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Article

Genome-Wide Identification of the TGA Gene Family and Expression Analysis under Drought Stress in Brassica napus L.

by
Yi Duan
1,
Zishu Xu
1,
Hui Liu
2,
Yanhui Wang
3,
Xudong Zou
3,
Zhi Zhang
3,
Ling Xu
1,* and
Mingchao Xu
3,*
1
College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou 310018, China
2
Institute of Agriculture, The University of Western Australia, Crawley, WA 6009, Australia
3
Leshan Academy of Agricultural Sciences, Leshan 614000, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(12), 6355; https://doi.org/10.3390/ijms25126355
Submission received: 7 May 2024 / Revised: 4 June 2024 / Accepted: 5 June 2024 / Published: 8 June 2024
(This article belongs to the Special Issue Advance in Plant Abiotic Stress)
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Abstract

:
TGA transcription factors belong to Group D of the bZIP transcription factors family and play vital roles in the stress response of plants. Brassica napus is an oil crop with rich economic value. However, a systematic analysis of TGA gene family members in B. napus has not yet been reported. In this study, we identified 39 full-length TGA genes in B. napus, renamed TGA1~TGA39. Thirty-nine BnTGA genes were distributed on 18 chromosomes, mainly located in the nucleus, and differences were observed in their 3D structures. Phylogenetic analysis showed that 39 BnTGA genes could be divided into five groups. The BnTGA genes in the same group had similar structure and motif compositions, and all the BnTGA genes had the same conserved bZIP and DOG1 domains. Phylogenetic and synteny analysis showed that the BnTGA genes had a close genetic relationship with the TGA genes of the Brassica juncea, and BnTGA11 and BnTGA29 may play an important role in evolution. In addition, qRT-PCR revealed that three genes (BnTGA14/17/23) showed significant changes in eight experimental materials after drought treatment. Meanwhile, it can be inferred from the results of drought treatment on different varieties of rapeseed that the stress tolerance of parental rapeseed can be transmitted to the offspring through hybridization. In short, these findings have promoted the understanding of the B. napus TGA gene family and will contribute to future research aimed at B. napus resistant breeding.

