformation method was similar to that of Aida et al. (2004) and Suh et al. (2015). Leaf explants (5 mm) for Agrobacterium inoculation were pre-cultured on MSIN pre-
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culture medium (MS medium with 0.8% [w/v] Phytagar containing 1.0 mg L−1 indole acetic acid and 1.0 mg L−1 6-benzylaminopurine) and co-cultured for 2 days in the dark at 23 ◦ C. The explants were submerged in LBA4404 suspension (OD600 = 0.8) for 10 min, transferred to sterilized filter paper to remove excess liquid, and cocultured for 2 days on MSIN at 23 ◦ C in the dark. The explants were placed on MSIN with 400 mg L−1 cefotaxime (MSINC) for 7 days, blot-dried, and transferred to MSINC with 10 mg L−1 kanamycin for the first selection of the transformants. Regenerated shoots were rooted on a solid selection MSINC medium supplemented with 15 mg L−1 kanamycin. After acclimation, the rooted plantlets were transplanted to soil and grown in a greenhouse. Transgenic chrysanthemums have been reproduced vegetatively by cutting stem with lateral meristems 4 times for two years in the green house. Transgenic plants with stable and high-level transgene expression were selected for further experimentation. 2.4. Verification of transgenic Arabidopsis and chrysanthemum plants To confirm the presence of the transgenes in the transgenic plants, we performed PCR analysis of genomic DNA using a genespecific primer set (Supplementary Table 1). The PCR reactions were performed using 35 cycles of 30 s at 95 ◦ C, 30 s at 55 ◦ C, and 45 s at 72 ◦ C, followed by a final extension for 10 min at 72 ◦ C. Total RNA was extracted from transgenic plant seedlings and tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) to analyze the expression of the transgenes using semiquantitative RT-PCR. cDNA was synthesized from 2 g total RNA using MMLV reverse transcriptase (RNaseH free; Toyobo, Osaka, Japan) according to the manufacturer’s instructions for the semiquantitative RT-PCR analysis. Each cDNA sample was diluted 1:3, and 1 L diluted cDNA and a gene-specific primer set (Supplementary Table 1) was used for PCR amplification (94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 45 s) in a 25 L reaction volume. The actin gene was used as the internal control for RNA quantification (Hwang et al., 2010). After a standard PCR of 30 cycles, the resulting PCR products (5 L each) were analyzed using electrophoresis and stained with ethidium bromide. All PCR products were sequenced and matched to individual gene sequences (data not shown). Relative expression levels were analyzed by RT-PCR amplification using the Quantity One program (Bio-Rad, Hercules, CA, USA; Hong et al., 2012). 2.5. Histochemical localization and fluorometric assays for GUS activity GUS activity was determined histochemically in various tissues and developmental and growth stages from transgenic plants using 5-bromo-4-chloro-3-indolyl--d-glucuroide (X-gluc; Duchefa, Haarlem, The Netherlands) as the substrate (Jefferson et al., 1987). Tissues were stained at 37 ◦ C in X-gluc reaction buffer (50 mM sodium phosphate buffer, pH 7.2, 0.05% Triton X-100, 1 mM potassium ferrocyanide and potassium ferricyanide, and 2 mM Xgluc) for 20 h. Subsequently, the samples were transferred to 70% ethanol to remove the chlorophyll prior to observations. Digital images were obtained using an Olympus SZX12 stereo microscope (Olympus, Tokyo, Japan). The GUS-stained tissue and plant images presented here represent typical results from at least three independent lines per construct. GUS enzyme activity was determined fluorometrically using the substrate 4-methylumbelliferyl -d-glucuronide (MUG; Duchefa), which is converted by the GUS enzyme into the fluorescent product 4-methylumbelliferone (4 MU) (Jefferson et al., 1987; Sessa et al., 1998). Various organs from transgenic Arabidopsis and chrysanthemum plants were homogenized in extraction buffer (50 mM sodium phosphate buffer pH 7.0, 10 mM 2-mercaptoethanol, 1 mM
disodium EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X-100). The enzymatic activity of GUS in the supernatants was assayed in buffer containing 1 mM MUG as the fluorescent substrate at 37 ◦ C using the FluorAceTM -glucuronidase Reporter Assay Kit (Bio-Rad). Fluorometric values were calculated using a 4 MU dilution series. The protein concentration in the supernatant was determined using the BioRad Protein Assay (BioRad), and bovine serum albumin as the standard. The 35S promoter-GUS transgenic plant line was used as a control to assess promoter strength, as described previously (Liang et al., 2009, 2011). At least three independent transgenic plants were analyzed from triplicate samples collected for each transgenic plant. GUS activity was normalized to the protein concentration of each supernatant extract and is expressed as nanomoles 4-MU per hour per microgram protein. 3. Results 3.1. Isolation and characterization of the actin gene promoter from chrysanthemum Genome walking is a simple method for finding unknown genomic DNA sequences adjacent to known coding sequences (Siebert et al., 1995). We isolated the 5 -upstream region of the actin gene to identify the promoter sequence that regulates the constitutive expression of the actin gene in chrysanthemum. A set of nested primers specific to the actin gene was designed and used in a 5 -DNA walking strategy that led to the isolation of the 1805 bp PCR fragment of the actin gene from chrysanthemum. The isolated 1.8 kb fragment was compared to ClActin and CsActin sequences from GenBank and confirmed to be the 1372 bp 5 -upstream sequence of the CmActin open reading frame (Supplementary Fig. 1). The cis-acting elements in promoter regions serve as binding sites for transcription factors to initiate transcription and modulate complex gene-regulation processes (Saha et al., 2007). We analyzed the putative promoter sequence using the PLACE (Higo et al., 1999) and PlantCARE (Rombauts et al., 1999) databases to identify the cis-acting elements in the CmActin promoter. The nucleotide sequence of the CmActin promoter region and putative regulatory motifs are shown in Fig. 1. We identified potential cis-acting elements associated with growth-, phytohormone-, and stress-related responses in the CmActin promoter, including a TATA-box, an auxinresponsive element (TGA-element), a cis-acting regulatory element essential for anaerobic induction, a cis-acting element required for endosperm expression (Skn-1 motif), a cis-acting element involved in low-temperature responsiveness (LTR), a gibberellin-responsive element (GARE), an MeJA-responsive motif (TGACG motif), an MYB binding site involved in drought inducibility, and the promoter enhancer region (CAAT-box). These findings suggest that these CmActin elements may modulate gene expression in organs and tissues of plants during development. 3.2. Expression of a reporter gene induced by the CmActin promoter during plant development We fused the 1.43 kb putative promoter region containing the first translation site of CmActin to a GUS reporter gene to analyze the CmActin promoter expression pattern (Figs. 2 A and 3 A ) and introduced the construct into Arabidopsis and chrysanthemum by Agrobacterium-mediated transformation (Aida et al., 2004; Hong et al., 2012). Three independent Arabidopsis transgenic plants that exhibited a progeny segregation ratio of 3:1 for resistance to selective agents consistent with expected Mendelian inheritance of one independent locus were selected for further analysis. Seventeen putative transformants derived from independent explants were obtained from transgenic chrysanthemum plants. Among these,
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Fig. 1. Schematic representation of the putative cis-acting elements in the CmActin promoter sequence. The 5 -flanking region sequence of CmActin is shown with the first translation site. Numbering is based on distance from the 5 -end of the nucleotide sequence (+1) of the open reading frame. The relative positions of potential cis-acting elements are underlined and annotated above. The deduced amino acid sequences of the first exon are indicated by single letter codes.
