Cloning and functional validation of DsWRKY6 gene from Desmodium styracifolium

ABSTRACT

The purpose of this study was to analyze the role of transcription factor in Desmodium styracifolium, proving that the DsWRKY6 transcription factor was related to the plant phenotypes of Desmodium styracifolium - cv. ‘GuangYaoDa1’ and it could be used in molecular-assisted breeding. ‘GuangYaoDa1’ was used as the material and its DNA was the template to clone DsWRKY6, the transgenic Arabidopsis thaliana line was constructed by agrobacterium tumefaciens‑mediated transformation. Transgenic Arabidopsis thaliana was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that DsWRKY6 transgenic Arabidopsis thaliana had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of DsWRKY6 increased and the abscisic acid content significantly increased in DsWRKY6 transgenic Arabidopsis thaliana compared with the control group. According to the above results, DsWRKY6 could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the DsWRKY6 transcription factor might be the cause of the upright growth of ‘GuangYaoDa1’.

KEYWORDS:

• Desmodium styracifolium
• New variety breeding
• transcription factor
• Arabidopsis thaliana

Introduction

Desmodium styracifolium (Osbeck.) Merr. is an herbaceous plant that belongs to the Desmodium genus in the Fabaceae family (Flora of China, www.iplant.cn/foc). As a traditional Chinese medicine, it is known for the efficacies of dampness-draining, anti-icteric, strangury-relieving, and diuretic.¹ The main chemical components of D. styracifolium are flavonoids and volatile components,² which are used widely to the treatments such as promoting urination,³ promoting gallbladder,^4,5 anti-stone,^6–8 anti-inflammatory,⁹ antioxidant^10,11 and protecting the cardiovascular system.^12,13 D. styracifolium is in great demand in the current medicinal materials market because of its high medicinal value. Unfortunately, a shortage of wild resources and incomplete technology of artificial cultivation can influence further development and comprehensive utilization of D. styracifolium.¹⁴ To utilize and develop D. styracifolium, some existing research has been conducted which includes the establishment of chemical composition fingerprint,¹⁵ evaluation of germplasm resources,¹⁶ Genetic diversity and molecular markers,^17,18 transcriptome sequencing and bioinformatics analysis.¹⁹ However, new cultivars are seldom reported and excellent cultivars on the market are not available up to now. Therefore, we have started breeding new cultivars,^18,20 managing to fill in the gaps in this area. A few plants of D. styracifolium with a nearly upright stem were found in the process of breeding accidentally,²¹ which were used as the parents to cultivate new varieties. Then, through several generations of cultivation and screening, the new cultivar - D. styracifolium cv. ‘GuangYaoDa1’ was eventually developed. Compared with the artificially planted and wild type, D. styracifolium cv. ‘GuangYaoDa1’ had many advantages which included upright phenotype, condensed planting, easy to harvest, and shorter growth cycle.

Plants have evolved throughout history and have developed transcription factors, which are integral parts of complex regulatory networks. These transcription factors are involved in plant growth, development, and stress response. They often act as a central regulator and molecular switch that activates or represses the transcription of multiple target genes.²² WRKY transcription factor family is one of the largest transcription factor families in higher plants. These transcription factors contain a highly conserved domain composed of 60 amino acids, with a highly conserved N-terminal heptapeptide sequence WRKYGQK and a zinc finger motif at the C-terminus, which is composed of C2H2 (C-X4-5-C-X22-23-H-X-H) or C2HC (C-X7-C-X23-H-X-C) type zinc finger structures. Based on the number of WRKY domains and the characteristics of zinc finger structures, WRKY transcription factors were classified into three classes. In them, Group I contained two WRKY conserved domains, and its C-terminal zinc finger structure was C2H2 type; Group II contained only one conserved WRKY domain and the zinc finger structure was the same as that of Group I, which was C2H2 type. The Group III zinc finger structure was C2HC type and contains one WRKY conserved domain.²³ WRKY functions as both repressor and activator²⁴ through its expression patterns and interactions with downstream target genes, as well as interactions with other WRKY ²⁴proteins to regulate important physiological processes in plants such as growth, development, secondary metabolism, responses to biotic, abiotic and hormonal stresses by inhibition or derepression.^25,26

