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Cloning, Characterization and Expression Analysis of GbWRKY11, a Novel Transcription Factor Gene in Ginkgo biloba.

Byline: Yong Ling Liao, Feng Xu, Wei Wei Zang, Shui Yuan Cheng, Yong Bao Shen and Jie Chang


WRKY transcription factors are involved in diverse life processes in plants. Thus far, these proteins have not been reported in Ginkgo biloba. In this study, we cloned GbWRKY11, a novel WRKY transcription factor gene, from G. biloba. The cDNA of GbWRKY11 is 1,707 bp in length and encodes a putative protein of 402 amino acids. The GbWRKY11 protein has an estimated molecular weight of 43.64 kDa and belongs to the WRKY IId group. The genomic DNA of GbWRKY11 contains two introns and three exons. GbWRKY11 gene is ubiquitously expressed in all tested tissues but preferentially expressed in female flowers. The GbWRKY11 transcript is upregulated in response to salicylic acid, ethephon, and abscisic acid but repressed by methyl jasmonate, salinity, cold and heat. Thus, GbWRKY11, a newly identified G. biloba WRKY transcription factor, is apparently involved in multiple signaling pathways in response to abiotic stresses. This study provides a basis for further research on the function of GbWRKY11.

Keywords: GbWRKY11; Ginkgo biloba; Transcription factor; Abiotic stress


Ginkgo biloba, usually referred to as a "living fossil," is the sole surviving species of the previously large plant division Ginkgophyta (Van Beek and Montoro, 2009). G. biloba, which experienced million years of complicated climate conditions, not only shows strong adaptability but also minor morphological changes; this characteristic could be due to the ability of G. biloba to adapt to the environment (Deng et al., 2006). G. biloba responds to environmental signals by modulating gene expression, regulating protein content or activity, altering metabolite levels, and changing the homeostasis of ions (Eyidogan et al., 2012). Thus, G. biloba could be a suitable model plant for studies on regulating a range of abiotic and biotic stresses.

A crucial aspect of plant responses to all types of stresses is the transcriptional control of defense-related gene expression (Singh et al., 2002). Transcription factors, including MYB, ethylene response factor, WRKY proteins, basic-domain leucine zipper, and other zinc finger factors, regulate the expression of diverse stress-responsive genes (Carlberg and Molnar, 2014).

The WRKY transcription factors are characterized by their DNA-binding domain of about 60 amino acids. The WRKY domain is composed of a conserved WRKYGQK peptide at the N-terminal and a typical zinc finger motif at the C-terminal, with the zinc finger structure as either C2H2 (CX4-5CX22-23HX1H) or C2HC (CX7CX23HX1C). The WRKYGQK motif and the C2H2 or C2HC zinc finger are essential for DNA-protein interaction (Llorca et al., 2014).

The WRKY protein contains one or two copies of the WRKY domain. These transcription factors are classified into three subgroups based on the number of WRKY domain and the structure of zinc finger. Group I transcription factors contain two WRKY domains and a C2H2 zinc finger motif. Groups II and III contain only one WRKY domain, with zinc finger motifs of C2H2 and C2HC, respectively (Eulgem et al., 2000). Group II WRKY transcription factors are further categorized into five small subgroups (IIa, IIb, IIc, IId, and IIe) based on their amino acid sequences (Rushton et al., 2010).

WRKY transcription factors mainly exist in plants and regulate multiple biological processes. These transcription factors are assumed to regulate multiple stress-responsive genes by interacting with the W box (TTGACC/T) in the promoters of their target genes. For example, single gene interference or co-silencing of NaWRKY3 and NaWRKY6 promotes the susceptibility of tobacco to herbivore damage by reducing the accumulation of volatile sesquiterpene and jasmonate (Skibbe et al., 2008). In Arabidopsis thaliana, overexpression of AtWRKY3 and AtWRKY4 improves the resistance of the plant to fungal diseases caused by Botrytis (Lai et al., 2008). Moreover, the expression of WRKY genes can be induced by drought, wounding, and plant hormones and triggers signal transduction cascades (Banerjee and Roychoudhury, 2015). The tolerance of rice to high temperatures and drought can be enhanced by overexpressing OsWRKY11 (Wu et al., 2009).

In Musa spp., the expression level of MusaWRKY71 is upregulated after the plant suffers from cold, drought, salt, ABA, H2O2, ethephon (ETH), salicylic acid (SA), and methyl jasmonate (MeJA) stresses (Shekhawat et al., 2011).

