# Isolation, Expression, and Promoter Analysis ofGbWRKY2: A Novel Transcription Factor Gene from Ginkgo biloba

Authors: Yong-Ling Liao; Yong-Bao Shen; Jie Chang; Wei-Wei Zhang; Shui-Yuan Cheng; Feng Xu
Journal: International Journal of Genomics
 (2015)

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## Abstract

WRKY transcription factor is involved in multiple life activities including plant growth and development as well as biotic and abiotic responses. We identified 28WRKY genes from transcriptome data of Ginkgo biloba according to conserved WRKY domains and zinc finger structure and selected three WRKY genes, which are GbWRKY2, GbWRKY16, and GbWRKY21, for expression pattern analysis. GbWRKY2 was preferentially expressed in flowers and strongly induced by methyl jasmonate. Here, we cloned the full-length cDNA and genomic DNA of GbWRKY2. The full-length cDNA of GbWRKY2 was 1,713 bp containing a 1,014 bp open reading frame encoding a polypeptide of 337 amino acids. The GbWRKY2 genomic DNA had one intron and two exons. The deduced GbWRKY2 contained one WRKY domain and one zinc finger motif. GbWRKY2 was classified into Group II WRKYs. Southern blot analysis revealed that GbWRKY2 was a single copy gene in G. biloba. Many cis-acting elements related to hormone and stress responses were identified in the 1,363 bp-length 5′-flanking sequence of GbWRKY2, including W-box, ABRE-motif, MYBCOREs, and PYRIMIDINE-boxes, revealing the molecular mechanism of upregulated expression of GbWRKY2 by hormone and stress treatments. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter.


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## Body

## 1. Introduction

Ginkgo biloba is the oldest relic plant among extant seed-bearing plants, usually referred to as a “living fossil” [1]. Experiencing million years of complicated climate,G. biloba not only shows strong adaptability but also exhibits minor changes in morphology. This phenomenon is possible becauseG. biloba can adapt to changing environments and tolerate harsh conditions [2]. Resistance genes encoding the enzymes in reactive oxygen-scavenging systems, including chloroplast copper/zinc-superoxide dismutase (GbCuZnSOD) [3], iron superoxide dismutase (GbFeSOD) [4], and manganese superoxide dismutase (GbMnSOD) [5], catalase (GbCAT1) [6], ascorbate peroxidases (GbAPX) [7], peroxidase (GbPOD1) [8], as well as dehydrin (GbDHN) [2], andGbASR [9] response to abscisic acid (ABA), stress, and ripening have been cloned fromG. biloba. Studies on these genes help us to elucidate the molecular mechanism by whichG. biloba tolerates severe conditions. Furthermore, stress conditions, such as drought, severe cold, harm, high temperature, and heavy metals, can stimulate accumulation and release of secondary metabolites of flavonoids and terpene lactones fromG. biloba. Flavonoids and terpene lactones play an important role in improving self-protection, promoting competitive capacity, and coordinating interaction with the environment.The WRKY family is among the ten largest families of transcription factor (TF) in higher plants and is found throughout the green lineage [10]. The defining feature of WRKY TFs is their DNA binding domain. This is called the WRKY domain containing the conserved WRKYGQK sequence at the N terminal, and as well as containing the WRKY signature it also has a typical zinc finger motif at the C terminal. The zinc finger structure is either C2H2 (CX4-5CX22-23HX1H) or C2HC (CX7CX23HX1C). Based on the number of the WRKY domains and the characteristic of a zinc finger motif, WRKY TFs can be divided into three groups: the first group contains two WRKY domains; the second group and the third group contain one WRKY domain each. The zinc finger motif of the second class is C2H2, which is the same as that of the first group; the zinc finger motif of the third group is C2HC [11].The WRKY domain binds to what is called W-box (TTGACC/T) in the promoters of target genes. This sequence is the minimal core element necessary for binding of a WRKY protein to DNA [12]. W-box is present in the promoter of many genes related to disease resistance, damage, growth, aging, and WRKY gene, which shows that the WRKY TF is closely related to biotic and abiotic stress responses [13]. Studies have successfully used the WRKY TF to improve the stress resistance of plants. For example, single or cosilence ofNaWRKY3 andNaWRKY6 can promote the susceptibility of tobacco to herbivore damage by weakening the accumulation of volatile sesquiterpene and jasmonate [14]. The resistance ofArabidopsis thaliana to the fungal diseaseBotrytis is also improved by overexpressingAtWRKY3 andAtWRKY4 [15]. The tolerance of rice to high temperature and drought can be improved by overexpressingOsWRKY11  [16]. The expression level ofMusaWRKY71 is improved after plants suffer from cold, drought, salt, ABA, H2O2, ethephon (ETH), salicylic acid (SA), and methyl jasmonate (MeJA) inMusa spp. [17]. The expression ofHbWRKY1 in latex is induced by ETH and MeJA, while the expression ofHbWRKY1 in the leaf is induced by drought and ABA fromHevea brasiliensis Müll. Arg. [18].The WRKY TF can also participate in the control of the secondary metabolism of plants except in plant defense against exogenous stress. For example, Kato et al. [19] isolated and identified theCjWRKY1 TF that controls the biosynthesis of berberine fromCoptis japonica. Ectopic expression ofCjWRKY1 cDNA inC.japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, indicating thatCjWRKY1 is a necessary positive regulator to control overall gene expression in berberine biosynthesis. Xu et al. [20] found thatGaWRKY1 gene, which encodes a protein containing a single WRKY domain, could combine with W-box in the promoter region of the key gene encoding (+)-δ-cadinene synthase to metabolize terpene and promote sesquiterpene biosynthesis. Ma et al. [21] also isolatedAaWRKY1 gene from glandular secretory trichomes ofArtemisia annua in which artemisinin is synthesized and sequestered. Transient expression ofAaWRKY1 cDNA inA. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes, suggesting the involvement of theAaWRKY1 TF in the regulation of artemisinin biosynthesis. Recently, Li et al. [22] and Sun et al. [23] demonstrated that aTcWRKY1 gene and a MeJA induciblePqWRKY1 gene participated in regulation of the biosynthesis of taxol inTaxus chinensis and triterpene ginsenoside inPanax quinquefolius, respectively.To date, the WRKY genes have been cloned from many species. A total of 5,936 WRKY genes are recorded in the database of plant TFs. These genes have also been reported in 83 species, such as algae, planus, needle-leaved plant, monocotyledons, and dicotyledons. However, no cloning and the WRKY gene inG. biloba has not been found. In this report, a total of 28 unigenes of WRKY transcription factor were identified using next-generation sequencing. Among these unigenes, a MeJA-inducibleWRKY gene, namedGbWRKY2, was cloned fromG.biloba using the rapid amplification of cDNA ends (RACE) method.GbWRKY2 is preferentially expressed in flowers and induced by salinity stress and phytohormones such as SA, ETH, MeJA, and ABA but repressed by heat. Promoter analysis provided the molecular mechanism of upregulation ofGbWRKY2 by phytohormones and abiotic stresses and allowed us to identify the region that can be considered as the minimal promoter. This work represents, to our knowledge, the first functional characterization of aG.biloba WRKY transcript factor.


## 2. Experimental Section

### 2.1. Plant Materials and Treatments

The 14-year-old grafts ofG. biloba were grown in an orchard at Yangtze University, China. For tissue expression ofGbWRKY2,GbWRKY16, andGbWRKY21, the roots, stems, leaves, and male and female flowers ofG. biloba grafts were collected, immediately frozen in liquid nitrogen, and kept at −80°C prior to total RNA extraction. The cultured callus lines, initiated from mature zygotic embryos ofG. biloba, were maintained on liquid MS basal medium supplementing with 1.5 mg/L naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA) on a rotary shaker at 100 rpm, in the light and at 25±1°C. The suspension cultures were subcultured every 2 weeks and after four subcultures the differential was omitted. In the experiments for investigating induction by various elicitors, the callus lines were dipped into the appropriate treatment such as 100 μmol L−1 MeJA, 100 μmol L−1 ABA, 100 μmol L−1 SA, 40 μmol L−1 ETH, and 200 mmol L−1 sodium chloride (NaCl), respectively, using ginkgo callus without any treatment as control. The cold and heat treatments were performed by placing the callus lines in a 4°C and 40°C growth room and the control in a 25°C growth room. The callus samples were harvested 0, 3, 6, 12, 24, 48, and 72 h after treatment and immediately frozen in liquid nitrogen, followed by storage at −80°C until use.


### 2.2. RNA and DNA Extraction

Total RNA was extracted from different tissues and all the treatments using CTAB method [42]. Genomic DNA was extracted from fresh leaves ofG. biloba grafts following the CTAB method described by Xu et al. [43]. The RNA and DNA quality and quantity were determined by agarose gel electrophoresis and spectrophotometer analysis before use.


### 2.3. RNA-Seq Library Construction for Illumina Sequencing and Identification of WRKY Transcription Factor Gene fromG. biloba Transcriptome

The extracted RNA was cleaned up using RNase-free DNaseI (Dalian TaKaRa, China) and purified using oligo-dT-attached magnetic beads. The purified mRNAs were cleaved into small pieces (200~500 bp) by super sonication. Cleaved mRNAs were used as templates to construct RNA-Seq library according to the manufacture’s protocol. The constructed libraries were purified by the AMPure beads and recovered from the low melting agarose (2%) at the length of about 300 base-pair by the Qiagen Nucleic acid purification kits (CA Qiagen, USA). Sequencing run was performed on Illumina HiSeq 2500 sequencer following the manufacturer’s protocols.To obtain the completeG. biloba WRKY gene family, the male and female flowers ofG. biloba transcriptome sequences were queried for 72A. thaliana WRKY protein sequences using TBLASTN searching program. All of the collectedG. biloba WRKY candidates were primarily analyzed using protein family database (Pfam) (http://pfam.xfam.org/) to confirm the presence of WRKY conserved domains on their protein structure.


### 2.4. Expression Analysis by Quantitative Real-Time PCR

Quantitative real-time PCR (qRT-PCR) was performed according to the instructions provided for the Applied Biosystems 7500 Real-Time PCR system (Foster City, CA, USA) and the SYBR Premix Ex Taq II (Dalian TaKaRa, China). The RNA samples were reversely transcribed using the PrimeScript RT-PCR kit (Dalian TaKaRa, China). Expression levels of WRKY genes inG. biloba were normalized using the glyceraldehyde-3-phosphate dehydrogenase (GbGAPDH, GenBank accession number L26924) as an internal reference gene. Based on the screened transcripts, three annotated WRKY transcripts (T2_Unigene_BMK.10348 namedGbWRKY2, T1_Unigene_BMK.13300 namedGbWRKY16, and T1_Unigene_BMK.18545 namedGbWRKY21) were selected for expression analysis. Primers were designed using the Sequence Detection System software and shown in Table 1. qRT-PCR was performed with 20 μL reaction consisting of 2x SYBR Premix Ex Ta 10 μL, 0.4 μL each primer and 50x ROX Reference Dye II, and 100 ng cDNA as the template. The thermal cycler conditions recommended by the manufacturer were used with first stage at 95°C for 30 s, and the second stage (40 cycles) at 95°C for 5 s followed by 60°C for 34 s, and the third stage at 95°C for 15 s followed by 60°C for 1 min and 95°C for 15 s. The amplification of the target genes was monitored every cycle by SYBR green fluorescence. The Ct (threshold cycle), defined as the real-time PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target gene. Three replicates of each experiment were performed, and data were analysed by the Livak method [44] and expressed as normalized relative expression level (2-ΔΔCT) of the respective genes in various samples. In each case, three technical replicates were performed for each of at least three independent biological replicates.Table 1
The primers used in this study.
Primers
Sequence (5′-3′)
Description
WRKY2-FP
CCCATAATCATCCTAAGCCTCTG
Forward primer forGbWRKY2 cDNA
WRKY2-RP
ATGGGACACTGTATCTTGAAGTTGAG
Reverse primer forGbWRKY2 cDNA
WRKY2-3
AGAAAGTAGTGAAAGGCAACCCTAACCCAA
Forward primer for 3′-RACE, outer
WRKY2-3N
CCATAGCACTGGCTGGTTCTCAACTTCAAG
Forward primer for 3′-RACE, nested
WRKY2-5
CAACTTTGATGACAGCACCGACGCC
Forward primer for 5′-RACE, outer
WRKY2-5N
CGGTTAGGCAGAGGCTTAGGATGATTA
Forward primer for 5′-RACE, nested
WRKY2-G1
ATGGGAGTTGGTGCTGCT
Forward primer forGbWRKY2 gDNA
WRKY2-G2
TCAATGAAAAGAGGTTTCAAAGC
Reverse primer forGbWRKY2 gDNA
WRKY2-SP1
CCCTTCCATCATCGTCGTCACTTGC
Reverse primer for 5′-promoter, outer
WRKY2-SP2
ACGCCTTCCGCCCTTTCTCCTGCTT
Reverse primer for 5′-promoter, nested
WRKY2-U
AGTGCCAGCAATCAGCGTT
Gene forward primer for qRT-PCR
WRKY2-D
AGCCCTTGATGGGTTTCTGT
Gene reverse primer for qRT-PCR
WRKY16-U
ATCAGTCCACAAGCAGACCTCA
Gene forward primer for qRT-PCR
WRKY16-D
TTGTCATTAGCAACACTGCCATC
Gene reverse primer for qRT-PCR
WRKY21-U
GAATAGTATGGGTTCCAGTAGGTCG
Gene forward primer for qRT-PCR
WRKY21-D
AACCTTTGATAGGCTTCTGTCCA
Gene reverse primer for qRT-PCR
GAPDH-U
GGTGCCAAAAAGGTGGTCAT
Gene forward primer for qRT-PCR
GAPDH-D
CAACAACGAACATGGGAGCAT
Gene reverse primer for qRT-PCR
WRKY2P-1363
AACTGCAGACTATAGGGCACGCGTGGTC
Forward primer for promoter deletion
WRKY2P-1018
AACTGCAGTTCTTACGAAAGTGGTGCTGCTA
Forward primer for promoter deletion
WRKY2P-668
AACTGCAGACGCATGTAGCCCTAAACCAAG
Forward primer for promoter deletion
WRKY2P-521
AACTGCAGTTTGCGGTGCTTGAGCTAATT
Forward primer for promoter deletion
WRKY2P-288
AACTGCAGTGTGGTGGATGCTACACCTGAG
Forward primer for promoter deletion
WRKY2P-137
AACTGCAGCATAATGCCCTGTTTCCTCCAA
Forward primer for promoter deletion
WRKY2P-48
AACTGCAGGCAGTGAGTATCCTCGGAGTTAT
Forward primer for promoter deletion
WRKY2-5UTR-F
AACTGCAGAACTAAGAAGAAGGTTGAACGGT
Forward primer for 5′-UTR
WRKY2P-anti
CGGGATCCGGACAATTCAAATGTGTGCACTT
Reverse primer for promoter deletion
WRKY2-5UTR-R
CGGGATCCTCGACGGTTAGGCAGAGGCTTA
Reverse primer for promoter deletion
FWR2PIN
TGAAGCAGGAGAAAGGGCGGAAGGCG
Forward primer for probe
RWR2PIN
CGCTGGTGCCCTTCCATCATCGTCG
Reverse primer for probe