To figure out what genes regulate the plant-type traits of D. styracifolium, the transcriptome of wild type and ‘GuangYaoDa1’ (Figure 1) were sequenced. Then the transcriptome libraries were established for gene function annotation and screening. Through sequence annotations, we identified the DsWRKY6 transcription factor as highly expressed with an upregulation trend among the differentially expressed genes associated with growth and development. Therefore, DsWRKY6 was cloned by us and transformed into Arabidopsis thaliana. The result of phenotype and physiological and biochemical indexes of transcribed Arabidopsis were used to elucidate its function and verify transcriptome data. RT-qPCR and WB experiments to ensure that the DsWRKY6 transcription factor was stably expressed. This research work provided a certain research basis for the molecular basis of D. styracifolium and points out a new direction.

Figure 1. Phenotype of Desmodium styracifolium. On the left was WT and on the right was ‘GuangYaoda1’.

2. Materials and methods

2.1. Plant materials

For transformation, plant materials of ‘GuangYaoDa1’ and wild D. styracifolium were obtained from Yunfu Base in Guangdong province.

2.2. Quantitative real-time PCR

Total RNAs from OEWRKY6 and CK were extracted, following the manufacturer’s instruction (TIANGENG, Beijing). After using AGE (120 V, 400 mA, 15 min) to examine the quality and measure the total RNA concentration, the total RNA was reverse-transcribed into single-stranded cDNA (Servicebio, Wuhan).

The primers for RT-qPCR were designed using Primer-BLAST based on the sequences retrieved from the RNA-Seq dataset of DsWRKY6. The specificity of the reactions was verified by melting curve analysis. Using AtACTIN2 as an inter-control, RT-qPCR was performed with Perfectstart Green qPCR superMix (TransGen Biotech, Beijing) and DsWRKY61-F, DsWRKY61-R, AtACT2F, AtACT2R (Table 1.) were provided by Qing Ke Co., LTD. The reaction system consisted of 0.4 μL of each primer, 1 μL of cDNA, 10 μL of 2×PerfectStart Green SuperMix, and 8.2 μL of deionized water. A two-step amplification protocol of 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 60°C for 30 s, was used. This experiment is repeated three times.

Table 1. Primer information. DsWRKY6_1-F, DsWRKY6_1-R, AtACT2_F, AtACT2_R were used to quantitative real-time PCR, DsWRKY6_2-F and DsWRKY6_2-R were used to PCR.

Prime name	Primer sequence
DsWRKY61-F	5’-TGCGAACTGGACAACCAACT-3’
DsWRKY61-R	5’-GATGGATGCCAATGGCGAAA-3’
AtACT2F	5’-TGCGAACTGGACAACCAACT-3’
AtACT2R	5’-GAATCCAGCACAATACCGGTTG-3’
DsWRKY62-F	5’-ggatcttccagagatggatccATGGACAAAGGATGGGGACTC-3’
DsWRKY62-R	5’-ctgccgttcgacgatgtcgacGTTTCCTGAAAAACTGCTAATGCTAG-3’

2.3. Development of transgenic Arabidopsis

DNA was extracted from the leaves of ‘GuangYaoDa1’ with a kit (Accurate Biology, China), then ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to perform an analysis of ORF and deduce the amino acid sequence. To clone DsWRKY6, DsWRKY6_2-F and DsWRKY6_2-R (Table 1.) were designed by Primer Premier 5 software and synthesized by Qing Ke Co., LTD. A DNA sample of 25 μL was used as a template, along with DsWRKY6 gene-specific primers (DsWRKY6_2-F/R) of 1 μL, PCR polymerase of 25 μL, and DEPC-treated water of 21 μL to amplify DsWRKY6.PCR reaction conditions were as follows: 95°C for 3 min, then 25 cycles at 98°C for 10 s, 55°C for 30 s, and 72°C for 2.5 min, with final extension at 72°C for 10 min. The PCR products were examined using AGE (120 V, 400 mA, 15 min) and recovered with the kit (TransGen Biotech, Beijing). They were inserted into the pEASY-T1 cloning vector (TransGen Biotech, Beijing) and then transformed into E. coli T-1 (TransGen Biotech, Beijing) and sequenced (QINGKE, Guangzhou). We used a blank LB medium to expand propagation and an LB with kanamycin (100 μg/ml) was used to select a positive single colony.

