Introduction

WRKY transcription factors (TFs) were widely distributed in plants, which were first discovered in sweet potato (Ipomoea batatas)1. With genome-wide analyses of different species, WRKY genes have been identified in more species, that included 66 WRKY genes in Arabidopsis2, 119 WRKY genes in maize3, 94 WRKY genes in sorghum4, 79 WRKY genes in potato5, 70 WRKY genes in chickpea6, and 61 WRKY genes in cucumber7. The WRKY protein contains a conserved WRKYGQK motif at its N-terminal and a 60-amino acid-long zinc finger motif at its C-terminus8. Zinc finger motifs can be classified as C2H2 or C2HC. WRKY proteins can be classified into three categories (I, II, III) based on the number of WRKY domains and the type of zinc finger motif9. Group I members have two WRKY domains and zinc fingers of type C2H2. Group II members have only one WRKY domain and one C2H2 zinc finger motif, and Group III members have one WRKY domain and one C2HC zinc finger. Group II can be further subdivided into five subgroups: IIa, IIb, IIc, IId, and IIe10,11.

Studies have shown that WRKY TFs play an important regulatory role in plant growth and development12. For example, AtWRKY12 and AtWRKY13 can exert important regulation on the flowering process of Arabidopsis13. The over-expression of GsWRKY20 from Glycine soja in Arabidopsis also altered the plant's flowering process14. However, OsWRKY11 in rice can control flowering time and plant height15. These depend on WRKY genes being expressed at different times in different parts of the plant. Similarly, the expression of these genes also affects various aspects of plants, such as hormone regulation and stress resistance16.

For example, overexpression of OsWRKY45 in rice enhances disease resistance and drought resistance17. The AtWRKY25 and AtWRKY33 in Arabidopsis thaliana enhance salt tolerance18. Some AcWRKY TFs of kiwifruit were up-regulated under salt stress19. Considering the important role of WRKY TFs in plant growth and development, the study of these genes is very important for agricultural production. The overexpression of TaWRKY2 in wheat enhanced the tolerance to drought stress and increased the yield20. Vitis amurensis VaWRKY12 gene enhanced the cold tolerance of transgenic grape callus21.

Most studies have focused on the genes of annual grasses, but few studies have focused on the genes of perennial grasses. Miscanthus is a perennial grass that has historically been cultivated as a papermaking material and as an ornamental plant. In recent years, Miscanthus has played a role in the direction of ecological restoration and sustainable bioenergy crops22. Therefore, the study has great significance for the genetic improvement of Miscanthus. The improvements can increase productivity and ensure that the crops remain robust despite persistent biological and abiotic stresses. As an essential stress resistance gene, WRKY TFs have high research value. The availability of the whole genome assembly of Miscanthus sinensis enabled identifying MsWRKY. The identification and study of MsWRKY are helpful in understanding the mechanism of plant stress resistance. The genomes of at least four Miscanthus species (M. sacchariflorus, M. sinensis, M. lutarioriparius, and M. floridulus) have been sequenced. M. sinensis was used as the object of this study. M. sacchariflorus has strong stress resistance and large biomass. However, the growth cycle of the plant is too long for commercial biomass production. M. lutarioriparius and M. floridulus have a narrow distribution range and are only found in parts of East Asia. Their biomass yield is low, and the fiber content is too high. Therefore, they are not suitable for the bioenergy development. The function of the Miscanthus WRKY family has been identified and characterized by various methods.

Material and methods

Identification of WRKY family genes in Miscanthus

The genomic data for Miscanthus were obtained from Phytozome 13 (https://phytozome-next.jgi.doe.gov/). From these data, the putative MsWRKY genes can be identified. The database contains the amino acid sequences of Miscanthus WRKY proteins. The Joint Genome Institute (JGI) (https://phytozome.jgi.doe.gov/pz/portal.html#) and the WRKY domain ID were used to identify the potential WRKY (PF03106) proteins of Miscanthus. The data used was Miscanthus sinensis v7.1. To ensure the quality of the data, the CD-HIT suite (https://github.com/weizhongli/cdhit) and Simple Modular Architecture the Research Tool (SMART) (http://smart.embl-heidelberg.de/#) were used to process the resulting sequence23. The duplicate sequences and incomplete sequences were removed.

