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Article

Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii

by
Xin He
1,2,
Meng-Meng Zhang
1,2,
Ye Huang
2,
Jiali Yu
1,2,
Xuewei Zhao
1,2,
Qinyao Zheng
2,
Zhong-Jian Liu
1,2,* and
Siren Lan
1,2,*
1
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture and Art, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(14), 7662; https://doi.org/10.3390/ijms25147662
Submission received: 21 June 2024 / Revised: 9 July 2024 / Accepted: 10 July 2024 / Published: 12 July 2024
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
As an important genus in Orchidaceae, Cymbidium has rich ecological diversity and significant economic value. DNA binding with one zinc finger (Dof) proteins are pivotal plant-specific transcription factors that play crucial roles in the growth, development, and stress response of plants. Although the Dof genes have been identified and functionally analyzed in numerous plants, exploration in Orchidaceae remains limited. We conducted a thorough analysis of the Dof gene family in Cymbidium goeringii, C. ensifolium, and C. sinensis. In total, 91 Dof genes (27 CgDofs, 34 CeDofs, 30 CsDofs) were identified, and Dof genes were divided into five groups (I–V) based on phylogenetic analysis. All Dof proteins have motif 1 and motif 2 conserved domains and over half of the genes contained introns. Chromosomal localization and collinearity analysis of Dof genes revealed their evolutionary relationships and potential gene duplication events. Analysis of cis-elements in CgDofs, CeDofs, and CsDofs promoters showed that light-responsive cis-elements were the most common, followed by hormone-responsive elements, plant growth-related elements, and abiotic stress response elements. Dof proteins in three Cymbidium species primarily exhibit a random coil structure, while homology modeling exhibited significant similarity. In addition, RT-qPCR analysis showed that the expression levels of nine CgDofs changed greatly under heat stress. CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23 showed varying degrees of upregulation. Most upregulated genes under heat stress belong to group I, indicating that the Dof genes in group I have great potential for high-temperature resistance. In conclusion, our study systematically demonstrated the molecular characteristics of Dof genes in different Cymbidium species, preliminarily revealed the patterns of heat stress, and provided a reference for further exploration of stress breeding in orchids.

1. Introduction

Plants have various strategies to regulate gene expression, among which transcription factors (TFs) play a vital role in regulating genes at the transcriptional level [1,2]. DNA binding with one zinc finger (Dof) proteins are one of the identified plant-specific TF families. The Dof proteins belong to C2H2-type zinc finger family proteins and have multiple roles such as stress response, phytohormones, photosynthesis, flower induction, seed germination, and light regulation [2,3,4,5]. Compared to the N-terminus, the C-terminal region of Dof proteins exhibits a high degree of variability. Dof transcription factors generally contain 200 to 400 amino acids, and the highly conserved Dof domain is usually located at the N-terminus, which has been identified as a DNA-binding domain and includes approximately 52 amino acids with a C2C2 zinc finger structure [3,6,7]. The Dof domain was proven to function as a Cys2/Cys2 zinc finger domain, which possesses a longer putative loop than that found in zinc finger domains of GATA1 and steroid hormone receptors [5]. The Dof domain is a multifunctional domain, not only involved in binding to DNA but also crucial for multiple protein–protein interactions, thus being regarded as a bi-functional domain that combines with DNA and interacts with other proteins [3,8,9,10].
Since the isolation of the first Dof gene, ZmDof1, from maize (Zea mays), homologs have been found in a variety of plants [11,12,13]. Dof genes have been shown to be highly expanded in terrestrial plants and increasingly identified in plants such as Dendrobium huoshanense (22), Selenicereus undatus (26), Oryza sativa (30), Arabidopsis thaliana (36), Solanum tuberosum (36), Populus trichocarpa (41), and Brassica rapa (76) [3,13,14,15,16,17,18,19]. Dof genes have been found in many plants, and their functions have aroused wide interest. First, Dof genes are involved in a range of crucial plant growth and developmental processes. Studies have shown that several Dof genes of Vitis vinifera perform their functional roles mainly during flower, berry, and seed development, highlighting their importance for V. vinifera growth and production [20]. Cai et al. found that the expression patterns of PsDof genes are consistently upregulated in various tissues and under different stress conditions, suggesting their pivotal involvement in both plant development and stress response [16]. Additionally, Dof proteins are also integral components in both biotic and abiotic stress responses. Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzyme activities in Z. mays [21]. Jin et al. identified five Cycling DOF Factors (CDFs) in potato, including StCDF1/StDof19, StCDF2/StDof4, StCDF3/StDof11, StCDF4/StDof24, and StCDF5/StDof15, which are homologs of Arabidopsis CDFs and may serve as the initial step in the abiotic stress response signaling cascade [19]. Furthermore, Chen et al. analyzed the induced expression of PeDofs under high-temperature stress during three periods, in which PeDof-11 was significantly induced with high expression [22]. Although Dof gene families in many plants have been extensively studied, the characteristics and roles of Dof genes in orchids have not been fully studied.
Cymbidium (Orchidaceae) has about 50 species and was divided into three subgenera (Cymbidium, Cyperorchis, and Jensoa). In addition, it is one of the most popular orchids in horticulture due to its high ornamental value [23]. Li et al. studied the response mechanisms of two different Cymbidiums (Cymbidium tracyanum and C. sinense) to drought stress and found that C. sinense (remedy strategy) and C. tracyanum (precaution strategy) adopt different adaptive strategies when facing drought stress [24]. The emergence of sequencing technologies has facilitated the discovery of many orchid genomes, laying a solid foundation for the in-depth analysis of Dof gene families in orchids. Therefore, it is significant that we study the Dof gene family in Cymbidium species.
This study systematically identified 27 Dof genes in C. goeringii, 34 in C. ensifolium, and 30 in C. sinense. We utilized bioinformatics methods to analyze the Dof gene family in three Cymbidium species. In addition, we also studied the response of CgDof genes to heat stress and provided a set of candidate genes for heat stress resistance. This is of great significance in identifying the key regulatory factors for stress resistance in C. goeringii. Our findings contribute to broadening our understanding of the evolutionary relationships and functional attributes of the orchid Dof gene family, providing some new insights for further investigating the role of Dof proteins and stress breeding.