The DNA fragment and pCAMBIA1300-mCherry vector (MIAOLING BIOLOGY, China) were digested by BamHI/SalI enzymes (TransGen Biotech, Beijing) and then purified by gel extraction kit (TransGen Biotech, Beijing) and ligated together (Takara, Japan). For cloning, the ligation products were transformed into E. coli T-1. Screening and expanding propagation as above. DsWRKY6-pCAMBIA1300-mCherry binary construct and pCAMBIA1300-mCherry were introduced into Agrobacterium strain GV3101 (Yu Bo Biotech, Shanghai) through heat shock. Expanding propagation was by blank and transformed clones were selected by resistance to kanamycin (50 mg/ml) at 28°C for 2–3 days. Arabidopsis thaliana (ecotype Col-0) seedlings were first vernalized at 4°C and surface-sterilized by 75% ethanol and 10% sodium hypochlorite, then sown in vitro on 1/2 MS medium with hygromycin (50 mg/ml). Once the seedlings had developed four true leaves, they were transferred to individual pots filled with commercial soil for the subsequent transformation process. The seedlings were cultivated under controlled conditions of 22°C, 70% humidity, and a 16-hour photoperiod with 3000 lux cool white fluorescent lighting. Introduced into the wild-type (WT) Arabidopsis thaliana a (Col-0) via a floral dip method when Arabidopsis was blossoming.

2.4. Identification of transgenic Arabidopsis thaliana

Agrobacterium single colonies containing recombinant plasmids were cultured to OD600 of 1, 5000 g for 5 minutes, the liquid supernatant was discarded and the bacterial solution was re-suspended with 1/2 MS. 4–7 layers of epidermis were taken from the onion bulb for sterilization, the inner epidermis of 1 cm² was torn and placed in agrobacterium solution for 30 minutes then spread on MS solid medium and cultured at 28°C in the dark for 3 days. Fluorescence was observed with a fluorescent inverted microscope. Two lines, CK (mCherry) and OEWRKY6 (DsWRKY6-mCherry) were obtained by the method described above. According to the instructions, abscisic acid (Sig Biotechnology, Shanghai) and protein (Boxbio, Beijing) were extracted from leaves of CK and OEWRKY6, collected three plants from each group, were selected randomly. This experiment is repeated three times.

In the western bolt experiment, the protein extracted from CK and OEWRKY6 were denatured by mixed with 5×SDS-PAGE loading buffer (with β-Mercaptoethanol) (Bioss, Beijing) and Water-DEPC that treated together, according to the instructions. After that, the SDS-PAGE gel preparation kit (Biosharp, Beijing) and Tris-Glycine-SDS Running Buffer,10× (EcoTop Bio, Guangzhou) were used for electrophoresis. After the transfer (NCM Biotech, Suzhou), mCherry-Tag and Plant actin (Bioss, Beijing) were selected as primary antibodies, Goat Anti-rabbit IgG/HRP and Goat Anti-rabbit IgG/HRP (Bioss, Beijing) served as secondary antibodies. In the end, those blots were displayed using BeyoECL Plus (Biosharp, Beijing). This experiment is repeated three times.

2.5. Bioinformatics analysis

Highly similar transcription factors were selected to compare with DsWRKY6 by using the NCBI (National Center for Biotechnology Information (nih.gov)) website then MAGE software was used to perform sequence alignment analysis. For inferring protein structure, physiological and biochemical properties, the following tools were used. SignalP-5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0), TMHMM Server v. 2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0), NPSA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_ automat.pl? page=/NPSA/npsa_sopm. html), SWISS-MODEL (https://swissmodel.expasy.org/interactive), Expasy (https://web.expasy.org/protparam/.) Multiple Sequence Alignment by CLUSTALW (https://www.genome.jp/tools-bin/clustalw) was used to picture a sequence comparison diagram.

3. Results

3.1. Phylogenetic and domain analyses of DsWRKY6 and subcellular localization of DsWRKY6 protein

The phylogenetic tree showed that D. styracifolium and other species shared a common ancestor, but D. styracifolium did not appear in the branch of the phylogenetic tree, and the phylogenetic tree was divided into two major branches. This suggests that D. styracifolium evolved earlier than other species in the evolutionary tree and independently evolved a new species (Figure 2).