1. Introduction

Plants are continuously subjected to an extensive array of biological and environmental pressures and have elaborated a variety of sophisticated defense mechanisms and complex signal networks to quickly perceive these challenges and regulate the expression of genes to successfully protect themselves from damage [1,2,3,4]. Transcription factors (TFs), as essential components of signal transduction, are prominently associated with the metabolic balance, growth, development, and multiple defense pathways of plants [5,6]. The TFs specifically activate or inhibit target gene expression by specifically interacting with the DNA cis-regulatory element sequence in the target gene promoter containing the binding site of the transcription factor, and guide the expression synchronously [7,8]. The bZIP transcription factor family is characterized by a vast array of members across various organisms and holds great significance in the TF families [9]. According to the amino acid sequence similarity and protein structure within the bZIP domain, the bZIP family of Arabidopsis thalianais is sorted into ten distinct groups, namely A, B, C, D, E, F, G, H, I, and S [10]. TGA (TGACG motif-binding factor) TF is classified within the D subfamily of the bZIP transcription factor family [11]. TGAs are composed of a typical bZIP domain, a highly variable N-terminal region, and a relatively conserved C-terminal region [12]. Within these domains, the bZIP domain can interact with specific DNA, serving as both a DNA-binding domain and a nuclear localization signal. The N-terminus exhibits significant variability in amino acid sequence and length. On the other hand, the C-terminus shows relative conservation, containing two glutamine-rich regions, Q1 and Q2. Additionally, the C-terminal region of TGAs also encompasses the DOG1 domain (Delay of Germination 1), which lacks DNA-binding ability but participates in the regulatory processes of TGAs [11,12,13,14]. TGA1a was the pioneering TGA gene to be cloned in plants and had performed as a pivotal landmark for characterizing the TGA gene family [15]. Subsequently, additional TGA TFs were discovered in wide-ranging plant species [10,16,17,18]. Based on sequence similarity, 10 TGA TFs in A. thaliana can be divided into five subgroups [14]. Within the TGA gene family, Group I consists of TGA1 and TGA4, which exhibit the highest similarity to TGA1a in tobacco. Group II comprises TGA2, TGA5, and TGA6, showing close relationships and functional redundancy. TGA3 and TGA7 form Group III, while Group IV is composed of TGA9 and TGA10. Group V consists solely of TGA8, which is also known as PERIANTHIA (PAN) [11,19]. Growing evidence suggests the vital involvement of TGA transcription factors in various biological processes, including pathogen defense and plant development [17,20,21,22,23,24], as well as in response to different stresses [25,26,27]. In addition, the role of TGA TFs is closely related to different plant hormone signaling pathways, including JA and SA [14,28].
Important crops such as rice, wheat, cotton, rapeseed, and sunflower have high economic and nutritional value [29,30,31,32,33]. Brassica napus, a globally cultivated oil crop, holds substantial economic significance due to its high commercial value [34]. B. napus originated through natural hybridization between Brassica rapa (ArAr, 2n = 20) and Brassica oleracea (CoCo, 2n = 18) approximately 7500 years ago [34]. It exhibits adaptability to various climatic conditions worldwide [35]. B. napus ranks as the third-largest contributor to vegetable oil production globally, accounting for approximately 13% of the total edible oil output [36]. The cultivation of B. napus is affected by many biotic and abiotic factors, such as drought, heat, and infection of Sclerotinia sclerotiorum [35,37,38]. Therefore, analyzing the resistance mechanism of B. napus varieties and excavating new resistance factors constitute important functions in B. napus resistance breeding. TGA TFS have been extensively investigated in multiple species; however, knowledge regarding TGA TFs in B. napus remains limited.
In this study, we accomplished an analysis of the TGA gene family in cruciferous plants, focused on B. napus. The phylogenetic relationship, structural characteristics, conserved motifs, chromosome localization, cis-element, and expression pattern of the BnTGA gene family under drought stress were described and analyzed. The comprehensive assessment of the BnTGA genes in this study offers potential candidate genes for clarifying the molecular regulatory mechanism of B. napus in the process of biotic and abiotic stress, which can be used to assist in the breeding of resistance varieties for B. napus.

2. Results

2.1. Identification and Location of Chromosomes of BnTGA Genes in Brassica napus

In this study, the protein sequences of 10 A. thaliana TGAs were used as query sequences in a BLAST search against the B. napus genome database. As a result, a total of thirty-nine putative TGA genes were identified in the B. napus genome. These genes were found to have entire bZIP and DOG1 domains based on the BLAST results. And these relevant BnTGA genes were renamed from BnTGA1 to BnTGA39. Thirty-nine BnTGA genes were located on the 18 chromosomes of B. napus (Figure 1). Detailed information about these genes, such as their full-length protein sequences, molecular weight (MW), putative subcellular localization, and isoelectric point (pI), is summarized in Table S1. The length of the protein sequences for these thirty-nine genes varies from 198 to 570. The theoretical isoelectric points (pI) and molecular weight (kDa) of putative TGA proteins ranged from 22.87 (BnTGA22) to 65.51 (BnTGA17) kDA and 5.72 (BnTGA18) to 9.36 (BnTGA33), respectively. Additionally, the prediction results of protein subcellular localization showed that BnTGA genes were concentrated within the cell nucleus.

