Drought stress is one of the important environmental factors affecting soybean growth and yield. In recent years, with the increase of drought events, the yield and quality of soybean have been seriously lost. This fact forced us to deeply analyze the mechanism of drought and salt tolerance of soybean, and obtain the key genes of salt tolerance, providing important genetic resources for breeding new varieties of drought-tolerant soybean (Dias et al., 2016). As one of the largest transcription factor families in plants, WRKY family plays an important role in various biotic and abiotic stress responses (Chen et al., 2018; Xie et al., 2019). WRKY transcription factor is named because its encoded protein contains domain WRKYGQK, which can be divided into three categories:WRKYⅠ, Ⅱ and Ⅲ. WRKYⅠ transcription factor has two WRKY conserved domains, and the zinc finger structure is C₂H₂; WRKYⅡ contains only one WRKY conserved domain, and the zinc finger structure is also C₂H₂; WRKY Ⅲ transcription factor contains only one WRKY conserved domain, but its zinc finger structure is C₂HC (Vives-Peris et al., 2017). At present, 72 and 100 members have been found in model plants Arabidopsis thaliana and Oryza sativa, respectively (Wang et al., 2015); 100 members were found in the woody plants Populus and Ziziphus jujuba respectively (Wang et al., 2015; Fu et al., 2018). The transcription factor directly or indirectly regulates the expression of the target gene by combining with cis-acting elements in the promoter region of the target gene or interacting with other proteins to participate in signal transduction pathways such as abscisic acid (ABA), jasmonic acid (JA) and brassinolide (BR), thereby improving the adaptability of plants to environmental stress (Hu et al., 2015). The widespread existence of WRKY transcription factors is closely related to plant stress resistance.

At present, WRKY family has been involved in the regulation of plant growth and development, biotic and abiotic stress responses by regulating downstream target genes or interacting with other proteins. For example, after overexpression of OsWRKY53 gene in Oryza sativa, the plant height increased significantly, and the resistance to Oryza sativa brown planthopper was also improved (Hu et al., 2016). In the leaves of Phyllostachys heterocycla, the expression of PhWRKY76 gene in old leaves is significantly higher than that in new leaves (Huang Rong et al., 2018). The expression of SdWRKY1/2 gene in Sorghum dochna increased significantly under drought stress, which indicated that the gene participated in the response of plants to drought stress (Xu et al., 2017). The expression of GmWRKY58 gene of Glycine max also increased significantly by 187.4 times under drought stress (Zhang et al., 2018). In addition, the study of soybean GmWRKY57B transcription factor showed that this gene participated in the response of plants to drought stress (Zhang et al., 2008). Previous studies have shown that members of WRKY transcription factor family participate in the regulation of growth and development and stress in many plant species.

At present, the molecular biology research on drought resistance of WRKY transcription factor mostly focuses on Arabidopsis thaliana, Oryza sativa, Gossypium spp and other model plants and cash crops, but there is no report that GmWRKY32 transcription factor is involved in drought resistance in soybean. In view of this, based on the soybean genome data, the soybean GmWRKY32 member was obtained by analyzing the conserved domain of WRKY protein. On the basis of cloning soybean GmWRKY32 gene in the early stage of our laboratory, bioinformatics and expression analysis of soybean transcription factor GmWRKY32 gene were carried out. The research results can provide scientific basis and key genes for analyzing the molecular mechanism of soybean drought resistance and breeding new varieties of drought-resistant soybean by molecular breeding.