2. Results

2.1. Identification and Protein Features of Dofs

A total of 91 Dof genes were identified in three Cymbidium species—27 in C. goeringii, 34 in C. ensifolium, and 30 in C. sinense. The genes were named CgDof1–27, CeDof1–34, and CsDof1–30, respectively, based on their distribution order (from top to bottom) on chromosomes (Table S1). The lengths of amino acids ranged from 112 aa to 634 aa, with an average of 267.54 aa. The molecular weight ranged from 12.39 to 70.48 kDa, with a mean of 29.17 kDa. A protein is considered alkaline if its isoelectric point exceeds 7, whereas a value below 7 indicates acidic properties [25]. Only around 15.38% (14/91) of the Dof proteins have low isoelectric points (pI ≤ 7), while the average pI is 8.43. Out of 91 Dof proteins, only five (CgDof19, CeDof13, CeDof28, and CsDof22) have an average instability index below 40, indicating stability in the majority, while the average instability index (II) stands at 56.89, with 44 Dof proteins falling below this index [26]. The average aliphatic index (AI) for 91 Dof proteins ranged from 41.29 to 83.09, with an average of 56.93. Moreover, the calculated mean hydrophilic index (GRAVY) of Dof proteins in the three orchids is negative, indicating a high degree of hydrophilicity. Furthermore, subcellular localization predictions indicate that the Dof proteins of the three orchids are primarily situated in the nucleus, suggesting a potential similarity with numerous transcription factors. Subcellular localization was predicted by WoLF PSORT [27]. The protein characteristics and sequences of Dofs in the three Cymbidium species are shown in Table S1.

2.2. Phylogeny and Classification of Dofs

The phylogenetic tree was constructed with 127 Dof genes from C. goeringii (27), C. ensifolium (34), C. sinense (30), and A. thaliana (36) (Figure 1). The phylogenetic tree showed that Dofs belonged to five categories, groups I–V, which is similar to the findings of previous studies [16]. We divided 127 Dof genes from four species into five groups: group I (47 genes), group II (24 genes), group III (25 genes), group IV (13 genes), and group V (18 genes). The results clearly showed that group I genes have a substantially higher number of genes than group IV genes.

2.3. Structure and Motif Analysis of Dofs

Conserved motifs of 127 Dof proteins in three Cymbidium species and A. thaliana were performed through the MEME Suite 5.5.5 online website (Figure 2B). Dof proteins within clades share similar motifs, while those from different clades differ. Ten motifs were discovered in Dof proteins. Motif 1 and motif 2 are present in all Dof proteins, which may be active regions for the exercise of functions; motif 4 and motif 5 only appeared in clade III; motif 3 is found in clades III and V. The roles of Dof proteins can be attributed to the specific distribution of different structures. We observed the number and distribution of Dof protein intron-exons to further reveal the gene structure in three Cymbidium species and A. thaliana (Figure 2C). The study showed that 49.45% of the genes lacked introns, and only 50.55% of the genes contained introns, of which 8.79% of the genes had two or more introns. Among 127 Dof proteins, the number of introns ranges from zero to ten and the number of exons ranges from one to eleven. The sequence information of motifs 1 to 10 are shown individually in Figure 3.

2.4. Chromosomal Localization and Collinearity of Dof Genes

After analysis of chromosome location, we found that 27 CgDof genes were distributed on 15 chromosomes, 30 CsDof genes were distributed on 15 chromosomes, and 34 CeDof genes were distributed on 17 chromosomes (Figure 4). In C. goeringii, Chr 09 has the most CgDof genes, totaling five genes; in C. sinensis, Chr 08 has the most CsDof genes, totaling six genes; in C. ensifolium, Chr 03 and Chr 08 have the most CeDof genes, with four genes each. In addition, we identified two, three, and six pairs of tandemly duplicated genes in the CgDof, CsDof, and CeDof groups, respectively. These tandemly duplicated genes are closely located on chromosomes and form clusters on phylogenetic trees, indicating that they have similar functions.
The intraspecific and interspecific collinearity of Dof gene sequences in three Cymbidium species was investigated. There are four pairs of segmentally duplicated genes in the C. goeringii genome (Figure 5A). The C. ensifolium genome had 11 pairs of segmentally duplicated genes (Figure 5B). Seven pairs of segmentally duplicated genes were present in the C. sinense genome (Figure 5C). There are 40 collinear gene pairs between C. goeringii and C. ensifolium, as well as 33 collinear gene pairs between C. goeringii and C. sinense (Figure 6). These results suggest that Dof genes in C. goeringii are more closely related to CeDofs than CsDofs.

2.5. Promoter Analysis of Dofs

At a distance of 2000 bp upstream of the CDS of 27 CgDof genes, 30 CsDof genes and 34 CeDof genes were extracted to identify cis-acting regulatory elements (CREs) and predict potential regulatory functions of Dof genes in three Cymbidium species (Figure 7). There are different types of cis-acting elements of the Dof gene family, which are related to plant growth and development, hormone response, light response, and abiotic stress response. The results found that the Dof genes of the three Cymbidium species contained more than 10 types of cis-acting elements, with light-responsive elements being the most prevalent, followed by hormone-responsive elements (Figure 8). In addition, some genes contain cis-acting elements related to abiotic stress, including low-temperature responsiveness, defense and stress responsiveness, and MYB binding sites involved in drought inducibility. A minority of genes possess cis-acting elements associated with plant secondary metabolism and growth development, including elements such as seed-specific regulation, zein metabolism regulation, and meristem expression. In summary, Dof genes contain various types of cis-acting elements, indicating that they may be involved in diverse biological processes. The types and numbers of CgDof genes, CeDof genes, and CsDof genes are listed in Table S2.

2.6. Prediction of Dof Protein Structure

An analysis of Dof proteins in three Cymbidium species, revealing random coils as the primary secondary structure (Table S3), offers crucial insights for further exploration into the biological functions of these proteins. Using SWISS-MODEL for homology modeling, the tertiary structures of Dof proteins from C. goeringii (Figure S1), C. ensifolium (Figure S2), and C. sinense (Figure S3) were predicted. Most instances of the 3D structure modeling results of Dof proteins were structurally similar in that they contained the extension chain (red part). The homology of Dof proteins in the three Cymbidium species and the modeling templates was almost 70%, indicating a strong structural similarity. Among SWISS-MODEL metrics, GMQE correlates positively with 3D model quality. The GMQE values of most Dof proteins were below 0.5, indicating that these proteins have high variability, while the GMQE values of relatively few proteins were above 0.5, indicating good modeling (Table S4) [28].

2.7. qRT-PCR Analysis of Dof Genes

To examine the expression patterns of Dof genes under heat stress, we selected and performed RT-qPCR analyses on the nine CgDof genes with the highest expression levels in the leaves of C. goeringii (Figure 9). The results of agarose gel electrophoresis are shown in Figure S4, while the RIN (RNA integrity number) is shown in Table S7. The results of the melt curve analysis are shown in Figures S5 and S6. The FPKM values of CgDof genes in leaves are listed in Table S5. Notably, the expression levels of CgDof03, CgDof22, and CgDof27 were observed to increase rapidly after 24 h under heat stress, exhibiting particularly significant upregulation. However, the expression levels of four CgDof genes (CgDof02, CgDof47, CgDof17, and CgDof23) were generally lower than in the control group (0 h) after heat stress. During heat treatment, the expression level of CgDof08 increased after 12 h but then decreased after 24 h, while the expression level of CgDof13 showed a similar pattern, increasing after 6 h and then decreasing after 18 h.