2.2. Conserved Motif, Conserved Domain, and Gene Structure Analysis of TGA Gene Family in Brassica napus

To analyze the evolutionary relationship of BnTGA gene members using B. napus and A. thaliana, we constructed a phylogenetic tree with the maximum likelihood (ML) method in TBtools V2.096 software for the full TGA protein sequence. Based on the conserved structural domains of AtTGA proteins, thirty-nine BnTGAs were classified into five subgroups, which are as follows: BnTGA1, BnTGA2, BnTGA3, BnTGA5, BnTGA7, BnTGA16, BnTGA23, BnTGA25, and BnTGA36 belonged to Group I; BnTGA4, BnTGA14, BnTGA17, BnTGA21, BnTGA24, BnTGA26, BnTGA34, and BnTGA35 to Group II; BnTGA8, BnTGA9, BnTGA10, BnTGA15, BnTGA20, BnTGA22, BnTGA27, BnTGA30, BnTGA33, and BnTGA37 to Group III; BnTGA18, and BnTGA39 to Group IV; and the rest to Group V. Except for the Group IV, the proportion of the other four groups is similar (Figure 2a). The results of the phylogenetic analysis (Figure 2a) were confirmed through the MEME online website (https://meme-suite.org/meme/ (accessed on 22 March 2023)). We detected 10 conserved motifs, ranging in length from 11 to 50 amino acids, in the thirty-nine BnTGA proteins, taking the AtTGA proteins as templates (Figure 2b and Figure S1). The quantity of motifs present in the TGA proteins ranged from three to nine, and all proteins exhibited the presence of three conserved motifs: motif 1, motif 2, and motif 5. This observation indicates that motifs 1, 2, and 5 demonstrate a high degree of conservation within the BnTGA proteins. In the same subgroup, the characteristics of motifs were highly consistent, including motif number, composition, and relative position. For example, the number and distribution characteristics of motifs of each member in Group I and IV were the same, and in subgroup V, the similarity of the characteristics of motifs reached 92%. The motif composition of members of the same subgroup was relatively conserved, which were more inclined to express identical or similar functions. Meanwhile, the distribution characteristics of motifs were different in different subgroups, such as motif 10 only existed in Group I, and motif 9 was only identified in Group III. And motif 9 and motif 10 exhibit a highly conserved amino acid sequence (Figure S1), indicating that these two motifs are likely to have important implications for the structural and functional properties of the corresponding protein members within their respective groups. The variation in motif distributions across different evolutionary branches can result in alterations in the structure of TGA genes. These structural changes play a crucial role in determining the function divergence among different evolutionary branches. DOG1 and bZIP domains were found in thirty-nine BnTGA proteins, and the relative positions of these domains were consistently observed across different sequences (Figure 2c), suggesting that the BnTGA genes share a conserved domain arrangement in their gene structure.
Analyzing the gene structure is crucial for unraveling the connection between genome evolution and function divergence. The exon count of BnTGA genes varies from 3 to 12, highlighting the diversity in gene structure within this gene family. Most subgroup members contained a range of exons between 7 and 11. The BnTGA15 group contained the largest number of exons, a total of 12, while BnTGA33 contained only 3 exons (Figure 3).
In summary, subgroup members were highly conserved in motifs and domain, which further validates the accuracy of subgroup classification in the phylogenetic analysis.