At the same time, the ExPASy proteomic server (http://web.expasy.org/protparam) was used to predict the physical and chemical properties of the proteins. Their isoelectric point (pI) and molecular weight (MW) were obtained.

Chromosome mapping and classification and phylogenetic analysis of MsWRKY genes

The chromosomal locations of all identified MsWRKY genes were obtained from the Phytozome BioMart tool (https://phytozome.jgi.doe.gov/biomart/martview/). And the MG2C v2.1 (mg2c.iask.in/mg2c_v2.1/) was used for Chromosome mapping of MsWRKY genes.

In constructing a phylogenetic tree to classify MsWRKY genes, we need to use Sorghum bicolor WRKY amino acid sequences. The data were obtained from The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/). They will be used with our MsWRKY sequences. The MEGA v7.0 (https://www.megasoftware.net/) for constructing a phylogenetic tree used multiple sequence alignments with ClustalW to process SbWRKY and MsWRKY protein sequences. The Neighbor-Joining method and the p-distance model were used in the process, and the pairwise deletion and 1000 bootstrap replicates were selected24. Eventually, the phylogenetic tree of SbWRKY and MsWRKY sequences was obtained. Thus, the unknown MsWRKY genes can be divided into different groups and subgroups. By using the sequence alignment data and the phylogenetic tree, the putative Miscanthus WRKY orthologs in Arabidopsis can be identified5.

Gene structure analysis and conserved motif distribution analysis of MsWRKY genes

The genomic sequence and coding sequence (CDS) of each MsWRKY gene can be used to predict the gene structure of the MsWRKY gene. The exon–intron structure of MsWRKYs was analyzed using TBtools25.

The Multiple Em for Motif Elicitation (MEME) (v.5.5.3; https://meme-suite.org/meme/tools/meme) used the parameters which are: maximum motif number: 20; site distributions: any number of repetitions; minimum and maximum width: 6 and 50, respectively to distinguish MsWRKY proteins with conserved motif.26.

Gene ontology annotation and analysis of cis-acting elements of MsWRKY genes

For the gene ontology (GO) annotation analysis of the obtained MsWRKY proteins, the eggNOG-mapper 2.1.12 (http://eggnog-mapper.embl.de/)27 was used. Then, the TBtools was used to map and annotate the obtained data. Ultimately, the biological processes, molecular functions and cellular components of these proteins were obtained.

The online website PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) analyzed 2000 bp of the upstream region for all MsWRKY genes to analyze the cis-acting elements of MsWRKY genes. It provided the cis-acting elements of MsWRKY genes.

Synteny analysis of MsWRKY genes

The Multiple Collinearity Scan toolkit (MCScanX) was used to examine the gene duplication events with the default parameters. The TBtools was the platform used to analyze the data. The Evalue of the blastP was 15. To explore the syntenic relationships of the WRKY genes obtained from Miscanthus and other selected species, syntenic analysis maps were constructed using MCScanX.

Digital expression pattern analysis of MsWRKY genes

The TBtools analyzed the Miscanthus transcriptomic array data to make heatmaps of the MsWRKY expression profiles to survey MsWRKY expression profiles. In the meantime, the Miscanthus transcriptomic array data was obtained from the JGI database (https://phytozome-next.jgi.doe.gov/).

Results

Identification of WRKY family members in Miscanthus

To identify members of the MsWRKY family, the WRKY domain consensus sequence (PF03106) and the keyword WRKY were used to search in the database. The most complete genome assembly for Miscanthus (Miscanthus sinensis v7.1) in the JGI database was selected. In the MsWRKY family, a conserved domain exists as the basic criterion for inclusion of genes. Using the conserved domain called the WRKYGQK or WRKYGQK-like conserved domain, 203 genes were identified in the JGI database. In these genes, the duplicates and incomplete were removed by the multiple sequence alignments of MEGA 7.0. At the same time, we removed the severely incomplete gene, identified the location of WRKY domain, and kept some of the incomplete genes because they clearly belonged to the MsWRKY family. It was done using the SMART database. In the end, there are a total of 179 non-redundant MsWRKY sequences. The sequences and proteins of these genes are summarized in Table 1.