three GUS-positive plants were selected for further analysis. The PCR analysis confirmed the presence of the CmActin promoter and GUS gene in each of the selected transgenic Arabidopsis and chrysanthemum plants (Supplementary Fig. 2A). GUS transcripts were expressed at high levels in leaves, indicating that GUS expression in leaves was conferred by the CmActin promoter (Supplementary Fig. 2B). GUS activity was monitored histochemically throughout plant growth to understand the spatiotemporal expression patterns of the CmActin promoter. GUS activity in different tissues and organs of transgenic Arabidopsis seedlings and maturing plants is shown in Fig. 2B. Histochemical staining showed moderate GUS activity in the cotyledons, hypocotyls, roots, and rosette leaves of transgenic plants but GUS expression in seeds was observed only in micropylar and chalazal endosperm. Histochemical staining of reproductive organs and tissues showed that the CmActin promoter directed moderate expression of the GUS reporter gene in all organ and tissues including stems, cauline leaves, flower buds, flowers, and
siliques (Fig. 2C). The GUS expression pattern driven by the CmActin promoter was similar between transgenic chrysanthemum and transgenic Arabidopsis in tissues of mature plants (Fig. 3). GUS was very highly expressed in leaves, stems, and flower buds and was also detected in roots, sepals, petals, receptacles, and pedicels, suggesting that the CmActin promoter was responsible for precise transcriptional regulation and determined the moderately constitutive expression of the GUS reporter in transgenic plants, although the intensity varied. Thus, GUS activities were similar in CmActin promoter-GUS transgenic Arabidopsis and chrysanthemum (Figs. 2 C and 3 B). To precisely determine the strength of CmActin promoter activity, we examined GUS activity via semiquantitative RT-PCR and fluorometry in various tissues of transgenic Arabidopsis and chrysanthemum. RT-PCR revealed that the GUS reporter gene was transcribed during all developmental stages and tissues of transgenic Arabidopsis (Fig. 4A and Supplementary Fig. 3). GUS transcript abundance was similar among the various tissues compared to that
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Fig. 2. GUS gene expression driven by the CmActin promoter in transgenic Arabidopsis. (A) Structure of the CmActin promoter-GUS construct used for Arabidopsis transformation. The hygromycin phosphotransferase gene (hpt), with the 35S promoter (35S-pro) and 3 terminator (35S-ter), served as the selectable marker for Arabidopsis transformation. GUS expression was driven by the CmActin promoter. Left and right T-DNA borders are indicated by LB and RB, respectively. (B) Promoter activity in CmActin promoter-GUS transgenic plants was measured during development. Bars are 1 mm. (C) Histochemical analysis of GUS expression driven by the CmActin promoter in reproductive organs of transgenic plants. Bars are 1 mm. Wt, untransformed wild-type; IS, inflorescence stem.
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Fig. 3. GUS expression driven by the CmActin promoter in transgenic chrysanthemum (Chrysanthemum morifolium Ramat. “White Wing”). (A) Structure of the CmActin promoter-GUS construct used for chrysanthemum transformation. The gene encoding bacterial neomycin phosphotransferase II (nptII), which is regulated by the 35S promoter (35S-pro) and the 3 terminator (35S-ter), served as a selectable marker for chrysanthemum transformation. GUS is regulated by the CmActin promoter. Left and right TDNA borders are indicated by LB and RB, respectively. (B) Histochemical analyses of GUS expression driven by the CmActin promoter in transgenic chrysanthemum. Wt, untransformed wild-type; R, root; L, leaf; S, stem; SC, stem cutting region; F, flower; Pt, petal; FC, flower cutting region; Sp, sepal. Bars are 1 cm.
of the AtActin gene. In addition, the GUS enzyme activity driven by the CmActin-promoter was equivalent among all tissues compared to that of 35S Pro:GUS transgenic plants (Fig. 4B). These results suggest that the chrysanthemum actin promoter may confer sim-
ilar regulation of gene expression in Arabidopsis as that of the 35S promoter. GUS transcripts were expressed at high levels in leaves and receptacles of transgenic chrysanthemum plants and were also expressed at low levels in roots, stems, flower buds, flowers, sepals,
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Fig. 4. Activity of the CmActin promoter driving GUS expression in different tissues of transgenic Arabidopsis. (A) Semiquantitative RT-PCR to detect GUS gene expression. A 2-g aliquot of total RNA was reverse-transcribed into first-strand cDNA for semiquantitative RT-PCR analysis of gene expression. Actin gene expression was used as a quantitative control. (B) Fluorometric analysis of GUS activity driven by the CmActin promoter. GUS activity driven by the 35S promoter in transgenic plants was used as a quantitative control. Data are mean ± standard deviation (SD) from at least three independent experiments. Wt, untransformed wild-type, Roots (lane R), leaves (lane L), shoot apical meristems (lane Sa), flower buds (lane Fb), flowers (lane F), stems (St), and siliques (Si) of Arabidopsis.
petals, and pedicels, compared to that of the CmActin gene (Fig. 5A). Furthermore, GUS enzyme activity was stronger in leaves, pedicels, and receptacles than in other tissues, which is very consistent with the GUS gene expression pattern (Fig. 5B). Interestingly, GUS enzyme activities driven by the CmActin promoter in chrysanthemum were more strongly induced in tissues than that of the 35S promoter, although their intensity varied. Therefore, these results indicate that the CmActin promoter induced constitutive expression and that intensity was significantly stronger than that of the 35S promoter at all levels in transgenic chrysanthemum.
caused by the 35S promoter (Hong et al., 2012). In order to confirm the activity of CmActin promoter in chrysanthemum, CmActin promoter-BrLRP1 construct was introduced into Chrysanthemum morifolium Ramat. ‘White Wing’ according to the Suh et al. (2015). The transgenic chrysanthemums which have been recutting four times for two years showed the 11–25% reduction of height at the same age compared to untransformed control (Supplementary Fig. 5).
4. Discussion 3.3. Phenotypic effects of a target gene expressed by the CmActin promoter in transgenic plants As the CmActin promoter induced constitutive expression of a reporter gene in Arabidopsis, we considered that the target gene expression driven by the CmActin promoter induced phenotypic effects similar to those of the 35S promoter (Hong et al., 2012). To confirm this, BrSRS cDNAs driven by the CmActin promoter were introduced into Arabidopsis by Agrobacterium-mediated transformation (Fig. 6A). Three independent transgenic plants were selected from T1 plants for further analysis. Genomic DNA PCR analysis confirmed the presence of the CmActin promoter and BrSRS genes in each of the selected transgenic Arabidopsis (Supplementary Fig. 4A). BrSRS transcripts were expressed at high levels in transgenic leaves (Supplementary Fig. 4B), indicating that BrSRS gene expression in leaves was conferred by the CmActin promoter. The CmActin promoter–BrSRS transgenic plants showed compact growth with reduced height, reduced apical dominance, dark green color, and upwardly curled leaves compared to those of wild-type plants at the same stage (Fig. 6B). In addition, transgenic plants had short and abnormally shaped siliques and poor fertility (data not shown). These results are strikingly similar to the phenotypes
A suitable promoter for strong and constitutive expression of a foreign gene is required for genetic engineering of plants (Shirasawa-Seo et al., 2002). Many constitutive promoters are used to generate useful transgenic plants with desirable phenotypes. Among these, the 35S promoter has been used as a constitutive and well-characterized promoter (Benfey and Chua, 1990; Samac et al., 2004). In addition, rice actin promoters are very effective at directing gene expression in cereal monocots (McElroy et al., 1991; Zhong et al., 2006). In addition, many promoters isolated from other plants are commonly used for their ability to drive constitutively high expression of foreign genes (Chen et al., 2013). However, most promoters available for such a purpose are limited to closely related species (Chen et al., 2013; Noda et al., 2013). The 35S promoter has strong constitutive activity in some plant species but is not strongly expressed in alfalfa or chrysanthemum (Noda et al., 2013; Samac et al., 2004; Shinoyama et al., 2012; Takatsu et al., 2000). Thus, a novel alternative to the 35S promoter is needed to direct strong constitutive expression in various plant species (Xiao et al., 2005). Based on these findings, we described the ability of the chrysanthemum actin promoter to drive foreign gene expression in homogeneous and heterogeneous transgenic systems.