Figure 2. Phylogenetic tree of DsWRKY6.

The present rate of the predicted signal peptide sequence was very low (only 0.001%) (Figure 3a), and (Figure 3e) there was no transmembrane domain and, this transcription factor was expressed throughout the cell (Figure 3b). Based on the subcellular localization results in onions, OEWRKY6 is expressed in various parts of onion cells, and CK is also found throughout the entire cell. These results confirm the prediction of DsWRKY6 protein localization in various parts of cells. In the Secondary structure prediction, it contained alpha helices at 34.04%, extended strands 6.17%, beta turns 15.17% and random coils 44.62% (Figure 3c). In the predicted three-dimensional structure, GMQE was 0.58 and Seq Identity was 90.28%, these showed a high degree of credibility (Figure 3d). As the physical and chemical properties of the DsWRKY6, the results showed that the molecular weight (MW) of DsWRKY6 was 61 kDa, the theoretical pI was 6.61, the protein instability index was 51.78 U, and the hydrophilic average coefficient was −0.657, the coefficient of fat was 63.19. We considered it to be an unstable hydrophilic protein (Figure 3e).Multiple alignments of the DsWRKY6 protein with other Fabaceae plants are presented in Figure 4. DsWRKY6 has 8.93% identity and 73.40% similarity with other species, indicating a high degree of similarity with other species, and the amino sequences are identical in regions such as 66-73 and 293-343 aa (Figure 4).

Figure 3. Properties and structure of the protein. Signal peptide prediction map(a), transmembrane domain prediction (b), secondary structure prediction (c), three-dimensional structure prediction (d), hydrophilicity and hydrophobicity (e).

Figure 4. Alignment of DsWRKY6 homologs in different plant species.

Figure 5a. The experiment of expression. They were transient expression CK (a) and OEWRKY6 (b) in infected onions with white light and green excitation light, RT-qPCR relative expression level (c), expression of protein mCherry and OEWRKY6 (d).

Figure 5b. The experiment of expression. They were transient expression CK (a) and OEWRKY6 (b) in infected onions with white light and green excitation light, RT-qPCR relative expression level (c), expression of protein mCherry and OEWRKY6 (d).

3.2. Overexpression of DsWRKY6 in transgenic Arabidopsis plants

RT-qPCR analysis was performed and the results were converted into relative gene expression by delta-delta Ct method p test analysis was performed to verify the correlation between the relative gene expression and the transcriptome data (TPM). The results showed significant up-regulation, indicating that DsWRKY6 was successfully transferred into Arabidopsis and expressed in it to regulate the growth of Arabidopsis (Figure 5).

3.3. Effect of DsWRKY6- overexpression on transgenic Arabidopsis

At the time of seedling growth, DsWRKY6 transgenic Arabidopsis thaliana showed stronger growth potential. We set the time of seedlings 4 true leaves emerged and they were transferred into spots as the beginning and noted the stages of growth, which contained bloting, flowering, setting, and ripening they needed. In contrast, the bolting time, flowering time, seed setting time and maturation time of DsWRKY6 were shorter than those of CK (Figure 6f–), indicating that DsWRKY6 had a faster growth rate (Figure 7). There were significant differences in length of main stem, length of lateral branches of cauline leaves, the number of lateral branches of cauline leaves, the number of cauline leaves and length of root. The main stem, side branches of stem leaves, and root length of stem leaves of DsWRKY6 were longer than those of CK, while the number of side branches of stem leaves and side branches of stem leaves was less than those of CK (Figure 6a–).

Figure 6. Comparison of agronomic traits in Arabidopsis (a-i). Comparison of abasic acid concentrations (j).

Figure 7. Phenotype of transgenic Arabidopsis. Both on the left were CK and on the right were OEWRKY6. It was shown two-week-old seedling plants (a) and seedling roots (b), rosette leaves (c) and stem leaves (d) of one-month-old plants, flowering Arabidopsis plants (e) and fruiting Arabidopsis plants (f), respectively.