Table 1 Characteristics of the identified MsWRKY genes.
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These MsWRKYs have been named from MsWRKY01 to MsWRKY179 according to their distribution on the chromosome. The starting point was the upper arm of chromosome 1, moving down to the lower arm (Table 1). The properties of this set of MsWRKY proteins were investigated. The MsWRKY proteins ranged from 98 to 1620 amino acids. Their average length was 368 amino acids. Their predicted MW and pI values ranged from 11,246 to 180,044 and 4.8 to 11.79, respectively.

Chromosome mapping and classification and phylogenetic analysis of MsWRKY genes

The locations of the 179 MsWRKY genes were determined by MG2C v2.1 (Fig. 1). MsWRKY genes were distributed on all 19 Miscanthus chromosomes (Chr). Chr01-Chr19 was the chromosome (Chr) number indicating the names and positions of the MsWRKY. Chr 5 had the highest number of MsWRKYs, with 23, representing 12.9% of the total gene family. It was followed by 18 genes on Chr 6, 14 on Chr 16, and 13 on Chr 17. Chromosomes 9, 12, and 13 had only four MsWRKYs each, the fewest.

Figure 1
figure 1

Distribution of 179 MsWRKY genes on Miscanthus chromosomes.

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An unrooted phylogenetic tree to study the evolution of MsWRKY family members was constructed by the multiple sequence alignment and neighbor-joining method in MEGA7.0. The data was full-length protein sequences of 40 SbWRKYs and 179 MsWRKYs. SbWRKYs are used as the basis for grouping. They were from Sorghum bicolor (L.) moench, a Poaceae plant that is similar to Miscanthus sinensis.

179 MsWRKYs could be divided into three major groups (I, II, and III) according to the constructed phylogenetic tree (Fig. 2). Of the 24 MsWRKYs in group I, all of them had two WRKYGQK motifs and 23 of them have two C2H2-type zinc finger motifs (C-X3-4-C-X22-23-H-X1-H), corresponding to two complete WRKY domains. Although the protein encoded by MsWRKY09 had only one zinc finger motif, it belonged to group I on the phylogenetic tree.

Figure 2
figure 2

Phylogenetic tree of WRKY members in Miscanthus and Sorghum. All MsWRKYs genes were further divided into subgroups I, II, and III, and group II was further divided into subgroups IIa, IIb, IIc, IId, and IIe.

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Group II had a total of 97 protein sequences and was the largest group, accounting for 54.2% of all putative MsWRKYs. Similar reports can also be found in sorghum, cucumber and chickpeas. Most of these proteins had one WRKY domain and the C2H2-type zinc finger motif (C-X4-5-C-X23-H-X1-H). This group was further divided into five subgroups, IIa, IIb, IIc, IId, and IIe, with 8, 16, 32, 21, and 20 members, respectively. Fifty-eight proteins belong to Group III. The proteins in this group had one WRKY domain and the C2HC-type zinc finger motif (C-X7-C-X23-H-X1-C)28. In summary, the classification of MsWRKYs indicated the diversity of these proteins. An extremely wide range of functions could be performed by these proteins.

Gene structure analysis and conserved motif distribution analysis of MsWRKY genes

The exon–intron structures of MsWRKY family members can illustrate the evolution of MsWRKY family members. The introns number of MsWRKY genes ranged from zero to five, while their size varied. The data showed that genes within the same group had certain similarities in the exon–intron distribution patterns. These results suggest that the MsWRKY genes had important structural diversity. It represented the functional diversity among closely related members of MsWRKYs (Fig. 3).

Figure 3
figure 3

Exon–intron structures of MsWRKY genes. Exon–intron structures of MsWRKY genes were obtained after analysed with TBtools for gene structure. Green bars indicate upstream and downstream UTRs, yellow bars indicate coding sequences (CDS), and black lines indicate introns in the gene diagrams.

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MEME (version 5.5.3), used to analyze the conserved motifs of all MsWRKY protein sequences, identified 20 distinct conserved motifs. The distribution of 20 conserved motifs identified by MEME in the different groups of MsWRKYs is shown in Fig. 4. Motifs 1 is the WRKY domain. Similar Motif structures can be found between the genes in the same group or subgroup through the results. Motifs 1, 2, 3, and 4 are found in almost all genes. Motifs 15 and 19 were unique to group I. Motifs 9 and 13 were unique to group IIb. Motifs 12 were unique to group IIe. Motifs 10, 16, and 18 were unique to group III. Some of the motifs shared by different groups. Motif 5 was shared by groups I and IIc, and motif 6 and 7 were shared by groups IIa and IIb.

