2.1 Physical and chemical properties of WRKY protein
A total of 135 sequences annotated as WRKY gene were preliminarily obtained from the transcriptome data of B. striata. After deletions of the sequences without typical WRKY domain and incomplete sequences, 29 WRKY sequences were finally reserved and renamed as BsWRKY 1-29. Physical and chemical properties analysis showed that the protein size of the BsWRKY members was between 159-703 aa, and the molecular weight was between 17546.1-76820.1 Da (Table 1). The theoretical isoelectric point of proteins ranged from 4.48 to 9.94, 11 of them were basic proteins with isoelectric point greater than 7.5, 13 of them were acidic proteins with isoelectric point less than 6.5, and 5 of them were neutral between 6.5 and 7.5. These results indicated that most proteins of BsWRKYs were acidic. The instability coefficients of the 29 WRKY proteins were all greater than 40, while fat index were less than 100, and GRVAY values were negative, indicating that the WRKY transcription factor family of B. striata was an unstable hydrophilic protein. The predicted secondary structure of the protein showed that α-helix, β-folding and elongation accounted for 21.07%, 4.07% and 11.84%, respectively, and random coil accounted for 63.02% (Table 1). Among the 29 proteins, the β-turn was normally more than the α-helix except in BsWRKY20.
Table 1
Physical and chemical properties of WRKY protein of Bletilla striata
Gene name
|
AA
|
MW/Da
|
PI
|
Instability index
|
GRAVY
|
Alpha helix
|
Extended
strand
|
Beta
turn
|
BsWRKY1
|
307
|
34243
|
5.4
|
43.37
|
-0.651
|
24.76%
|
10.10%
|
3.58%
|
BsWRKY2
|
481
|
52166.7
|
7.01
|
46.65
|
-0.672
|
10.19%
|
14.35%
|
3.74%
|
BsWRKY3
|
406
|
44754.8
|
5.5
|
47.5
|
-0.695
|
25.62%
|
6.40%
|
4.43%
|
BsWRKY4
|
702
|
76105.4
|
5.67
|
48
|
-0.627
|
11.97%
|
11.11%
|
3.42%
|
BsWRKY5
|
500
|
54510.7
|
8.97
|
48.21
|
-0.752
|
12.00%
|
12.80%
|
3.60%
|
BsWRKY6
|
469
|
50789.1
|
6.98
|
50.9
|
-0.729
|
10.45%
|
14.07%
|
3.41%
|
BsWRKY7
|
185
|
21037.2
|
6.26
|
51.01
|
-0.97
|
24.86%
|
18.92%
|
10.27%
|
BsWRKY8
|
194
|
51538.4
|
9.59
|
52.71
|
-0.865
|
11.34%
|
13.92%
|
6.19%
|
BsWRKY9
|
335
|
35817.5
|
9.69
|
53.06
|
-0.484
|
21.49%
|
9.85%
|
6.87%
|
BsWRKY10
|
431
|
47054.3
|
6.57
|
54.84
|
-0.703
|
14.39%
|
12.30%
|
2.55%
|
BsWRKY11
|
341
|
36780.7
|
9.67
|
55.85
|
-0.484
|
18.18%
|
10.26%
|
6.45%
|
BsWRKY12
|
329
|
37045.9
|
8.57
|
56.08
|
-0.632
|
32.22%
|
10.33%
|
1.52%
|
BsWRKY13
|
347
|
38224.3
|
9.94
|
56.17
|
-0.688
|
25.07%
|
9.22%
|
5.48%
|
BsWRKY14
|
270
|
29189.3
|
5.64
|
56.66
|
-0.0743
|
17.04%
|
11.11%
|
3.70%
|
BsWRKY15
|
217
|
24804.6
|
5.91
|
57.12
|
-0.86
|
11.06%
|
15.21%
|
3.69%
|
BsWRKY16
|
233
|
25270
|
4.93
|
57.21
|
-0.642
|
27.90%
|
10.30%
|
3.43%
|
BsWRKY17
|
412
|
45995.3
|
8.62
|
57.49
|
-0.617
|
31.31%
|
8.98%
|
1.94%
|
BsWRKY18
|
595
|
64961.1
|
6.55
|
57.8
|
-0.65
|
28.57%
|
10.92%
|
1.51%
|
BsWRKY19
|
402
|
43491.8
|
8.58
|
59.9
|
-0.829
|
9.95%
|
11.44%
|
3.23%
|
BsWRKY20
|
159
|
17546.1
|
4.48
|
60.03
|
-0.82
|
40.25%
|
11.32%
|
8.81%
|
BsWRKY21
|
427
|
47131.5
|
5.25
|
60.59
|
-0.63
|
23.89%
|
6.56%
|
3.51%
|
BsWRKY22
|
174
|
19581.5
|
4.55
|
60.82
|
-0.506
|
21.84%
|
18.39%
|
5.17%
|
BsWRKY23
|
595
|
64961.1
|
6.55
|
62.18
|
-0.65
|
28.57%
|
10.92%
|
1.51%
|
BsWRKY24
|
335
|
35367.1
|
7.61
|
62.24
|
-0.354
|
25.37%
|
13.73%
|
3.88%
|
BsWRKY25
|
530
|
56563.4
|
6.36
|
62.43
|
-0.622
|
20.94%
|
10.94%
|
2.45%
|
BsWRKY26
|
703
|
76820.1
|
5.61
|
62.54
|
-0.648
|
13.23%
|
10.53%
|
3.27%
|
BsWRKY27
|
363
|
41768.4
|
8.52
|
63.93
|
-0.781
|
31.68%
|
13.50%
|
2.75%
|
BsWRKY28
|
526
|
57802.3
|
6.2
|
66.1
|
-0.657
|
13.50%
|
12.93%
|