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Fig. 5. Activity of the CmActin promoter driving GUS expression in different tissues of transgenic chrysanthemum. (A) Semiquantitative RT-PCR to detect GUS gene expression. A 2 g aliquot of total RNA was reverse-transcribed into first-strand cDNA for the semiquantitative RT-PCR analysis of gene expression. CmActin gene expression was used as a quantitative control. (B) Fluorometric analysis of GUS activity driven by the CmActin promoter. GUS activity driven by the 35S promoter in transgenic plants was used as a quantitative control. Data are mean ± standard deviation (SD) from at least three independent experiments. Wt, untransformed wild-type, Roots (lane R), leaves (lane L), stems (lane S), flower buds (lane Fb), flowers (lane F), petals (Pt), sepals (Sp), receptacle (Rc), and pedicel (Pd) of chrysanthemum.
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Fig. 6. Phenotypic analysis of CmActin promoter-BrSRS transgenic Arabidopsis. (A) Structure of the CmActin promoter-BrSRS constructs used for Arabidopsis transformation. The gene encoding bacterial neomycin phosphotransferase II (npt II), which is regulated by the 35S promoter (35S-pro) and the 3 terminator (35S-ter), served as a selectable marker for Arabidopsis transformation. BrSRS expression was driven by the CmActin promoter. Left and right T-DNA borders are indicated by LB and RB, respectively. (B) Phenotypes of T1 transgenic Arabidopsis plants transformed with CmActin promoter-BrSRS genes, respectively. Seedlings were grown for 7 days in the same Petri-dish, transplanted to soil, and photographed 20 days later. Lanes: Wt, untransformed wild-type; lanes #1 to #3, transgenic line 1, 2 and 3. Bars are 1 cm.
A 1.43 kb promoter region was isolated and analyzed to investigate the expression mechanism of the CmActin promoter. This promoter was isolated using the genome-walking method at a time when the complete chrysanthemum sequence was not available in the database (Kuriakose et al., 2009). The isolated CmActin promoter fragment was incorporated 1372 bp CmActin promoter with a 60bp, encoding the N-terminal 20 amino acid (Supplementary Fig. 1). The signal peptide of first exon was not detected in the results of signalP (data not shown). Several investigators reported that the first exon containing 5 - untranslated region enhances transgene expression driven by promoter in both dicotyledonous and monocotyledonous plants (Sugio et al., 2008; Suzuki et al., 2001). However, the function of first exon in CmActin promoter is not completely understood. To monitor the activity of CmActin promoter, the promoter region of CmActin gene with its starting 20 codons was fused in frame to the coding region of GUS reporter gene, since addition of the first exon containing 5 - untranslated region
might be effective for transgene expression. The spatiotemporal expression pattern of this promoter was clarified by expressing the GUS reporter gene in transgenic Arabidopsis and chrysanthemum plants containing the CmActin promoter-GUS and 35S promoterGUS constructs to examine whether the function of this promoter is conserved between different species. The histochemical analysis of transgenic Arabidopsis showed constitutive GUS activity in all tissues during development except seeds, where activity was detected only in micropylar and chalazal endosperm (Fig. 2B). In addition, GUS activity was also observed in all tissues during reproduction (Fig. 2C). These results agree with the RT-PCR and fluorometric GUS enzyme activity analyses; the GUS staining pattern was strikingly similar to the GUS expression and enzyme activity patterns during the seedling and growth stages in transgenic Arabidopsis plants (Fig. 4 and Supplementary Fig. 3). Furthermore, the CmActin promoter conferred similar or better gene expression in Arabidopsis than that of the 35S promoter, suggesting that this promoter can
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drive higher levels of foreign gene expression in a heterogeneous transgenic system. GUS was strongly expressed in leaves, stems, flowers, and other tissues of transgenic chrysanthemum plants (Fig. 3B). These results were strikingly similar to the GUS transcript expression patterns (Fig. 5A). Interestingly, gene expression driven by the CmActin promoter in transgenic chrysanthemum was similar to that of transgenic Arabidopsis with constitutive expression in tissues of mature plants, although their intensity varied. These observations indicate that CmActin promoter function is presumably conserved among species. Thus, both the histochemical and transcript abundance analyses suggest that the CmActin promoter was capable of conferring a high-level of constitutive foreign gene expression in different tissues of Arabidopsis and chrysanthemum. It is possible that the CmActin promoter is regulated by similar regulatory factors and cis-elements during plant growth and development in different species (Saha et al., 2007; Yi et al., 2010). Furthermore, the GUS enzyme activity driven by the CmActin promoter was higher than that of the 35S promoter in transgenic chrysanthemum tissues (Fig. 5B). Although the 35S promoter had strong constitutive activity in Arabidopsis, it was not strongly expressed in chrysanthemum, suggesting that the CmActin promoter confers strong constitutive expression of transgenes compared to that of the 35S promoter in chrysanthemum. However, the different activity patterns of the CmActin promoter in tissues of transgenic Arabidopsis and chrysanthemum plants may reflect differences in the combination of transcriptional regulatory elements that uniquely control gene expression in different species (Yi et al., 2010). As the CmActin promoter induced constitutive GUS expression in transgenic Arabidopsis, we considered that the target gene expression driven by the CmActin promoter induced phenotypic effects similar to those of the 35S promoter (Hong et al., 2012). Hence, we assessed the phenotypes of transgenic Arabidopsis and chrysanthemum containing CmActin promoter-BrSRS constructs (Fig. 6 and Supplementary Fig. 5). In a previous study, constitutive expression of BrSRS genes driven by 35S promoter induced phenotypic characteristics, such as dwarfism and compactness in Arabidopsis (Hong et al., 2012). Similarly, overexpression of the BrSRS genes driven by the CmActin promoter induced dwarf and compact plants with narrow upwardly curled leaves and short petioles (Fig. 6). In addition, BrLRP1 transgenic chrysanthemum was shorter than that of the wild type (Supplementary Fig. 5). Thus, the dwarf phenotypes in BrSRS over-expressing transgenic Arabidopsis and chrysanthemum were due to the accumulation of BrSRS transcripts by the CmActin promoter (Supplementary Figs. 4 and 5). This indicates that the CmActin promoter may be useful for studies aimed at modifying phenotypic characters in plants. As the CmActin promoter induced constitutive expression of transgenes in Arabidopsis, we considered that the constitutive induction of target genes may be due to the presence of specific promoter elements. Therefore, we analyzed the promoter sequence of the CmActin gene to determine whether it contains potential cisacting regulatory elements (Fig. 1). This analysis revealed that the CmActin promoter contains a TATA box element necessary for accurately initiating basal transcription in promoters of other plants. In addition, four CAAT enhancer elements were identified in the promoter, which represent binding sites for CAAT enhancer binding proteins (Clarke et al., 1997). The Skn-1 motif is required for high levels of endosperm expression (Washida et al., 1999). This motif was found in the ChAcin promoter region and may modulate gene expression in the endosperm of seeds. Furthermore, this promoter contains several putative hormone- and stress-related cis-elements, including ARE, GARE, LTR, the TGA motif, and the TGACG motif. These findings suggest that the CmActin promoter may regulate gene expression under control of various cis-acting regulatory elements within the promoter as well as corresponding
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trans-acting factors and may be controlled by complex regulatory mechanisms that respond to developmental and environmental cues (Hong and Hwang, 2009; Hwang et al., 2010). Although our data do not indicate whether the putative cis-acting elements directly or indirectly influence gene expression, they do indicate that the promoter is capable of driving high and constitutive expression of transgenes. In addition, the CmActin promoter may confer similar regulation of gene expression in other plants and could be a valuable new tool for plant genetic engineering. However, the detailed function of the CmActin promoter remains to be elucidated, so further experiments are required to define specific elements responsible for conferring constitutive expression. 5. Conclusions In conclusion, we identified and characterized the actin promoter region from chrysanthemum. The 1.43 kb promoter region of the chrysanthemum actin gene directed constitutive expression of a reporter gene during all developmental stages and various tissues except seeds. Moreover, target gene expression driven by the CmActin promoter induced phenotypic characters similar to those of the 35S promoter in transgenic plants. Taken together, these findings suggest that the CmActin gene promoter is an actively functional promoter that confers high-level constitutive expression and may be a useful tool for modulating the production of biomass, quality, and plant form via genetic engineering in floriculture and other plant species. Acknowledgments This work was supported by a grant from the National Institute of Agricultural Sciences (PJ010881), the Rural Development Administration, Republic of Korea. J.K. Hong was supported by a 2016 Post Doctoral Course Program from the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2016. 08.006. References Aida, R., Ohira, K., Tanaka, Y., Yoshida, K., Kishimoto, S., Shibata, M., Ohmiya, A., 2004. Efficient transgene expression in chrysanthemum, Dendranthema grandiflorum (Ramat.) Kitamura, by using the promoter of a gene for chrysanthemum chlorophyll a/b binding protein. Breed. Sci. 54, 51–58. Benfey, P.N., Chua, N.-H., 1990. The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250, 959–966. Burge, C.B., Karlin, S., 1998. Finding the genes in genomic DNA. Curr. Opin. Struct. Biol. 8, 346–354. Chen, Z., Wang, J., Ye, M.-X., Li, H., Ji, L.-X., Li, Y., Cui, D.-Q., Liu, J.-M., An, X.-M., 2013. A novel moderate constitutive promoter derived from Poplar (Populus tomentosa Carrière). Int. J. Mol. Sci. 14, 6187–6204. Clarke, S.L., Robinson, C.E., Gimble, J.M., 1997. CAAT/Enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor ␥2 promoter. Biochem. Biophys. Res. Commun. 240, 99–103. Cornejo, M.J., Luth, D., Blankenship, K.M., Anderson, O.D., Blechl, A.E., 1993. Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol. Biol. 23, 567–581. Dong, Y., von Arnim, A.G., 2003. Novel plant activation-tagging vectors designed to minimize 35S enhancer-mediated gene silencing. Plant Mol. Biol. Rep. 21, 349–358. Foster, E., Hattori, J., Labbe, H., Ouellet, T., Foster, P.R., James, L.E., Lyer, V.N., Miki, B.L., 1999. A tobacco crypic constitutive promoter, tCUP, revealed by T-DNA tagging. Plant Mol. Biol. 41, 45–55. Garbarino, J.E., Oosumi, T., Belknap, W.R., 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109, 1371–1378. He, C., Lin, Z., McElroy, D., Wu, R., 2009. Identification of a rice Actin2 gene regulatory region for high-level expression of transgenes in monocots. Plant Biotechnol. J. 7, 227–239.