Several gene studies have implicated WRKY proteins in plant secondary metabolism. Kato et al. (2007) isolated and identified the transcription factor CjWRKY1 from Coptis japonica and found a positive correlation between the expression of this gene and all the examined berberine biosynthesis genes, indicating that CjWRKY1 is involved in berberine biosynthesis. Ma et al. (2009) also isolated AaWRKY1 from the glandular secretory trichomes of Artemisia annua, in which artemisinin is synthesized and sequestered. AaWRKY1 activates the expression of the majority of artemisinin biosynthesis genes, thereby suggesting that AaWRKY1 is a necessary positive regulator in artemisinin biosynthesis. Recently, Li et al. (2013) have reported that TcWRKY1 regulates taxol biosynthesis in Taxus chinensis.

Thus far, no WRKY genes have been isolated and characterized from G. biloba because of limited sequence information. We previously performed transcriptome analysis of G. biloba by using Illumina HiSeqTM2500 sequencing platform. The dataset provides abundant information on the sequences of transcription factor genes, which can be used for full-length cDNA cloning and functional studies. In the present study, we cloned and characterized GbWRKY11, a novel WRKY transcription factor gene, from G. biloba. GbWRKY11 is preferentially expressed in female flowers and induced by phytohormones, such as SA, ETH, and ABA, but repressed by MeJA, salinity, cold, and heat. This work is the first to report a WRKY transcription factor in G. biloba.

Materials and Methods

Plant Material and Stress Treatments

G. biloba grafts of 14 years old were grown in Botanical Garden of Yangtze University, China. In order to study the tissue expression patterns of GbWRKY11, the leaves, stems, roots, male and female flowers of G. biloba graft were collected for RNA extraction.

The cultured callus, initiated from mature zygotic embryos of G. biloba, were cultured on liquid MS basal medium supplementing with 2 mg/L 6-benzyladenine (6-BA) and 1.5 mg/L naphthaleneacetic acid (NAA) on a rotary shaker at 100 rpm, in the light and at 25 1C. The suspension cultures were subcultured every 2 weeks and after four subcultures the differential was omitted. In the experiments for testing induction by various elicitors, the callus were dipped into the treatment such as 100 umol/L methyl jasmonate (MeJA), 100 umol/L abscisic acid (ABA), 100 umol/L salicylic acid (SA), 40 umol/L ethephon (ETH) and 200 mmol/L sodium chloride (NaCl), respectively, using the callus without any treatment as control. The cold and heat treatments were applied by placing the callus lines in a 4C and 40C rotary shaker while the control in a 25C growth room.

The callus samples were harvested 0, 3, 6, 12, 24, 48 and 72 h after treatment and immediately frozen in liquid nitrogen, and kept at -80C prior to total RNA extraction.

RNA and DNA Extraction

Total RNA was extracted from different organs and callus of all the treatments using the CTAB method (Cai et al., 2007; Liao et al., 2004). Genomic DNA was extracted from the leaves of ginkgo grafts following the CTAB method (Xu et al., 2008).The quantity and quality of DNA and RNA were detected by spectrophotometer analysis and agarose gel electrophoresis.

Cloning of cDNA and Genomic DNA of GbWRKY11

First-strand cDNA was synthesized using PrimeScriptTM 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China) according to the instruction book. Using a pair of GbWRKY11-specific primers WRKY11-FP (5'-GGAGGAGAATGACGAAGAGTGC-3') and WRKY11-RP (5'-GTCTTTCTGCTCCTACATTTGATT-3'), the cDNA and genomic DNA of GbWRKY11 containing full open reading frame were amplified using the first-strand cDNA and genomic DNA as the temple, respectively. The PCR product was purified and cloned into pMD19-T vector (TaKaRa, Dalian, China), followed by sequencing for confirmation.

GbWRKY11 Transcript Analysis by Real-time PCR (qRT-PCR)

The RNA samples were reversely transcribed using the PrimeScript RT-PCR kit (TaKaRa, Dalian, China). The housekeeping gene, namely glyceraldehyde-3-phospate dehydrogenase gene (GbGAPDH, L26924), was used as a reference gene with primers of GAPDH-U (5'-GGTGCCAAAAAGGTGGTCAT-3') and GAPDH-D (5'-CAACAACGAACATGGGAGCAT-3'). Gene-specific primers of WRKY11-U (5'-TGTAAGAGGTTGCCCTGCTAGA-3') and WRKY11-D (5'-TGGTTATGTTCTCCCTCGTATGT-3') for GbWRKY11. The experiments were repeated independently with three biological replicates using SYBR(r) Premix Ex Taq II (TaKaRa, Dalian, China) in the Applied Biosystems 7500 Realtime PCR systems (Foster City, CA, USA). Relative expression level was calculated using the method described by Livak and Schmittgen (2001).

