Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema / 中国中药杂志

WRKY transcription factor family plays an important role in plant growth and development, secondary metabolite synthesis, and biotic and abiotic stress responses. The present study performed full-length transcriptome sequencing of Polygonatum cyrtonema by virtue of the PacBio SMRT high-throughput platform, identified the WRKY family by bioinformatics methods, and analyzed the physicochemical properties, subcellular localization, phylogeny, and conserved motifs. The results showed that 30.69 Gb nucleotide bases and 89 564 transcripts were obtained after redundancy removal. These transcripts had a mean length of 2 060 bp and an N50 value of 3 156 bp. Based on the full-length transcriptome sequencing data, 64 candidate proteins were selected from the WRKY transcription factor family, with the protein size of 92-1 027 aa, the relative molecular mass of 10 377.85-115 779.48 kDa, and the isoelectric point of 4.49-9.84. These WRKY family members were mostly located in the nucleus and belonged to the hydrophobic proteins. According to the phylogenetic analysis of WRKY family in P. cyrtonema and Arabidopsis thaliana, all WRKY family members were clustered into seven subfamilies and WRKY proteins from P. cyrtonema were distributed in different numbers in these seven subgroups. Expression pattern analysis confirmed that 40 WRKY family members had distinct expression patterns in the rhizomes of 1-and 3-year-old P. cyrtonema. Except for PcWRKY39, the expression of 39 WRKY family members was down-regulated in 3-year-old samples. In conclusion, this study provides abundant reference data for genetic research on P. cyrtonema and lays a foundation for the in-depth investigation of the biological functions of the WRKY family.

The role of plant WRKY transcription factors against salt stress: a review / 生物工程学报

High salt content in soils severely hampers plant growth and crop yields. Many transcription factors in plants play important roles in responding to various stresses, but their molecular mechanisms remain unclear. WRKY transcription factors are one of the largest families of transcription factors in higher plants that are involved in and influence many aspects of plant growth and development. They play important roles in responding to salt stress. The regulation of gene expression by WRKY proteins is mainly achieved by binding to the DNA's specific cis-regulatory elements, the W-box elements (TTGACC). In recent years, there have been many studies revealing the roles and mechanisms of WRKY family members, from model plant Arabidopsis to agricultural crops. This paper reviews the latest research progress on WRKY transcription factors in response to salt stress and discusses the current challenges and future perspectives of WRKY transcription factor research.

Bioinformatics and expressional analysis of WRKY transcription factor family in Baphicacanthus cusia / 药学学报

WRKY, a class of conserved transcription factors in plants, plays important roles in plant growth, development and secondary metabolism. In the present study, 65 WRKY members were identified from de novo transcriptome sequencing data of three different tissues (root, stems and leaves) of Baphicacanthus cusia. BcWRKY proteins contained from 221 to 706 amino acids and the isoelectric point is from 4.68 to 9.68. Molecular weights range from 25 711.8 to 75 475 Da. The main secondary structures of BcWRKYs protein are random coil. A subcellular localization prediction indicated that the putative BcWRKY proteins were enriched in the nuclear region. Phylogenetic analysis showed that BcWRKYs could be categorized into three groups and five subgroups (Group IIa, Group IIb, Group IIc, Group IId and Group IIe) in Group II. Structural analysis found that all BcWRKY proteins contained a highly conserved motif WRKYGQK. Finally, the transcriptional profiles of ten BcWRKY genes highly expressed in root, stem and leaf tissues under abscisic acid (ABA), methyl jasmonate (MeJA), or salicylic acid (SA) treatment were systematically investigated using qRT-PCR analysis. Results showed that a total of ten BcWRKY genes were differentially expressed in response to ABA, MeJA, and SA treatment. This work would be provided a basis for further elucidating the molecular mechanism of WRKY transcription factors in the biosynthesis of indole alkaloids in B. cusia.