### 2.5. Cloning of the Full-Length cDNA and Genomic DNA ofGbWRKY2

Two specific primers WRKY2-FP and WRKY2-RP (Table1) were designed and synthesized (Shanghai Sangon, China) based on the transcriptome data to obtain the internal fragment. One step RT-PCR was carried out and a fragment of 689 bp was obtained by using one step RT-PCR kit (Dalian TaKaRa, China) under the following program: 50°C for 30 min and 94°C for 3 min, followed by 35 cycles of amplification (94°C for 1 min, 54°C for 1 min, and 72°C for 1 min), and then followed by extension for 10 min at 72°C. The PCR product was purified and cloned into pMD18-T vector (Dalian TaKaRa, China) followed by sequencing. Subsequent BLAST results confirmed that amplified product was partial fragment of WRKY gene.Based on the sequence of the internal fragment ofGbWRKY2 gene, the specific primers WRKY2-3/WRKY2-5 and the nested primer WRKY2-3N/WRKY2-5N (Table 1) were designed to amplify the 3′ and 5′ end of theGbWRKY2 gene using the SMART RACE cDNA Amplification kit (Clontech, USA). The 3′-RACE-PCR and 5′-RACE-PCR were performed according to the manufacturer’s instructions. The PCR products were purified and cloned into pMD18-T vector for sequencing. After comparing and aligning the sequence of 3′-RACE, 5′-RACE, and the internal region products, the full-length cDNA sequence ofGbWRKY2 was obtained and verified through PCR amplification using 3′-Ready cDNA as the template and a pair of primers WRKY2-G1 and WRKY2-G2 (Table 1) under the following conditions: 94°C for 3 min, followed by 35 cycles of amplification (94°C for 20 s, 56°C for 30 s, and 72°C for 2 min). After sequencing, the full-length cDNA ofGbWRKY2 was subsequently analyzed for molecular characterization. Two gene-specific primers, WRKY2-G1 and WRKY2-G2, designed based on the cDNA sequence were used to amplify the genomic sequence ofGbWRKY2.


### 2.6. Amplification of the Promoter Region ofGbWRKY2

Ginkgo genome walker libraries were constructed using the Genome Walker Universal Kit User (Clontech, USA). To clone the promoter region ofGbWRKY2, two round PCR were performed using gene-specific primers (WRKY2-SP1 and WRKY2-SP2) that were designed according to the sequence ofGbWRKY2 cDNA and the adapter primers (AP1 and AP2) provided by the kit. Their sequences were shown in Table 1. After the nested PCR was carried out, amplified fragments were cloned and then sequenced. The sequences that extended upstream of the cDNA ofGbWRKY2 were isolated as the 5′-upstream region ofGbWRKY2 gene and used for further analysis.


### 2.7. Promoter Deletion-GUS Constructs and Agrobacterium-Mediated Transient Expression Assay

For construction of theGbWKRY2 promoter-driven GUS fusion genes, theGbWRKY2 promoter fragment-covering regions were amplified by PCR. Forward primers (Table 1) were designed to correspond to the −1363, −1018, −721, −668, −521, −288, −137, and −48 ofGbWRKY2 promoter and reverse primers, WRKY2P-anti and WRKY2-5UTR-R, were complementary to the 3′-end sequence ofGbWRKY2 promoter and 5′-UTR region, respectively. In addition, a supplementary fragment was amplified using primers WRKY2-1363 and WRKY2P-anti lacking the 5′-UTR region ofGbWRKY2. Each fragment was digested withPstI/BamHI and subcloned intoPstI/BamHI-digested pBI121 to generate seven promoter deletion derivatives. The promoterless construct (pBI121) was used as negative control. All constructs were verified by sequence analysis. Each promoter-GUS fusion construct was introduced intoA.tumefaciens LBA4404 via electroporation.Agrobacterium-mediated transient expression assays were performed according to the method of An et al. [45]. Agrobacterium-mediated transient expression was conducted on fully expanded tobacco leaves, and the intercellular spaces of the intact leaves were infiltrated with the bacterial suspension. After agroinfiltration, tobacco plants were maintained in a moist chamber at 24°C for 48 h. The GUS activity was examined in the tobacco leaves coinfiltrated withAgrobacterium strains harboring differentGbWRKY2 promoter-GUS fusion constructs.


### 2.8. GUS Activity Assay

The seedlings of tobacco transformant were ground in liquid nitrogen. The crude proteins were extracted with 50 mM phosphate buffer, pH 7.0, containing 10 mMβ-mercaptoethanol, 10 mM EDTA, and 0.1% Trition X-100. Homogenates were cleared by centrifugation, and the supernatants were assayed by Bradford assay for total protein (DU700, Beckman, Germany). GUS activity was measured as described by Jefferson et al. [46].


### 2.9. Southern Blot Analysis

Aliquots of DNA (30μg/sample) were digested overnight at 37°C withBamHI andPstI, respectively, which does not cut within the probe region, fractionated by 0.85% agarose gel electrophoresis and transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia, UK). The 283-bp probe was generated by PCR using genomic sequence ofGbWRKY2 including intron as template with primers FWR2PIN and RWR2PIN (Table 1). Probe labeling (biotin), hybridization, and signal detection were performed using Gene Images random prime labeling module and CDP-Star detection module following the manufacturer’s instructions (Amersham Pharmacia, UK). The film was washed under stringent conditions (60°C) and signals were visualized by exposure to Fuji X-ray film at room temperature for 1.5 h.


### 2.10. Bioinformatic Analysis and Molecular Evolution Analyses

The obtained sequences were analyzed using bioinformatic tools at websites (http://www.ncbi.nlm.nih.gov and http://www.expasy.org/). The software vector NTI Suite 10 was used for sequence multialignment. The isolated 5′-upstream sequence was analyzed for the putativecis-acting regulatory elements using the PLACE (http://www.dna.affrc.go.jp/PLACE/) and the Signal Scan Program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database. Phylogenetic tree analysis ofGbWRKY2 and known WRKYs from other plant species retrieved from GenBank were aligned with CLUSTAL W. Subsequently, a phylogenetic tree was constructed by neighbor-joining (NJ) method. The reliability of the tree was measured by bootstrap analysis with 1000 replicates.


## 2.1. Plant Materials and Treatments

The 14-year-old grafts ofG. biloba were grown in an orchard at Yangtze University, China. For tissue expression ofGbWRKY2,GbWRKY16, andGbWRKY21, the roots, stems, leaves, and male and female flowers ofG. biloba grafts were collected, immediately frozen in liquid nitrogen, and kept at −80°C prior to total RNA extraction. The cultured callus lines, initiated from mature zygotic embryos ofG. biloba, were maintained on liquid MS basal medium supplementing with 1.5 mg/L naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA) on a rotary shaker at 100 rpm, in the light and at 25±1°C. The suspension cultures were subcultured every 2 weeks and after four subcultures the differential was omitted. In the experiments for investigating induction by various elicitors, the callus lines were dipped into the appropriate treatment such as 100 μmol L−1 MeJA, 100 μmol L−1 ABA, 100 μmol L−1 SA, 40 μmol L−1 ETH, and 200 mmol L−1 sodium chloride (NaCl), respectively, using ginkgo callus without any treatment as control. The cold and heat treatments were performed by placing the callus lines in a 4°C and 40°C growth room and the control in a 25°C growth room. The callus samples were harvested 0, 3, 6, 12, 24, 48, and 72 h after treatment and immediately frozen in liquid nitrogen, followed by storage at −80°C until use.


## 2.2. RNA and DNA Extraction

Total RNA was extracted from different tissues and all the treatments using CTAB method [42]. Genomic DNA was extracted from fresh leaves ofG. biloba grafts following the CTAB method described by Xu et al. [43]. The RNA and DNA quality and quantity were determined by agarose gel electrophoresis and spectrophotometer analysis before use.


## 2.3. RNA-Seq Library Construction for Illumina Sequencing and Identification of WRKY Transcription Factor Gene fromG. biloba Transcriptome

The extracted RNA was cleaned up using RNase-free DNaseI (Dalian TaKaRa, China) and purified using oligo-dT-attached magnetic beads. The purified mRNAs were cleaved into small pieces (200~500 bp) by super sonication. Cleaved mRNAs were used as templates to construct RNA-Seq library according to the manufacture’s protocol. The constructed libraries were purified by the AMPure beads and recovered from the low melting agarose (2%) at the length of about 300 base-pair by the Qiagen Nucleic acid purification kits (CA Qiagen, USA). Sequencing run was performed on Illumina HiSeq 2500 sequencer following the manufacturer’s protocols.To obtain the completeG. biloba WRKY gene family, the male and female flowers ofG. biloba transcriptome sequences were queried for 72A. thaliana WRKY protein sequences using TBLASTN searching program. All of the collectedG. biloba WRKY candidates were primarily analyzed using protein family database (Pfam) (http://pfam.xfam.org/) to confirm the presence of WRKY conserved domains on their protein structure.