Figure 4
figure 4

Motif analysis of MsWRKYs.

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Gene ontology annotation and analysis of cis-acting elements of MsWRKY genes

The Blast2GO analyzed the Gene ontology (GO) annotations of 179 MsWRKY proteins. The MsWRKY target genes can be categorized into three main categories according to different functional groups. The biological processes, molecular functions, and cellular components together make up the Gene ontology (GO) annotations. Through the enrichment analysis, the involvement of MsWRKY in biological processes, molecular functions, and cellular components is in Fig. 5. Most MsWRKYs are involved in regulating cellular processes, biosynthetic processes, and different metabolic processes. Further analysis showed that most MsWRKYs were involved in the plant's stress to external adversity. Many MsWRKYs have been linked to bacterial infections and environmental stress29. The molecular functions of MsWRKYs are mainly a variety of DNA-binding and gene expression regulation. The cellular component of this protein family is mainly organelle and intracellular organelle. Most of the MsWRKY proteins are located in the cell nucleus.

Figure 5
figure 5

Gene ontology analysis of identified MsWRKYs.

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Cis-acting elements are essential sequences in regulating gene expression by transcription factors. An online tool PlantCARE was used to analyze all MsWRKY cis-acting elements and extracted 2000 bp promoter regions upstream of all MsWRKY genes. The result shows that every MsWRKY have many cis-acting elements.

Firstly, many transcription-related cis-acting elements can be found including TATA-box, CAAT-box, A-box, HD-Zip, and W-box. The stress-responsive elements formed an important part in the cis-acting elements. This suggests that MsWRKY plays an important role in plants' resistance to external stress. These cis-acting elements include MBS (Anti-drought stress), LTR (anti-low temperature stress), and WUN-motif (wound-responsive elements). In addition, many of the cis-acting elements were regulated by phytohormones. ABA regulates the ABA-responsive element (ABRE). Methyl jasmonate (MeJA) responsive element (TGACG-motif and CGTCA-motif) is regulated by jasmonate phytohormones30. Finally, there are auxin-responsive elements (AuxRR-core and TGA-element), salicylic acid-responsive elements (TCA-element), etc. Finally, there are many light-responsive elements and other regulatory elements. At least one stress-responsive element is on all of the MsWRKY genes and reflects the potential functional variation of the MsWRKY gene.

Synteny analysis of MsWRKY genes

The segmental duplication events occurring in the Miscanthus WRKY family were investigated by conducting a synteny analysis of the MsWRKY genes using BLASTP and MCScanX in TBtools. As shown in Fig. 6, 19 segmental duplication events involving 53 WRKY genes were observed. Tandem duplication events, which were defined by a chromosomal region within 200 kb containing two or more genes, were widely identified for Miscanthus WRKY genes. A very large tandem duplication event was observed in the chromosome 14. These results suggested that some MsWRKYs were possibly generated by segmental duplication events and that the evolution of MsWRKY genes may have been driven, at least in part, by segmental duplication events.

Figure 6
figure 6

Schematic representations for the interchromosomal relationships of MsWRKYs. Blue lines show duplicated WRKY gene pairs in the Miscanthus genome.

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The phylogenetic mechanisms of the Miscanthus WRKY family were further explored by constructing comparative syntenic maps of cucumber associated with four representative species, including two dicots (Arabidopsis and cucumber) and two monocots (sorghum and maize) (Fig. 7). 249, 231, 28, and 27 pairs of genes showed syntenic relationships between the other four species: cucumber, Arabidopsis, sorghum and maize, respectively. A total of 249 WRKY collinear gene pairs between Miscanthus and maize were identified, followed by Miscanthus and sorghum (231), Miscanthus and cucumber (28), and Miscanthus and Arabidopsis (27). Both Miscanthus and maize belong to the Poaceae family, and more than 75.4% of the MsWRKY genes showed a syntenic relationship with WRKYs in maize. But some of MsWRKY genes were associated with more than one syntenic gene pair, indicating that WRKY genes in Poaceae family have gone through multiple rounds of duplication events. This may be the reason why monocotyledonous plants have far more WRKY genes than dicotyledonous plants. Importantly, collinear MsWRKY09/11/60/64/83/85/112/179 genes pairs were observed between Miscanthus and all of the other four species, suggesting that these orthologous pairs may have formed before the divergence of dicotyledonous and monocotyledonous plants.