Identification and expression analysis of WRKY gene family in eukaryotic algae / 生物工程学报

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.

Molecular mechanism of WRKY12 in regulating plant development / 生物工程学报

WRKY transcription factors are one of the largest families of transcription factors in higher plants and involved in regulating multiple and complex growth and development processes in plants. WRKY12 is a typical member of WRKY family. This article summarizes recent research progresses on the regulatory mechanism of WRKY12 in multiple growth and development processes, and analyzes the functional differences between WRKY12 and WRKY13. It provides a useful reference for further studying the molecular mechanism of WRKY12 in plant complex developments. It also provides clearer research ideas and reference strategies for exploring the self-regulation of other WRKY member and the mutual regulatory relationships between different WRKY family genes.

Cloning and expression analysis of WRKY family member SmWRKY14 in Salvia miltiorrhiza / 中草药

Objective: To clone a WRKY protein gene SmWRKY14 with full length cDNA from Salvia miltiorrhiza and carry out bioinformatics and expression analysis in different tissues and response to environmental factors and phytohomone. Methods: The PCR was preformed based on the sequence of Unigene (c50007_g1) searched from our transcriptome database, and characteristics of physiochemical properties, conserved domains and structure prediction of the protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNAMAN and MEGA 6.0, respectively. Real-time quantitative PCR was used for gene expression analysis. Results: In this study, the full length cDNA of SmWRKY14 was 1103 bp in size, encoding a 244-aa protein with a molecular weight of 27.6 KDa and an isoelectric point of 8.19. SmWRKY14 was an unstable hydrophilic protein containing characteristic and conserved WRKY domain without signal peptide or transmembrane domain. The main secondary structure of the amino acid sequences was random coil. Moreover, multiple sequence alignments and phylogenetic trees showed that SmWRKY14 protein had high homology with WRKY14 of Diospyros kaki. Quantitative real-time PCR indicated that SmWRKY14 constitutively expressed in the roots, stems, leaves and flowers of S. miltiorrhiza and was strongly induced by methyl jasmonate, abscisic acid, gibberellins, and mechanical wound, which indicated SmWRKY14 could participate in regulation of biosynthesis of tanshinones and defense process. Conclusion: The gene sequences of SmWRKY14 was successfully cloned and the bioinformatics and expression pattern analysis was carried out, which will provide a foundation for further research on the molecular mechanism of regulation of tanshinones synthesis and response to defense process in S. miltiorrhiza.

Cloning of AhWRKY33 from Asarum heterotropoides and its genetic transformation in Arabidopsis / 中国中药杂志

The WRKY family genes, which play an important role in plant morphogenesis and stress response, were selected based on the data of the full-length transcriptome of Asarum heterotropoides. Using AtWRKY33, which regulates the synthesis of the camalexin in the model plant Arabidopsis to compare homologous genes in A. heterotropoides, primers were designed to amplify the open reading frame(ORF) fragment of AhWRKY33 gene by RT-PCR using total RNA of A. heterotropoides leaves as template. Real-time PCR results showed that there was a significant difference between the aerial part and the underground part of A. heterotropoides, the toxic aristolochic acid content is highly expressed in the leaves higher than the root. After verification, the WRKY33 gene of A. heterotropoides is ORF long 1 686 bp, encoding 561 amino acids.AhWRKY33 had two conserved WRKYGQK domains. According to the classical classification, it belongs to group Ⅰ WRKY transcription factor. A. heterotropoides WRKY33 had some homology with amino acids of other species. The study successfully constructed the plant eukaryotic expression vector PHG-AhWRKY33 and transformed Arabidopsis thaliana, the transgenic Arabidopsis was obtained by PCR detection and hygromycin resistant plate screening. It found that the germination of transgenic Arabidopsis seeds was accelerated and the stress resistance was increased. It laid a foundation for further analysis of WRKY transcription factor in the growth and development of A. heterotropoides and the synthesis of secondary metabolites.