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Promoter of chrysanthemum actin confers high-level constitutive gene expression in Arabidopsis and chrysanthemum Joon Ki Hong a,1 , Eun Jung Suh a,1 , Soo-Jin Kwon b , Seung Bum Lee a , Jin A. Kim a , Soo In Lee a , Yeon-Hee Lee a,∗ a Agricultural Biotechnology Department, National Institute of Agricultural Sciences, Rural Development Administration, 370 Nongsaengmyeong-ro, Jeonju-si, Jeollabuk-do, Republic of Korea b R&D Coordination Division, Rural Development Administration, 300, Nongsaengmyeong-ro, Jeonju-si, Jeollabuk-do, Republic of Korea
a r t i c l e
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Article history: Received 13 May 2016 Received in revised form 2 August 2016 Accepted 7 August 2016 Keywords: Actin Chrysanthemum GUS Histochemical analysis Promoter Transgenic plant
a b s t r a c t A promoter that confers high-level constitutive expression of transgenes in crop plants, designated the CmActin promoter, was isolated from chrysanthemum (Chrysanthemum morifolium Ramat. ‘White Wing’). We characterized Arabidopsis and chrysanthemum transformants bearing a CmActin promoter-glucuronidase (GUS) reporter construct. Analysis of GUS expression demonstrated that the promoter directed reporter gene expression during all developmental stages and tissues of transgenic Arabidopsis except in seeds. Higher expression of GUS was observed in organs and tissues of transgenic chrysanthemum compared to those of 35S promoter-GUS transgenic plants. In addition, BrSRS expression driven by the CmActin promoter induced dwarf and compact plants with narrow upwardly curled leaves and short petioles. Transgene expression driven by the CmActin promoter was very similar to that of the 35S promoter in Arabidopsis and was significantly stronger than that of the 35S promoter in chrysanthemum, suggesting that the CmActin promoter can induce constitutive gene expression in various plants and that its function is presumably conserved in different species. The CmActin promoter may provide a practical choice for high-level constitutive expression of target genes and could be a useful tool in floriculture plant genetic engineering. © 2016 Elsevier B.V. All rights reserved.
1. Introduction A promoter initiates and regulates transcription, which is the most important step in gene expression (Xiao et al., 2005). A promoter that strongly and constitutively expresses a foreign gene is required to generate useful transgenic plants with desirable phenotypes to genetically engineer plants (Shirasawa-Seo et al., 2002). A number of promoters confer constitutive expression of foreign genes in transgenic plants regardless of developmental and environmental cues. Among them, the 35S promoter from cauliflower mosaic virus has been the most widely used in various configurations as a strong and constitutive promoter to introduce foreign genes into various plant species (Benfey and Chua, 1990; Samac et al., 2004; Shirasawa-Seo et al., 2002). The foreign gene under the
∗ Corresponding author. E-mail addresses: [email protected] (J.K. Hong), [email protected] (E.J. Suh), [email protected] (S.-J. Kwon), [email protected] (S.B. Lee), [email protected] (J.A. Kim), [email protected] (S.I. Lee), [email protected] (Y.-H. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2016.08.006 0304-4238/© 2016 Elsevier B.V. All rights reserved.
35S promoter is expressed in all tissues and organs during plant growth and development. The 35S promoter is a useful tool that functions as a promoter element for constitutive high-level expression of foreign genes in plants. Several studies have shown that reporter genes driven by the 35S promoter display unequal tissue and developmental expression patterns, and that transgene expression from the 35S promoter in other plants causes loss of the transgenic phenotypic characteristics (Chen et al., 2013; Noda et al., 2013). In addition, it is less effective for monocots, such as cereals (Morita et al., 2012). The 35S promoter gives rise to the gene-silencing phenomenon, which inactivates genes at the post-transcriptional level (Chen et al., 2013; Dong and von Arnim, 2003). Thus, many constitutive promoters isolated from various plants have an activity similar to that of the 35S promoter in transgenic plants, including Gmubi from soybean (Hernandez-Garcia et al., 2009), tCUP from tobacco (Foster et al., 1999), RUBQ1 and OsAct2 from rice (He et al., 2009; Wang and Oard, 2003), ubi4 and ubi9 from sugarcane (Wei et al., 2003), ubi7 from potato (Garbarino et al., 1995), and ZmUbi1 from maize (Cornejo et al., 1993). These promoters are commonly used to drive ectopic gene expression and generate transgenic plants (Chen et al., 2013).