## 2.4. Expression Analysis by Quantitative Real-Time PCR

Quantitative real-time PCR (qRT-PCR) was performed according to the instructions provided for the Applied Biosystems 7500 Real-Time PCR system (Foster City, CA, USA) and the SYBR Premix Ex Taq II (Dalian TaKaRa, China). The RNA samples were reversely transcribed using the PrimeScript RT-PCR kit (Dalian TaKaRa, China). Expression levels of WRKY genes inG. biloba were normalized using the glyceraldehyde-3-phosphate dehydrogenase (GbGAPDH, GenBank accession number L26924) as an internal reference gene. Based on the screened transcripts, three annotated WRKY transcripts (T2_Unigene_BMK.10348 namedGbWRKY2, T1_Unigene_BMK.13300 namedGbWRKY16, and T1_Unigene_BMK.18545 namedGbWRKY21) were selected for expression analysis. Primers were designed using the Sequence Detection System software and shown in Table 1. qRT-PCR was performed with 20 μL reaction consisting of 2x SYBR Premix Ex Ta 10 μL, 0.4 μL each primer and 50x ROX Reference Dye II, and 100 ng cDNA as the template. The thermal cycler conditions recommended by the manufacturer were used with first stage at 95°C for 30 s, and the second stage (40 cycles) at 95°C for 5 s followed by 60°C for 34 s, and the third stage at 95°C for 15 s followed by 60°C for 1 min and 95°C for 15 s. The amplification of the target genes was monitored every cycle by SYBR green fluorescence. The Ct (threshold cycle), defined as the real-time PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target gene. Three replicates of each experiment were performed, and data were analysed by the Livak method [44] and expressed as normalized relative expression level (2-ΔΔCT) of the respective genes in various samples. In each case, three technical replicates were performed for each of at least three independent biological replicates.Table 1
The primers used in this study.
Primers
Sequence (5′-3′)
Description
WRKY2-FP
CCCATAATCATCCTAAGCCTCTG
Forward primer forGbWRKY2 cDNA
WRKY2-RP
ATGGGACACTGTATCTTGAAGTTGAG
Reverse primer forGbWRKY2 cDNA
WRKY2-3
AGAAAGTAGTGAAAGGCAACCCTAACCCAA
Forward primer for 3′-RACE, outer
WRKY2-3N
CCATAGCACTGGCTGGTTCTCAACTTCAAG
Forward primer for 3′-RACE, nested
WRKY2-5
CAACTTTGATGACAGCACCGACGCC
Forward primer for 5′-RACE, outer
WRKY2-5N
CGGTTAGGCAGAGGCTTAGGATGATTA
Forward primer for 5′-RACE, nested
WRKY2-G1
ATGGGAGTTGGTGCTGCT
Forward primer forGbWRKY2 gDNA
WRKY2-G2
TCAATGAAAAGAGGTTTCAAAGC
Reverse primer forGbWRKY2 gDNA
WRKY2-SP1
CCCTTCCATCATCGTCGTCACTTGC
Reverse primer for 5′-promoter, outer
WRKY2-SP2
ACGCCTTCCGCCCTTTCTCCTGCTT
Reverse primer for 5′-promoter, nested
WRKY2-U
AGTGCCAGCAATCAGCGTT
Gene forward primer for qRT-PCR
WRKY2-D
AGCCCTTGATGGGTTTCTGT
Gene reverse primer for qRT-PCR
WRKY16-U
ATCAGTCCACAAGCAGACCTCA
Gene forward primer for qRT-PCR
WRKY16-D
TTGTCATTAGCAACACTGCCATC
Gene reverse primer for qRT-PCR
WRKY21-U
GAATAGTATGGGTTCCAGTAGGTCG
Gene forward primer for qRT-PCR
WRKY21-D
AACCTTTGATAGGCTTCTGTCCA
Gene reverse primer for qRT-PCR
GAPDH-U
GGTGCCAAAAAGGTGGTCAT
Gene forward primer for qRT-PCR
GAPDH-D
CAACAACGAACATGGGAGCAT
Gene reverse primer for qRT-PCR
WRKY2P-1363
AACTGCAGACTATAGGGCACGCGTGGTC
Forward primer for promoter deletion
WRKY2P-1018
AACTGCAGTTCTTACGAAAGTGGTGCTGCTA
Forward primer for promoter deletion
WRKY2P-668
AACTGCAGACGCATGTAGCCCTAAACCAAG
Forward primer for promoter deletion
WRKY2P-521
AACTGCAGTTTGCGGTGCTTGAGCTAATT
Forward primer for promoter deletion
WRKY2P-288
AACTGCAGTGTGGTGGATGCTACACCTGAG
Forward primer for promoter deletion
WRKY2P-137
AACTGCAGCATAATGCCCTGTTTCCTCCAA
Forward primer for promoter deletion
WRKY2P-48
AACTGCAGGCAGTGAGTATCCTCGGAGTTAT
Forward primer for promoter deletion
WRKY2-5UTR-F
AACTGCAGAACTAAGAAGAAGGTTGAACGGT
Forward primer for 5′-UTR
WRKY2P-anti
CGGGATCCGGACAATTCAAATGTGTGCACTT
Reverse primer for promoter deletion
WRKY2-5UTR-R
CGGGATCCTCGACGGTTAGGCAGAGGCTTA
Reverse primer for promoter deletion
FWR2PIN
TGAAGCAGGAGAAAGGGCGGAAGGCG
Forward primer for probe
RWR2PIN
CGCTGGTGCCCTTCCATCATCGTCG
Reverse primer for probe


## 2.5. Cloning of the Full-Length cDNA and Genomic DNA ofGbWRKY2

Two specific primers WRKY2-FP and WRKY2-RP (Table1) were designed and synthesized (Shanghai Sangon, China) based on the transcriptome data to obtain the internal fragment. One step RT-PCR was carried out and a fragment of 689 bp was obtained by using one step RT-PCR kit (Dalian TaKaRa, China) under the following program: 50°C for 30 min and 94°C for 3 min, followed by 35 cycles of amplification (94°C for 1 min, 54°C for 1 min, and 72°C for 1 min), and then followed by extension for 10 min at 72°C. The PCR product was purified and cloned into pMD18-T vector (Dalian TaKaRa, China) followed by sequencing. Subsequent BLAST results confirmed that amplified product was partial fragment of WRKY gene.Based on the sequence of the internal fragment ofGbWRKY2 gene, the specific primers WRKY2-3/WRKY2-5 and the nested primer WRKY2-3N/WRKY2-5N (Table 1) were designed to amplify the 3′ and 5′ end of theGbWRKY2 gene using the SMART RACE cDNA Amplification kit (Clontech, USA). The 3′-RACE-PCR and 5′-RACE-PCR were performed according to the manufacturer’s instructions. The PCR products were purified and cloned into pMD18-T vector for sequencing. After comparing and aligning the sequence of 3′-RACE, 5′-RACE, and the internal region products, the full-length cDNA sequence ofGbWRKY2 was obtained and verified through PCR amplification using 3′-Ready cDNA as the template and a pair of primers WRKY2-G1 and WRKY2-G2 (Table 1) under the following conditions: 94°C for 3 min, followed by 35 cycles of amplification (94°C for 20 s, 56°C for 30 s, and 72°C for 2 min). After sequencing, the full-length cDNA ofGbWRKY2 was subsequently analyzed for molecular characterization. Two gene-specific primers, WRKY2-G1 and WRKY2-G2, designed based on the cDNA sequence were used to amplify the genomic sequence ofGbWRKY2.


## 2.6. Amplification of the Promoter Region ofGbWRKY2

Ginkgo genome walker libraries were constructed using the Genome Walker Universal Kit User (Clontech, USA). To clone the promoter region ofGbWRKY2, two round PCR were performed using gene-specific primers (WRKY2-SP1 and WRKY2-SP2) that were designed according to the sequence ofGbWRKY2 cDNA and the adapter primers (AP1 and AP2) provided by the kit. Their sequences were shown in Table 1. After the nested PCR was carried out, amplified fragments were cloned and then sequenced. The sequences that extended upstream of the cDNA ofGbWRKY2 were isolated as the 5′-upstream region ofGbWRKY2 gene and used for further analysis.


## 2.7. Promoter Deletion-GUS Constructs and Agrobacterium-Mediated Transient Expression Assay

For construction of theGbWKRY2 promoter-driven GUS fusion genes, theGbWRKY2 promoter fragment-covering regions were amplified by PCR. Forward primers (Table 1) were designed to correspond to the −1363, −1018, −721, −668, −521, −288, −137, and −48 ofGbWRKY2 promoter and reverse primers, WRKY2P-anti and WRKY2-5UTR-R, were complementary to the 3′-end sequence ofGbWRKY2 promoter and 5′-UTR region, respectively. In addition, a supplementary fragment was amplified using primers WRKY2-1363 and WRKY2P-anti lacking the 5′-UTR region ofGbWRKY2. Each fragment was digested withPstI/BamHI and subcloned intoPstI/BamHI-digested pBI121 to generate seven promoter deletion derivatives. The promoterless construct (pBI121) was used as negative control. All constructs were verified by sequence analysis. Each promoter-GUS fusion construct was introduced intoA.tumefaciens LBA4404 via electroporation.Agrobacterium-mediated transient expression assays were performed according to the method of An et al. [45]. Agrobacterium-mediated transient expression was conducted on fully expanded tobacco leaves, and the intercellular spaces of the intact leaves were infiltrated with the bacterial suspension. After agroinfiltration, tobacco plants were maintained in a moist chamber at 24°C for 48 h. The GUS activity was examined in the tobacco leaves coinfiltrated withAgrobacterium strains harboring differentGbWRKY2 promoter-GUS fusion constructs.


## 2.8. GUS Activity Assay

The seedlings of tobacco transformant were ground in liquid nitrogen. The crude proteins were extracted with 50 mM phosphate buffer, pH 7.0, containing 10 mMβ-mercaptoethanol, 10 mM EDTA, and 0.1% Trition X-100. Homogenates were cleared by centrifugation, and the supernatants were assayed by Bradford assay for total protein (DU700, Beckman, Germany). GUS activity was measured as described by Jefferson et al. [46].


## 2.9. Southern Blot Analysis

Aliquots of DNA (30μg/sample) were digested overnight at 37°C withBamHI andPstI, respectively, which does not cut within the probe region, fractionated by 0.85% agarose gel electrophoresis and transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia, UK). The 283-bp probe was generated by PCR using genomic sequence ofGbWRKY2 including intron as template with primers FWR2PIN and RWR2PIN (Table 1). Probe labeling (biotin), hybridization, and signal detection were performed using Gene Images random prime labeling module and CDP-Star detection module following the manufacturer’s instructions (Amersham Pharmacia, UK). The film was washed under stringent conditions (60°C) and signals were visualized by exposure to Fuji X-ray film at room temperature for 1.5 h.


## 2.10. Bioinformatic Analysis and Molecular Evolution Analyses

The obtained sequences were analyzed using bioinformatic tools at websites (http://www.ncbi.nlm.nih.gov and http://www.expasy.org/). The software vector NTI Suite 10 was used for sequence multialignment. The isolated 5′-upstream sequence was analyzed for the putativecis-acting regulatory elements using the PLACE (http://www.dna.affrc.go.jp/PLACE/) and the Signal Scan Program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database. Phylogenetic tree analysis ofGbWRKY2 and known WRKYs from other plant species retrieved from GenBank were aligned with CLUSTAL W. Subsequently, a phylogenetic tree was constructed by neighbor-joining (NJ) method. The reliability of the tree was measured by bootstrap analysis with 1000 replicates.


## 3. Results

### 3.1.WRKY Candidate Genes Were Identified inG. biloba Transcriptome

Transcriptomes of male and female flowers fromG. biloba were sequenced using Illumina RNA-seq technology, yielding 20 and 19 million transcript reads, respectively. Using the PFAM protein family database with the WRKY domain (PF03106), we identified a total of 28 WRKY transcription factors (Table 2). There were differences in the lengths of proposed sequences of 28WRKY genes, ranging from 258 bp to 2325 bp. These differences may have been due largely to assembly error in partial chromosomal regions and require further confirmation. Gene expression level was estimated using RPKM (reads per kilobase per million mapped reads) value. Among these 28 WRKY transcripts, 20 WRKY genes were identified to be highly expressed in male flowers and the others were highly expressed in female flowers. Subsequently, we selectedGbWRKY2,GbWRKY16, andGbWRKY21 for expression analysis.Table 2
The WRKY transcription factors inG. biloba.
Sequence ID∗
RPKM
CDs (bp)
Annotation to the Arabidopsis WRKYs
Male flower
Female flower
T1_Unigene_BMK.14363
10.97
13.00
2583
WRKY2
CL11186Contig1
21.20
5.54
1977
WRKY4
T1_Unigene_BMK.20451
15.23
38.68
2019
WRKY3
CL8351Contig1
7.37
10.07
2037
WRKY4
CL1197Contig1
7.25
14.22
2325
WRKY20
CL6861Contig1
50.14
3.48
2121
WRKY31
T1_Unigene_BMK.5610
1.92
0.00
783
WRKY61
CL6024Contig1
3.55
2.24
1338
WRKY57
CL10281Contig1
12.46
3.65
744
WRKY28
T1_Unigene_BMK.13300
23.85
18.55
1068
WRKY21
CL7955Contig1
68.18
44.85
1206
WRKY11
T1_Unigene_BMK.24659
7.59
0.45
1281
WRKY42
CL10826Contig1
2.54
1.29
1374
WRKY14
T1_Unigene_BMK.18545
25.53
3.26
642
WRKY16
T2_Unigene_BMK.20037
15.38
39.75
1440
WRKY4
CL7159Contig1
11.59
6.68
1086
WRKY21
T1_Unigene_BMK.7872
3.06
1.19
900
WRKY31
T2_Unigene_BMK.15740
11.05
13.52
1806
WRKY2
CL2657Contig1
2.31
23.10
804
WRKY71
CL3411Contig1
5.22
1.97
1527
WRKY48
T2_Unigene_BMK.8142
21.95
18.36
1068
WRKY21
CL11312Contig1
32.03
14.08
1101
WRKY7
T2_Unigene_BMK.4532
0.08
1.46
258
WRKY12
T2_Unigene_BMK.10348
10.05
2.10
808
WRKY2
T2_Unigene_BMK.10347
18.22
4.04
915
WRKY2
CL1517Contig1
22.31
6.87
1080
WRKY21
T2_Unigene_BMK.9572
0.13
2.84
1404
WRKY49
CL4085Contig1
1.76
0.69
378
WRKY23
∗The candidate gene is indicated in bold.