Bioinformatic Analysis

The sequence was analyzed by the bioinformatic software on websites ( and Plant WRKY protein sequences were retrieved from NCBI GenBank. Sequence alignment was performed using ClustalX 2.0 and phylogenetic tree was constructed by neighbor-joining method using MEGA version 5. A bootstrap statistical analysis was carried out with 1000 replicates.


Cloning and Sequence Analysis of GbWRKY11

The WRKY domain (PF03106) was used to search for the transcriptome data of G. biloba. We obtained a sequence with high similarity to the known WRKY transcription factors of other plants. Using a pair of specific primers based on this sequence, we performed PCR using cDNA as the template and a 1,707 bp GbWRKY11 clone was obtained. Sequence analysis revealed that GbWRKY11 cDNA contained 1,209 bp-length open reading frame, as well as 201 and 297 bp long 5' and 3' untranslated regions, respectively. We compared the identities of this transcription factor with the nucleotide sequences of other plants in the NCBI database and found 92, 88, 82 and 83% similarities to PmWRKY104 in Pinus monticola, GhWRKY61 in Gossypium hirsutum, BrWRKY15 in Brassica rapa, and AtWRKY7 in A. thaliana, respectively.

The specific primers WRKY11-FP and WRKY11-RP were synthesized using genomic DNA as template and used for PCR to obtain the genomic DNA of GbWRKY11. The amplification product is 2,794 bp long (Fig. 1). Comparison of the cDNA and DNA sequences revealed that the genomic DNA of GbWRKY11 contains two introns with lengths of 258 and 829 bp. The two introns contain the conserved GT/AG splicing site and high AT (67.0% of intron 1 and 68.2% of intron 2), which are typical characteristics of plant introns. In addition, both introns are of R-type and their insertion position was found in the codon of arginine between the nucleotide AG and A (Wu et al., 2005). The second intron interrupted the encoded region of the WRKY domain, and this feature is common in Arabidopsis WRKY (Eulgem et al., 2000). These results demonstrated that the typical intron splicing site may play a crucial role in evolution.

Characterization and Phylogenetic Analysis of the GbWRKY11 Protein

The GbWRKY11 protein encodes a 402 amino acid residue, harbors one WRKY domain containing 60 amino acids, and presents one C2H2-type zinc-finger motif.

Hence, GbWRKY11 belongs to the group II of the WRKY transcription factor family (Rushton et al., 2010). The molecular weight of the GbWRKY11 protein is 43.64 kDa, and the theoretical isoelectric point is 9.19. The GbWRKY11 protein shared 66%, 67%, 68%, 76% and 77% identities to GsWRKY11 of Glycine soja, GhWRKY11-1 of G. hirsutum, VpWRKY11 of Vitis pseudoreticulata, EgWRKY11 of Elaeis guineensis, and PdWRKY11 of Phoenix dactylifera, respectively. As this G. biloba WRKY is closely correlated with WRKY11, we named the protein as GbWRKY11 (GenBank accession number: KP987206).

Sequence analysis showed that the predicted GbWRKY11 protein contains two putative nuclear localization signal (NLS) sequences (83KKVVSLLSRTGHARFRR100 and 306KRRK310, Fig. 2). Hence, GbWRKY11 is likely to localize to the nucleus.

GbWRKY11 showed high protein sequence identities (41%-48%) to Group IId WRKY proteins, including GhWRKY32 of G. hirsutum, JcWRKY42 of Jatropha curcas, PqWRKY1 of Panax quinquefolius, NbWRKY17 of Nicotiana benthamiana, PsWRKY46 of Papaver somniferum, and NnWRKY7 of Nelumbo nucifera. As shown in Fig. 2, the WRKY domain and nuclear localization sites were strictly conserved but litter sequence conservation existed outside these regions.