2.3. Secondary and Tertiary Structural Analysis of TGA Proteins in Brassica napus

Proteins serve various functions in organisms, such as regulating gene expression and catalyzing biochemical reactions. The structural integrity of proteins plays a crucial role in enabling them to fulfill their functions. The functional significance of proteins heavily relies on their intricate 3D structure, so we conducted predictions regarding the secondary and tertiary structure of the BnTGA proteins. The predominant types of secondary structure in the BnTGA proteins were the α-helix (48.74–75.23%), followed by the random coil (17.22–41.24%), the extended strand (3.99–12.44%) and β-turn (1.53–4.56%) (Table S2). These results indicated that the dominant components of the secondary structure in the BnTGA proteins were α-helix and random coil, while β-turn and extended strand were relatively less abundant. By using AlphaFold2, the 3D structure of the BnTGA protein was predicted. The structure of the BnTGA family protein primarily consisted of α-helix and random coil, as demonstrated in Figure S2, which was consistent with the secondary structure prediction of the BnTGA protein. Based on the evolutionary relationship between B. napus and A. thaliana TGA full proteins, we classified 39 TGA proteins into five groups according to the classification of TGA proteins in A. thaliana. In particular, the majority of TGA proteins in Group I, Group II, and Group V exhibited high structural similarity in their 3D structures, suggesting that these proteins likely have redundant or comparable functions in B. napus. Some TGA proteins in Group III and Group IV showed significant structural differences compared to other proteins within the same subgroup, indicating that they may have distinct functions. Furthermore, variations in 3D structures were observed among proteins from different subgroups (Figure S2).
When proteins play important roles in complex organisms, they often require interactions with other proteins or molecules, leading to the formation of protein networks and signaling pathways, enabling proteins to accomplish intricate and coordinated cellular processes in organisms. To investigate the protein interaction network of TGA genes of B. napus, we employed the String web tool for functional protein interaction prediction. A. thaliana was selected as a suitable organism for comparison with B. napus. The predicted results showed that the core members of the interaction network were members of the TGA gene family. There are interactions between different TGA proteins, and TGA proteins also interact with other transcription factors (Figure 4). They primarily interact with NPR proteins and GRXC proteins. The interactions among these proteins collectively regulate various metabolic reactions in organisms.
It is worth noting that PAN (TGA8), as a core member of the protein interaction network, interacted with multiple proteins, such as NPR1, NPR3, NPR4, NPR5, NPR6, GRXC7, and GRXC8, etc. PAN, as a single subfamily, has been confirmed to have a significant impact on plant flowering and root growth. Based on the predicted protein interaction network results, we aimed to investigate the functions of the PAN gene further. We used purchased A. thaliana mutant seeds to study the response of PAN mutant to drought stress. Col-0 and PAN mutant were germinated on 1/2MS medium and 1/2MS medium supplemented with PEG6000. After 3 days, the germination rates of both seeds were recorded (Table S3). Regardless of the presence of PEG in the medium, the germination rate of the PAN mutant was consistently lower than that of the Col-0. As the concentration of PEG increased, both the Col-0 and PAN mutant germination rates decreased, with the PAN mutant showing a more pronounced decrease. After 7 days of growth in the medium, the phenotypic changes of both materials were documented, with the PAN mutant exhibiting shorter root lengths compared to the Col-0, consistent with previous research findings (Figure S3). Following drought treatment, the root length of A. thaliana seedlings displayed a decrease with increasing PEG concentration, and the change was more evident at 30% PEG concentration. Particularly, the PAN mutant showed a significantly shorter root length than the Col-0 after drought treatment. From this, we inferred that the PAN gene plays a crucial role in plant drought response. BnTGA18 and BnTGA39, belonging to the same subfamily as PAN, may also have important roles in plants’ responses to drought stress.

2.4. Phylogenetic Analysis of TGA Gene Family in Brassica napus

To investigate the evolutionary relationship among TGA genes in various species, based on protein homology and cluster analysis, this study employed TBtools V2.096 software to analyze the TGA protein sequences of B. napus, Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, and Camelina sativa, and constructed a phylogenetic tree (Table S4). In total, 105 TGA genes were identified across the five species, including 10 AtTGAs, 39 BnTGAs, 10 AIyTGAs, 13 ArhTGAs, and 33 CsaTGAs. Multi-sequence alignment of protein sequences was performed, and the phylogenetic tree categorized the TGAs into five distinct subgroups: Group I, Group II, Group III, Group IV, and Group V (Figure 5). In these five subgroups, except that Group III contained only two BnTGAs, BnTGA18 and BnTGA39, in the remaining Group I, Group II, Group IV, and Group V, the distribution of BnTGAs was relatively average, with 9, 8, 10, and 10, respectively. The five species selected were all belonging to the cruciferous family. Overall, the BnTGAs protein had the highest sequence similarity to the TGAs protein in Camelina sativa. Therefore, TGAs in B. napus may have similar functions to TGAs in Camelina sativa.