1 Results and Analysis

1.1 Bioinformatics analysis of soybean transcription factor GmWRKY32 gene

1.1.1 Analysis of conservative structural domain of soybean GmWRKY32 gene protein sequence

In this study, Prosite online analysis software (http://prosite.expasy.org/) was used to learn that there was a WRKY conserved domain containing 62 amino acids through the protein sequence encoded by GmWRKY32, and there was a conserved amino acid sequence WRKYGQK at the N-terminus (Figure 1A). According to the amino acid sequence of the conserved domain and the conserved domain of GmWRKY32 protein, the zinc finger structure type of GmWRKY32 protein was obtained: CX₇CX₂₂HXXC (C₂HC) (Figure 1B). Thus, GmWRKY32 has one WRKY conserved domain and one C₂HC zinc finger structure class, which can preliminarily judge that the transcription factor belongs to WRKY Ⅲ in gene family.

Figure 1 Analysis of conservative structural domain of soybean GmWRKY32 gene protein sequence Note: A: Conservative structural domain; B: Structural analysis of zinc finger of protein

1.1.2 Analysis of hydrophilicity and hydrophobicity of soybean GmWRKY32 protein polypeptide chain

In this study, the hydrophobicity/hydrophilicity of soybean GmWRKY32 transcription factor protein was analyzed by using Protscale online software. The analysis results (Figure 2) showed that most peptide chains of the protein were above 0 and belonged to hydrophobic region, while the regions below 0 belonged to hydrophilic region. Therefore, GmWRKY32 protein has strong hydrophobicity and is a hydrophobic protein.

Figure 2 Analysis of GmWRKY32 peptide chain hydrophilicity/ hydrophobicity distribution curve

1.1.3 Subcellular localization prediction of soybean transcription factor GmWRKY32 protein

In this study, the subcellular localization prediction analysis of soybean transcription factor GmWRKY32 protein showed that the predicted localization score of this transcription factor GmWRKY32 protein was 6.1 (Table 1). It is speculated that this soybean transcription factor protein GmWRKY32 protein may be localized in subcellular nucleus.

Table 1 Prediction of soybean GmWRKY32 subcellular localization

1.1.4 Homology comparison of soybean GmWRKY32 protein sequence

The homologous genes were searched by NCBI, and then the homology of GmWRKY32 protein with Cajanus cajan CcWRKY70, Vitis amurensis VaWRKY70 and wild soybean GsWRKY70 was 79%, 72% and 85%, respectively. The amino acid sequence alignment analysis by DNAMAN software shows that GmWRKY32 includes a highly conserved WRKY domain, and the conserved domain has the structural characteristics of the WRKY gene family conserved domain, that is, the WRKY domain at the n end of WRKY protein contains a highly conserved WRKYGQK heptapeptide sequence in the red frame (Figure 3).

Figure 3 GmWRKY32 is compared by protein homology sequence Note: WRKY conserved domain was marked by red frame

1.1.5 Construction of evolutionary tree between soybean GmWRKY32 and arabidopsis WRKY family

According to the multiple alignment of amino acid sequences, the phylogenetic tree of soybean GmWRKY32 and Arabidopsis WRKY transcription factor family members was constructed (Figure 4). the soybean transcription factor GmWRKY32 and Arabidopsis AtWRKY70, which have the function of resisting drought stress, converge on one branch, which shows that the transcription factor GmWRKY32 may have the same regulatory function as Arabidopsis AtWRKY70 members.

Figure 4 Evolutionary tree of WRKY sequence of GmWRKY32 and Arabidopsis thaliana family

1.2 Expression patterns of soybean transcription factor GmWRKY32 gene under drought treatment

After 15% PEG and 50 mmol/L NaCl treatment, the gene expression of GmWRKY32 transcription factor was analyzed (Figure 5). Under the two treatment conditions, the gene expression increased first and then decreased. After 12 h of drought stress, the gene expression of GmWRKY32 in soybean reached the highest level, which increased by 4.5 times compared with the control (Figure 5A). After NaCl treatment for 12 h, the expression level of this gene also reached the maximum, which was 4.2 times higher than that of the control (Figure 5B). Therefore, GmWRKY32 is involved in the response to drought stress such as NaCl and PEG.