A phylogenetic tree was constructed with 18 WRKY proteins, including GbWRKY11 and other WRKY transcription factors. The results showed that GbWRKY11 was closely related to PsWRKY of Picea sitchensis, AtWRKY11 of A. thaliana, NnWRKY7 of N. nucifera, and PqWTKY1 of P. quinquefolius. Thus, GbWRKY11 was clustered in the group IId of the WRKY family (Fig. 3).

Expression Profile of GbWRKY11 in Different Tissues

Some studies revealed that WRKY transcripts were present and exhibited varied expression levels in all tissues (Zheng et al., 2011; Wang et al., 2013; Song and Nan, 2014; Yang et al., 2015). As such, GbWRKY11 expression patterns in tissues were systematically analyzed through qRT-PCR method. As shown in Fig. 4, GbWRKY11 transcripts were detected in all tissues but their expression levels varied in tissues of different organs. The highest gene expression level was observed in the female flowers, whereas the lowest level was detected in the roots and male flowers. The expression level of GbWRKY11 was significantly higher in the stems than that in the leaves but still lower than that in the female flowers.

Expression Patterns of GbWRKY11 under Abiotic Stress

The callus lines of Ginkgo were subjected to cold (4C), heat (40C), and salt (NaCl) treatments to investigate the expression of GbWRKY11 under various abiotic stresses. The results are shown in Fig. 5. The transcript level of GbWRKY11 minimally changed in response to salt stress at 3 h post treatment (hpt) and then sharply decreased thereafter (Fig. 5A). Cold stress gradually reduced the mRNA level of GbWRKY11 until the end of the experiment (Fig. 5B). Similarly, heat stress progressively repressed GbWRKY11 expression (Fig. 5C). These results showed that abiotic stresses, except salt stress, repressed GbWRKY11 expression. Thus, we deduced that GbWRKY11 can repress salt-, cold- and heat-response genes.

Effects of Signaling Molecules on GbWRKY11 Transcription

Signaling molecules, such as SA, MeJA, ABA, and ETH, can regulate the expression of defense-related genes in response to biotic and abiotic stresses (Bari and Jones, 2009). The expression levels of GbWRKY11 were analyzed through qRT-PCR in Ginkgo callus treated with SA, MeJA, ABA, and ETH to determine the involvement of the gene in signaling pathways. The expression of GbWRKY11 was rapidly upregulated in response to exogenous application of ETH at 3 hpt, reached the highest level at 12 hpt (8.8-fold relative to mock-treated control samples), and reduced thereafter until the end of the experiment (Fig. 6A). The transcript level of GbWRKY11 minimally changed in response to SA treatment at 3 hpt, sharply decreased between 6 and 12 hpt, reached the highest level at 24 hpt (6.3-fold relative to the mock-treated control samples), and then gradually decreased until the end of the experiment (Fig. 6B).

GbWRKY11 transcript levels were sharply enhanced in response to ABA treatment at 6 hpt, reached the highest level at 6 hpt (3.1-fold relative to mock-treated control samples), and then decreased until the completion of the treatment (Fig. 6C). Meanwhile, the transcript levels of GbWRKY11 gradually decreased in response to MeJA until the end of the experiment (Fig. 6D). These results show that GbWRKY11 may regulate the responses of G. biloba to SA, ETH, and ABA treatments. Basing from these results, we speculated that GbWRKY11 may play a crucial role in the phytohormone-regulated defense-related responses of G. biloba.


Numerous studies indicate that plant WRKYs play an intricate role in plant adaptation to various stresses (Banerjee and Roychoudhury, 2015). A large number of WRKY genes have been isolated and characterized in various plant species, such as in Arabidopsis (Eulgem et al., 2000), Zea mays (Wei et al., 2012), Gossypium (Dou et al., 2014), Medicago truncatula ( and Nan, 2014), Cucumis sativus (Ling et al., 2011), B. rapa (Tang et al., 2014), Vitis vinifera (Wang et al., 2014), Carica papaya (Pan and Jiang, 2014), Coffea arabica (Ramiro et al., 2010), Populus (Jiang et al., 2014), Hevea brasiliensis (Li et al., 2014), Coptis japonica (Kato et al., 2007), and Catharanthus roseus (Yang et al., 2013). However, knowledge on WRKYs of G. biloba remains limited. WRKY is a large family of transcription factors, most of which have not yet been elucidated, particularly in non-model plants.