### 3.2. Expression Analysis in Different Tissues

The expression profile ofGbWRKY2,GbWRKY16, andGbWRKY21 was assessed by qRT-PCR in leaves, roots, stems, and male and female flowers of 14-year-old graft ginkgo trees (Figure 1). The results showed that all of three genes transcripts could be detected in all tissues, but at different expression levels. The highest expression level ofGbWRKY2 was observed in flowers and significantly higher than in the leaves and roots. The lowest expression level ofGbWRKY2 was observed in stems and was significantly lower (P<0.05) in other tissues. In contrast, the expression level ofGbWRKY16 was very low in flowers, and the expression level ofGbWRKY21 was significantly higher in leaves, stems, and female flowers than in roots and male flowers.Figure 1
The expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 inGinkgo biloba different tissue. Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 in different tissues withGbGAPDH gene as internal reference gene. Total RNA samples were isolated from leaves, roots, stems, male flowers, and female flowers, respectively. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three plants and the error bars indicated the standard errors of the mean.


### 3.3. Effect of Abiotic Stresses on the Expression of ThreeWRKY Genes

To determine the function ofGbWRKY2,GbWRKY16, andGbWRKY21 in response to abiotic stress, we investigated the time-course expression patterns of these three genes in gingko callus treated with NaCl, cold, and heat. As shown in Figure 2(a),GbWRKY2 andGbWRKY16 transcript levels were enhanced by NaCl, showing a 1.9-fold and 2.8-fold, respectively, compared with the control at 3 hpt (hours after treatment). Afterwards, the transcript levels of these two genes were decreased sharply after 6 hpt.GbWRKY21 transcript level was increased slightly and then decreased along with the treatment.GbWRKY2 transcript levels were also somewhat enhanced by low temperature (4°C) at 12 hpt (Figure 2(b)) but were decreased slightly along with the treatment time under high temperature (40°C) stress (Figure 2(c)). The relative expressions ofWRKY16 andGbWRKY21 were significantly decreased under both low and high temperature treatments (Figure 2(c)).Figure 2
Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 under salinity (a), cold (b), and heat (c) treatments. Relative expression ofGbWRKY2,GbWRKY16, andGbWRKY21 at various hours after treatment under salinity, cold, and heat treatments withGbGAPDH gene as internal reference gene. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three treated samples and the error bars indicated the standard errors of the mean.
(a)
(b)
(c)


### 3.4. Effect of Hormones on the Expression of ThreeWRKY Genes

Phytohormones, such as SA, MeJA, ABA, and ETH, serve as important signaling molecules and play crucial roles in controlling the expression of downstream defense genes and physiological reactions against biotic and abiotic stresses. To assess the possible involvement of threeWRKY genes in signaling pathways utilized by these hormones, the transcript level ofGbWRKY2,GbWRKY16, andGbWRKY21 in ginkgo callus was determined by qRT-PCR treated with ABA, SA, ETH, and MeJA. All the transcript levels of theGbWRKY2,GbWRKY16, andGbWRKY21 were significantly (P<0.05) increasingly treated with ABA compared with the control. The strongest response ofGbWRKY2 (5.1-fold compared with the control),GbWRKY16 (2.9-fold compared with the control), andGbWRKY21 (1.8-fold compared with the control) to ABA treatment was observed at 12, 6, and 24 hpt, respectively (Figure 3(a)).Figure 3
Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 with ABA (a), SA (b), ETH (c), and MeJA (d) treatments. Relative expressions ofGbWRKY2,GbWRKY16, andGbWRKY21 at various hours after treatment under ABA, SA, ETH, and MeJA treatments withGbGAPDH gene as internal reference gene. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three treated samples and the error bars indicated the standard errors of the mean.
(a)
(b)
(c)
(d)In regard to SA,GbWRKY2 transcript level was increased between 3 and 24 hpt (Figure 3(b)). The highest levels ofGbWRKY2 transcript level (3.9-fold compared with the control) were reached at 6 hpt.GbWRKY16 transcript level was sustainably upregulated during SA treatment and the highest level ofGbWRKY16 (2.5-fold compared with the control) was reached at 12 hpt. In contrast,GbWRKY21 transcript level was firstly decreased after treatment and increased between 12 and 24 hpt, treated with SA compared with the control. The strongest response to SA (4.2-fold compared with the control) was observed at 24 hpt.Figure3(c) showed that the transcript levels ofGbWRKY2 andGbWRKY16 were decreased sharply before 12 hpt but enhanced rapidly and reached the peak, nearly 3.5-fold and 5.6-fold compared with the control, respectively, under ETH treatment.GbWRKY21 transcript level was continuously upregulated until 12 hpt reaching a 6.5-fold compared with the control and then was decreased.For MeJA treatment,GbWRKY2 transcript level was significantly (P<0.05) induced between 0 and 3 hpt, after which they decreased to the similar level to the control (Figure 3(d)). In contrast to GbWRKY2, no significant effect of MeJA was observed inGbWRKY16 transcript level, whileGbWRKY21 transcript level was significantly (P<0.05) decreased by MeJA.


### 3.5. Cloning ofGbWRKY2 and Its Sequence Analysis

The WRKY genes have previously been determined to respond to MeJA and are involved in many life processes, including stress resistance [47], secondary metabolism [23], and plant defense [48]. The exploration of the function of MeJA-inducible WRKY genes inG. biloba would be beneficial for discovering genes involved in stress resistance and in secondary metabolite biosynthesis inG. biloba. Since qRT-PCR analysis indicated thatGbWRKY2 might be involved in the signal transduction of MeJA in stress resistance of ginkgo, we cloned the full-length cDNA, genomic DNA, and promoter region ofGbWRKY2 to further study the function ofGbWRKY2 in stress defense.The full-length cDNA ofGbWRKY2 (GenBank accession number KP987204) was 1,713 bp and contained a 1,014 bp open reading frame encoding a 337 amino acid proteins. One possible polyadenylation signal (AATAA) was found at 194 bp position downstream from the stop codon (Figure 4). A pair of specific primers was designed to synthesize cDNA between the start codon and the terminator codon ofGbWRKY2. The genomic sequence ofGbWRKY2 (GenBank accession number KP987205) with a length of 1,137 bp was amplified and exhibited 100% identity of the coding region of the full-length cDNA sequence. The genomic sequence ofGbWRKY2 contained one intron with a length of 123 bp. This intron was smaller than that ofArabidopsis (241 bp) and rice (868 bp). The putative splicing site obeyed the GT/AG rule [11] (Figure 4). Compared with the identities of the nucleotides of other plants in NCBI, the identities ofGbWRKY2 with the nucleotide sequence of the WRKY gene fromPicea glauca,P. sitchensis,Amborella trichopoda,Medicago truncatula, andMorus notabilis were 83%, 82%, 81%, 81%, and 83%, respectively.Figure 4
The full-length cDNA, intron, and deduced amino acid sequence ofGbWRKY2 gene. The exons sequence is indicated in capital letter and the intron is indicated in lowercase. The start codon (ATG), the stop (TAG), and putative exon-intron splicing sites (gt/ag) are shown by bold letters. One putative polyadenylation signal is bold and italic. A putative nuclear localization signal is underlined. The WRKY domain and the C and H residues in the zinc finger motif (CX4CX23HX1H) are boxed. The zinc finger motif (CX4CX23HX1H) is marked by underline.


### 3.6. Southern Blot Analysis

To investigate the genomic organization ofGbWRKY2, we carried out a Southern blot analysis by digesting genomic DNA ofG.biloba withBamHI andPstI, followed by hybridization with a 283 bp probe generated by PCR using the genomic sequence ofGbWRKY2 as template. As shown in Figure 5, only one hybridized band was detected in each lane, indicating thatGbWRKY2 was a single copy gene inG. biloba.Figure 5
Southern blot analysis ofGbWRKY2. Genomic DNA ofG. biloba was digested withBamHI andPstI, separated on a 0.85% agarose gel, blotted onto a positively charged nylon membrane and probed with a biotin-labeledGbWRKY2 fragment.


### 3.7. Analysis ofGbWRKY2 Protein

TheGbWRKY2 was predicted to encode a protein of 337 amino acid residues. The relative molecular mass and theoretical isoelectric point (pI) of the predicted protein were 36.25 kDa and 6.16, respectively. BLASTP analysis in NCBI revealed that the deduced GbWRKY2 amino acid showed high identity to known WRKY TFs from other plants (Figure 6). The analysis of the deduced amino acid revealed that GbWRKY2 contained one conserved WRKY domain and a zinc finger motif of C2H2 (Figure 6), suggesting GbWRKY2 is a member of the WRKY II family [10, 11]. Like other members of WRKY II family, the conserved domain zinc finger motif for binding Zn+2 ion required for protein function presents at similar positions in GbWRKY2. In addition, sequence analysis using WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html) indicated that the predicted GbWRKY2 protein contains a putative nuclear localization signal (78KRRKK82). All the observed conservations of these domains and motifs in all aligned sequences, especially inG.biloba, suggested the function of the GbWRKY2 protein.Figure 6
Sequence multialignment of the deduced GbWRKY2 protein with other WRKYs. The completely identical amino acids are indicated with white foreground and black background. The conserved amino acids are indicated with white foreground and grey background. Nonsimilar amino acids are indicated with black foreground and white background. The WRKY domain and zinc finger motif (CX4CX23HX1H) are underlined. A putative nuclear localization signal (KRRKK) is shown by bold letters and boxed. The GenBank accession numbers of WRKY proteins and translation of their names are shown as follows: GbWRKY2:Ginkgo biloba; CjWRKY1:Coptis japonica var. dissecta BAF41990.1; PsWRKY:Picea sitchensis ADE77055.1; AaWRKY2:Artemisia annua AGR40498.1.To investigate the evolutionary relationships among GbWRKY2 and other WRKY TF proteins, the phylogenetic tree was constructed using neighbor-joining method (Figure7). The results showed a total of 19 WRKY proteins divided into three classes. Among these proteins, MnWRKY2, NtWRKY4, GsWRKY2, and AtWRKY2 belonged to class I; AtWRKY53, AtWRKY70, and CrWRKY1 belonged to class III. GbWRKY2, HbWRKY1, CjWRKY1, TwWRKY, and GaWRKY1, as well as PqWTKY1, clustered in Group II. Furthermore, GbWRKY2 and PsWRKY of gymnospermae clustered in the same branch, implying that these two WRKY TFs may exhibit a close genetic relationship and display similar protein function.Figure 7
Phylogenetic tree of the sequences of GbWRKY2 and other plants WRKY protein. The numbers at each node represented the bootstrap values (with 1000 replicates). The GenBank accession numbers of WRKY proteins and translation of their names are shown as follows: GbWRKY2:Ginkgo biloba; MnWRKY2:Morus notabilis XP_010092241.1; NtWRKY4:Nicotiana tabacum BAA86031.1; AtWRKY2:Arabidopsis thaliana AED96743.1; GsWRKY2:Glycine soja KHN40472.1; PsWRKY:Picea sitchensis ADE77055.1; HbWRKY1:Hevea brasiliensis ADF45433.1; CjWRKY1:Coptis japonica var. dissecta BAF41990.1; AtWRKY13:Arabidopsis thaliana AEE87071.1; TwWRKY:Taxus wallichiana var. chinensis AEW91476.1; AtWRKY6:Arabidopsis thaliana AEE33948.1; GaWRKY1:Gossypium arboreum AAR98818.1; AtWRKY18:Arabidopsis thaliana AEE85961.1; AtWRKY14:Arabidopsis thaliana AEE31256.1; PqWRKY1:Panax quinquefolius AEQ29014.1; AtWRKY11:Arabidopsis thaliana AEE85928.1; AtWRKY53:Arabidopsis thaliana AEE84809.1; CrWRKY1:Catharanthus roseus HQ646368; AtWRKY70:Arabidopsis thaliana AEE79517.1.