3.42%
|
BsWRKY29
|
343
|
38673.7
|
9.76
|
66.63
|
-1.146
|
23.32%
|
12.83%
|
4.37%
|
2.2 Subcellular localization, signal peptide and transmembrane structure
The subcellular localization prediction showed that 24 WRKY proteins were all located in nucleus, except that BSWRKY16 was located in chloroplast, BSWRKY14 and BSWRKY29 were located in mitochondria, BSWRKY17 was located in vacuole, and BSWRKY28 was located in endoplasmic reticulum. Subcellular location determines its specific biological effects. WRKYs can form a net that contributes to various cytoplasmic and nuclear processes including signaling events from organelles or the cytoplasm to the nucleus (Bakshi and Oelmüller, 2014). Studies had shown that WRKY TFs on the ABAR-ABA complex in the downstream chloroplast envelope, regulates seed germination and other processes, and is one of the key nodes of abscisic acid signaling pathways (Rushton et al., 2012). These results indicated that WRKY genes might be involved in the regulation of plant growth and development and is an important node in metabolic regulation.
2.3 Promoter cis- regulatory elements of BsWRKY genes
The upstream of these BsWRKY genes were detected for finding cis-regulatory elements, like promoter and other cis-acting elements related to hormone regulation and stress-response (Fig. 1). The result showed that the cis-regulatory elements of the promoters of BsWRKY genes were related to growth and development (meristem expression, specific to the endosperm, seed-specific regulation and regulates circadian rhythm), plant hormones (auxin, abscisic acid, methyl jasmonate (MeJA), gibberellin, and salicylic aci), and stress (drought, low temperature, oxygen specificity induced response element and anaerobic induced indispensable cis function adjustment). It also showed that all the 29 BsWRKY genes had light response elements (LRE), and 14 of them had the drought-inducibility response elements. However, the elements of cell cycle regulatory elements, elements involved in defense and stress response, flavonoid synthesis and seed germination were only existed in BsWRKY28, BsWRKY7, BsWRKY16 and BsWRKY24, respectively. This not only indicated that BsWRKY genes are associated with plant growth, but also playing a vital role in drought stress regulatory networks. Collectively, these results indicated that WRKY family members participate in embryonic development, meristem growth and environmental stress regulation during the growth and development of B. striata.
2.4 Conservative motif of WRKY protein
A total of 10 conserved motifs were obtained by using online MEME for motif analysis of WRKY transcription factors in B. striata (Fig. 2A, B, C). Among them, motif3 were contained in 28 members except BsWRKY7. Motif1 and motif2 existed in 26 members, motif8 was found in 6 members, motif4 was detected from 4 members. The CD-search analysis found that motif1, motif2, and motif4 belong to WRKY domains, motif8 was a zinc finger domain relates to WRKY. Interestingly, the motif3 had no function record in the database currently, which needs to be further studied.
2.5 Conserved domains identification and evolutionary analysis
Through the online server CD-search, the structure domains of the WRKY genes family were analyzed for comparison. The results showed that 24 members of the 29 BsWRKY had typical WRKYGQK heptapeptide domain and W-box, but there was different degree of variation which mainly occurred in the N-terminal. Five transcription factors, i.e. BsWRKY 7, 16, 17, 22 and 27, had incomplete domains, like missing N or C terminus. It was speculated that the deletion may occur in the evolutionary process.