Transcriptome-wide Identification and Expression Profiles of WRKY Transcription Factor in Prunella vulgaris / 中国实验方剂学杂志

Objective:To identify WRKY genes from the transcriptome dataset of Prunella vulgaris by bioinformatics method, and analyze the protein characteristics and expression level of these genes. Method:WRKY transcription factor were identified from the P. vulgaris transcriptome database，their motif，physical and chemical properties，functional annotations，family evolution and expression patterns were analyzed, and their functions were predicted. Result:A total of 23 WRKY transcription factors were identified from P. vulgaris in this study by computational prediction method.Structural analysis found that WRKY proteins contained a highly conserved motif WRKYGQK. Phylogenetic analysis of WRKYs together with the homologous genes from Arabidopsis thaliana could be divided into two groups（group Ⅰ-Ⅱ）. There were 7 members in group Ⅰ，and 16 members in group Ⅱ, group Ⅱ was subdivided into five subgroups，namely group Ⅱb （3 members），Ⅱc（5 members），Ⅱd（3 members），Ⅱe（5 members）. The physical and chemical properties of WRKY protein showed that the amino acid number was between 85 and 599，the molecular weight was between 9 527.5-66 438.45 Da，the theoretical isoelectric point was between 5.01-9.83.Among them, c13719.graph_c0，c32199.graph_c0，c24547.graph_c0，c37881.graph_c0 may play a role in the regulation of secondary metabolitessynthesis of P. vulgaris.And c32199.graph_c0，c26537.graph_c0，c23728.graph_c0 may has an effect in identifying and defensing pathogens in P. vulgaris.The transcriptional profiles of these 23 WRKY genes in various tissues were investigated using transcriptome dataset.The results showed that the expression level of WRKY genes varied significantly in different tissues. Conclusion:This study identifies the organization and transcriptional profiles of PmWRKY genes for the first time, so as to provide the helpful information for further studies of functions of WRKYs.

Cloning and expression analysis of eight WRKY genes in Camellia sinensis / 中草药

Objective: To clone eight members of WRKY of transcription factor family in Camellia sinensis, and analyze their bioinformatics and expression under abiotic stress. Methods: Eight WRKY transcription factor genes were cloned from Tieguanyin cultivar by RT-PCR, and the physicochemical properties of the eight WRKY protein were analyzed by bioinformatics Methods:. At the same time, the establishment of phylogenetic tree, comparison of multiple sequences, and analysis of conserved motifs were carried out by comparing WRKY of C. sinensis with homologous genes of Arabidopsis thaliana. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of eight WRKY genes under low temperature, drought, and ABA stress treatment. Results: The ORF lengths of eight WRKY genes were 1 407, 2 208, 1 302, 849, 978, 879, 1 443, and 810 bp, encoding 468, 735, 433, 282, 325, 292, 480, and 269 amino acids, respectively. GenBank accession numbers were MG298951, MG298952, MG298955, MG298956, MG298957, MG298959, MG298960, and MG298963, respectively. Phylogenetic tree and sequence alignment analysis showed that eight CsWRKYs could be divided into two groups and contained WRKYGQK conserved domain and zinc finger structures, except that CsWRKY39 lacked zinc finger structure. The expression pattern of CsWRKYs was induced under the condition of low temperature, drought, and ABA stress. The expression of CsWRKY2, CsWRKY21, CsWRKY23, CsWRKY44 and CsWRKY65 increased to more than 2 after low temperature treatment with significant response to low temperature stress. The expression of CsWRKY21, CsWRKY23, CsWRKY3,9 and CsWRKY65 was up-regulated under 12 h of drought stress and 6 h of ABA treatment. This result indicated that CsWRKYs might be closely related to stress response in C. sinensis. Conclusion: Eight CsWRKY genes from different groups were cloned, and this result indicated that CsWRKYs might be closely related to stress response in C. sinensis.