### 3.8. Analysis of 5′-Flanking Sequence ofGbWRKY2

Based on the DNA sequence ofGbWRKY2 gene, the nested primer is designed, and the 5′-flanking sequence (KP987205) with 1,363 bp ofGbWRKY2 was isolated by genome walking method. Taking into account the result of 5-RACE PCR, we concluded the initiation site ofGbWRKY2 transcription at −97 bp in the upstream of ATG (Figure 1S in the Supplementary Material available online at http://dx.doi.org/10.1155/2015/607185). The promoter region ofGbWRKY2 possessed a typically high A + T content of 58.0%, which was commonly found in other plant promoters. The promoter region ofGbWRKY2 was then submitted to PLACE and PlantCARE databases for the analysis of putativecis-element. The result showed that the TATA box was located at −26 bp in the upstream of the transcription initiation site. Another conserved eukaryoticcis-element, the CAAT box, was also found at 232, 787, and 824 bp (Figure 1S), consistent with typical characteristics of the plant promoter. Other stress-relatedcis-elements were also observed in the promoter region ofGbWRKY2 (Table 3). For example, one low temperature response (CRTDREHVCBF2) [30], two drought stress response and aging response (ACGTATERD1) [27], and four disease defense (BIHD1OS) [28]cis-elements were found. Motifs related to light regulation, including three GT1 [49] and one IBOX [36], were also detected. Furthermore,cis-acting elements related to hormones were also predicted (Figure 1S). For example, we found one core motif for binding DPBF TF, three GT1 motifs, seven ARR1AT TF binding sites, and four PYRIMIDINE-boxes that participate in the signal response of ABA, salicylic acid, cytokinin, and gibberellin [26, 32, 39, 49], respectively, in the promoter region ofGbWRKY2. Two ABA responsecis-elements were also found at 10 and 585 bp position [24, 25]. Moreover, some TF binding sites were also detected. For example, five Dof TF binding sites were found in the promoter region ofGbWRKY2, and these motifs participate in the signal response of auxin [50], jasmonic acid, or ethylene [51] in other plants. In addition, we found ten E-boxes, five MYB-boxes, nine MYC-boxes, and one W-box inGbWRKY2 promoter, which is conservative binding motif of bHLH [33], MYB [38], MYC [37], and WRKY [12] TFs, respectively. Interestingly, the presence of a putative W-box binding site within theGbWRKY2 promoter might indicate that this ginkgo gene can be subjected to autoregulation or can be modulated by other WRKY members [52, 53]. Prediction results showed that the regulatorycis-elements ofGbWRKY2 promoter were related to stress-induced response, indicating that the promoter may play an important role in response to external environmental stress and biological defense process.Table 3
Putativecis-acting regulatory elements identified in the promoter of GbWRKY2 using PLACE  (http://www.dna.affrc.go.jp/PLACE/) and the Signal Scan Program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database.
Name ofcis-element
Position
Signal sequence
Function
References
ABRELATERD1
585
ACGTG
ABA-responsive elements
[24]
ABRERATCAL
10
MACGYGB
Ca2+-responsive and ABA upregulated genes
[25]
ARR1AT
629, 634, 564, 90, 751, 883
NGATT
ARR1 binding element involved in cytokinin signaling
[26]
ACGTATERD1
262, 585
ACGT
Involved in upregulation by dehydration stress and dark-induced senescence
[27]
BIHD1OS
595, 772, 863, 1307
TGTCA
BELL recognition site involved in disease resistance responses
[28]
CAATBOX1
232, 787, 824
CAAT
Commoncis-acting element in promoter and enhancer regions
[29]
CRTDREHVCBF2
18
GTCGAC
Low-temperature responsive
[30]
DOFCOREZM
354, 621, 687, 1258, 1301
AAAG
Dof1 and Dof2 binding element involved in carbon metabolism
[31]
DPBFCOREDCDC3
1089
ACACNNG
DPBF-1 binding core sequence involved in ABA signaling
[32]
EBOXBNNAPA
86, 232, 318, 714, 729, 775, 1090, 1246, 1280, 1350
CANNTG
Cis-element binding BHLH factor is dispensable for light responsiveness
[33]
GATABOX
124, 222, 290, 640, 681, 1253, 1276
GATA
Commoncis-acting element in promoter
[34]
GT1CONSENSUS
475, 803, 928
GRWAAW
Consensus GT-1 binding site in many light-regulated genes and influence the level of SA-inducible gene expression
[35]
IBOX
124
GATAAG
Conserved sequence upstream of light-regulated genes
[36]
MYB2CONSENSUSAT
318, 1246, 1280, 591
YAACKG
MYB recognition site involved in dehydration responsiveness
[37]
MYCCONSENSUSAT
86, 232, 318, 714, 729, 775, 1090, 1246, 1280
CANNTG
MYC recognition site involved in dehydration responsiveness and cold responsiveness
[38]
PYRIMIDINE-box
214, 840, 869, 890
CCTTTT
Gibberellin-response cis-element
[39]
ROOTMOTIFTAPOX1
178, 223, 546, 641, 722
ATATT
Root specific expression
[40]
TATA box
94
TTATTT
Commoncis-acting element in promoter and enhancer regions
[41]
WBOXATNPR1
887
TTGACY
WRKY binding site, involved in many physiological processes
[10]


### 3.9. Deletion Analysis of theGbWRKY2 Promoter in Tobacco Leaf Tissues

To gain insight into the functional role of theGbWRKY2 promoter region, series deletions were sequential of thecis-elements and fusion of the remaining promoter to the GUS reporter gene was constructed. A promoterless construct (pBI121) was used as a negative control (Figure 8). Sequential 5′-deletions of the promoter were performed by PCR, and the responsiveness of the deleted versions of theGbWRKY2 promoter was analyzed by transient assays in tobacco leaves. As shown in Figure 8, the GUS activity of the maximum-length containing 5′-UTR ofGbWRKY2 (construct −1363) was 358.36 nM 4-methylumbelliferone (MU) mg−1 protein min−1, although the GUS activity (352.19 nM 4-mg−1 protein min−1) of 5′-UTR deletion of full-length promoter (−1363Δ) was slightly less than that of construct −1363 but did not reach significant level (P>0.05), suggesting that 5′-UTR had no effect on the transcript level ofGbWRKY2 promoter under our experimental condition. The highest level of GUS activity was observed in transgenic tobacco leaves harboring the −1018 promoter construct and significantly higher (P<0.05) than that of the full-length promoter (construct −1363), implying that negativecis-elements may be present in the promoter region between −1363 and −1018. By contrast, deletion of the GbWRKY2 promoter from −1018 to −668 caused a significant (P<0.05) reduction of the promoter expression. Successive deletion from −668 to −521 had no additional significant effect on GUS activity while further deletion to −228 containing a W-box causes a significant (P<0.05) decrease of GUS activity. Likewise, both further deletions from −228 to −137 and from −137 to −48 led to 39.3% and 16.7% decrease of promoter expression, respectively. Finally, both the constructs containing 5′-UTR and negative constructs present a quasicomplete disappearance of GUS activity, suggesting 5′-UTR region had no contribution to GbWRKY2 expression.Figure 8
Deletion analysis of the GbWRKY2 promoter-driven GUS activity. Schematic diagram of the constructs used for GUS activity assays in leaves of transgenic tobacco plants is shown at left. Dash line pinpoints the deletion of the 5′-UTR region for the construct −1363Δ. Quantitative analyses of GUS activity of transgenic plants driven by deletion constructs of theGbWRKY2 promoter are shown at right. Error bars represent standard deviation (SD). Data are mean ± SD from triplicate experiments. Values with different letters show significant differences at P=0.05 according to the Fisher’s least significant difference (LSD) test.


## 3.1.WRKY Candidate Genes Were Identified inG. biloba Transcriptome

Transcriptomes of male and female flowers fromG. biloba were sequenced using Illumina RNA-seq technology, yielding 20 and 19 million transcript reads, respectively. Using the PFAM protein family database with the WRKY domain (PF03106), we identified a total of 28 WRKY transcription factors (Table 2). There were differences in the lengths of proposed sequences of 28WRKY genes, ranging from 258 bp to 2325 bp. These differences may have been due largely to assembly error in partial chromosomal regions and require further confirmation. Gene expression level was estimated using RPKM (reads per kilobase per million mapped reads) value. Among these 28 WRKY transcripts, 20 WRKY genes were identified to be highly expressed in male flowers and the others were highly expressed in female flowers. Subsequently, we selectedGbWRKY2,GbWRKY16, andGbWRKY21 for expression analysis.Table 2
The WRKY transcription factors inG. biloba.
Sequence ID∗
RPKM
CDs (bp)
Annotation to the Arabidopsis WRKYs
Male flower
Female flower
T1_Unigene_BMK.14363
10.97
13.00
2583
WRKY2
CL11186Contig1
21.20
5.54
1977
WRKY4
T1_Unigene_BMK.20451
15.23
38.68
2019
WRKY3
CL8351Contig1
7.37
10.07
2037
WRKY4
CL1197Contig1
7.25
14.22
2325
WRKY20
CL6861Contig1
50.14
3.48
2121
WRKY31
T1_Unigene_BMK.5610
1.92
0.00
783
WRKY61
CL6024Contig1
3.55
2.24
1338
WRKY57
CL10281Contig1
12.46
3.65
744
WRKY28
T1_Unigene_BMK.13300
23.85
18.55
1068
WRKY21
CL7955Contig1
68.18
44.85
1206
WRKY11
T1_Unigene_BMK.24659
7.59
0.45
1281
WRKY42
CL10826Contig1
2.54
1.29
1374
WRKY14
T1_Unigene_BMK.18545
25.53
3.26
642
WRKY16
T2_Unigene_BMK.20037
15.38
39.75
1440
WRKY4
CL7159Contig1
11.59
6.68
1086
WRKY21
T1_Unigene_BMK.7872
3.06
1.19
900
WRKY31
T2_Unigene_BMK.15740
11.05
13.52
1806
WRKY2
CL2657Contig1
2.31
23.10
804
WRKY71
CL3411Contig1
5.22
1.97
1527
WRKY48
T2_Unigene_BMK.8142
21.95
18.36
1068
WRKY21
CL11312Contig1
32.03
14.08
1101
WRKY7
T2_Unigene_BMK.4532
0.08
1.46
258
WRKY12
T2_Unigene_BMK.10348
10.05
2.10
808
WRKY2
T2_Unigene_BMK.10347
18.22
4.04
915
WRKY2
CL1517Contig1
22.31
6.87
1080
WRKY21
T2_Unigene_BMK.9572
0.13
2.84
1404
WRKY49
CL4085Contig1
1.76
0.69
378
WRKY23
∗The candidate gene is indicated in bold.


## 3.2. Expression Analysis in Different Tissues

The expression profile ofGbWRKY2,GbWRKY16, andGbWRKY21 was assessed by qRT-PCR in leaves, roots, stems, and male and female flowers of 14-year-old graft ginkgo trees (Figure 1). The results showed that all of three genes transcripts could be detected in all tissues, but at different expression levels. The highest expression level ofGbWRKY2 was observed in flowers and significantly higher than in the leaves and roots. The lowest expression level ofGbWRKY2 was observed in stems and was significantly lower (P<0.05) in other tissues. In contrast, the expression level ofGbWRKY16 was very low in flowers, and the expression level ofGbWRKY21 was significantly higher in leaves, stems, and female flowers than in roots and male flowers.Figure 1
The expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 inGinkgo biloba different tissue. Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 in different tissues withGbGAPDH gene as internal reference gene. Total RNA samples were isolated from leaves, roots, stems, male flowers, and female flowers, respectively. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three plants and the error bars indicated the standard errors of the mean.