Figure 5 The expression of GmWRKY32 gene under different treatments at different time Note: A: PEG treatment; B: NaCl treatmen

1.3 Hormone-induced expression pattern of GmWRKY32

After induction with 100 mmol/L ABA and JA hormones, the expression of GmWRKY32 was found to be in an increasing trend, and ABA induction had a significant effect on the expression of GmWRKY32 gene in soybean. Particularly, 24 h after induction, the expression of GmWRKY32 reached the highest level, which was six times higher than that of the control (Figure 6A). After 12 h of JA induction treatment, the expression level of this gene also reached the highest, which was 3.2 times higher than that of the control (Figure 6B). Thus, both ABA and JA were involved in that expression of soybean GmWRKY32.

Figure 6 The expression of GmWRKY32 gene under different treatments at different time Note: A: ABA treatment; B: JA treatment

2 Discussion

The WRKY transcription factor has always been an important research field in plant responses to stress (Chen et al., 2018; Xie et al., 2019). The information about the WRKY gene family in all kinds of databases is more and more comprehensive, which is helpful for us to study gene sequence, structure and functional characteristics, and plays a huge role in promoting the smooth development of the experiment. In this study, the cDNA sequence of transcription factor GmWRKY32 was screened from the published soybean genome database, and the homology between the soybean GmWRKY32 protein sequence and GsWRKY70 in wild soybean was found to be as high as 85% by homology comparison. At the same time, sequence analysis of the protein revealed that GmWRKY32 contained a conserved domain WRKYGQK and C₂HC zinc finger structure at the N-terminus. These results clarified that the soybean GmWRKY32 transcription factor belongs to the third large subfamily of the WRKY gene family. In addition, it could be seen from the evolutionary tree that the AtWRKY70 genes with the function of resisting drought stress in arabidopsis were clustered together, and AtWRKY70 was a positive factor for resisting drought abiotic stress in arabidopsis. Therefore, it is speculated that the GmWRKY32 gene of soybean may have the biological function of resisting abiotic stress of drought.

WRKY transcription factor has many biological functions during plant growth and development, and it is one of the important transcription factor families involved in plant stress regulation (Sun et al., 2014; Chen et al., 2017; Li et al., 2017). At present, WRKY transcription factor has been found to be widely involved in the process of plant stress response, and overexpression of AtWRKY44 can improve the drought performance of Arabidopsis plants by regulating sugar signal pathway; Overexpression of ArabidopsisAtWRKY57 transcription factor can improve the salt tolerance and drought tolerance of plants (Asai et al., 2002; Jiang et al., 2016). In addition, overexpression of soybean GmWRKY35 in tobacco can also improve the stress tolerance of tobacco under drought (Li et al., 2017). Previous studies have found that SbWRKY1/2 gene is involved in drought resistance of sweet sorghum (Xu et al., 2017). In this study, under the abiotic stress of PEG and NaCl and the induction of hormones ABA and JA, the expression of soybean transcription factor GmWRKY32 showed an increasing trend in different degrees. Therefore, it is speculated that this transcription factor regulates the process of plant response to drought stress.

On the basis of soybean genome database, the related bioinformatics and gene expression were studied in this study. Using the WRKY sequence in the soybean genome database, the open reading frame (ORF) of soybean GmWRKY32 gene was cloned as 903 bp, which encoded 300 amino acids. According to bioinformatics analysis, GmWRKY32 contains a highly conserved WRKY domain, and has C₂HC zinc lipid structure, belonging to class III of WRKY gene family. Subcellular localization of the protein showed that GmWRKY32 gene was located in the nucleus and had the highest homology with wild soybean (GsWRKY23). In addition, the protein encoded by GmWRKY32 is insoluble and has no signal peptide. The results of fluorescence quantitative PCR showed that GmWRKY32 gene was involved in the regulation of drought and hormone induction in soybean.