2.5. Synteny and Duplication Analysis of TGA Gene Family in Brassica napus

Gene duplication could cause the amplification of the gene family, and two primary forms of duplication are tandem and segmental duplications. To comprehend the amplification mechanisms of BnTGA genes, we employed MCScanX to explore gene duplication events in B. napus. Consequently, a total of 73 pairs of gene duplication events were identified (Figure 6a). For a deeper analysis of the evolutionary connection between TGA genes in B. napus and various other cruciferous species, we also investigated the syntenic relationship of TGAs across six different plants. We identified orthologous gene pairs between B. napus TGA and several other plants, namely Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, Camelina sativa, Brassica juncea, and Brassica oleracea (Figure 6b). B. napus and other six species have gene pairs reaching 54 pairs with Arabidopsis thaliana, 53 pairs with Arabidopsis lyrata, 49 pairs with Arabidopsis halleri, 170 pairs with Brassica juncea, 106 pairs with Brassica oleracea, and 162 pairs with Camelina sativa. The findings indicated that the TGAs in B. napus exhibited a close genetic relationship with those in Brassica juncea and Camelina sativa, while showing distant phylogenetic relationships with Arabidopsis halleri, Arabidopsis lyrata, and Arabidopsis thaliana. Notably, BnTGA11 and BnTGA29, reaching 26 gene pairs in B. napus and other six species, displayed significant homology with other species (Table S5). This suggests that these two genes may serve as a major factor in the evolution of the TGA gene family.

2.6. Cis-Element in the Promoter Region of TGA Gene Family in Brassica napus

The cis-elements play a vital role in controlling transcription initiation and gene expression. To explore the transcriptional regulation of BnTGA genes, the cis-elements within their promoter regions were analyzed (Figure 7). The analysis of cis-elements revealed significant variations in the types and quantities of core components present in BnTGA genes (Table S6). A total of 36 distinct cis-elements were identified to have potential functions. Among these cis-elements, 20 were related to growth and development, 6 were associated with stress reactions, and 10 were involved in hormone reactions. Most BnTGA genes contained Box 4 elements, G-box elements, TCT-motif, ARE, and ABRE elements. Among all TGA genes, the Box 4 element was the most abundant element, followed by the ARE core element. The SARE element was only included in BnTGA33. The BnTGA2 promoter contained 18 cis-elements, which was the most diverse of all genes, but the BnTGA24 promoter exhibited the lowest diversity in terms of cis-elements, with a mere 7 elements identified. Among the cis-elements of the BnTGA genes, the number of cis-elements that respond to different hormones varied greatly. The number of elements that respond to jasmonic acid and abscisic acid was similar, but the number of cis-elements that respond to salicylic acid was less than that of jasmonic acid and abscisic acid. The variations in hormone reaction elements suggested the significance of BnTGA genes in hormone signaling pathways. Furthermore, the diverse types and quantities of cis-elements across different genes implied the potential involvement of BnTGA genes in plant responses to biological and environmental pressures, as well as the regulation of environmental adaptation.