## 3.3. Effect of Abiotic Stresses on the Expression of ThreeWRKY Genes

To determine the function ofGbWRKY2,GbWRKY16, andGbWRKY21 in response to abiotic stress, we investigated the time-course expression patterns of these three genes in gingko callus treated with NaCl, cold, and heat. As shown in Figure 2(a),GbWRKY2 andGbWRKY16 transcript levels were enhanced by NaCl, showing a 1.9-fold and 2.8-fold, respectively, compared with the control at 3 hpt (hours after treatment). Afterwards, the transcript levels of these two genes were decreased sharply after 6 hpt.GbWRKY21 transcript level was increased slightly and then decreased along with the treatment.GbWRKY2 transcript levels were also somewhat enhanced by low temperature (4°C) at 12 hpt (Figure 2(b)) but were decreased slightly along with the treatment time under high temperature (40°C) stress (Figure 2(c)). The relative expressions ofWRKY16 andGbWRKY21 were significantly decreased under both low and high temperature treatments (Figure 2(c)).Figure 2
Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 under salinity (a), cold (b), and heat (c) treatments. Relative expression ofGbWRKY2,GbWRKY16, andGbWRKY21 at various hours after treatment under salinity, cold, and heat treatments withGbGAPDH gene as internal reference gene. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three treated samples and the error bars indicated the standard errors of the mean.
(a)
(b)
(c)


## 3.4. Effect of Hormones on the Expression of ThreeWRKY Genes

Phytohormones, such as SA, MeJA, ABA, and ETH, serve as important signaling molecules and play crucial roles in controlling the expression of downstream defense genes and physiological reactions against biotic and abiotic stresses. To assess the possible involvement of threeWRKY genes in signaling pathways utilized by these hormones, the transcript level ofGbWRKY2,GbWRKY16, andGbWRKY21 in ginkgo callus was determined by qRT-PCR treated with ABA, SA, ETH, and MeJA. All the transcript levels of theGbWRKY2,GbWRKY16, andGbWRKY21 were significantly (P<0.05) increasingly treated with ABA compared with the control. The strongest response ofGbWRKY2 (5.1-fold compared with the control),GbWRKY16 (2.9-fold compared with the control), andGbWRKY21 (1.8-fold compared with the control) to ABA treatment was observed at 12, 6, and 24 hpt, respectively (Figure 3(a)).Figure 3
Relative expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 with ABA (a), SA (b), ETH (c), and MeJA (d) treatments. Relative expressions ofGbWRKY2,GbWRKY16, andGbWRKY21 at various hours after treatment under ABA, SA, ETH, and MeJA treatments withGbGAPDH gene as internal reference gene. Each sample was individually assayed in triplicate. Values shown represent the mean reading from three treated samples and the error bars indicated the standard errors of the mean.
(a)
(b)
(c)
(d)In regard to SA,GbWRKY2 transcript level was increased between 3 and 24 hpt (Figure 3(b)). The highest levels ofGbWRKY2 transcript level (3.9-fold compared with the control) were reached at 6 hpt.GbWRKY16 transcript level was sustainably upregulated during SA treatment and the highest level ofGbWRKY16 (2.5-fold compared with the control) was reached at 12 hpt. In contrast,GbWRKY21 transcript level was firstly decreased after treatment and increased between 12 and 24 hpt, treated with SA compared with the control. The strongest response to SA (4.2-fold compared with the control) was observed at 24 hpt.Figure3(c) showed that the transcript levels ofGbWRKY2 andGbWRKY16 were decreased sharply before 12 hpt but enhanced rapidly and reached the peak, nearly 3.5-fold and 5.6-fold compared with the control, respectively, under ETH treatment.GbWRKY21 transcript level was continuously upregulated until 12 hpt reaching a 6.5-fold compared with the control and then was decreased.For MeJA treatment,GbWRKY2 transcript level was significantly (P<0.05) induced between 0 and 3 hpt, after which they decreased to the similar level to the control (Figure 3(d)). In contrast to GbWRKY2, no significant effect of MeJA was observed inGbWRKY16 transcript level, whileGbWRKY21 transcript level was significantly (P<0.05) decreased by MeJA.


## 3.5. Cloning ofGbWRKY2 and Its Sequence Analysis

The WRKY genes have previously been determined to respond to MeJA and are involved in many life processes, including stress resistance [47], secondary metabolism [23], and plant defense [48]. The exploration of the function of MeJA-inducible WRKY genes inG. biloba would be beneficial for discovering genes involved in stress resistance and in secondary metabolite biosynthesis inG. biloba. Since qRT-PCR analysis indicated thatGbWRKY2 might be involved in the signal transduction of MeJA in stress resistance of ginkgo, we cloned the full-length cDNA, genomic DNA, and promoter region ofGbWRKY2 to further study the function ofGbWRKY2 in stress defense.The full-length cDNA ofGbWRKY2 (GenBank accession number KP987204) was 1,713 bp and contained a 1,014 bp open reading frame encoding a 337 amino acid proteins. One possible polyadenylation signal (AATAA) was found at 194 bp position downstream from the stop codon (Figure 4). A pair of specific primers was designed to synthesize cDNA between the start codon and the terminator codon ofGbWRKY2. The genomic sequence ofGbWRKY2 (GenBank accession number KP987205) with a length of 1,137 bp was amplified and exhibited 100% identity of the coding region of the full-length cDNA sequence. The genomic sequence ofGbWRKY2 contained one intron with a length of 123 bp. This intron was smaller than that ofArabidopsis (241 bp) and rice (868 bp). The putative splicing site obeyed the GT/AG rule [11] (Figure 4). Compared with the identities of the nucleotides of other plants in NCBI, the identities ofGbWRKY2 with the nucleotide sequence of the WRKY gene fromPicea glauca,P. sitchensis,Amborella trichopoda,Medicago truncatula, andMorus notabilis were 83%, 82%, 81%, 81%, and 83%, respectively.Figure 4
The full-length cDNA, intron, and deduced amino acid sequence ofGbWRKY2 gene. The exons sequence is indicated in capital letter and the intron is indicated in lowercase. The start codon (ATG), the stop (TAG), and putative exon-intron splicing sites (gt/ag) are shown by bold letters. One putative polyadenylation signal is bold and italic. A putative nuclear localization signal is underlined. The WRKY domain and the C and H residues in the zinc finger motif (CX4CX23HX1H) are boxed. The zinc finger motif (CX4CX23HX1H) is marked by underline.


## 3.6. Southern Blot Analysis

To investigate the genomic organization ofGbWRKY2, we carried out a Southern blot analysis by digesting genomic DNA ofG.biloba withBamHI andPstI, followed by hybridization with a 283 bp probe generated by PCR using the genomic sequence ofGbWRKY2 as template. As shown in Figure 5, only one hybridized band was detected in each lane, indicating thatGbWRKY2 was a single copy gene inG. biloba.Figure 5
Southern blot analysis ofGbWRKY2. Genomic DNA ofG. biloba was digested withBamHI andPstI, separated on a 0.85% agarose gel, blotted onto a positively charged nylon membrane and probed with a biotin-labeledGbWRKY2 fragment.


## 3.7. Analysis ofGbWRKY2 Protein

TheGbWRKY2 was predicted to encode a protein of 337 amino acid residues. The relative molecular mass and theoretical isoelectric point (pI) of the predicted protein were 36.25 kDa and 6.16, respectively. BLASTP analysis in NCBI revealed that the deduced GbWRKY2 amino acid showed high identity to known WRKY TFs from other plants (Figure 6). The analysis of the deduced amino acid revealed that GbWRKY2 contained one conserved WRKY domain and a zinc finger motif of C2H2 (Figure 6), suggesting GbWRKY2 is a member of the WRKY II family [10, 11]. Like other members of WRKY II family, the conserved domain zinc finger motif for binding Zn+2 ion required for protein function presents at similar positions in GbWRKY2. In addition, sequence analysis using WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html) indicated that the predicted GbWRKY2 protein contains a putative nuclear localization signal (78KRRKK82). All the observed conservations of these domains and motifs in all aligned sequences, especially inG.biloba, suggested the function of the GbWRKY2 protein.Figure 6
Sequence multialignment of the deduced GbWRKY2 protein with other WRKYs. The completely identical amino acids are indicated with white foreground and black background. The conserved amino acids are indicated with white foreground and grey background. Nonsimilar amino acids are indicated with black foreground and white background. The WRKY domain and zinc finger motif (CX4CX23HX1H) are underlined. A putative nuclear localization signal (KRRKK) is shown by bold letters and boxed. The GenBank accession numbers of WRKY proteins and translation of their names are shown as follows: GbWRKY2:Ginkgo biloba; CjWRKY1:Coptis japonica var. dissecta BAF41990.1; PsWRKY:Picea sitchensis ADE77055.1; AaWRKY2:Artemisia annua AGR40498.1.To investigate the evolutionary relationships among GbWRKY2 and other WRKY TF proteins, the phylogenetic tree was constructed using neighbor-joining method (Figure7). The results showed a total of 19 WRKY proteins divided into three classes. Among these proteins, MnWRKY2, NtWRKY4, GsWRKY2, and AtWRKY2 belonged to class I; AtWRKY53, AtWRKY70, and CrWRKY1 belonged to class III. GbWRKY2, HbWRKY1, CjWRKY1, TwWRKY, and GaWRKY1, as well as PqWTKY1, clustered in Group II. Furthermore, GbWRKY2 and PsWRKY of gymnospermae clustered in the same branch, implying that these two WRKY TFs may exhibit a close genetic relationship and display similar protein function.Figure 7
Phylogenetic tree of the sequences of GbWRKY2 and other plants WRKY protein. The numbers at each node represented the bootstrap values (with 1000 replicates). The GenBank accession numbers of WRKY proteins and translation of their names are shown as follows: GbWRKY2:Ginkgo biloba; MnWRKY2:Morus notabilis XP_010092241.1; NtWRKY4:Nicotiana tabacum BAA86031.1; AtWRKY2:Arabidopsis thaliana AED96743.1; GsWRKY2:Glycine soja KHN40472.1; PsWRKY:Picea sitchensis ADE77055.1; HbWRKY1:Hevea brasiliensis ADF45433.1; CjWRKY1:Coptis japonica var. dissecta BAF41990.1; AtWRKY13:Arabidopsis thaliana AEE87071.1; TwWRKY:Taxus wallichiana var. chinensis AEW91476.1; AtWRKY6:Arabidopsis thaliana AEE33948.1; GaWRKY1:Gossypium arboreum AAR98818.1; AtWRKY18:Arabidopsis thaliana AEE85961.1; AtWRKY14:Arabidopsis thaliana AEE31256.1; PqWRKY1:Panax quinquefolius AEQ29014.1; AtWRKY11:Arabidopsis thaliana AEE85928.1; AtWRKY53:Arabidopsis thaliana AEE84809.1; CrWRKY1:Catharanthus roseus HQ646368; AtWRKY70:Arabidopsis thaliana AEE79517.1.


## 3.8. Analysis of 5′-Flanking Sequence ofGbWRKY2

Based on the DNA sequence ofGbWRKY2 gene, the nested primer is designed, and the 5′-flanking sequence (KP987205) with 1,363 bp ofGbWRKY2 was isolated by genome walking method. Taking into account the result of 5-RACE PCR, we concluded the initiation site ofGbWRKY2 transcription at −97 bp in the upstream of ATG (Figure 1S in the Supplementary Material available online at http://dx.doi.org/10.1155/2015/607185). The promoter region ofGbWRKY2 possessed a typically high A + T content of 58.0%, which was commonly found in other plant promoters. The promoter region ofGbWRKY2 was then submitted to PLACE and PlantCARE databases for the analysis of putativecis-element. The result showed that the TATA box was located at −26 bp in the upstream of the transcription initiation site. Another conserved eukaryoticcis-element, the CAAT box, was also found at 232, 787, and 824 bp (Figure 1S), consistent with typical characteristics of the plant promoter. Other stress-relatedcis-elements were also observed in the promoter region ofGbWRKY2 (Table 3). For example, one low temperature response (CRTDREHVCBF2) [30], two drought stress response and aging response (ACGTATERD1) [27], and four disease defense (BIHD1OS) [28]cis-elements were found. Motifs related to light regulation, including three GT1 [49] and one IBOX [36], were also detected. Furthermore,cis-acting elements related to hormones were also predicted (Figure 1S). For example, we found one core motif for binding DPBF TF, three GT1 motifs, seven ARR1AT TF binding sites, and four PYRIMIDINE-boxes that participate in the signal response of ABA, salicylic acid, cytokinin, and gibberellin [26, 32, 39, 49], respectively, in the promoter region ofGbWRKY2. Two ABA responsecis-elements were also found at 10 and 585 bp position [24, 25]. Moreover, some TF binding sites were also detected. For example, five Dof TF binding sites were found in the promoter region ofGbWRKY2, and these motifs participate in the signal response of auxin [50], jasmonic acid, or ethylene [51] in other plants. In addition, we found ten E-boxes, five MYB-boxes, nine MYC-boxes, and one W-box inGbWRKY2 promoter, which is conservative binding motif of bHLH [33], MYB [38], MYC [37], and WRKY [12] TFs, respectively. Interestingly, the presence of a putative W-box binding site within theGbWRKY2 promoter might indicate that this ginkgo gene can be subjected to autoregulation or can be modulated by other WRKY members [52, 53]. Prediction results showed that the regulatorycis-elements ofGbWRKY2 promoter were related to stress-induced response, indicating that the promoter may play an important role in response to external environmental stress and biological defense process.Table 3
Putativecis-acting regulatory elements identified in the promoter of GbWRKY2 using PLACE  (http://www.dna.affrc.go.jp/PLACE/) and the Signal Scan Program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database.
Name ofcis-element
Position
Signal sequence
Function
References
ABRELATERD1
585
ACGTG
ABA-responsive elements
[24]
ABRERATCAL
10
MACGYGB
Ca2+-responsive and ABA upregulated genes
[25]
ARR1AT
629, 634, 564, 90, 751, 883
NGATT
ARR1 binding element involved in cytokinin signaling
[26]
ACGTATERD1
262, 585
ACGT
Involved in upregulation by dehydration stress and dark-induced senescence
[27]
BIHD1OS
595, 772, 863, 1307
TGTCA
BELL recognition site involved in disease resistance responses
[28]
CAATBOX1
232, 787, 824
CAAT
Commoncis-acting element in promoter and enhancer regions
[29]
CRTDREHVCBF2
18
GTCGAC
Low-temperature responsive
[30]
DOFCOREZM
354, 621, 687, 1258, 1301
AAAG
Dof1 and Dof2 binding element involved in carbon metabolism
[31]
DPBFCOREDCDC3
1089
ACACNNG
DPBF-1 binding core sequence involved in ABA signaling
[32]
EBOXBNNAPA
86, 232, 318, 714, 729, 775, 1090, 1246, 1280, 1350
CANNTG
Cis-element binding BHLH factor is dispensable for light responsiveness
[33]
GATABOX
124, 222, 290, 640, 681, 1253, 1276
GATA
Commoncis-acting element in promoter
[34]
GT1CONSENSUS
475, 803, 928
GRWAAW
Consensus GT-1 binding site in many light-regulated genes and influence the level of SA-inducible gene expression
[35]
IBOX
124
GATAAG
Conserved sequence upstream of light-regulated genes
[36]
MYB2CONSENSUSAT
318, 1246, 1280, 591
YAACKG
MYB recognition site involved in dehydration responsiveness
[37]
MYCCONSENSUSAT
86, 232, 318, 714, 729, 775, 1090, 1246, 1280
CANNTG
MYC recognition site involved in dehydration responsiveness and cold responsiveness
[38]
PYRIMIDINE-box
214, 840, 869, 890
CCTTTT
Gibberellin-response cis-element
[39]
ROOTMOTIFTAPOX1
178, 223, 546, 641, 722
ATATT
Root specific expression
[40]
TATA box
94
TTATTT
Commoncis-acting element in promoter and enhancer regions
[41]
WBOXATNPR1
887
TTGACY
WRKY binding site, involved in many physiological processes
[10]