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Chrysanthemum is a member of the Asteraceae family and among the most popular plants in the global floriculture industry (Noda et al., 2013). Various promoters have been used to introduce useful agronomic traits into chrysanthemums. Several efficient promoters have been developed for high-level transgene expression in chrysanthemums using the -glucuronidase (GUS) reporter gene, including Lhca3.St.1 from potato; UEP1, rbcS1, and cab from chrysanthemum; and elongation factor 1 (EF1) from tobacco (Shinoyama et al., 2012; Takatsu et al., 2000). However, introduced transgenes have occasionally been expressed at lower levels or silenced in chrysanthemum transformants (Shinoyama et al., 2012). In a previous study, induction of the heterogeneous 3 , 5 -hydroxylase gene under control of the Nicotiana tabacumm EF-1a promoter failed to yield delphinidin in transgenic chrysanthemum (Noda et al., 2013). Furthermore, GUS activity under control of the 35S promoter decreases in the next generation of nearly all transgenic plants (Takatsu et al., 2000). It appears that these constitutive promoters are not suitable for expressing transgenes in chrysanthemum, and that the expression of the transgenes and the resultant phenotypes may differ depending on the target species (Togami et al., 2006). Accordingly, novel plant sequences are needed that function as promoter elements suitable for the constitutive highlevel expression of target genes in chrysanthemum (Xiao et al., 2005). Here, we cloned and analyzed an actin gene promoter sequence from chrysanthemum (Chrysanthemum morifolium Ramat. ‘White Wing’) that functions as a promoter element for moderate constitutive expression. The CmActin gene promoter led to constitutive GUS reporter gene expression in tissues and organs during growth and development of transgenic Arabidopsis and chrysanthemum. In addition, constitutive expression of Brassica rapa SHI-related sequence SRS genes by the CmActin promoter induced identical phenotypic characteristics in transgenic Arabidopsis, such as dwarfism and upwardly curled leaves. These results suggest that the CmActin promoter is a useful and powerful constitutive promoter for baseline and practical studies in various plants including floral crops. 2. Materials and methods 2.1. Plant materials and growth conditions Chrysanthemum plants (Chrysanthemum morifolium Ramat. ‘White Wing’) were grown in a growth chamber under long-day conditions (16 h light/8 h dark photoperiod) at 25 ◦ C (Suh et al., 2015). Young leaves from 4-week-old plants with identical growth status were collected and prepared for cloning the CmActin promoter. Surface-sterilized Arabidopsis thaliana Columbia (Col-0) seeds were placed on MS (Murashige and Skoog) medium supplemented with vitamins, 1% sucrose, and 0.3% phytagel (Sigma, St. Louis, MO, USA) and stratified at 4 ◦ C for 3 days in the dark to induce synchronous germination. The stratified seeds were germinated under long-day conditions. The plants were transferred to horticultural substrate and grown at 23 ◦ C under long-day conditions for transformation. Samples were collected throughout the growth period. 2.2. Cloning and characterization of the CmActin promoter The chrysanthemum actin promoter was isolated using the genome-walking method according to the manufacturer’s instructions (Siebert et al., 1995; GenomeWalker Universal kit; Clontech, Palo Alto, CA, USA). Genomic DNA was isolated from chrysanthemum leaves using the cetyltrimethylammonium bromide (CTAB) method. Genomic DNA was digested with DraI, EcoRV, PvuII, and
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StuI blunt-end restriction enzymes and ligated to GenomeWalker adaptors. The desired genomic region was amplified via polymerase chain reaction (PCR) using the specific AP1 primer and actin-specific primer1 (GSP1; Supplementary Table 1). The PCR products were diluted 1:5000 and 1 L was used as template for the second PCR amplification (94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 2 min) in a 25 L reaction volume using nested GSP2 and AP2 primers. PCR reactions contained 1 × PCR buffer, 1.5 mM MgCl2 , 0.5 M each primer, 0.2 mM each dNTP, and 2.5 units Taq DNA polymerase (Takara, Shiga, Japan). The PCR amplicon was cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced to confirm the fidelity of amplification. Exon-intron splice sites were analyzed and confirmed by comparing the Chrysanthemum lavandulifolium actin (ClActin) partial coding sequence (accession no. JN638568) and Chrysanthemum seticuspe actin (CsActin) coding sequence (accession no. AB679277) using GENSCAN software (http://genes.mit.edu/GENSCAN.html; Burge and Karlin, 1998) and GeneMark software (http://opal.biology.gatech.edu/GeneMark/; Lukashin and Borodovsky, 1998). The cis-acting elements of the promoter sequence were analyzed using plantCARE (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PLACE (http://www.dna.affrc.go.jp/PLACE/) databases (Higo et al., 1999; Rombauts et al., 1999). Signal peptide analysis was performed with the SignalP Version 4.1 (http://www.cbs.dtu.dk/services/SignalP/) program. The upstream sequence containing the first translation site (CmActin promoter, −1372 to +60) was retrieved from the isolated 1.8 kb CmActin gene fragment for analysis in transgenic plants. The CmActin gene promoter region was amplified by PCR from the isolated clone using the specific primer set (Supplementary Table 1). The CmActin promoter was digested with BamHI and SpeI and subcloned into the same restriction sites of the pCAMBIA1381 vector. The pCAMBIA2300 vector containing kanamycin-resistance gene (nptII) as a selectable marker was digested with XhoI to select transgenic chrysanthemum plants. Subsequently, the digested nptII fragment was subcloned into the CmActin promoter-GUS vector, which had previously been digested with the same enzymes. We also generated CmActin promoter-BrSRS constructs. The CmActin promoter was digested with PstI and BamHI and subcloned into the same restriction sites of the pCAMBIA1390 vector. The SpeI and EcoRI fragments of three BrSRS cDNAs were inserted into the sense orientations between the CmActin promoter and the nopaline synthase terminator. Correct insertion of the promoter region was confirmed by full vector sequencing. The constructs were transformed into Agrobacterium tumefaciens strains GV3101 and LBA4404 using the freeze-thaw shock method (Holsters et al., 1978). 2.3. Generation of transgenic Arabidopsis and chrysanthemum plants The binary vectors containing the CmActin promoter-GUS or CmActin promoter-BrSRS expression constructs were introduced stably into Arabidopsis plants using the Agrobacterium tumefaciensmediated spray method (Hong et al., 2012; Kim et al., 2007). Arabidopsis flowers were sprayed with A. tumefaciens GV3101 containing the binary vectors suspended in 5% sucrose and 0.05% Silwet-L77, the plants were incubated in a growth chamber at 23 ◦ C and 100% humidity for 1 day, and then they were grown in a growth chamber under a 16 h light/8 h dark photoperiod at 23 ◦ C. T1 seeds and progeny were germinated on MS medium containing 30 mg L−1 hygromycin or 50 mg L−1 kanamycin to select the transformants. T3 generation transgenic plants were selected for analysis. The chrysanthemum transformation method was similar to that of Aida et al. (2004) and Suh et al. (2015). Leaf explants (5 mm) for Agrobacterium inoculation were pre-cultured on MSIN pre-
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culture medium (MS medium with 0.8% [w/v] Phytagar containing 1.0 mg L−1 indole acetic acid and 1.0 mg L−1 6-benzylaminopurine) and co-cultured for 2 days in the dark at 23 ◦ C. The explants were submerged in LBA4404 suspension (OD600 = 0.8) for 10 min, transferred to sterilized filter paper to remove excess liquid, and cocultured for 2 days on MSIN at 23 ◦ C in the dark. The explants were placed on MSIN with 400 mg L−1 cefotaxime (MSINC) for 7 days, blot-dried, and transferred to MSINC with 10 mg L−1 kanamycin for the first selection of the transformants. Regenerated shoots were rooted on a solid selection MSINC medium supplemented with 15 mg L−1 kanamycin. After acclimation, the rooted plantlets were transplanted to soil and grown in a greenhouse. Transgenic chrysanthemums have been reproduced vegetatively by cutting stem with lateral meristems 4 times for two years in the green house. Transgenic plants with stable and high-level transgene expression were selected for further experimentation. 