3 Materials and Methods

3.1 Test materials

The experimental material was cultivated Cangdou 11, which was provided by Hebei Key Laboratory of Saline-alkali Tolerance Evaluation and Genetic Improvement of Crops. The leaf total RNA extraction kit, DNaseI and reverse transcription kit were all purchased from Bao Bioengineering (Dalian) Co., Ltd. The procedures and system for the qRT-PCR reaction were purchased from USEverbright® Inc.. with reference to the 2×FastSuperEvaGreen® qPCRMasterMix fluorescent quantitative reagent (Code, S2008).

3.2 Bioinformatics analysis of soybean transcription factor GmWRKY32 sequence

In this study, the conservative domain prediction analysis of the gene WRKY32 protein was performed by online Prosite analysis software. The zinc finger structure type of GmWRKY32 protein can be obtained by using the amino acid sequence of the conservative domain of GmWRKY32 protein. Prediction of protein tertiary structure using SWISS-MODEL online software (http://SWISS Model.expasy.org/); The hydrophobic/hydrophilic analysis of the soybean transcription factor GmWRKY32 protein was performed using ProtScale software. Prediction analysis of whet that signal peptide exists is carry out by SignalP- bioinformatics software; The transmembrane domain of the transcription factor GmWRKY32 protein was analyzed by the http://www.cbs.dtu.dk/services/TMHMM-2.0/ website. And subcellular localization of GmWRKY32 protein was predicted by Cell-PLoc website. The NCBI database and the Phytozome database (http://www.phytozome.net/) were used to search for homologous genes in near-source species, and the searched sequences were subjected to sequence alignment of homologous genes by DNAMAN software. Finally, the phylogenetic tree of soybean GmWRKY32 gene and members of arabidopsis AtWRKY gene family was constructed by using DNAMAN software.

3.3 Changes of GmWRKY32 gene expression under drought stress

Soybean seedlings with 7~8 leaves grown for about one month were treated with 150 mmol/L NaCl, 15% PEG, 100 mmol/L ABA and 100 mmol/L JA respectively. Samples were taken at 0, 4 h, 8 h, 12 h, 24 h and 48 h respectively. 3 leaves were taken from the bottom, middle and upper parts of each plant. Each treatment was repeated three times. The samples were quickly frozen in liquid nitrogen and then transferred to a refrigerator at -80℃.

The total RNA of soybean leaves after different treatments is extracted, and then is reversely transcribed into a total single-stranded cDNA by a reverse transcription kit, and then ACTIN gene (F: 5'-GTCAGCACATACTGTCCCCATTT-3'; R: 5'-GTTTCAAGCTCTCTCTCGTAATCA-3') in a soybean plant body is taken as an internal reference gene, Fluorescent quantitative qRT-PCR analysis was performed to analyze the expression pattern of GmWRKY32 (F: AGAAGAAGGAGATGG; R: CTTTCAACATGTGTGAAGG). The procedure of qRT-PCR and the instruction of the 2×FastSuperEvaGreen® qPCRMasterMix fluorescent quantitative kit are referred. The system is 20 μL, the template cDNA is about 50 ng, the primers added are 200 nmol/L respectively, 10 μL qRT-PCRMasterMix is added, and the 20 μL system is supplemented with sterilized water ddH2O. The PCR reaction conditions were: pre-denaturation at 95℃ for 5 min 95℃ The melting curve was analyzed after 40 cycles at 20 s 94℃, 30 s 58℃, 45 s 72℃ (95℃→65℃, 0.1℃/s). Every biological treatment was repeated three times, and the result data were calculated by the method of 2^－ΔΔCt.

Authors’ contributions

ZL is the executor of the experimental design and research of this study; ZJ finished data analysis and writing the first draft of the paper; SYY participated in the experimental design and analyzed the experimental results; CJF and LJF are the architects and leaders of the project, guiding experimental design, data analysis, thesis writing and revision. All authors have read and agreed to the final text.

Acknowledgements

The research is supported by the innovative ability training project of Hebei University (hbu2020ss036).

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