Molecular cloning, prokaryotic expression and characterization of a transcription factor AsWRKY62 in Aquilaria sinensis / 中草药

Objective: The molecular cloning and prokaryotic expression of the transcription factor AsWRKY62 of Aquilaria sinensis were carried out, at the same time, the bioinformatics analysis and expression pattern analysis were also performed. The purpose of this study was to lay a foundation for further study on the role of AsWRKY62 in the growth and development of A. sinensis and the formation of agarwood. Methods: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY62 was amplified using RT-PCR and PCR method. The recombinant vector pET-21a-AsWRKY62 which was built and verified by gene recombination technique was transformed into E. coli BL21 (DE3) for prokaryotic expression and purification. The characteristics of physiochemical properties, conserved domains and subcellular localization of AsWRKY62 were calculated by a series of bioinformatics tools. The analyses of multiple sequence alignment of amino acid and phylogenetic tree were performed using DNAMAN and MEGA 5.0, respectively. The gene expression pattern in different tissues was detected by RNA-seq data. Results: The full length CDS of AsWRKY62 (GenBank accession MH925301) was 1581 bp, encoding a 526-aa protein which belongs to WRKY group I. The optimized induction conditions of recombinant pET-21a-AsWRKY62 were 0.5 mmol/L IPTG at 37 ℃ for 4 h. According to the tissue-specific expression pattern analysis, the AsWRKY62 gene in A. sinensis is mainly expressed in roots and stems, followed by agarwood and flowers. Conclusion: Cloning, expression and characterization of the AsWRKY62 gene for the first time indicated that it may be related to the formation of agarwood, which provided a theoretical basis for further study of its biological function.

Gene cloning and expression analysis of benzoic acid stress responsive transcription factor WRKY7 in Panax ginseng / 中国中药杂志

WRKY transcription factor is one of the most important transcription factor families widely existing in higher plants, which playing critical role in plant morphogenesis, development, biotic (including phytopathogens, pests etc.) and abiotic (drought, salt, chilling, high temperature, etc.) stress. In the present work, primers used to amplify full-length gene encoding WRKY transcription factor were designed based on the transcriptome data of P. ginseng that induced by benzoic acid, one of the most important autotoxins identified from root exudates and rhizosphere soil of P. ginseng. Then, a WRKY gene, temporarily named as WRKY7, was confirmed by RT-RCR. Furthermore, sequencing and sequence analysis of WRKY7 was conducted. Results indicated that, the full length cDNA of WRKY7 was 1 216 bp, the open reading frame (ORF) of which was 1 014 bp, encodes 337 amino acids. Homologous analysis and phylogenetic tree showed that, WRKY7 belonged to the Ⅲ category of WRKY families, which showing 87% similarity to WRKY6 in P. quinquefolius. Real-time PCR results showed that the expression of WRKY7 in P. ginseng induced by benzoic acid was up-regulated markedly than the control, so we speculated that WRKY7 was involved in the response to benzoic acid stress, which will be helpful for further research on the molecular mechanism of ginseng plant response to benzoic acid stress.

Identification of WRKY Transcription Factors Related to Saikosaponin Biosynthesis in Adventitious Roots of Bupleurum chinense / 中草药·英文版

Objective To identify the genes of WRKY transcription factors (TFs) from roots of Bupleurum chinense and genes that potentially regulate saikosaponin (SS) biosynthesis. Methods Firstly, the subfamily cluster analysis was mainly based on Arabidopsis thaliana WRKYs for 27 putative WRKY TFs selected from previous transcriptome sequencing data. Secondly, qPCR was used to screen such genes of WRKY TFs that could be induced by NaCl and PEG6000 in adventitious roots of B. chinense. Meanwhile, saikosaponins (SSs) in treated adventitious roots were determined by HPLC. The roots were collected at 0, 2, 4, 8, 12, 24, 48, and 72 h after treatments, and 120 h only for PEG. Finally, the tissue-specific expression was analyzed on screened genes by qPCR. Results The 27 genes were grouped into three categories: There were nine in Group I, 15 in Group II, and two in Group III. Four genes of WRKYTFs, BCWRKY6, BCWRKY16, BCWRKY32, and BCWRKY35 were obviously induced by NaCl in adventitious roots of B. chinense, while only BCWRKY32 was induced by PEG. The content of SSs increased at different levels in NaCl and PEG6000 treatment. Three genes including BCWRKY6, BCWRKY32, and BCWRKY35, expressed most in roots, were similar to the accumulation pattern of SS. Conclusion The three WRKY genes, BCWRKY6, BCWRKY32, and BCWRKY35, may be involved in the biosynthesis of SS.