The concentration of ABA was found in DsWRKY6 higher than CK, the result indicated that DsWRKY6 transcription factor positively regulated ABA expression and increased ABA content in plants (Figure 6j).

4. Discussion

The WRKY gene family belongs to a widespread class in flowering plants that play essential roles in diverse stress responses, and developmental, and physiological processes, as one of the biggest transcription factors.²² At the same time, the synthesis and response of plant hormones such as abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) are also regulated by WRKY.²⁷ ABA is one of the important plant hormones, playing a significant physiological role in plant growth, development, and stress responses,²⁸ like promoting flowering and fruiting^29,30 as well as root elongation.^31–34 It is shown that DsWRKY6 may regulate ABA, comparing with their expression results and agronomic trait. The ABA signaling network is composed of a large number of signaling components, such as FCA, ChlH, G-Protein-Coupled receptor, PYR/PYL/RCAR receptors, protein phosphatases, targets of PP2Cs, ABA-regulated genes.³⁵ WRKY transcription factors were also involved^35,36 and even a critical part of abscisic acid transduction.³⁷ It has also been reported widely that DsWRKY6 regulates ABA in other species of overexpression of Sweet Potato³⁸， Eriobotrya japonica³⁹， Gossypium barbadense⁴⁰， banana fruit.⁴¹ Therefore, we suggested that DsWRKY6 may be a link in the ABA signaling network, which directly or indirectly regulates ABA.

It appears that the overexpression of DsWRKY6 not only regulates ABA but also has other functions that may directly or indirectly regulate IAA. In addition to its antagonistic effect, ABA affects the tropic movement of plants or regulates other genes, which in turn alters the transport pathway of IAA, directly or indirectly regulating IAA. This ultimately leads to the concentration and distribution of IAA and ABA. In synergy with other transcription factors, it ultimately leads to the phenotype of Cv. ‘GuangYaoDa1’.IAA is one of the significant hormones in plant development as well as a factor in tropic movement.⁴² The stems curve upwards, while the roots curve downwards due to their respective concentration gradients.⁴³ There are two aspects to the tropic movement. On the one hand, ABA can influence the ability to respond to gravity.⁴⁴ The content and distribution of IAA hormones were changed due to gravity stimulation, which led to the creeping growth characteristics.^45,46 On the other hand, ABA can influence the water response that will affect transportation. It has also been reported that WRKY family transcription factors can regulate ABA. For example, OsWRKY72 changes the normal transport of IAA and OsWRKY31 can transform the reaction and transport of IAA by inducing OsIAA4 and OsCrl1.⁴⁷ Besides, some genes that are related to stems turn up, like sgr1, and sgr7 genes can inhibit stem gravity bending.^48–50 PIN2, PIN3, and PIN7 are members of PIN family proteins, which are IAA export vectors and are involved in the formation of IAA asymmetric gradients in gravity-stimulated organs.⁵¹

Through the results, we consider the DsWRKY6 transcription factor may have the function of changing plants creeping growth characteristics and breeding new cultivation. For some plants would reduce the growth cycle and improve harvest with the effect of promoting flowering and fruiting. DsWRKY6 will take part in cultivation and screening in the future, it can speed up the progress of breeding and increase efficiency. The research on function and molecular mechanisms of DsWRKY6 regulates ABA and IAA is still shallow, further investigation of the mechanism of action is needed.

List of Abbreviations
ABA	=	abscisic acid
RT-qPCR	=	Quantitative real-time PCR
ORF	=	open reading frame
MS	=	Murashige and Skoog
WB	=	western blot
TPM	=	transcriptome data
WT	=	wild type
AGE	=	agarose gel electrophoresis
OEWRKY6	=	overexpression WRKY6
CK	=	control check

Author contribution statement

Qilin Yang performed the experiment and drafted the manuscript; Jinheng Huang participated in the experiments; Xiaofeng Nie sampled the material in the field and treated samples; Peiran Liao provided ideas, designed the research, and edited the manuscript; XiaoMin Tang provided technical support; Quan Yang provided funding support; all authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Key-Area Research and Development Program of Guangdong Province (NO. 2020B020221002) and the Guangdong Provincial Rural Revitalization Strategy Special Fund - Seed Industry Revitalization Project.