2.4. Verification of transgenic Arabidopsis and chrysanthemum plants To confirm the presence of the transgenes in the transgenic plants, we performed PCR analysis of genomic DNA using a genespecific primer set (Supplementary Table 1). The PCR reactions were performed using 35 cycles of 30 s at 95 ◦ C, 30 s at 55 ◦ C, and 45 s at 72 ◦ C, followed by a final extension for 10 min at 72 ◦ C. Total RNA was extracted from transgenic plant seedlings and tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) to analyze the expression of the transgenes using semiquantitative RT-PCR. cDNA was synthesized from 2 g total RNA using MMLV reverse transcriptase (RNaseH free; Toyobo, Osaka, Japan) according to the manufacturer’s instructions for the semiquantitative RT-PCR analysis. Each cDNA sample was diluted 1:3, and 1 L diluted cDNA and a gene-specific primer set (Supplementary Table 1) was used for PCR amplification (94 ◦ C for 30 s, 55 ◦ C for 30 s, and 72 ◦ C for 45 s) in a 25 L reaction volume. The actin gene was used as the internal control for RNA quantification (Hwang et al., 2010). After a standard PCR of 30 cycles, the resulting PCR products (5 L each) were analyzed using electrophoresis and stained with ethidium bromide. All PCR products were sequenced and matched to individual gene sequences (data not shown). Relative expression levels were analyzed by RT-PCR amplification using the Quantity One program (Bio-Rad, Hercules, CA, USA; Hong et al., 2012). 2.5. Histochemical localization and fluorometric assays for GUS activity GUS activity was determined histochemically in various tissues and developmental and growth stages from transgenic plants using 5-bromo-4-chloro-3-indolyl--d-glucuroide (X-gluc; Duchefa, Haarlem, The Netherlands) as the substrate (Jefferson et al., 1987). Tissues were stained at 37 ◦ C in X-gluc reaction buffer (50 mM sodium phosphate buffer, pH 7.2, 0.05% Triton X-100, 1 mM potassium ferrocyanide and potassium ferricyanide, and 2 mM Xgluc) for 20 h. Subsequently, the samples were transferred to 70% ethanol to remove the chlorophyll prior to observations. Digital images were obtained using an Olympus SZX12 stereo microscope (Olympus, Tokyo, Japan). The GUS-stained tissue and plant images presented here represent typical results from at least three independent lines per construct. GUS enzyme activity was determined fluorometrically using the substrate 4-methylumbelliferyl -d-glucuronide (MUG; Duchefa), which is converted by the GUS enzyme into the fluorescent product 4-methylumbelliferone (4 MU) (Jefferson et al., 1987; Sessa et al., 1998). Various organs from transgenic Arabidopsis and chrysanthemum plants were homogenized in extraction buffer (50 mM sodium phosphate buffer pH 7.0, 10 mM 2-mercaptoethanol, 1 mM
disodium EDTA, 0.1% sodium lauryl sarcosine, and 0.1% Triton X-100). The enzymatic activity of GUS in the supernatants was assayed in buffer containing 1 mM MUG as the fluorescent substrate at 37 ◦ C using the FluorAceTM -glucuronidase Reporter Assay Kit (Bio-Rad). Fluorometric values were calculated using a 4 MU dilution series. The protein concentration in the supernatant was determined using the BioRad Protein Assay (BioRad), and bovine serum albumin as the standard. The 35S promoter-GUS transgenic plant line was used as a control to assess promoter strength, as described previously (Liang et al., 2009, 2011). At least three independent transgenic plants were analyzed from triplicate samples collected for each transgenic plant. GUS activity was normalized to the protein concentration of each supernatant extract and is expressed as nanomoles 4-MU per hour per microgram protein. 3. Results 3.1. Isolation and characterization of the actin gene promoter from chrysanthemum Genome walking is a simple method for finding unknown genomic DNA sequences adjacent to known coding sequences (Siebert et al., 1995). We isolated the 5 -upstream region of the actin gene to identify the promoter sequence that regulates the constitutive expression of the actin gene in chrysanthemum. A set of nested primers specific to the actin gene was designed and used in a 5 -DNA walking strategy that led to the isolation of the 1805 bp PCR fragment of the actin gene from chrysanthemum. The isolated 1.8 kb fragment was compared to ClActin and CsActin sequences from GenBank and confirmed to be the 1372 bp 5 -upstream sequence of the CmActin open reading frame (Supplementary Fig. 1). The cis-acting elements in promoter regions serve as binding sites for transcription factors to initiate transcription and modulate complex gene-regulation processes (Saha et al., 2007). We analyzed the putative promoter sequence using the PLACE (Higo et al., 1999) and PlantCARE (Rombauts et al., 1999) databases to identify the cis-acting elements in the CmActin promoter. The nucleotide sequence of the CmActin promoter region and putative regulatory motifs are shown in Fig. 1. We identified potential cis-acting elements associated with growth-, phytohormone-, and stress-related responses in the CmActin promoter, including a TATA-box, an auxinresponsive element (TGA-element), a cis-acting regulatory element essential for anaerobic induction, a cis-acting element required for endosperm expression (Skn-1 motif), a cis-acting element involved in low-temperature responsiveness (LTR), a gibberellin-responsive element (GARE), an MeJA-responsive motif (TGACG motif), an MYB binding site involved in drought inducibility, and the promoter enhancer region (CAAT-box). These findings suggest that these CmActin elements may modulate gene expression in organs and tissues of plants during development. 3.2. Expression of a reporter gene induced by the CmActin promoter during plant development We fused the 1.43 kb putative promoter region containing the first translation site of CmActin to a GUS reporter gene to analyze the CmActin promoter expression pattern (Figs. 2 A and 3 A ) and introduced the construct into Arabidopsis and chrysanthemum by Agrobacterium-mediated transformation (Aida et al., 2004; Hong et al., 2012). Three independent Arabidopsis transgenic plants that exhibited a progeny segregation ratio of 3:1 for resistance to selective agents consistent with expected Mendelian inheritance of one independent locus were selected for further analysis. Seventeen putative transformants derived from independent explants were obtained from transgenic chrysanthemum plants. Among these,
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Fig. 1. Schematic representation of the putative cis-acting elements in the CmActin promoter sequence. The 5 -flanking region sequence of CmActin is shown with the first translation site. Numbering is based on distance from the 5 -end of the nucleotide sequence (+1) of the open reading frame. The relative positions of potential cis-acting elements are underlined and annotated above. The deduced amino acid sequences of the first exon are indicated by single letter codes.
three GUS-positive plants were selected for further analysis. The PCR analysis confirmed the presence of the CmActin promoter and GUS gene in each of the selected transgenic Arabidopsis and chrysanthemum plants (Supplementary Fig. 2A). GUS transcripts were expressed at high levels in leaves, indicating that GUS expression in leaves was conferred by the CmActin promoter (Supplementary Fig. 2B). GUS activity was monitored histochemically throughout plant growth to understand the spatiotemporal expression patterns of the CmActin promoter. GUS activity in different tissues and organs of transgenic Arabidopsis seedlings and maturing plants is shown in Fig. 2B. Histochemical staining showed moderate GUS activity in the cotyledons, hypocotyls, roots, and rosette leaves of transgenic plants but GUS expression in seeds was observed only in micropylar and chalazal endosperm. Histochemical staining of reproductive organs and tissues showed that the CmActin promoter directed moderate expression of the GUS reporter gene in all organ and tissues including stems, cauline leaves, flower buds, flowers, and
siliques (Fig. 2C). The GUS expression pattern driven by the CmActin promoter was similar between transgenic chrysanthemum and transgenic Arabidopsis in tissues of mature plants (Fig. 3). GUS was very highly expressed in leaves, stems, and flower buds and was also detected in roots, sepals, petals, receptacles, and pedicels, suggesting that the CmActin promoter was responsible for precise transcriptional regulation and determined the moderately constitutive expression of the GUS reporter in transgenic plants, although the intensity varied. Thus, GUS activities were similar in CmActin promoter-GUS transgenic Arabidopsis and chrysanthemum (Figs. 2 C and 3 B). To precisely determine the strength of CmActin promoter activity, we examined GUS activity via semiquantitative RT-PCR and fluorometry in various tissues of transgenic Arabidopsis and chrysanthemum. RT-PCR revealed that the GUS reporter gene was transcribed during all developmental stages and tissues of transgenic Arabidopsis (Fig. 4A and Supplementary Fig. 3). GUS transcript abundance was similar among the various tissues compared to that
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Fig. 2. GUS gene expression driven by the CmActin promoter in transgenic Arabidopsis. (A) Structure of the CmActin promoter-GUS construct used for Arabidopsis transformation. The hygromycin phosphotransferase gene (hpt), with the 35S promoter (35S-pro) and 3 terminator (35S-ter), served as the selectable marker for Arabidopsis transformation. GUS expression was driven by the CmActin promoter. Left and right T-DNA borders are indicated by LB and RB, respectively. (B) Promoter activity in CmActin promoter-GUS transgenic plants was measured during development. Bars are 1 mm. (C) Histochemical analysis of GUS expression driven by the CmActin promoter in reproductive organs of transgenic plants. Bars are 1 mm. Wt, untransformed wild-type; IS, inflorescence stem.