3. Discussion

Cymbidium in Orchidaceae is widely known for its unique floral morphology and floral scent characteristics [29]. Previous studies have focused on the molecular mechanisms of its growth and development, as well as complex floral development regulation processes [30,31,32]. However, there is limited research on the strategies employed by Cymbidium for regulating gene expression in response to abiotic stress. Dof transcription factors are a significant family of plant-specific transcription factors that play key roles in many plant biological processes, including responses to abiotic stress [13]. This study used bioinformatics analysis to investigate the evolutionary and functional characteristics of CgDof, CeDof, and CsDof genes, comprehensively profiling the Dof genes in three Cymbidium species. Furthermore, RT-qPCR experiments on nine CgDofs revealed variations in the expression levels of nine genes under heat stress, laying the foundation for further exploring the molecular regulation mechanism of Dof genes in plant stress resistance.
In this study, we identified 27 CgDof genes in C. goeringii, 34 CeDof genes in C. ensifolium, and 30 CsDof genes in C. sinense. The number of Dof genes in three Cymbidium species was higher than in Passiflora edulis (13) [22]; D. huoshanense (22) [17]; Prunus avium cv. ‘Tieton’ (23) [33]; S. undatus (26) [18]; Cerasus serrulata (27) [33]. However, it is relatively lower in other plants, such as S. tuberosum (36) [19]; Medicago polymorpha (36) [34]; A. thaliana (36) [14]; Populus simonii (41) [16]; Akebia trifoliata via (41) [35]; Helianthus annuus (50) [36]; Cerasus × yedoensis (53) [33]; Prunus cerasus (88) [33]. The results suggest that the Dof genes of these three Cymbidium species were not overextended during evolution [37]. Such variation in the numbers of Dof genes may stem from different gene duplication events or the impact of overall genome size in these diverse species [38].
In three Cymbidium species, 91 Dof genes were identified and their physicochemical properties were analyzed. Most proteins had instability index values above 40.0, except for four Dof proteins, including CgDof19, CeDof13, CeDof28, and CsDof22, each of which had instability index values below 40.0. In addition, the isoelectric point of most proteins (84.62%) is greater than 7. This indicates that most of the Dof proteins in these three orchid genera are stable alkaline proteins, which is similar to the Dof proteins of D. huoshanense [17,25,26]. Subcellular localization analysis revealed that most Dof proteins can be transported from the cytoplasm to the nucleus via nuclear transport signal domains, and some may play functions through modifications, enabling them to bind to target promoters for transcriptional activation or regulation [39]. The 127 Dofs are typically classified into groups I–V based on phylogenetic relationships with the Dof proteins in A. thaliana. The number of members of group I has far exceeded that of group IV in the course of evolution. Likewise, the Dof genes in P. simonii and Triticum aestivum also followed this classification [16,40].
The exploration of Dof protein motifs in three Cymbidium species is expected to provide valuable insights into their unique roles in developmental processes and stress adaptation [16]. All Dof proteins possess motif 1 and motif 2, indicating the strong conservatism of motifs 1 and 2 and their importance in determining the functional role of Dof proteins. Similar studies have also been conducted in Spinacia oleracea, where motifs 1 and 2 were found to be present in every Dof protein [41]. This finding parallels our research results, suggesting that motifs 1 and 2 may be key motifs within the Dof gene family, playing crucial roles in the functionality. Furthermore, the discovery that motifs 4 and 5 only exist in group III indicates the unique evolutionary significance of these motifs in this group. Gene structure analysis of Dof genes will provide valuable insights into its distinct functional role in the evolutionary process [42,43]. The majority of Dof genes belonging to the same group exhibit similar intron-exon structures. In this study, the number of introns in Dof genes ranged from zero to ten, with most members having no introns, a result similar to that of many other plant species, including Solanum lycopersicum, P. trichocarpa, Cucumis sativus, Cajanus cajan, and Passiflora edulis [22,44,45,46,47]. Research has found that plant genes with fewer introns exhibit stronger adaptability to the external environment and often respond quickly to stress [48]. Therefore, Dof genes in three Cymbidium species may be able to respond quickly to environmental changes, and these intron-free genes are the main driving force for plant tissue-specific evolution.
Gene duplication is a major factor in plant novelty and diversity and gene family expansion [49,50,51]. There are differences in the quantity and distribution of Dof genes among the three Cymbidium species. There are two, three, and six pairs of tandemly duplicated genes in CgDof, CsDof, and CeDof genes, respectively. Tandem duplications are known as a source of genetic novelty and can contribute to new genes with novel functions [52]. The collinearity analysis showed that C. goeringii and C. ensifolium had four and eleven pairs of segmentally duplicated genes, respectively, while C. sinense had seven. In addition, C. goeringii and C. ensifolium share 40 collinear gene pairs, and C. goeringii and C. sinense share 33 gene pairs. This suggests that C. goeringii is more closely related to C. ensifolium than C. senense.
Cis-elements play a vital part in the life cycle of plants [41]. Most cis-elements in the Dof gene family were associated with responses to stress, light, and hormones (Table S2). Among them, the light element is the most numerous, which is consistent with the Dof gene family of Passiflora edulis, indicating that the growth of these plants requires a high level of light [22]. In our study, the number of hormone-related elements in the Dofs ranked second. Phytohormones play a critical role in helping plants adapt to adverse environmental conditions. The elaborate hormone signaling networks and their ability to crosstalk make them ideal candidates for mediating defense responses [53]. Therefore, it is speculated that Dof genes may impact the stress resistance of Cymbidium species.
Previous research has shown that Dof genes can respond to various abiotic stresses. For example, the expression level of AtDof1.1 is upregulated 2–3 times after the induction of Me-JA and AtDof5.8 in response to salt stress [54,55]. Ma et al. found that the expression levels of BraDof023, BraDof045, and BraDof074 were all upregulated under drought and salt stress. Low temperatures may induce the expression of BraDof003, BraDof023, BraDof045, and BraDof053, and inhibit the expression of BraDof072 [13]. In addition, Corrales et al. found that high salt, drought, and ABA can induce CDF3 gene expression [56]. However, there is limited research into the role of the Dof gene family in heat stress. This study showed that under heat stress, the expression levels of CgDof02, CgDof07, CgDof17, and CgDof23 were significantly downregulated, while the expression levels of most CgDof genes were markedly upregulated, indicating that the CgDof genes are sensitive to high-temperature stress. High temperatures can inhibit the expression of some genes and also induce the expression of CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23. These five genes showed varying degrees of upregulation, with CgDof03, CgDof22, and CgDof27 reaching their highest expression levels after 24 h of high-temperature treatment. The expression level of CgDof13 increased significantly shortly after high-temperature stress, while CgDof08 only increased significantly in the middle stage of high-temperature stress. Remarkably, most upregulated genes under high-temperature stress belong to group I, indicating that CgDof genes in group I have great potential for high-temperature resistance. In conclusion, our study provides new clues for the further exploration of regulatory mechanisms governing the response of orchids to heat stress.