Figure 7
figure 7

Synteny analysis of WRKYs between Miscanthus and other plant species. The collinear blocks are marked by gray lines, while the collinear gene pairs with WRKY genes are highlighted in the red lines. ‘M. sinensis’, ‘A. thaliana’, ‘C. sativus’, ‘S. bicolor’ and ‘Z. mays’ indicate Miscanthus sinensis, Arabidopsis thaliana, Cucumis sativus, Sorghum bicolor, and Zea mays, respectively.

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Digital expression analysis of MsWRKY genes at different seasons and in different tissues

The study of the temporal and spatial expression profiles of MsWRKY genes used the microarray data provided by the JGI database and presented the results as heatmaps by TBtools. the microarray datasets used gene expression data for M. sinensis and included a total of 22 samples. The samples were taken from leaf (7), rhizome (9) and stem (6). The samples were collected from plants at different times of the year and reflected the expression of the MsWRKY gene at different stages of plant growth. 175 of the 179 genes showed differential expression in plants. Most MsWRKY genes are highly expressed in rhizome. The expression patterns of MsWRKY in different growth and development stages were also analyzed. Firstly, the MsWRKY gene is first expressed in rhizome in large quantities during plant growth. Then, some MsWRKY genes were heavily expressed in the leaf. Eventually, some MsWRKY genes are over-expression in the stem when plants wither. The results showed that these genes may be involved in stress response at sensitive developmental stages to improve plant tolerance (Fig. 8).

Figure 8
figure 8

Heatmaps of MsWRKY gene expression. MsWRKY expression levels in different tissues and at different seasons.

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By analyzing the gene expression heat map and cis-acting elements together, it can be found that genes activated in different periods have different characteristics. In the early stage of plant development, the expression of the MsWRKY gene is mostly controlled by plant hormones and light regulatory elements. In the middle stage of plant development, the MsWRKY gene expressed in leaves is more regulated by infection and injury. At the end of plant development, MsWRKY genes expressed were mostly regulated by ABA and jasmonic acid, and some were stressed by environmental conditions such as drought. This suggests that the MsWRKY gene plays an important role in the growth of the perennial plant Miscanthus sinensis.

Discussion

WRKY transcription factors (TFs) are widely distributed in the plant kingdom and play a crucial role in stress tolerance. The WRKY gene family comprises 66 genes in Arabidopsis, 119 in maize, 94 in sorghum, 79 in potatoes, 70 in chickpeas, and 61 in cucumbers. By analyzing the genomic assembly of Miscanthus sinensis, 179 WRKY genes were identified. As the Miscanthus sinensis is a paleotetraploid31, the amount of the WRKY genes is much higher than that of normal plants, but this has not received much attention in previous studies. The MsWRKY gene is distributed across all 19 chromosomes of Miscanthus, and we have identified MsWRKY92, located on chromosome 7, as a gene that promotes flowering32. Conserved WRKY domains bind to the W-box motif in the promoter of WRKY target genes, which is the most important feature of the WRKY family33,34. A phylogenetic analysis of all the obtained MsWRKY genes has been performed. We performed a phylogenetic analysis of all the obtained MsWRKY genes and classified them into groups I, II, and III based on the number of WRKY domains and the type of zinc finger motif. group II is further subdivided into five subgroups: IIa, IIb, IIc, IId, and IIe. Group I had 24 MsWRKY genes, group II had 97, and group III had 58. In group II, group IIc had the most MsWRKY genes, with 32. The proportions of these genes are similar to those found in other plants35,36,37.

Most MsWRKY genes have a very conserved WRKYGQK motif. However, other similar sequences have been found in many genes. (MsWRKY07 MsWRKY12 MsWRKY17 MsWRKY24 MsWRKY26 MsWRKY34 MsWRKY40 MsWRKY46 MsWRKY51 MsWRKY58 MsWRKY67 MsWRKY71 MsWRKY103 MsWRKY104 MsWRKY105 MsWRKY108 MsWRKY109 MsWRKY124 MsWRKY137 MsWRKY138 MsWRKY145 MsWRKY149 MsWRKY152 MsWRKY158 MsWRKY161 MsWRKY164 MsWRKY173) Furthermore, there are some genes that are clearly WRKY genes but are missing this sequence (MsWRKY44 MsWRKY65 MsWRKY106). These differences can seriously affect the ability of MsWRKY proteins to bind to W-box elements, which in turn affects the biological function of these proteins. Similar heptapeptide motif variations have been found in other plants, such as sorghum4. In soybeans, for example, two WRKY genes with WRKYGKK motif do not bind to normal W-box elements and do not work33. Therefore, further studies are necessary to confirm the biological function of these WRKY motif aberrant genes.