Molecular cloning and characterization of a transcription factor gene dowrky3 in dendrobium officinale / 中草药

Objective To isolate and characterize a WRKY transcription factor encoding gene DoWRKY3 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. Methods RT-PCR and RACE technologies were used to isolate the full length cDNA of DoWRKY3. Characteristics of physiochemical properties, conserved domains, and subcellular localization of the deduced DoWRKY3 protein were determined by a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. Results The full length cDNA of DoWRKY3 (GenBank accession KT957549) was 2 065 bp in length, and encoded a 509-aa protein with a molecular weight of 55 580 and an isoelectric point of 6.58; The deduced DoWRKY3 protein sequence had two WRKYGQK motifs, two WRKY domains (217-279, 381-449), and two C2H2-type zinc-finger signatures (C-X4-C-X22-23-H-X1-H), which are all conserved among the WRKY proteins.DoWRKY3 protein did not contain a signal peptide or a transmembrane region, and was predicted to locate in nucleus; DoWRKY3 had high identities (46.3%-57.4%) with various WRKY proteins from several plants; DoWRKY3 was closely related to Arabidopsis AtWRKY3, AtWRKY4, and Salvia miltiorrhiza SmWRKY54 proteins, and belonged to the Group 1 of the WRKY evolutionary tree; DoWRKY3 gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots and leaves, with 2.32 and 1.69 fold, respectively, over that in the stems. Conclusion Molecular cloning and characterization of the full length DoWRKY3 gene will be useful for further functional determination of the gene involving in the growth and development, physiological stress adaptations, and secondary metabolic regulations of D. officinale.

Cloning and expression analysis of WRKY5 gene in Dendrobium officinale / 中草药

Objective: In order to isolate and analysize the bioinformatics and expression pattern of DoWRKY5 gene from Dendrobium officinale. Methods: A WRKY gene was first obtained by transcriptome sequencing and reverse transcription-polymerase chain reaction (RT-PCR) from D. officinale and analyzed by bioinformatics tools. The tissue expression pattern and the low temperature stress, abscisic acid (ABA) stress, and sucrose stress responses were analyzed by qRT-PCR. Results: The cDNA sequence of DoWRKY5 gene was isolated, which was 1 336 bp in length, with an open reading frame (ORF) of 834 bp and an encoded polypeptide of 277 amino acid. The amino acid sequence contained a conserved WRKY domains and a zinc finger structures (C2H2), belonging to Group II of WRKY family. Expression analysis by qRT-PCR showed that DoWRKY5 was expressed in the roots, stems, and leaves of D. officinale, and the most abundant in leaves. The amount of DoWRKY5 expression were significantly increased under low temperature of 4℃ and different time. Moreover, the expression of DoWRKY5 could be induced by ABA and source. Conclusion: DoWRKY5 may be an important transcription factor to response cold stress and other abiotic stresses in D. officinale, which provides a foundation for further study of cold tolerance mechanism and cold-resistant breeding of D. officinale.