2.7. Expression Patterns of TGA Gene Family in Brassica napus

Examining the expression patterns of BnTGA genes offers valuable insights into their regulatory mechanisms. By studying RNA-Seq data that has been published in the NCBI database, we assessed the expression levels of BnTGA genes during various stress conditions. Specifically, under heat stress, all BnTGA genes exhibited increased expression, except for BnTGA5, BnTGA7, BnTGA16, and BnTGA25, which displayed lower expression compared to the control group, among which the transcription levels of BnTGA4 (2.37-fold), BnTGA14 (8.37-fold), BnTGA28 (2.47-fold) and BnTGA34 (4.00-fold) increased significantly (Figure 8a). Under drought stress, the expression of BnTGA14 (2.11-fold), BnTGA17 (1.54-fold), BnTGA32 (1.84-fold), and BnTGA35 (1.53-fold) increased significantly, while the expression level of the BnTGA23 was markedly diminished (2.75-fold) in comparison to the control group (Figure 8a). Westar and ZY821 were Sclerotinia sclerotiorum susceptibility and resistance varieties, respectively. When the leaves of susceptible and resistant B. napus were infected by Sclerotinia sclerotiorum, respectively, most TGA gene expression levels had similar trends in susceptible and resistant B. napus. Notably, the expression of BnTGA4, BnTGA6, and BnTGA11 in the Westar variety, infected by Sclerotinia sclerotiorum, was almost unchanged compared with the control group, while these three gene expression levels in the ZY821 variety were significantly reduced after Sclerotinia sclerotiorum infection (Figure 8b). The distinct expression levels of BnTGA genes under different conditions provide evidence for the involvement of TGA genes in the response of B. napus to both biotic and abiotic stresses, and it is speculated that BnTGA4, BnTGA6, and BnTGA11 may be important genes for B. napus to respond to Sclerotinia sclerotiorum.

2.8. Expression Analysis of the BnTGAs under Drought Stress

Drought stress poses significant challenges to the growth, productivity, and quality of B. napus. To explore how TGA genes respond to drought stress in eight experimental materials, LY8, WJY520, LY07, LY1008H, DY6, ZS11, LY31AB, and ZD630, we subjected eight experimental materials to drought treatment. LY8 and WJY520 were crossbred using LY31AB as the female parent, while LY07 and LY1008H were used as the male parents in the crossbreeding (Figure 9). To facilitate phenotypic recording and photography, we assigned numerical labels to the experimental materials as follows: LY8, WJY520, LY07, LY1008H, DY6, ZS11, LY31AB, and ZD630 were labeled as 1–8, respectively. We selected three genes, BnTGA14, BnTGA17, and BnTGA23, which showed significant changes in expression levels in response to drought treatment according to transcriptomic data. We conducted qRT-PCR analysis to validate the results, and the findings were consistent with the transcriptomic analysis results (Figure 10a). Specific primers are shown in Table S7. The expression levels of the three BnTGA genes after drought stress were significantly different. Following stress treatment, there was a noteworthy increase in the expression of BnTGA14 and BnTGA17 in the eight rapeseed species used in the experiment, whereas BnTGA23 exhibited a markedly lower expression compared to the control group. This finding suggests that these three genes exhibit contrasting roles in response to drought stress. The increased expression of BnTGA14 and BnTGA17 may enhance the drought resistance of B. napus, while the increased expression level of BnTGA23 may have an inhibitory effect. It is worth noting that although BnTGA14, BnTGA17, and BnTGA23 showed significant changes compared to the control group after drought treatment, and their expression patterns were generally consistent among the eight experimental materials, LY8 exhibited the most superior performance among the eight experimental materials after drought treatment, presenting not only robust plant phenotypes (Figure S4a,b) but also extremely significant expression differences in BnTGA14 and BnTGA17 (Figure 10b). In addition to LY8, LY31AB and ZD630 also demonstrated varying degrees of drought tolerance, but leaf wilting occurred in a small area along the leaf margins. Therefore, we speculate that LY8 may possess stronger adaptability under drought conditions, thereby promoting growth. The specific mechanisms underlying this phenomenon require further investigation.