4. Materials and Methods

4.1. Data Source and Plant Materials

The full-genome data of C. goeringii (NCBI: PRJNA749652) [57], C. sinense (NCBI: PRJNA743748) [58], and C. ensifolium (NCBI: PRJCA005355) [29] were obtained from the National Center for Biotechnology Information database (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 18 February 2024). Additionally, we downloaded the protein sequence of the Dofs of A. thaliana from The Arabidopsis Information Resource (TAIR, http://arabidopsis.org, accessed on 18 February 2024) [59]. Furthermore, the C. goeringii obtained in this study were cultivated in the “Forest Orchid Garden” at Fujian Agricultural and Forestry University (26°05′ N, 119°13′ E) under the shade of trees with natural light and temperature. In the cultivation environment of C. goeringii, the shade in summer was controlled at about 70% to simulate the light conditions of its natural growth. Three pots of mature C. goeringii were selected under natural growth conditions and subjected to heat stress treatments in an artificial climate culture box. Under the photoperiod of 16 h of light/8 h of darkness and 30 °C/38 °C, samples were taken at 0 h, 6 h, 12 h, 18 h, and 24 h, respectively. Subsequently, the collected samples were quickly frozen in liquid nitrogen and stored in a freezer room at −80 °C for later use.

4.2. Identification and Physicochemical Properties of Dof Genes

After retrieving the complete genomic sequence files of C. goeringii, C. ensifolium, and C. sinense from the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 February 2024) [60], the protein and CDS sequence files were extracted utilizing the TBtools v1.120 software. Subsequently, 47 AtDof protein sequences were procured from the plantTFDB database [61]. Furthermore, the HMM (Hidden Markov Model) profile specific to the Dof domain (PF02701) was extracted from the Pfam database [62]. Utilizing the AtDofs as queries, Basic Local Alignment Search Tool (BLAST) analysis was conducted to identify putative Dof proteins in three Cymbidium species. The physicochemical properties of the identified Dof proteins, including amino acids, molecular weight, theoretical isoelectric points, instability index, aliphatic indexes, and Grand average of hydropathicity (GRAVY), were calculated using the ProtParam tool from ExPasy3.0 (https://www.expasy.org/, accessed on 20 February 2024) [63]. Subcellular localization prediction of proteins was performed using the WOLF PSORT website (https://wolfpsort.hgc.jp/, accessed on 20 February 2024) [27].

4.3. Phylogenetic Analysis of Dofs

The protein sequences of Dofs from C. goeringii (27 CgDofs), C. ensifolium (34 CeDofs), C. sinense (30 CsDofs), and A. thaliana (36 AtDofs) were merged and imported into the MEGA 7.0 software for analysis. The phylogenetic relationship of the aligned sequences was estimated using the neighbor-joining (NJ) method by MEGA 11 software. The bootstrap method was executed with 500 replicates, setting partial deletion to 50%. Then, the Phylogeny test was performed using 1000 replications of the bootstrap method. The phylogenetic tree was visualized using Evolview (http://www.evolgenius.info/evolview/#/treeview, accessed on 3 March 2024) [64].

4.4. Gene Structures and Conserved Motif Analysis of Dofs

Using NCBI’s CDD tool, we analyzed the conserved domains of Dof genes. In addition, MEME Suite 5.5.5 online software (http://meme-suite.org/, accessed on 3 March 2024) was employed to analyze the conserved sequence patterns of Dof genes in three Cymbidium species and A. thaliana, with a predicted number of ten [63]. TBtools v1.120 was employed to integrate phylogenetic trees, conserved protein motifs, and comparative maps of gene structures in this study [65].

4.5. Collinearity and Chromosomal Localization of Dofs

Multiple Synteny plot and Advanced Circos tools from TBtools v1.120 was utilized to examine the interspecific collinear relationships between C. goeringii, C. ensifolium, and C. sinense. Additionally, the intraspecific collinearity of these three orchid species was analyzed using the One Step MCScanX-Super Fast tool.
To analyze the distribution of Dof genes on chromosomes, the TBtools v1.120 software was employed. For collinearity analysis among the chromosomes of the three Cymbidium species, the One Step MCScanx command within TBtools v1.120 was utilized. Additionally, the Advance Circos package program in TBtools v1.120 was applied for displaying segmental duplications among the Dof genes.

4.6. Cis-Element Analysis of Dofs

To identify potential cis-acting elements in Dof gene promoters in three Cymbidium species and A. thaliana, we employed TBtools v1.120 software to extract 2000 bp upstream regions of these genes. Subsequently, we used PlantCARE online software (https://bioinformatics.psb.ugent.be/webtools/plantcare/html, accessed on 20 March 2024) to perform a detailed analysis of cis-acting regulatory elements in the extracted promoter regions of the Dof genes [66]. Data processing was carried out using Excel 2019 software for organization, and visualization was achieved with TBtools v1.120 software.

4.7. Protein Structure Prediction

The secondary structure of the protein is predicted by the SOPMA procedure [67]. SOPMA is a well-established method that helps us to predict the presence of various secondary structural elements, such as alpha-helices and beta-sheets, within the protein sequence. By utilizing SWISS-MODEL (https://swissmodel.expasy.org/interactive, accessed on 15 March 2024), we were able to generate a three-dimensional representation of the Cymbidium Dof proteins.

4.8. Analysis of Expression and RT-qPCR

Using a FastPure Plant Total RNA Isolation Kit (for polysaccharide- and polyphenol-rich tissues) (Vazyme Biotech Co., Ltd., Nanjing, China), RNA was isolated from the leaves of C. goeringii. Transcriptome sequencing and library construction were completed by Bgi Genomics Co., Ltd. (Shenzhen, China). Calculations of the gene expression level of each sample were performed using the software RSEMv1.2.8 to obtain the fragments per kilobase of transcript per million fragments (FPKM) values.
Then we employed Hifair® AdvanceFast One-step RT-gDNA Digestion SuperMixfor qPCR (Yeasen Biotechnology Co., Ltd., Shanghai, China) for reverse transcription to remove the contaminated genomic DNA and generate cDNA. Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix was used for qRT-PCR. Primers for RT-qPCR targeting CgDofs were designed using Primer Premier 5 software, and their specificity was confirmed through primer blast on the NCBI website. RT-qPCR analysis was conducted using PerfectStart™ Green qPCR SuperMax (TransGen Biotech, Beijing, China). The actin gene from C. goeringii served as the reference gene. The expression levels of each gene were normalized to the actin internal control gene, and the relative gene expression levels were calculated by using the 2−∆∆CT method [68].