To place the evolution role and further identify the functions of BsWRKY genes, these 29 WRKY sequences from B. striata, 22 WRKY from Arabidopsis thaliana and 20 WRKY from Dendrobium catenatum were used to perform a phylogenetic analysis (Fig.3). The 29 BsWRKY transcription factors were grouped into three major groups. Group I included 6 members as BsWRKY 4, 17, 19, 10, 25, 26 and 28, which contains two WRKY conserved domains. Group III had members of BsWRKY1, 15 and 22, which containing only one WRKY domain with the zinc finger structure of type C2HC. In addition, Group II had only one domain with the zinc finger structure of C2H2, which could be further divided into 5 subgroups. In which, subgroup II-a had only member of BsWRKY12, subgroup II-b had four as BsWRKY 18, 23, 24 and 27, subgroup Ⅱ-c had BsWRKY2, 5, 6, 7, 10 and 29, subgroupⅡ-d was consisted by six members i.e. BsWRKY8, 9, 11, 13, 14 and 16, subgroupⅡ-e hold three members as BsWRKY3, 20 and 21.
According to the evolutionary tree, event of domain’s gain or loss might occur in the process of evolution, and group I was closer the real ancestor. The evolutionary relationship of WRKY genes among B. striata, A. thaliana and D. catenatum indicated that the three may have similar roles in certain biological functions. The results showed that BsWRKY5, BsWRKY12 and BsWRKY3 had the highest similarity with AtWRKY3, AtWRKY40 and AtWRKY14, respectively.
2.6 GO enrichment and KEGG functional cluster of WRKY genes
The GO and KEGG analysis results (Table 3) showed that most of BsWRKY genes were divided into three categories with functions of cellular components, biological processes and molecular functions, except BsWRKY8, 14, 16, 20 and 22. Among the biological processes, 19 genes were annotated for transcriptional regulation, BsWRKY18 and BsWRKY27 for cell cycle, BsWRKY25 for DNA repair and BsWRKY17 for meiosis prophase. In the molecular function category, 16 proteins were annotated for sequence specific DNA binding and 20 proteins for sequence specific DNA binding transcription factor activity. Among the large group of cellular components, only BsWRKY12 was annotated into the troponin complex, and BsWRKY25 had DNA ligase (NAD+) activity. KEGG analysis showed that BsWRKY19, 20, 26 and BsWRKY28 were involved in environmental information processing, signal transduction, MAPK signal transduction pathway, environmental adaptation, plant pathogen interaction and biological system.
Table 3
GO function classification of WRKY protein of B. striata
Gene name
|
Biological processes
|
|
Molecular function
|
|
Cellular components
|
Transcriptional regulation
|
DNA repair
|
Cell cycle
|
Meiosis prophase
|
Sequence-specific DNA binding
|
Sequence-specific DNA binding transcription factor activity
|
DNA ligase (NAD+) activity
|
Troponin complex
|
BsWRKY 1
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 2
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 3
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 4
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 5
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 6
|
|
|
|
|
|
√
|
|
|
BsWRKY 7
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 8
|
|
|
|
|
|
|
|
|
BsWRKY 9
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 10
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 11
|
|
|
|
|
|
√
|
|
|
BsWRKY 12
|
√
|
|
|
|
√
|
√
|
|
√
|
BsWRKY 13
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 14
|
|
|
|
|
|
|
|
|
BsWRKY 15
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 16
|
|
|
|
|
|
|
|
|
BsWRKY 17
|
√
|
|
|
√
|
|
|
|
|
BsWRKY 18
|
√
|
|
√
|
|
|
|
|
|
BsWRKY 19
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 20
|
|
|
|
|
|
|
|
|
BsWRKY 21
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 22
|
|
|
|
|
|
|
|
|
BsWRKY 23
|
|
|
|
|
|
√
|
|
|
BsWRKY 24
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 25
|
|
√
|
|
|
|
|
√
|
|
BsWRKY 26
|
√
|
|
|
|
√
|
√
|
|
|
BsWRKY 27
|
√
|
|
√
|
|
|
|
|
|
BsWRKY 28
|
|
|
|
|
|
√
|
|
|
BsWRKY 29
|
√
|
|
|
|
√
|
√
|
|
|
2.7 EST-SSR polymorphism of WRKY genes in B. striata
The EST-SSR markers have the advantages of high polymorphism and variability, high reproducibility, accurate and rapid detection (Li et al., 2015). A total of 10 among the 29 sequences were detected with SSR sites by NWISRL, of which 3 sequences were dinucleotide repeats and 7 sequences were trinucleotide repeats. The lowest number of replicates was 5, and the highest number was 18. The primer pairs of the 10 SSR sites were designed by DNAMAN software which could be amplified stably in all the four landraces (Table A), and the length of the amplified products ranged from 100 to 200 bp (Fig. 4). These results indicated that WRKY gene family was probably high-conserved in different B. striata germplasms. These newly found SSR primers could be used as molecular markers to identify the members of BsWRKY gene families in different germplasm (Zhong et al., 2021).