## 3.9. Deletion Analysis of theGbWRKY2 Promoter in Tobacco Leaf Tissues

To gain insight into the functional role of theGbWRKY2 promoter region, series deletions were sequential of thecis-elements and fusion of the remaining promoter to the GUS reporter gene was constructed. A promoterless construct (pBI121) was used as a negative control (Figure 8). Sequential 5′-deletions of the promoter were performed by PCR, and the responsiveness of the deleted versions of theGbWRKY2 promoter was analyzed by transient assays in tobacco leaves. As shown in Figure 8, the GUS activity of the maximum-length containing 5′-UTR ofGbWRKY2 (construct −1363) was 358.36 nM 4-methylumbelliferone (MU) mg−1 protein min−1, although the GUS activity (352.19 nM 4-mg−1 protein min−1) of 5′-UTR deletion of full-length promoter (−1363Δ) was slightly less than that of construct −1363 but did not reach significant level (P>0.05), suggesting that 5′-UTR had no effect on the transcript level ofGbWRKY2 promoter under our experimental condition. The highest level of GUS activity was observed in transgenic tobacco leaves harboring the −1018 promoter construct and significantly higher (P<0.05) than that of the full-length promoter (construct −1363), implying that negativecis-elements may be present in the promoter region between −1363 and −1018. By contrast, deletion of the GbWRKY2 promoter from −1018 to −668 caused a significant (P<0.05) reduction of the promoter expression. Successive deletion from −668 to −521 had no additional significant effect on GUS activity while further deletion to −228 containing a W-box causes a significant (P<0.05) decrease of GUS activity. Likewise, both further deletions from −228 to −137 and from −137 to −48 led to 39.3% and 16.7% decrease of promoter expression, respectively. Finally, both the constructs containing 5′-UTR and negative constructs present a quasicomplete disappearance of GUS activity, suggesting 5′-UTR region had no contribution to GbWRKY2 expression.Figure 8
Deletion analysis of the GbWRKY2 promoter-driven GUS activity. Schematic diagram of the constructs used for GUS activity assays in leaves of transgenic tobacco plants is shown at left. Dash line pinpoints the deletion of the 5′-UTR region for the construct −1363Δ. Quantitative analyses of GUS activity of transgenic plants driven by deletion constructs of theGbWRKY2 promoter are shown at right. Error bars represent standard deviation (SD). Data are mean ± SD from triplicate experiments. Values with different letters show significant differences at P=0.05 according to the Fisher’s least significant difference (LSD) test.


## 4. Discussion

Given the important roles that WRKY transcription factors play in response to various stresses, the traditional gene-by-gene research was not a high-efficiency method to study the plant that had no genome sequence. High throughput sequencing data have been used for functional gene mining and have proven to be an effective method for metabolic gene discovery and others [23]. In this study, the transcriptome dataset was searched for WRKY transcription factors inG. biloba and a total of 28 candidate WRKY genes were identified. Of these WRKY genes, we selectedGbWRKY2,GbWRKY16, andGbWRKY21 to expression analysis. Interestingly, our data showed that the transcript level ofGbWRKY2 was predominantly observed in ginkgo inflorescence and strongly induced by MeJA, implyingGbWRKY2 might play dual roles in the development of ginkgo inflorescence and defense responses by mediating MeJA signaling.


### 4.1.GbWRKY2 Is a Group II WRKY Transcription Factor

A ginkgo gene (GbWRKY2) encoding a protein with sequence homology to members of the WRKY family has been characterized. This newly characterized gene is the first WRKY factor described inG. biloba and presents structural hallmarks that allow us to classify it within Group II of WRKY TFs [11]. The phylogenetic analysis also clearly showed thatGbWRKY2 belong to Group II WRKY family. Numerous members of plant WRKY genes belong to Group II family and play roles in transcriptional reprogramming associated with resistance to various stresses [11]. A predicted nuclear localization signal (NLS) (Figure 4) strongly suggests that GbWRKY2 protein is translocated into the nucleus to control gene expression. These data suggested thatGbWRKY2 might play an important role in defense response of ginkgo to biotic and abiotic stresses.


### 4.2. Multiple cis-acting Elements Response to Stress and Hormone in the Promoter Region ofGbWRKY2

Stress-inducible gene expression is transcriptionally regulated by a change in the level and/or activity of sequence-specific DNA-binding transcription factors bound to specificcis-acting elements of promoter regions [54]. These regulatory factors are involved in the activation, suppression, and modulation of various signaling pathways in plant cells on biotic and abiotic stresses. Many plant promoters have therefore been identified and isolated, and genetic engineering in plants has been greatly enhanced using individual promoters [55]. Thus, we isolated a 1,363 bp-length promoter ofGbWRKY2 (Figure 1S). Bioinformatics analysis showed that multiplecis-acting elements including fundamental and special elements associated with abiotic stresses and hormone regulations were found in theGbWRKY2 promoter (Table 3), indicating thatGbWRKY2 gene might respond to various environment stimulus. Furthermore, a series of 5′ end deletion fragments of this promoter were detected using transient expression assays, which demonstrated that the activity of a truncated promoter from position −1 to −48 bp retained the ability to initiate the expression ofGUS in transgenic tobacco leaves. This region is enough to keep the basic function ofGbWRKY2 promoter. Bioinformatics analysis showed a TATA box was located in the region between −1 and −48 bp. Together taken into the results of bioinformatics and deletion analysis, we conclude that the region of −1 to −48 bp is the equal of core promoter and positions of TATA box correspond to the typical core promoter model [56]. Moreover, our experiments showed that deletion of the promoter regions between positions −1018 and −668 or between positions −288 and −137 caused very significant (P<0.01) reductions in promoter activity suggesting that these regions likely contain potential transcription-enhancingcis-acting elements. Sequence analysis discussed above indicates that both regions contain three and two Dof TF binding sites, respectively, which may be important for the hormonal responsiveness ofGbWRKY2 promoter.


### 4.3.GbWRKY2 Might Participate in the Development of Ginkgo Inflorescence

Similar to expression patterns observed in other plant species, threeGbWRKYs were found to be expressed in all tissues we used, but at different expression levels. Moreover, the transcription ofGbWRKY2 was observed abundant in flowers. In contrast, expression levels ofGbWRKY16 were very low in flowers. And the expression levels ofGbWRKY21 were significantly higher in leaves, stems, and female flowers than in roots and male flowers. The result may indicate thatGbWRKY2,GbWRKY16, andGbWRKY21 played different roles involved in regulating plant developmental and physiological processes. The highest expression level ofGbWRKY2 is in flowers while the lowest expression level of in stems, consistent with the spatial expression profile ofChWRKY2 fromCorylus heterophylla [57]. Given highest transcript level ofGbWRKY2 in flowers, it can be speculated thatGbWRKY2 might play an important role in tolerance to low temperature in inflorescence, which developed in cold early spring. Thus, we deduced thatGbWKY2 likely exhibited similar function ofChWRKY2 TF to participate in the developmental process of ginkgo inflorescence.


### 4.4.GbWRKYs Might Play Role in Stress-Related Signal Pathways

Salinity is an important abiotic stress factor, usually affecting plant growth, development, survival, and crop productivity. Thus, understanding the complex mechanism of salinity tolerance is important for agriculture production. Interestingly, several WRKY proteins were shown to be involved in plant salinity stress response. For example, the expression ofCmWRKY17 was induced by salinity inChrysanthemum morifolium and overexpression ofCmWRKY17 in Chrysanthemum and Arabidopsis increased the sensitivity to salinity stress [58]. The transcripts of two closely related WRKY TFs (AtWRKY25 andAtWRKY33) from Arabidopsis were increased by NaCl treatment and both the Atwrky33 null mutants and Atwrky25Atwrky33 double mutants showed moderately increased NaCl sensitivity [59]. Poplar species increase expressions of transcription factors to deal with salt environments. Salinity increased heat-shock transcription factor (HSF) transcription inP. euphratica andPeHSF binds the cis-acting heat shock element (SHE) of thePeWRKY1 promoter, thus activatingPeWRKY1 expression [60]. Similarly, the first evidence pointing towards a role forGbWRKY2 andGbWRKY16 in defense comes from the data showing that theGbWRKY2 andGbWRKY16 transcript is dramatically upregulated in ginkgo in response to NaCl. Further study on suppression/overexpression ofGbWRKY2 andGbWRKY16 was required for unveiling the molecular mechanism ofGbWRKY2 andGbWRKY16 gene participation in ginkgo tolerance to salt.Temperature that exceeds an organism’s optimal tolerance range is considered as an important abiotic stress factor. Tremendous work has been done in the past two decades to reveal the complex molecular mechanism in plants’ responses to extreme temperature and there is increasing evidence that WRKY proteins are involved in responses to both heat and cold stresses. For example, a WRKY TF inNicotiana tabacum responds to a combination of drought and heat stress [61]. Another example is that transgenic Arabidopsis plants overexpressingGmWRKY21 exhibited increased tolerance to cold stress when compared with wild-type plants [62]. In our studies, the expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 were all repressed by heat stress. The expression level ofGbWRKY2 was upregulated by cold stress and there was no significant effect of cold stress onGbWRKY16 transcript levels. In contrast, the expression level ofGbWRKY21 was downregulated by cold stress. We also reported for the first time an increase ofGbWRKY2 transcript level by cold but decrease by heat, consistent with the observation of cold responsivecis-elements (CRTDREHVCBF2 and MYC recognition site) (Table 3). Although low-temperature related WRKYs were isolated in several species [62–64]. The mechanism of how WRKYs respond to cold signals and regulated the expression downstream genes is still largely unknown. Further work is needed to elucidate the function of these important genes in low-temperature related signal pathways.ABA is an important signal molecule related to abiotic stress. Previous studies had demonstrated that WRKY proteins may act as activator in ABA signaling [65]. It was reported that the expression ofLtWRKY21 inLarrea tridentata was shown to function as an activator to control ABA-regulated expression of genes [66]. Recently, Sun et al. also [67] reported that 13 numbers of WRKY family inO. sativa were upregulated by ABA, indicating that theseOsWRKY genes may play an important role in the response to abiotic stress, particularly as a key regulatory factor in ABA signaling pathway. MYBCORE, DPBF, and ABRE are known to involve in ABA and drought responses. Some MYBCORE, DPBF, and ABRE motifs were detected in theGbWRKY2 promoter by bioinformatic analysis (Figure 1S). The upregulated expression ofGbWRKY2,GbWRKY16, andGbWRKY21 in ginkgo callus by ABA gave the direct evidence, suggesting that these WRKY genes probably function as key components during ABA signaling. Our results also showed that all threeGbWRKYs were upregulated by ABA, indicating that theseGbWRKY genes may participate in ABA signaling pathway responding to abiotic stress.The upregulation ofGbWRKY2 by SA is expected because threecis-elements (GT1 motifs) associated with SA were identified in theGbWRKY2 promoter region. Interestingly,GbWRKY2 also responds with a strong induction to a wide range of molecules such as MeJA and ETH, which constitute key signaling elements modulating defense responses to pathogens. This suggests thatGbWRKY2 is a common component in the signaling pathways mediated by these hormones. Numerous studies report the induction of WRKY gene expression in response to SA and MeJA [13, 68, 69], but few analyses report the effect of other signal molecules such as ETH [38, 70]. Pathways involving MeJA and ET are considered to be mainly effective against necrotrophic pathogens, insects, and wounding, whereas those involving SA are more effective against biotrophs [71]. Thus, little attack of pathogens and insects in ginkgo may be due to the fact that WRKY protein, such asGbWRKY2, played important roles in defense responses by mediating SA, MeJA, and ETH signaling.Taken together,GbWRKY2,GbWRKY16, andGbWRKY21 were activated by more than one type of stress condition.GbWRKY2 was observed to be upregulated in response to many different sources of stress, including salinity, ABA, SA, ETH, and MeJA treatment.GbWRKY16 was observed to be upregulated in response to salinity, ABA, SA, and ETH treatment.GbWRKY21 was observed to be upregulated in response to ABA, SA, and ETH treatment.GbWRKYs were upregulated in response to more than two types of stress which supported the occurrence of cross-talk between signal transduction pathways in response to different stress conditions in plants. Moreover,GbWRKYs displayed complex expression patterns in response to the stress. For example,GbWRKY2 andGbWRKY16 were upregulated at 3 hpt in response to salinity treatment and then downregulated quickly, demonstrating thatGbWRKYs could be quickly and instantaneously responded to the stress.GbWRKY16 was not disturbed by the cold and MeJA treatment.GbWRKY16 was sustainably upregulated during the whole SA treatment. All of these showed thatGbWRKYs played complicated and essential roles in defense in response to the stress.