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Fig. 3. GUS expression driven by the CmActin promoter in transgenic chrysanthemum (Chrysanthemum morifolium Ramat. “White Wing”). (A) Structure of the CmActin promoter-GUS construct used for chrysanthemum transformation. The gene encoding bacterial neomycin phosphotransferase II (nptII), which is regulated by the 35S promoter (35S-pro) and the 3 terminator (35S-ter), served as a selectable marker for chrysanthemum transformation. GUS is regulated by the CmActin promoter. Left and right TDNA borders are indicated by LB and RB, respectively. (B) Histochemical analyses of GUS expression driven by the CmActin promoter in transgenic chrysanthemum. Wt, untransformed wild-type; R, root; L, leaf; S, stem; SC, stem cutting region; F, flower; Pt, petal; FC, flower cutting region; Sp, sepal. Bars are 1 cm.
of the AtActin gene. In addition, the GUS enzyme activity driven by the CmActin-promoter was equivalent among all tissues compared to that of 35S Pro:GUS transgenic plants (Fig. 4B). These results suggest that the chrysanthemum actin promoter may confer sim-
ilar regulation of gene expression in Arabidopsis as that of the 35S promoter. GUS transcripts were expressed at high levels in leaves and receptacles of transgenic chrysanthemum plants and were also expressed at low levels in roots, stems, flower buds, flowers, sepals,
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Fig. 4. Activity of the CmActin promoter driving GUS expression in different tissues of transgenic Arabidopsis. (A) Semiquantitative RT-PCR to detect GUS gene expression. A 2-g aliquot of total RNA was reverse-transcribed into first-strand cDNA for semiquantitative RT-PCR analysis of gene expression. Actin gene expression was used as a quantitative control. (B) Fluorometric analysis of GUS activity driven by the CmActin promoter. GUS activity driven by the 35S promoter in transgenic plants was used as a quantitative control. Data are mean ± standard deviation (SD) from at least three independent experiments. Wt, untransformed wild-type, Roots (lane R), leaves (lane L), shoot apical meristems (lane Sa), flower buds (lane Fb), flowers (lane F), stems (St), and siliques (Si) of Arabidopsis.
petals, and pedicels, compared to that of the CmActin gene (Fig. 5A). Furthermore, GUS enzyme activity was stronger in leaves, pedicels, and receptacles than in other tissues, which is very consistent with the GUS gene expression pattern (Fig. 5B). Interestingly, GUS enzyme activities driven by the CmActin promoter in chrysanthemum were more strongly induced in tissues than that of the 35S promoter, although their intensity varied. Therefore, these results indicate that the CmActin promoter induced constitutive expression and that intensity was significantly stronger than that of the 35S promoter at all levels in transgenic chrysanthemum.
caused by the 35S promoter (Hong et al., 2012). In order to confirm the activity of CmActin promoter in chrysanthemum, CmActin promoter-BrLRP1 construct was introduced into Chrysanthemum morifolium Ramat. ‘White Wing’ according to the Suh et al. (2015). The transgenic chrysanthemums which have been recutting four times for two years showed the 11–25% reduction of height at the same age compared to untransformed control (Supplementary Fig. 5).
4. Discussion 3.3. Phenotypic effects of a target gene expressed by the CmActin promoter in transgenic plants As the CmActin promoter induced constitutive expression of a reporter gene in Arabidopsis, we considered that the target gene expression driven by the CmActin promoter induced phenotypic effects similar to those of the 35S promoter (Hong et al., 2012). To confirm this, BrSRS cDNAs driven by the CmActin promoter were introduced into Arabidopsis by Agrobacterium-mediated transformation (Fig. 6A). Three independent transgenic plants were selected from T1 plants for further analysis. Genomic DNA PCR analysis confirmed the presence of the CmActin promoter and BrSRS genes in each of the selected transgenic Arabidopsis (Supplementary Fig. 4A). BrSRS transcripts were expressed at high levels in transgenic leaves (Supplementary Fig. 4B), indicating that BrSRS gene expression in leaves was conferred by the CmActin promoter. The CmActin promoter–BrSRS transgenic plants showed compact growth with reduced height, reduced apical dominance, dark green color, and upwardly curled leaves compared to those of wild-type plants at the same stage (Fig. 6B). In addition, transgenic plants had short and abnormally shaped siliques and poor fertility (data not shown). These results are strikingly similar to the phenotypes
A suitable promoter for strong and constitutive expression of a foreign gene is required for genetic engineering of plants (Shirasawa-Seo et al., 2002). Many constitutive promoters are used to generate useful transgenic plants with desirable phenotypes. Among these, the 35S promoter has been used as a constitutive and well-characterized promoter (Benfey and Chua, 1990; Samac et al., 2004). In addition, rice actin promoters are very effective at directing gene expression in cereal monocots (McElroy et al., 1991; Zhong et al., 2006). In addition, many promoters isolated from other plants are commonly used for their ability to drive constitutively high expression of foreign genes (Chen et al., 2013). However, most promoters available for such a purpose are limited to closely related species (Chen et al., 2013; Noda et al., 2013). The 35S promoter has strong constitutive activity in some plant species but is not strongly expressed in alfalfa or chrysanthemum (Noda et al., 2013; Samac et al., 2004; Shinoyama et al., 2012; Takatsu et al., 2000). Thus, a novel alternative to the 35S promoter is needed to direct strong constitutive expression in various plant species (Xiao et al., 2005). Based on these findings, we described the ability of the chrysanthemum actin promoter to drive foreign gene expression in homogeneous and heterogeneous transgenic systems.
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Fig. 5. Activity of the CmActin promoter driving GUS expression in different tissues of transgenic chrysanthemum. (A) Semiquantitative RT-PCR to detect GUS gene expression. A 2 g aliquot of total RNA was reverse-transcribed into first-strand cDNA for the semiquantitative RT-PCR analysis of gene expression. CmActin gene expression was used as a quantitative control. (B) Fluorometric analysis of GUS activity driven by the CmActin promoter. GUS activity driven by the 35S promoter in transgenic plants was used as a quantitative control. Data are mean ± standard deviation (SD) from at least three independent experiments. Wt, untransformed wild-type, Roots (lane R), leaves (lane L), stems (lane S), flower buds (lane Fb), flowers (lane F), petals (Pt), sepals (Sp), receptacle (Rc), and pedicel (Pd) of chrysanthemum.
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Fig. 6. Phenotypic analysis of CmActin promoter-BrSRS transgenic Arabidopsis. (A) Structure of the CmActin promoter-BrSRS constructs used for Arabidopsis transformation. The gene encoding bacterial neomycin phosphotransferase II (npt II), which is regulated by the 35S promoter (35S-pro) and the 3 terminator (35S-ter), served as a selectable marker for Arabidopsis transformation. BrSRS expression was driven by the CmActin promoter. Left and right T-DNA borders are indicated by LB and RB, respectively. (B) Phenotypes of T1 transgenic Arabidopsis plants transformed with CmActin promoter-BrSRS genes, respectively. Seedlings were grown for 7 days in the same Petri-dish, transplanted to soil, and photographed 20 days later. Lanes: Wt, untransformed wild-type; lanes #1 to #3, transgenic line 1, 2 and 3. Bars are 1 cm.