3. Discussion

Transcriptional regulation is fundamental to various physiological processes in plants throughout their life cycle, including growth, development, and response to environmental stresses [39,40]. Gene regulation goes through a series of complex processes [41], in which transcription factors play an important role. TFs exhibit a wide range of functions in plant growth, including plant development and the formation of overall morphological diversity of plants [42,43]. Herein, gaining insights into the structure and function of TFs is essential to unravel the regulatory mechanisms that govern plant growth and development. TGA TFs play an important role in various physiological processes of plants, and many important studies have been carried out on their structure and function [14,44]. Nevertheless, there is a dearth of extensive studies on the TGA gene family of B. napus. In this study, we utilized the AtTGA genes as a reference to investigate the BnTGA gene family members throughout the entire genome. Additionally, we analyzed the gene structure and the gene evolutionary relationship of the TGA gene in different species. The expression patterns of the BnTGA genes were explored, including the differences in expression under heat and drought stress, as well as differences in expression after Sclerotinia sclerotiorum infection. This provides useful data for disease resistance breeding of B. napus.
Within this research, we identified 39 full-length TGA genes in B. napus. Through an analysis of gene structure and conserved motifs, we classified these TGA proteins into five distinct subgroups. Notably, this subgroup classification in B. napus aligns with the grouping observed in A. thaliana for the TGA gene family. Subgroup members were not only highly conserved in protein motifs, but also highly similar in gene structure [45]. Exon/intron and conserved motif analysis showed that there were significant differences in gene structure and sequence lengths of BnTGA members in different subgroups. It has been reported that introns can improve the content of mRNA by affecting transcription, and can also enhance mRNA translation efficiency [46]. The BnTGA gene may have different biological activities due to its different intron structures. Conserved domain and motif results showed the presence of typical motifs in all BnTGAs. High sequence similarity among motif sequences within each subgroup indicated that TGA members of each subgroup potentially share similar functions [47]. All BnTGA proteins contain bZIP and DOG1 domains, and relevant studies have demonstrated that the alkaline region within the bZIP domain utilized N-x7-R/K-x9, the fixed nuclear localization signal structure, to bind to DNA, thereby determining DNA specificity and nuclear localization [11]. To further explore their structural properties, we employed AlphaFold2 online website (https://alphafold.com/ (accessed on 16 May 2024)) for the prediction of the 3D structures of BnTGA proteins. The results indicated that the protein structure of 39 BnTGAs was in line with the secondary structure results and corroborated the phylogenetic classification results. The tertiary structure of members within the same subgroup is highly akin. Additionally, the protein interaction prediction results showed that the BnTGAs interacted with multiple proteins—primarily NPR proteins. This further confirmed the involvement of TGA genes in the NPR signaling pathway and it was consistent with the existing research results in other species [16,17,48]. Putative subcellular localization revealed that BnTGA TFs in B. napus were present in the cell nucleus, indicating that BnTGA TFs play a vital role in the cell nucleus. This indicated that although the functions of genes in each subgroup were different, the overall gene family functions remained conserved.
In evolutionary relationships, the sequences of proteins belonging to the same subgroup were highly similar, so they were very likely to perform similar functions. Based on sequence similarity, the BnTGA genes can be divided into five subgroups, which was consistent with A. thaliana. There were a total of 10 TGA TFs in the A. thaliana genome, and the classification and function of each member of the AtTGA have been thoroughly studied. The evolutionary relationship between B. napus and A. thaliana was utilized to infer the biological function of the BnTGA genes. It has been previously observed that AtTGA01-AtTGA07 belong to three distinct subgroups; these seven TGA TFs play crucial roles in plant disease resistance [43]. Specifically, AtTGA02, AtTGA05, and AtTGA06 serve as essential regulators in systemic acquired resistance (SAR) reactions, which are involved in plant defense against diseases [49,50,51]. Therefore, we hypothesize that