## 4.1.GbWRKY2 Is a Group II WRKY Transcription Factor

A ginkgo gene (GbWRKY2) encoding a protein with sequence homology to members of the WRKY family has been characterized. This newly characterized gene is the first WRKY factor described inG. biloba and presents structural hallmarks that allow us to classify it within Group II of WRKY TFs [11]. The phylogenetic analysis also clearly showed thatGbWRKY2 belong to Group II WRKY family. Numerous members of plant WRKY genes belong to Group II family and play roles in transcriptional reprogramming associated with resistance to various stresses [11]. A predicted nuclear localization signal (NLS) (Figure 4) strongly suggests that GbWRKY2 protein is translocated into the nucleus to control gene expression. These data suggested thatGbWRKY2 might play an important role in defense response of ginkgo to biotic and abiotic stresses.


## 4.2. Multiple cis-acting Elements Response to Stress and Hormone in the Promoter Region ofGbWRKY2

Stress-inducible gene expression is transcriptionally regulated by a change in the level and/or activity of sequence-specific DNA-binding transcription factors bound to specificcis-acting elements of promoter regions [54]. These regulatory factors are involved in the activation, suppression, and modulation of various signaling pathways in plant cells on biotic and abiotic stresses. Many plant promoters have therefore been identified and isolated, and genetic engineering in plants has been greatly enhanced using individual promoters [55]. Thus, we isolated a 1,363 bp-length promoter ofGbWRKY2 (Figure 1S). Bioinformatics analysis showed that multiplecis-acting elements including fundamental and special elements associated with abiotic stresses and hormone regulations were found in theGbWRKY2 promoter (Table 3), indicating thatGbWRKY2 gene might respond to various environment stimulus. Furthermore, a series of 5′ end deletion fragments of this promoter were detected using transient expression assays, which demonstrated that the activity of a truncated promoter from position −1 to −48 bp retained the ability to initiate the expression ofGUS in transgenic tobacco leaves. This region is enough to keep the basic function ofGbWRKY2 promoter. Bioinformatics analysis showed a TATA box was located in the region between −1 and −48 bp. Together taken into the results of bioinformatics and deletion analysis, we conclude that the region of −1 to −48 bp is the equal of core promoter and positions of TATA box correspond to the typical core promoter model [56]. Moreover, our experiments showed that deletion of the promoter regions between positions −1018 and −668 or between positions −288 and −137 caused very significant (P<0.01) reductions in promoter activity suggesting that these regions likely contain potential transcription-enhancingcis-acting elements. Sequence analysis discussed above indicates that both regions contain three and two Dof TF binding sites, respectively, which may be important for the hormonal responsiveness ofGbWRKY2 promoter.


## 4.3.GbWRKY2 Might Participate in the Development of Ginkgo Inflorescence

Similar to expression patterns observed in other plant species, threeGbWRKYs were found to be expressed in all tissues we used, but at different expression levels. Moreover, the transcription ofGbWRKY2 was observed abundant in flowers. In contrast, expression levels ofGbWRKY16 were very low in flowers. And the expression levels ofGbWRKY21 were significantly higher in leaves, stems, and female flowers than in roots and male flowers. The result may indicate thatGbWRKY2,GbWRKY16, andGbWRKY21 played different roles involved in regulating plant developmental and physiological processes. The highest expression level ofGbWRKY2 is in flowers while the lowest expression level of in stems, consistent with the spatial expression profile ofChWRKY2 fromCorylus heterophylla [57]. Given highest transcript level ofGbWRKY2 in flowers, it can be speculated thatGbWRKY2 might play an important role in tolerance to low temperature in inflorescence, which developed in cold early spring. Thus, we deduced thatGbWKY2 likely exhibited similar function ofChWRKY2 TF to participate in the developmental process of ginkgo inflorescence.


## 4.4.GbWRKYs Might Play Role in Stress-Related Signal Pathways

Salinity is an important abiotic stress factor, usually affecting plant growth, development, survival, and crop productivity. Thus, understanding the complex mechanism of salinity tolerance is important for agriculture production. Interestingly, several WRKY proteins were shown to be involved in plant salinity stress response. For example, the expression ofCmWRKY17 was induced by salinity inChrysanthemum morifolium and overexpression ofCmWRKY17 in Chrysanthemum and Arabidopsis increased the sensitivity to salinity stress [58]. The transcripts of two closely related WRKY TFs (AtWRKY25 andAtWRKY33) from Arabidopsis were increased by NaCl treatment and both the Atwrky33 null mutants and Atwrky25Atwrky33 double mutants showed moderately increased NaCl sensitivity [59]. Poplar species increase expressions of transcription factors to deal with salt environments. Salinity increased heat-shock transcription factor (HSF) transcription inP. euphratica andPeHSF binds the cis-acting heat shock element (SHE) of thePeWRKY1 promoter, thus activatingPeWRKY1 expression [60]. Similarly, the first evidence pointing towards a role forGbWRKY2 andGbWRKY16 in defense comes from the data showing that theGbWRKY2 andGbWRKY16 transcript is dramatically upregulated in ginkgo in response to NaCl. Further study on suppression/overexpression ofGbWRKY2 andGbWRKY16 was required for unveiling the molecular mechanism ofGbWRKY2 andGbWRKY16 gene participation in ginkgo tolerance to salt.Temperature that exceeds an organism’s optimal tolerance range is considered as an important abiotic stress factor. Tremendous work has been done in the past two decades to reveal the complex molecular mechanism in plants’ responses to extreme temperature and there is increasing evidence that WRKY proteins are involved in responses to both heat and cold stresses. For example, a WRKY TF inNicotiana tabacum responds to a combination of drought and heat stress [61]. Another example is that transgenic Arabidopsis plants overexpressingGmWRKY21 exhibited increased tolerance to cold stress when compared with wild-type plants [62]. In our studies, the expression levels ofGbWRKY2,GbWRKY16, andGbWRKY21 were all repressed by heat stress. The expression level ofGbWRKY2 was upregulated by cold stress and there was no significant effect of cold stress onGbWRKY16 transcript levels. In contrast, the expression level ofGbWRKY21 was downregulated by cold stress. We also reported for the first time an increase ofGbWRKY2 transcript level by cold but decrease by heat, consistent with the observation of cold responsivecis-elements (CRTDREHVCBF2 and MYC recognition site) (Table 3). Although low-temperature related WRKYs were isolated in several species [62–64]. The mechanism of how WRKYs respond to cold signals and regulated the expression downstream genes is still largely unknown. Further work is needed to elucidate the function of these important genes in low-temperature related signal pathways.ABA is an important signal molecule related to abiotic stress. Previous studies had demonstrated that WRKY proteins may act as activator in ABA signaling [65]. It was reported that the expression ofLtWRKY21 inLarrea tridentata was shown to function as an activator to control ABA-regulated expression of genes [66]. Recently, Sun et al. also [67] reported that 13 numbers of WRKY family inO. sativa were upregulated by ABA, indicating that theseOsWRKY genes may play an important role in the response to abiotic stress, particularly as a key regulatory factor in ABA signaling pathway. MYBCORE, DPBF, and ABRE are known to involve in ABA and drought responses. Some MYBCORE, DPBF, and ABRE motifs were detected in theGbWRKY2 promoter by bioinformatic analysis (Figure 1S). The upregulated expression ofGbWRKY2,GbWRKY16, andGbWRKY21 in ginkgo callus by ABA gave the direct evidence, suggesting that these WRKY genes probably function as key components during ABA signaling. Our results also showed that all threeGbWRKYs were upregulated by ABA, indicating that theseGbWRKY genes may participate in ABA signaling pathway responding to abiotic stress.The upregulation ofGbWRKY2 by SA is expected because threecis-elements (GT1 motifs) associated with SA were identified in theGbWRKY2 promoter region. Interestingly,GbWRKY2 also responds with a strong induction to a wide range of molecules such as MeJA and ETH, which constitute key signaling elements modulating defense responses to pathogens. This suggests thatGbWRKY2 is a common component in the signaling pathways mediated by these hormones. Numerous studies report the induction of WRKY gene expression in response to SA and MeJA [13, 68, 69], but few analyses report the effect of other signal molecules such as ETH [38, 70]. Pathways involving MeJA and ET are considered to be mainly effective against necrotrophic pathogens, insects, and wounding, whereas those involving SA are more effective against biotrophs [71]. Thus, little attack of pathogens and insects in ginkgo may be due to the fact that WRKY protein, such asGbWRKY2, played important roles in defense responses by mediating SA, MeJA, and ETH signaling.Taken together,GbWRKY2,GbWRKY16, andGbWRKY21 were activated by more than one type of stress condition.GbWRKY2 was observed to be upregulated in response to many different sources of stress, including salinity, ABA, SA, ETH, and MeJA treatment.GbWRKY16 was observed to be upregulated in response to salinity, ABA, SA, and ETH treatment.GbWRKY21 was observed to be upregulated in response to ABA, SA, and ETH treatment.GbWRKYs were upregulated in response to more than two types of stress which supported the occurrence of cross-talk between signal transduction pathways in response to different stress conditions in plants. Moreover,GbWRKYs displayed complex expression patterns in response to the stress. For example,GbWRKY2 andGbWRKY16 were upregulated at 3 hpt in response to salinity treatment and then downregulated quickly, demonstrating thatGbWRKYs could be quickly and instantaneously responded to the stress.GbWRKY16 was not disturbed by the cold and MeJA treatment.GbWRKY16 was sustainably upregulated during the whole SA treatment. All of these showed thatGbWRKYs played complicated and essential roles in defense in response to the stress.


## 5. Conclusion

This study presents the isolation and expression profile of the novel WRKY transcription factor geneGbWRKY2, which encodes a protein of 337 amino acid residues. The protein domain and phylogenetic analysis also showed thatGbWRKY2 belong to Group II WRKY family. Bioinformatics analysis showed that multiplecis-acting elements including fundamental and special elements associated with abiotic stresses and hormone regulations were found in theGbWRKY2 promoter. Expression pattern analysis suggested thatGbWRKY2 might play an important role in tolerance to salt and cold stresses and defense responses by mediating ABA, SA, MeJA, and ETH signaling. Studies on downstream function genes regulated by WRKY TF and mutual regulation between WRKY TFs inG. biloba have not been reported. On the basis of response abiotic adversity ofGbWRKY2, we establish the binary overexpression vector of this gene. Studies on the genetic transformation of this gene in the callus ofG. biloba are underway. This study will provide a basis to further reveal that the upregulated gene expression can strengthen the ability of plants to resist abiotic stress and identify some targeted genes regulated by WRKY TF inG. biloba. The present work on cloning and characterization ofGbWRKY2 provided new clues for future studies on Ginkgo response to various stresses, such as sanity, drought, cold, and disease.



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*Source: 607185-2015-08-16.xml*