5. Conclusions

This study examined the Dof gene family in three Cymbidium species. A total of 27 CgDofs, 34 CeDofs, and 30 CsDofs were identified for the first time, and several aspects such as the physicochemical properties, conserved motifs, exon-intron structure, and secondary/tertiary structure of proteins were analyzed. These findings indicated a high degree of conservation in Dof genes. The chromosomal localization and collinearity analysis of Dof genes in three Cymbidium species provided crucial information about their evolutionary relationships. The identification of hormone-responsive cis-acting elements in the promoter region of Dof genes helped expand the knowledge of the abiotic stress pathway in orchids. We explored the performance of Dof genes at five high-temperature stages and validated their expression patterns in leaves. Five Dof genes (CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23) were speculated to have potential functions in the heat stress response of C. goeringii. These findings not only expand our understanding of the Dof gene family but also lay the foundation for a deeper understanding of their contribution to plant stress tolerance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25147662/s1.

Author Contributions

X.H. finalized the manuscript and integrated all other authors’ comments; M.-M.Z., Y.H. and J.Y. analyzed the data; X.Z. and Q.Z. provided the data. S.L. and Z.-J.L. conceived the study, coordinated with all coauthors, and supervised the whole project. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by scientific funding from the Technical Services for Introduction and Domestication of Orchids in Sanjiangkou Botanical Garden, Fuzhou (KH240047A).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original data presented in the study are openly available in the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 18 February 2024) genome database. The BioProject numbers for C. goeringii, C. ensifolium, and C. sinense are PRJNA749652, PRJCA005355, and PRJNA743748, respectively.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Floris, M.; Mahgoub, H.; Lanet, E.; Robaglia, C.; Menand, B. Post-transcriptional regulation of gene expression in plants during abiotic stress. Int. J. Mol. Sci. 2009, 10, 3168–3185. [Google Scholar] [CrossRef] [PubMed]
  2. Gupta, S.; Malviya, N.; Kushwaha, H.; Nasim, J.; Bisht, N.C.; Singh, V.K.; Yadav, D. Insights into structural and functional diversity of Dof (DNA binding with one finger) transcription factor. Planta 2015, 241, 549–562. [Google Scholar] [CrossRef] [PubMed]
  3. Yanagisawa, S. The Dof family of plant transcription factors. Trends Plant Sci. 2002, 7, 555–560. [Google Scholar] [CrossRef] [PubMed]
  4. De Paolis, A.; Sabatini, S.; De Pascalis, L.; Costantino, P.; Capone, I. A rolB regulatory factor belongs to a new class of single zinc finger plant proteins. Plant J. Cell Mol. Biol. 1996, 10, 215–223. [Google Scholar] [CrossRef] [PubMed]
  5. Umemura, Y.; Ishiduka, T.; Yamamoto, R.; Esaka, M. The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain. Plant J. Cell Mol. Biol. 2004, 37, 741–749. [Google Scholar] [CrossRef] [PubMed]
  6. Imaizumi, T.; Schultz, T.F.; Harmon, F.G.; Ho, L.A.; Kay, S.A. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science 2005, 309, 293–297. [Google Scholar] [CrossRef] [PubMed]
  7. Yanagisawa, S. A novel DNA-binding domain that may form a single zinc finger motif. Nucleic Acids Res. 1995, 23, 3403–3410. [Google Scholar] [CrossRef] [PubMed]
  8. Yanagisawa, S. Dof DNA-binding domains of plant transcription factors contribute to multiple protein-protein interactions. Eur. J. Biochem. 1997, 250, 403–410. [Google Scholar] [CrossRef] [PubMed]
  9. Yanagisawa, S. Dof domain proteins: Plant-specific transcription factors associated with diverse phenomena unique to plants. Plant Cell Physiol. 2004, 45, 386–391. [Google Scholar] [CrossRef]
  10. Cavalar, M.; Möller, C.; Offermann, S.; Krohn, N.M.; Grasser, K.D.; Peterhänsel, C. The interaction of DOF transcription factors with nucleosomes depends on the positioning of the binding site and is facilitated by maize HMGB5. Biochemistry 2003, 42, 2149–2157. [Google Scholar] [CrossRef]
  11. Yanagisawa, S.; Izui, K. Molecular cloning of two DNA-binding proteins of maize that are structurally different but interact with the same sequence motif. J. Biol. Chem. 1993, 268, 16028–16036. [Google Scholar] [CrossRef] [PubMed]
  12. Moreno-Risueno, M.A.; Martínez, M.; Vicente-Carbajosa, J.; Carbonero, P. The family of DOF transcription factors: From green unicellular algae to vascular plants. Mol. Genet. Genom. MGG 2007, 277, 379–390. [Google Scholar] [CrossRef] [PubMed]
  13. Ma, J.; Li, M.Y.; Wang, F.; Tang, J.; Xiong, A.S. Genome-wide analysis of Dof family transcription factors and their responses to abiotic stresses in Chinese cabbage. BMC Genom. 2015, 16, 33. [Google Scholar] [CrossRef] [PubMed]
  14. Lijavetzky, D.; Carbonero, P.; Vicente-Carbajosa, J. Genome-wide comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol. Biol. 2003, 3, 17. [Google Scholar] [CrossRef]
  15. Yang, X.; Tuskan, G.A.; Cheng, M.Z. Divergence of the Dof gene families in poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after duplication. Plant Physiol. 2006, 142, 820–830. [Google Scholar] [CrossRef] [PubMed]
  16. Cai, K.; Xie, X.; Han, L.; Chen, J.; Zhang, J.; Yuan, H.; Shen, J.; Ren, Y.; Zhao, X. Identification and functional analysis of the DOF gene family in Populus simonii: Implications for development and stress response. Front. Plant Sci. 2024, 15, 1412175. [Google Scholar] [CrossRef]
  17. Gu, F.; Zhang, W.; Wang, T.; He, X.; Chen, N.; Zhang, Y.; Song, C. Identification of Dof transcription factors in Dendrobium huoshanense and expression pattern under abiotic stresses. Front. Genet. 2024, 15, 1394790. [Google Scholar] [CrossRef] [PubMed]