Studying the exon–intron structure of MsWRKY genes can reveal population-specific patterns. Similar MsWRKY genes on the evolutionary tree tend to have similar exon–intron patterns. The number of introns in the MsWRKY gene ranges from 0 to 5, and some MsWRKY genes do not contain introns, indicating that some WRKY genes have experienced intron loss events38. Intron-free genes have been found in other organisms. There are three main mechanisms by which intron-free genes are produced: reverse transcription (the integration of sequences from RNA into the genome), duplication of existing intron-free genes, and horizontal gene transfer39. Differences in the intron size of MsWRKY genes may result from gene duplication, inversion, and/or fusion events40. In conclusion, the diverse exon–intron structure of MsWRKY genes reflects the evolutionary diversity of the MsWRKY gene family.

Motif structural studies of the MsWRKY gene family reveal both structural conservatism and diversity. Motifs 1, 2, 3, and 4 correspond to WRKY domains and zinc finger domains, and they are found in most MsWRKY genes. Although most motifs' functions are unclear, their distribution also has certain rules. Motifs 15 and 19 were unique to group I. Motifs 9 and 13 were unique to group IIb. Motifs 12 were unique to group IIe. Motifs 10, 16, and 18 were unique to group III. Some of the motifs shared by different groups included motif 5, shared by groups I and IIc, and motifs 6 and 7, shared by groups IIa and IIb. Motif 8 is a nuclear localization signal (NLS), mainly in groups IId, IIe, and III41. In conclusion, motif analysis clearly demonstrates the structural differences of genes in different groups in MsWRKY. These motifs may reflect that these genes participate in specific biological processes and play similar biological functions.

Studying the cis-acting elements of MsWRKY can obtain more information about the gene expression of the MsWRKY gene family. Firstly, many transcription-related cis-acting elements42 including TATA-box, CAAT-box, A-box and HD-Zip are essential for gene expression, and most are involved in constructing transcription complexes. In addition, there is also a batch of cis-acting elements regulated by phytohormones. ABA regulates the ABA-responsive element (ABRE). Methyl jasmonate (MeJA) responsive element (TGACG-motif and CGTCA-motif) is regulated by jasmonate phytohormones. In addition, there are auxin-responsive elements (AuxRR-core and TGA-element), salicylic acid-responsive elements (TCA-element), and so on. A variety of biological and abiotic stresses also regulate these genes. These cis-acting elements include MBS (Anti-drought stress), LTR (anti-low temperature stress), and WUN-motif (wound-responsive elements). At the same time, W-box was also found in the promoter region of many MsWRKY genes. This suggests that there is also mutual regulation between WRKY genes43,44. Studies on cis-acting elements of MsWRKY reflect the diversity of the MsWRKY gene in gene expression regulation.

Existing transcriptome data of Miscanthus sinensis can be used to analyze the expression of MsWRKY gene in different stages of Miscanthus development and expression patterns in different tissues. Future studies should investigate the function of the MsWRKY gene by studying homologous genes in other model plants (such as Arabidopsis) and related plants (other grasses). Future research should focus on the effect of MsWRKY on plant growth and development and against adverse winter environments. Additionally, exploring the role of the MsWRKY gene in plant stress response would be valuable. Our research results provide a foundation for future studies in this field.

Conclusion

In this study, 179 WRKY genes were identified from Miscanthus sinensis. The identification, chromosome mapping, classification, phylogenetic analysis, gene structure analysis, conserved motif distribution analysis, gene ontology annotation, analysis of cis-acting elements, and digital expression pattern analysis have been performed. Through digital expression pattern analysis, the specific expression of the MsWRKY gene in different developmental stages and different parts of plants was found. At the same time, some MsWRKY genes may play an important role in plant stress resistance. In conclusion, this study is conducive to further research on the important functions of the WRKY gene in response to abiotic and biological stresses.