In this study, we isolated GbWRKY11, a novel WRKY gene, from G. biloba. Phylogenetic analysis results demonstrated that GbWRKY11 belongs to the group IId of the WRKY transcription factor family. An increasing number of transcription factors belonging to group IId WRKY have been isolated and identified from several plants. For example, PqWRKY1, which responds to MeJA, regulated osmotic stress responses and triterpene ginsenoside biosynthesis in P. quinquefolius (Sun et al., 2013). In Brassica rapa, BrWRKY7, which is the homolog of AtWRKY7, was induced 6 h after SA treatment (Kim et al., 2008). Further research revealed that all Arabidopsis WRKY group IId proteins, including AtWRKY11, contain the calmodulin-binding and RTGHARFRR[A/G]P domains (Park et al., 2005). AtWRKY11 is a negative regulator that resists Pseudomonas syringae pv. tomato and is involved in jasmonic acid (JA)-dependent responses (Journot-Catalino et al., 2006).

Although these two domains were not found in GbWRKY11, the possibility that AtWRKY11 and GbWRKY11 exhibit similar functions cannot be excluded. Two predicted NLS sequences were also uncovered using the PSORT program, which implied that GbWRKY11 may function in the nucleus.

GbWRKY11 is highly expressed in female flowers and stems. The expression pattern of GbWRKY11 is consistent with that of HbWRKY1, a negative transcription regulator of small rubber particle protein, which is involved in biosynthesis of natural rubber in H. brasiliensis (Wang et al., 2013). This result implied that GbWRKY11 may play a crucial role in the growth of the female flower and stems in G. biloba.

The expression of WRKY proteins, which regulate diverse developmental and defense-related responses, can be activated by a series of signaling molecules, such as ETH, SA, MeJA, and ABA (Jiang and Yu, 2015). The current study revealed that GbWRKY11 expression was induced by ETH, SA, and ABA but was repressed by MeJA. Increasing studies have demonstrated that WRKY genes play vital roles in both JA- and SA-mediated signaling pathways. For example, in Capsicum, CaWRKY30 expression was rapidly upregulated by SA treatment but downregulated by MeJA (Zheng et al., 2011).

In Helianthus, the expression of HaWRKY3 and HaWRKY76 was significantly upregulated by SA but downregulated by MeJA (Giacomelli et al., 2010). Similarly, AtWRKY70 expression in Arabidopsis was induced by SA but repressed by MeJA (Li et al., 2004). Consistent with these findings, GbWRKY11 expression was also upregulated by SA and downregulated by MeJA, suggesting that GbWRKY11 may play similar roles to that of HaWRKY3, HaWRKY76, CaWRKY30, or AtWRKY70. Previous studies have demonstrated that WRKY proteins may act as activators in ABA signaling (Antoni et al., 2011). For example, in Larrea tridentate, LtWRKY21 is an activator that controls the expression of ABA-regulated genes (Zou et al., 2007). Sun et al. (2014) also reported that 13 numbers of WRKY family in O. sativa were upregulated by ABA. Pathways involving MeJA and ET are considered to be mainly effective against necrotrophic pathogens, insects, and wounding (Kunkel and Brooks, 2002).

Thus, little attack of pathogens and insects in ginkgo may be due that WRKY protein, such as GbWRKY11, played important roles in defense responses by mediating MeJA and ET signaling. However, the molecular mechanism of GbWRKY11 in response to signaling molecules requires further study.


As GbWRKY11 expression was upregulated by SA, ETH, and ABA and repressed by MeJA, we infer that GbWRKY11 regulates defense-related signaling pathways. We established the binary expression vector for overexpression of GbWRKY11 for further studies. Further research on the genetic transformation of this gene in G. biloba callus should be performed. The present study provides a basis to elucidate whether the upregulated gene expression can enhance the ability of plants to resist abiotic stresses.


This work was supported by the National Natural Science Foundation of China (31370680 and 31400603), the Natural Science Foundation of Hubei Province (2013CFA039), Key Project of Chinese Ministry of Education (212112), Foundation for Innovative Research Groups of Natural Science of Hubei Province (2011CDA117), International Science and Technology Cooperation Project of Hubei Province (2013BHE029 and 2013BHE039), and Innovative Research Project of Ordinary University Graduate Student of China Jiangsu Province (2013CX2213-0551).


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Author:Liao, Yong Ling; Xu, Feng; Zang, Wei Wei; Cheng, Shui Yuan; Shen, Yong Bao; Chang, Jie
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Feb 29, 2016
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