  18. Alam, O.; Khan, L.U.; Khan, A.; Salmen, S.H.; Ansari, M.J.; Mehwish, F.; Ahmad, M.; Zaman, Q.U.; Wang, H.F. Functional characterisation of Dof gene family and expression analysis under abiotic stresses and melatonin-mediated tolerance in pitaya (Selenicereus undatus). Funct. Plant Biol. FPB 2024, 51, FP23269. [Google Scholar] [CrossRef]
  19. Jin, X.; Wang, Z.; Ai, Q.; Li, X.; Yang, J.; Zhang, N.; Si, H. DNA-Binding with One Finger (Dof) Transcription Factor Gene Family Study Reveals Differential Stress-Responsive Transcription Factors in Contrasting Drought Tolerance Potato Species. Int. J. Mol. Sci. 2024, 25, 3488. [Google Scholar] [CrossRef]
  20. da Silva, D.C.; da Silveira Falavigna, V.; Fasoli, M.; Buffon, V.; Porto, D.D.; Pappas, G.J., Jr.; Pezzotti, M.; Pasquali, G.; Revers, L.F. Transcriptome analyses of the Dof-like gene family in grapevine reveal its involvement in berry, flower and seed development. Hortic. Res. 2016, 3, 16042. [Google Scholar] [CrossRef]
  21. Cao, L.; Ye, F.; Fahim, A.M.; Ma, C.; Pang, Y.; Zhang, X.; Zhang, Q.; Lu, X. Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzymes activities in maize (Zea mays L.). Theor. Appl. Genet. 2024, 137, 132. [Google Scholar] [CrossRef] [PubMed]
  22. Chen, G.; Xu, Y.; Gui, J.; Huang, Y.; Ma, F.; Wu, W.; Han, T.; Qiu, W.; Yang, L.; Song, S. Characterization of Dof Transcription Factors and the Heat-Tolerant Function of PeDof-11 in Passion Fruit (Passiflora edulis). Int. J. Mol. Sci. 2023, 24, 12091. [Google Scholar] [CrossRef] [PubMed]
  23. Yang, J.B.; Tang, M.; Li, H.T.; Zhang, Z.R.; Li, D.Z. Complete chloroplast genome of the genus Cymbidium: Lights into the species identification, phylogenetic implications and population genetic analyses. BMC Evol. Biol. 2013, 13, 84. [Google Scholar] [CrossRef] [PubMed]
  24. Li, J.W.; Chen, X.D.; Hu, X.Y.; Ma, L.; Zhang, S.B. Comparative physiological and proteomic analyses reveal different adaptive strategies by Cymbidium sinense and C. tracyanum to drought. Planta 2018, 247, 69–97. [Google Scholar] [CrossRef] [PubMed]
  25. Zhao, X.; Liu, D.K.; Wang, Q.Q.; Ke, S.; Li, Y.; Zhang, D.; Zheng, Q.; Zhang, C.; Liu, Z.J.; Lan, S. Genome-wide identification and expression analysis of the GRAS gene family in Dendrobium chrysotoxum. Front. Plant Sci. 2022, 13, 1058287. [Google Scholar] [CrossRef] [PubMed]
  26. Enany, S. Structural and functional analysis of hypothetical and conserved proteins of Clostridium tetani. J. Infect. Public Health 2014, 7, 296–307. [Google Scholar] [CrossRef] [PubMed]
  27. Horton, P.; Park, K.J.; Obayashi, T.; Fujita, N.; Harada, H.; Adams-Collier, C.J.; Nakai, K. WoLF PSORT: Protein localization predictor. Nucleic Acids Res. 2007, 35, W585–W587. [Google Scholar] [CrossRef]
  28. Liang, T.; Jiang, C.; Yuan, J.; Othman, Y.; Xie, X.Q.; Feng, Z. Differential performance of RoseTTAFold in antibody modeling. Brief. Bioinform. 2022, 23, bbac152. [Google Scholar] [CrossRef]
  29. Ai, Y.; Li, Z.; Sun, W.H.; Chen, J.; Zhang, D.; Ma, L.; Zhang, Q.H.; Chen, M.K.; Zheng, Q.D.; Liu, J.F.; et al. The Cymbidium genome reveals the evolution of unique morphological traits. Hortic. Res. 2021, 8, 255. [Google Scholar] [CrossRef]
  30. Liu, D.K.; Zhang, C.; Zhao, X.; Ke, S.; Li, Y.; Zhang, D.; Zheng, Q.; Li, M.H.; Lan, S.; Liu, Z.J. Genome-wide analysis of the TCP gene family and their expression pattern in Cymbidium goeringii. Front. Plant Sci. 2022, 13, 1068969. [Google Scholar] [CrossRef]
  31. Su, S.; Shao, X.; Zhu, C.; Xu, J.; Lu, H.; Tang, Y.; Jiao, K.; Guo, W.; Xiao, W.; Liu, Z.; et al. Transcriptome-Wide Analysis Reveals the Origin of Peloria in Chinese Cymbidium (Cymbidium sinense). Plant Cell Physiol. 2018, 59, 2064–2074. [Google Scholar] [CrossRef]
  32. Li, X.; Jin, F.; Jin, L.; Jackson, A.; Ma, X.; Shu, X.; Wu, D.; Jin, G. Characterization and comparative profiling of the small RNA transcriptomes in two phases of flowering in Cymbidium ensifolium. BMC Genom. 2015, 16, 622. [Google Scholar] [CrossRef]
  33. Hou, Q.; Yu, R.; Shang, C.; Deng, H.; Wen, Z.; Qiu, Z.; Qiao, G. Molecular characterization and evolutionary relationships of DOFs in four cherry species and functional analysis in sweet cherry. Int. J. Biol. Macromol. 2024, 263 Pt 2, 130346. [Google Scholar] [CrossRef]
  34. Yang, L.; Min, X.; Wei, Z.; Liu, N.; Li, J.; Zhang, Y.; Yang, Y. Genome-Wide Identification and Expression Analysis of the Dof Transcription Factor in Annual Alfalfa Medicago polymorpha. Plants 2023, 12, 1831. [Google Scholar] [CrossRef]
  35. Zhang, Q.; Zhong, S.; Dong, Q.; Yang, H.; Yang, H.; Tan, F.; Chen, C.; Ren, T.; Shen, J.; Cao, G.; et al. Identification of Photoperiod- and Phytohormone-Responsive DNA-Binding One Zinc Finger (Dof) Transcription Factors in Akebia trifoliata via Genome-Wide Expression Analysis. Int. J. Mol. Sci. 2023, 24, 4973. [Google Scholar] [CrossRef]
  36. Song, H.; Ji, X.; Wang, M.; Li, J.; Wang, X.; Meng, L.; Wei, P.; Xu, H.; Niu, T.; Liu, A. Genome-wide identification and expression analysis of the Dof gene family reveals their involvement in hormone response and abiotic stresses in sunflower (Helianthus annuus L.). Gene 2024, 910, 148336. [Google Scholar] [CrossRef] [PubMed]
  37. Rashid, A.; Khajuria, M.; Ali, V.; Faiz, S.; Jamwal, S.; Vyas, D. Genome-wide identification and expression profiling of WRKY family suggest their potential role in cannabinoid regulation in Cannabis sativa L. Ind. Crops Prod. 2023, 206, 117706. [Google Scholar] [CrossRef]
  38. Grimplet, J.; Agudelo-Romero, P.; Teixeira, R.T.; Martinez-Zapater, J.M.; Fortes, A.M. Structural and Functional Analysis of the GRAS Gene Family in Grapevine Indicates a Role of GRAS Proteins in the Control of Development and Stress Responses. Front. Plant Sci. 2016, 7, 353. [Google Scholar] [CrossRef]
  39. Le Hir, R.; Bellini, C. The plant-specific dof transcription factors family: New players involved in vascular system development and functioning in Arabidopsis. Front. Plant Sci. 2013, 4, 164. [Google Scholar] [CrossRef]
  40. Liu, Y.; Liu, N.; Deng, X.; Liu, D.; Li, M.; Cui, D.; Hu, Y.; Yan, Y. Genome-wide analysis of wheat DNA-binding with one finger (Dof) transcription factor genes: Evolutionary characteristics and diverse abiotic stress responses. BMC Genom. 2020, 21, 276. [Google Scholar] [CrossRef]