Identification and bioinformatic analysis on rhizomes of Dioscorea zingiberensis WRKY transcription factor / 中草药

Objective: To identify WRKY genes from the rhizomes of Dioscorea zingiberensis and analyze the protein characteristics and expression level of these genes. Methods: The transcriptional EST database of the rhizomes of D. zingiberensis was used to search the analogs of AtWRKY genes by BLASTn, the full-length open reading frames (ORF) of DzWRKY and its protein characteristics were studied using bioinformatic method, and the expression levels of DzWRKY genes in rhizomes and leaves were detected from transcriptional data of the rhizomes of D. zingiberensis. Results: Twenty-seven DzWRKY transcription factors family genes with full length ORF were isolated from the rhizomes of D. zingiberensis, and two WRKY domains were confirmed in six WRKY genes. All the DzWRKY proteins were predicted as hydrophilic proteins and nucleoproteins with highly conserved WRKY domains. The 27 DzWRKY and 19 AtWRKY proteins were divided into three groups by phylogenetic analysis. All DzWRKY genes showed higher expression level in the leaves compared to the rhizomes, and highly expressed genes were mainly in groups I and IId. DzWRKY proteins exhibited lower sequence identity. Conclusion: The DzWRKY genes are successfully isolated for the first time, which would provide a reference for the study on the roles of WRKY in development and active components biosynthesis of the rhizomes of D. zingiberensis.

Arabidopsis FLOWERING LOCUS D influences systemic-acquiredresistance- induced expression and histone modifications of WRKY genes.

A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections – a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that FLOWERING LOCUS D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysinespecific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD’s involvement in epigenetic regulation of SAR.

GbWRKY1, a novel cotton (Gossypium barbadense) WRKY gene isolated from a bacteriophage full-length cDNA library, is induced by infection with Verticillium dahliae.

WRKY transcription factor proteins play important roles in diverse stress responses. In this study, we first cloned a novel WRKY from our constructed bacteriophage full-length cDNA library for cotton (Gossypium barbadense). The plants were stressed by exposure to a defoliating strain of Verticillium dahliae. The capacity of primary cDNA library was 1.28 × 106 PFU and the titer of the amplified cDNA library was >1010 PFU mL–1. The recombination rate of the library was 94% and average insert size was about 1.1 kb. This novel gene, named GbWRKY1 was 1971 bp long and encodes a protein of 489 amino acids. It contains two characteristic WRKY domains and two zinc finger motifs. The sub-cellular assay indicated that GbWRKY1–GFP fusion protein was localized in the nucleus. Furthermore, Northern blot analysis showed that expression pattern of GbWRKY1 was similar among tissue types (roots, stems and leaves), but differed between pathogen-infiltrated and Czapek medium-infiltrated (untreated control) plants. Quantitative real-time PCR showed that GbWRKY1 could also be induced by salicylic acid (SA), methyl jasmonate (MeJA) and 1-aminocyclopropane-1-carboxylic acid (ACC). These findings clearly suggest that as a pathogen-inducible transcription factor GbWRKY1 plays an important role in plant defense responses.

Isolation of a Rice WRKY Gene OsWRKY52,Whose Expression Is Induced by Magnaporthe grisea / 生物化学与生物物理进展

WRKY proteins, a big family of transcription factors, are involved in regulation diverse developmental and physiological processes in plants. Here, a novel WRKY gene, OsWRKY52, was isolated from a rice cDNA library. This gene included an open reading frame of 1 719 bp in length, and the deduced polypeptide contained 572 amino acids,sharing 54% identity with a WRKY1 protein from Avena sativa. Expression of OsWRKY52 gene was induced rapidly by Magnaporthe grisea in the incompatible interaction with rice plant. OsWRKY52 protein, expressed prokaryotically bound specifically to W box cis elements derived from the promoter of a rice PR1a. Transcriptional activation assay was performed by a yeast one- hybrid method. Regions of transactivation were identified to be the N-terminal serine- and threonine-rich islands and the C-terminal acidic domain of OsWRKY52. These results suggest that OsWRKY52, as a transcription activator, may be involved in defense responses against Magnaporthe grisea in rice plants.