A 1.43 kb promoter region was isolated and analyzed to investigate the expression mechanism of the CmActin promoter. This promoter was isolated using the genome-walking method at a time when the complete chrysanthemum sequence was not available in the database (Kuriakose et al., 2009). The isolated CmActin promoter fragment was incorporated 1372 bp CmActin promoter with a 60bp, encoding the N-terminal 20 amino acid (Supplementary Fig. 1). The signal peptide of first exon was not detected in the results of signalP (data not shown). Several investigators reported that the first exon containing 5 - untranslated region enhances transgene expression driven by promoter in both dicotyledonous and monocotyledonous plants (Sugio et al., 2008; Suzuki et al., 2001). However, the function of first exon in CmActin promoter is not completely understood. To monitor the activity of CmActin promoter, the promoter region of CmActin gene with its starting 20 codons was fused in frame to the coding region of GUS reporter gene, since addition of the first exon containing 5 - untranslated region
might be effective for transgene expression. The spatiotemporal expression pattern of this promoter was clarified by expressing the GUS reporter gene in transgenic Arabidopsis and chrysanthemum plants containing the CmActin promoter-GUS and 35S promoterGUS constructs to examine whether the function of this promoter is conserved between different species. The histochemical analysis of transgenic Arabidopsis showed constitutive GUS activity in all tissues during development except seeds, where activity was detected only in micropylar and chalazal endosperm (Fig. 2B). In addition, GUS activity was also observed in all tissues during reproduction (Fig. 2C). These results agree with the RT-PCR and fluorometric GUS enzyme activity analyses; the GUS staining pattern was strikingly similar to the GUS expression and enzyme activity patterns during the seedling and growth stages in transgenic Arabidopsis plants (Fig. 4 and Supplementary Fig. 3). Furthermore, the CmActin promoter conferred similar or better gene expression in Arabidopsis than that of the 35S promoter, suggesting that this promoter can
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drive higher levels of foreign gene expression in a heterogeneous transgenic system. GUS was strongly expressed in leaves, stems, flowers, and other tissues of transgenic chrysanthemum plants (Fig. 3B). These results were strikingly similar to the GUS transcript expression patterns (Fig. 5A). Interestingly, gene expression driven by the CmActin promoter in transgenic chrysanthemum was similar to that of transgenic Arabidopsis with constitutive expression in tissues of mature plants, although their intensity varied. These observations indicate that CmActin promoter function is presumably conserved among species. Thus, both the histochemical and transcript abundance analyses suggest that the CmActin promoter was capable of conferring a high-level of constitutive foreign gene expression in different tissues of Arabidopsis and chrysanthemum. It is possible that the CmActin promoter is regulated by similar regulatory factors and cis-elements during plant growth and development in different species (Saha et al., 2007; Yi et al., 2010). Furthermore, the GUS enzyme activity driven by the CmActin promoter was higher than that of the 35S promoter in transgenic chrysanthemum tissues (Fig. 5B). Although the 35S promoter had strong constitutive activity in Arabidopsis, it was not strongly expressed in chrysanthemum, suggesting that the CmActin promoter confers strong constitutive expression of transgenes compared to that of the 35S promoter in chrysanthemum. However, the different activity patterns of the CmActin promoter in tissues of transgenic Arabidopsis and chrysanthemum plants may reflect differences in the combination of transcriptional regulatory elements that uniquely control gene expression in different species (Yi et al., 2010). As the CmActin promoter induced constitutive GUS expression in transgenic Arabidopsis, we considered that the target gene expression driven by the CmActin promoter induced phenotypic effects similar to those of the 35S promoter (Hong et al., 2012). Hence, we assessed the phenotypes of transgenic Arabidopsis and chrysanthemum containing CmActin promoter-BrSRS constructs (Fig. 6 and Supplementary Fig. 5). In a previous study, constitutive expression of BrSRS genes driven by 35S promoter induced phenotypic characteristics, such as dwarfism and compactness in Arabidopsis (Hong et al., 2012). Similarly, overexpression of the BrSRS genes driven by the CmActin promoter induced dwarf and compact plants with narrow upwardly curled leaves and short petioles (Fig. 6). In addition, BrLRP1 transgenic chrysanthemum was shorter than that of the wild type (Supplementary Fig. 5). Thus, the dwarf phenotypes in BrSRS over-expressing transgenic Arabidopsis and chrysanthemum were due to the accumulation of BrSRS transcripts by the CmActin promoter (Supplementary Figs. 4 and 5). This indicates that the CmActin promoter may be useful for studies aimed at modifying phenotypic characters in plants. As the CmActin promoter induced constitutive expression of transgenes in Arabidopsis, we considered that the constitutive induction of target genes may be due to the presence of specific promoter elements. Therefore, we analyzed the promoter sequence of the CmActin gene to determine whether it contains potential cisacting regulatory elements (Fig. 1). This analysis revealed that the CmActin promoter contains a TATA box element necessary for accurately initiating basal transcription in promoters of other plants. In addition, four CAAT enhancer elements were identified in the promoter, which represent binding sites for CAAT enhancer binding proteins (Clarke et al., 1997). The Skn-1 motif is required for high levels of endosperm expression (Washida et al., 1999). This motif was found in the ChAcin promoter region and may modulate gene expression in the endosperm of seeds. Furthermore, this promoter contains several putative hormone- and stress-related cis-elements, including ARE, GARE, LTR, the TGA motif, and the TGACG motif. These findings suggest that the CmActin promoter may regulate gene expression under control of various cis-acting regulatory elements within the promoter as well as corresponding
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trans-acting factors and may be controlled by complex regulatory mechanisms that respond to developmental and environmental cues (Hong and Hwang, 2009; Hwang et al., 2010). Although our data do not indicate whether the putative cis-acting elements directly or indirectly influence gene expression, they do indicate that the promoter is capable of driving high and constitutive expression of transgenes. In addition, the CmActin promoter may confer similar regulation of gene expression in other plants and could be a valuable new tool for plant genetic engineering. However, the detailed function of the CmActin promoter remains to be elucidated, so further experiments are required to define specific elements responsible for conferring constitutive expression. 5. Conclusions In conclusion, we identified and characterized the actin promoter region from chrysanthemum. The 1.43 kb promoter region of the chrysanthemum actin gene directed constitutive expression of a reporter gene during all developmental stages and various tissues except seeds. Moreover, target gene expression driven by the CmActin promoter induced phenotypic characters similar to those of the 35S promoter in transgenic plants. Taken together, these findings suggest that the CmActin gene promoter is an actively functional promoter that confers high-level constitutive expression and may be a useful tool for modulating the production of biomass, quality, and plant form via genetic engineering in floriculture and other plant species. Acknowledgments This work was supported by a grant from the National Institute of Agricultural Sciences (PJ010881), the Rural Development Administration, Republic of Korea. J.K. Hong was supported by a 2016 Post Doctoral Course Program from the National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2016. 08.006. References Aida, R., Ohira, K., Tanaka, Y., Yoshida, K., Kishimoto, S., Shibata, M., Ohmiya, A., 2004. Efficient transgene expression in chrysanthemum, Dendranthema grandiflorum (Ramat.) Kitamura, by using the promoter of a gene for chrysanthemum chlorophyll a/b binding protein. Breed. Sci. 54, 51–58. Benfey, P.N., Chua, N.-H., 1990. The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250, 959–966. Burge, C.B., Karlin, S., 1998. Finding the genes in genomic DNA. Curr. Opin. Struct. Biol. 8, 346–354. Chen, Z., Wang, J., Ye, M.-X., Li, H., Ji, L.-X., Li, Y., Cui, D.-Q., Liu, J.-M., An, X.-M., 2013. A novel moderate constitutive promoter derived from Poplar (Populus tomentosa Carrière). Int. J. Mol. Sci. 14, 6187–6204. Clarke, S.L., Robinson, C.E., Gimble, J.M., 1997. CAAT/Enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor ␥2 promoter. Biochem. Biophys. Res. Commun. 240, 99–103. Cornejo, M.J., Luth, D., Blankenship, K.M., Anderson, O.D., Blechl, A.E., 1993. Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol. Biol. 23, 567–581. Dong, Y., von Arnim, A.G., 2003. Novel plant activation-tagging vectors designed to minimize 35S enhancer-mediated gene silencing. Plant Mol. Biol. Rep. 21, 349–358. Foster, E., Hattori, J., Labbe, H., Ouellet, T., Foster, P.R., James, L.E., Lyer, V.N., Miki, B.L., 1999. A tobacco crypic constitutive promoter, tCUP, revealed by T-DNA tagging. Plant Mol. Biol. 41, 45–55. Garbarino, J.E., Oosumi, T., Belknap, W.R., 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109, 1371–1378. He, C., Lin, Z., McElroy, D., Wu, R., 2009. Identification of a rice Actin2 gene regulatory region for high-level expression of transgenes in monocots. Plant Biotechnol. J. 7, 227–239.
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