  41. Yu, H.; Ma, Y.; Lu, Y.; Yue, J.; Ming, R. Expression profiling of the Dof gene family under abiotic stresses in spinach. Sci. Rep. 2021, 11, 14429. [Google Scholar] [CrossRef] [PubMed]
  42. Li, X.; Cai, K.; Pei, X.; Li, Y.; Hu, Y.; Meng, F.; Song, X.; Tigabu, M.; Ding, C.; Zhao, X. Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening. Int. J. Mol. Sci. 2021, 22, 12414. [Google Scholar] [CrossRef] [PubMed]
  43. Koralewski, T.E.; Krutovsky, K.V. Evolution of exon-intron structure and alternative splicing. PLoS ONE 2011, 6, e18055. [Google Scholar] [CrossRef] [PubMed]
  44. Rojas-Gracia, P.; Roque, E.; Medina, M.; López-Martín, M.J.; Cañas, L.A.; Beltrán, J.P.; Gómez-Mena, C. The DOF Transcription Factor SlDOF10 Regulates Vascular Tissue Formation During Ovary Development in Tomato. Front. Plant Sci. 2019, 10, 216. [Google Scholar] [CrossRef]
  45. Wang, H.; Zhao, S.; Gao, Y.; Yang, J. Characterization of Dof Transcription Factors and Their Responses to Osmotic Stress in Poplar (Populus trichocarpa). PLoS ONE 2017, 12, e0170210. [Google Scholar] [CrossRef]
  46. Wen, C.L.; Cheng, Q.; Zhao, L.; Mao, A.; Yang, J.; Yu, S.; Weng, Y.; Xu, Y. Identification and characterisation of Dof transcription factors in the cucumber genome. Sci. Rep. 2016, 6, 23072. [Google Scholar] [CrossRef]
  47. Malviya, N.; Gupta, S.; Singh, V.K.; Yadav, M.K.; Bisht, N.C.; Sarangi, B.K.; Yadav, D. Genome wide in silico characterization of Dof gene families of pigeonpea (Cajanus cajan (L) Millsp.). Mol. Biol. Rep. 2015, 42, 535–552. [Google Scholar] [CrossRef]
  48. Sang, Y.; Liu, Q.; Lee, J.; Ma, W.; McVey, D.S.; Blecha, F. Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity. Sci. Rep. 2016, 6, 29072. [Google Scholar] [CrossRef]
  49. Demuth, J.P.; Hahn, M.W. The life and death of gene families. BioEssays 2009, 31, 29–39. [Google Scholar] [CrossRef]
  50. Magadum, S.; Banerjee, U.; Murugan, P.; Gangapur, D.; Ravikesavan, R. Gene duplication as a major force in evolution. J. Genet. 2013, 92, 155–161. [Google Scholar] [CrossRef]
  51. Soltis, P.S.; Soltis, D.E. Ancient WGD events as drivers of key innovations in angiosperms. Curr. Opin. Plant Biol. 2016, 30, 159–165. [Google Scholar] [CrossRef]
  52. Conant, G.C.; Wolfe, K.H. Turning a hobby into a job: How duplicated genes find new functions. Nat. Reviews. Genet. 2008, 9, 938–950. [Google Scholar] [CrossRef]
  53. Verma, V.; Ravindran, P.; Kumar, P.P. Plant hormone-mediated regulation of stress responses. BMC Plant Biol. 2016, 16, 86. [Google Scholar] [CrossRef]
  54. Skirycz, A.; Reichelt, M.; Burow, M.; Birkemeyer, C.; Rolcik, J.; Kopka, J.; Zanor, M.I.; Gershenzon, J.; Strnad, M.; Szopa, J.; et al. DOF transcription factor AtDof1.1 (OBP2) is part of a regulatory network controlling glucosinolate biosynthesis in Arabidopsis. Plant J. Cell Mol. Biol. 2006, 47, 10–24. [Google Scholar] [CrossRef]
  55. He, L.; Su, C.; Wang, Y.; Wei, Z. ATDOF5.8 protein is the upstream regulator of ANAC069 and is responsive to abiotic stress. Biochimie 2015, 110, 17–24. [Google Scholar] [CrossRef]
  56. Corrales, A.R.; Carrillo, L.; Lasierra, P.; Nebauer, S.G.; Dominguez-Figueroa, J.; Renau-Morata, B.; Pollmann, S.; Granell, A.; Molina, R.V.; Vicente-Carbajosa, J.; et al. Multifaceted role of cycling DOF factor 3 (CDF3) in the regulation of flowering time and abiotic stress responses in Arabidopsis. Plant Cell Environ. 2017, 40, 748–764. [Google Scholar] [CrossRef]
  57. Chen, J.; Bi, Y.Y.; Wang, Q.Q.; Liu, D.K.; Zhang, D.; Ding, X.; Liu, Z.J.; Chen, S.P. Genome-wide identification and analysis of anthocyanin synthesis-related R2R3-MYB genes in Cymbidium goeringii. Front. Plant Sci. 2022, 13, 1002043. [Google Scholar] [CrossRef]
  58. Wang, L.; Zhao, X.; Zheng, R.; Huang, Y.; Zhang, C.; Zhang, M.M.; Lan, S.; Liu, Z.J. Genome-Wide Identification and Drought Stress Response Pattern of the NF-Y Gene Family in Cymbidium sinense. Int. J. Mol. Sci. 2024, 25, 3031. [Google Scholar] [CrossRef]
  59. Lamesch, P.; Berardini, T.Z.; Li, D.; Swarbreck, D.; Wilks, C.; Sasidharan, R.; Muller, R.; Dreher, K.; Alexander, D.L.; Garcia-Hernandez, M.; et al. The Arabidopsis Information Resource (TAIR): Improved gene annotation and new tools. Nucleic Acids Res. 2012, 40, D1202–D1210. [Google Scholar] [CrossRef]
  60. Schoch, C.L.; Ciufo, S.; Domrachev, M.; Hotton, C.L.; Kannan, S.; Khovanskaya, R.; Leipe, D.; McVeigh, R.; O’Neill, K.; Robbertse, B.; et al. NCBI Taxonomy: A comprehensive update on curation, resources and tools. Database 2020, 2020, baaa062. [Google Scholar] [CrossRef]
  61. Jin, J.; Tian, F.; Yang, D.C.; Meng, Y.Q.; Kong, L.; Luo, J.; Gao, G. PlantTFDB 4.0: Toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res. 2017, 45, D1040–D1045. [Google Scholar] [CrossRef]
  62. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.E.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021, 49, D412–D419. [Google Scholar] [CrossRef]
  63. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef] [PubMed]
  64. Subramanian, B.; Gao, S.; Lercher, M.J.; Hu, S.; Chen, W.H. Evolview v3: A webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res. 2019, 47, W270–W275. [Google Scholar] [CrossRef]
  65. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  66. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  67. Geourjon, C.; Deléage, G. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. CABIOS 1995, 11, 681–684. [Google Scholar] [CrossRef]
  68. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic tree of 127 Dof genes found in C. goeringii, C. ensifolium, C. sinense, and A. thaliana. The phylogenetic relationship of aligned sequences was estimated using the neighbor-joining (NJ) method by MEGA 11 software, and the phylogeny test was performed using 1000 replications of the bootstrap method.
Figure 1. Phylogenetic tree of 127 Dof genes found in C. goeringii, C. ensifolium, C. sinense, and A. thaliana. The phylogenetic relationship of aligned sequences was estimated using the neighbor-joining (NJ) method by MEGA 11 software, and the phylogeny test was performed using 1000 replications of the bootstrap method.
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Figure 2. Motif and gene structure analysis of the Dof gene family in three Cymbidium species and A. thaliana. (A) Phylogenetic tree of 127 Dof genes; (B) The conserved motif of Dof proteins; (C) The structure of Dof proteins. TBtools v1.120 was used to integrate phylogenetic trees, conserved protein motifs, and comparative maps of gene structures.
Figure 2. Motif and gene structure analysis of the Dof gene family in three Cymbidium species and A. thaliana. (A) Phylogenetic tree of 127 Dof genes; (B) The conserved motif of Dof proteins; (C) The structure of Dof proteins. TBtools v1.120 was used to integrate phylogenetic trees, conserved protein motifs, and comparative maps of gene structures.
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Figure 3. The sequence information of motifs 1 to 10. MEME Suite 5.5.5 online software was employed to analyze conserved sequence patterns of Dof genes.
Figure 3. The sequence information of motifs 1 to 10. MEME Suite 5.5.5 online software was employed to analyze conserved sequence patterns of Dof genes.
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Figure 4. Distribution of Dofs on the chromosomes of three Cymbidium species. (A) C. goeringii, (B) C. sinense, and (C) C. ensifolium. The black designates the names of chromosomes, the red is employed to indicate the names of Dofs.
Figure 4. Distribution of Dofs on the chromosomes of three Cymbidium species. (A) C. goeringii, (B) C. sinense, and (C) C. ensifolium. The black designates the names of chromosomes, the red is employed to indicate the names of Dofs.
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Figure 5. The interspecific collinearity of C. goeringii, C. ensifolium, and C. sinense. Red lines represent segmental duplicated gene pairs. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense.
Figure 5. The interspecific collinearity of C. goeringii, C. ensifolium, and C. sinense. Red lines represent segmental duplicated gene pairs. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense.
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Figure 6. Intraspecific collinearity of three Cymbidium species. Blue lines depict Dof genes that exhibit collinear relationships across different species.
Figure 6. Intraspecific collinearity of three Cymbidium species. Blue lines depict Dof genes that exhibit collinear relationships across different species.
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Figure 7. Promoter analysis of Dof genes in three Cymbidium species. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense.
Figure 7. Promoter analysis of Dof genes in three Cymbidium species. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense.
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Figure 8. Statistics on the number of Dofs in different categories. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense. The types and numbers of Dof genes in three Cymbidium species are listed in Table S2.
Figure 8. Statistics on the number of Dofs in different categories. (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense. The types and numbers of Dof genes in three Cymbidium species are listed in Table S2.
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Figure 9. Real-time reverse transcription quantitative PCR (RT-qPCR) validation of nine CgDofs under high-temperature stress. The Y-axis represents the relative expression values (2−∆∆CT) and the X-axis represents the time of high-temperature stress. This analysis examined the status of the control sample at 0 h before high-temperature stress, and then recorded its condition after being subjected to high-temperature stress for 6, 12, 18, and 24 h. A total of three biological replicates, each with three technical repeats, were used in the experiment. Bars represent the mean values of three technical replicates ± SE. For data analysis, a student-t test was performed to identify differentially expressed genes. The red asterisk serves as an indicator denoting the significance level of the p-value in the respective test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Primers are shown in Table S6.
Figure 9. Real-time reverse transcription quantitative PCR (RT-qPCR) validation of nine CgDofs under high-temperature stress. The Y-axis represents the relative expression values (2−∆∆CT) and the X-axis represents the time of high-temperature stress. This analysis examined the status of the control sample at 0 h before high-temperature stress, and then recorded its condition after being subjected to high-temperature stress for 6, 12, 18, and 24 h. A total of three biological replicates, each with three technical repeats, were used in the experiment. Bars represent the mean values of three technical replicates ± SE. For data analysis, a student-t test was performed to identify differentially expressed genes. The red asterisk serves as an indicator denoting the significance level of the p-value in the respective test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Primers are shown in Table S6.
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He, X.; Zhang, M.-M.; Huang, Y.; Yu, J.; Zhao, X.; Zheng, Q.; Liu, Z.-J.; Lan, S. Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii. Int. J. Mol. Sci. 2024, 25, 7662. https://doi.org/10.3390/ijms25147662

AMA Style

He X, Zhang M-M, Huang Y, Yu J, Zhao X, Zheng Q, Liu Z-J, Lan S. Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii. International Journal of Molecular Sciences. 2024; 25(14):7662. https://doi.org/10.3390/ijms25147662

Chicago/Turabian Style

He, Xin, Meng-Meng Zhang, Ye Huang, Jiali Yu, Xuewei Zhao, Qinyao Zheng, Zhong-Jian Liu, and Siren Lan. 2024. "Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii" International Journal of Molecular Sciences 25, no. 14: 7662. https://doi.org/10.3390/ijms25147662

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