BnTGA6, BnTGA11-BnTGA13, BnTGA19, BnTGA28-BnTGA29, BnTGA31-BnTGA32, and BnTGA38, through the NPR1 signal transduction pathway, may enhance plant disease resistance. Additionally, it has been observed that in the presence of cytokinin (CTK), AtTGA03 interacts with ARR2 and binds to the PR1 promoter, thereby enhancing plant resistance against diseases [20,52]. Furthermore, AtTGA01 and AtTGA04 interact with the ethylene reaction factor (ERF) to enhance plant resistance [53]. It is speculated that BnTGA1-BnTGA5, BnTGA7, BnTGA14, BnTGA16-BnTGA17, BnTGA21, BnTGA23-BnTGA26, and BnTGA34-BnTGA36 can also coordinate plant disease resistance through NPR 1 signaling pathways and other signaling pathways. AtTGA09, AtTGA10, and AtPAN play an important role in regulating the development of flower organs [54,55,56]. Moreover, studies have indicated a correlation between AtPAN and root growth [57]. It is speculated that the remaining BnTGA genes belonging to the same subgroup as AtTGA09, AtTGA10, and AtPAN may be related to flower organ development. Furthermore, members belonging to the same subgroup as AtPAN may be associated with root growth. Synteny analysis showed that among the six selected cruciferous species, BnTGAs had the closest genetic relationship and the most orthologous gene pairs with Brassica juncea, followed by Camelina sativa, but a more distant relationship with Arabidopsis halleri, so it can be postulated that the TGA genes in B. napus and Brassica juncea might demonstrate comparable functions, in line with the phylogenetic relationship between B. napus and other species. BnTGA11 and BnTGA29 display the highest number of homologous gene pairs across other species, suggesting their potential significance in TGA gene evolution [58].
The biological function of a gene product is often closely associated with cis-elements within its promoter region [59]. In this study, the analysis of cis-element showed the presence of diverse hormone-related cis-elements and stress-related cis-elements within the promoter region of the BnTGA gene family, including “WUN-motif”, “drought-inducibility”, and “defense and stress responsiveness”, etc. This showed that the BnTGA genes participate in the growth, development, and disease resistance of plants, through a variety of hormone-regulatory pathways and physiological processes that respond to stress. The different expression levels of the BnTGA genes under heat stress, drought stress, and Sclerotinia sclerotiorum infection further confirmed that the BnTGA genes play an important role in responding to stress. After the infection of Sclerotinia sclerotiorum, BnTGA4, BnTGA6, and BnTGA11 changed significantly in resistant variety. Therefore, it is speculated that these BnTGA genes play an important role in responding to the infection of Sclerotinia sclerotiorum. In addition, the results of qRT-PCR were consistent with the trend of transcriptome data, and TGA14, TGA17, and TGA23 changed significantly after drought treatment. It is further confirmed that TGA TFs play a vital role in the biotic stress response of B. napus.
The goal of rapeseed breeding is to develop varieties with good quality, high oil yield, and strong stress resistance. In preliminary experiments, we identified LY31AB and ZD630 as two excellent varieties. Using LY31AB as the maternal parent, we conducted hybrid breeding and obtained the first filial generation. To verify whether the progeny LY8 and WJY520 still possessed excellent traits and stress resistance, we subjected different rapeseed varieties to drought treatment and selected three genes with significant changes in the transcriptome data for validation. The qRT-PCR results showed that the LY8 exhibited strong stress resistance, manifested by its sturdy leaves in the phenotype, as well as significant changes in qRT-PCR data. Therefore, this variety is considered to be a superior variety, and further analysis of its stress resistance mechanism can be conducted in the future.
Overall, this study establishes the foundation for future study endeavors focused on unraveling the regulatory function of the TGA gene family in the growth and disease resistance mechanisms of B. napus. Furthermore, these results offer a valuable resource for utilizing the BnTGA gene family in resistance breeding programs for B. napus.

4. Materials and Methods

4.1. Plant Material and Stress Treatment

The B. napus seeds were from commercial varieties (DY6, ZS11 and ZD630) or our own groups (LY07, LY1008H, LY31AB, LY8 and WJY520). Seeds were germinated in a low-light environment, ensuring adequate moisture. Three days after seed germination, the seedlings were moved to a hydroponic tank filled with Hoagland nutrient solution, and the seedlings were kept growing at 24/20 °C (day/night), 14/10 h (light/night) and 60–70% relative humidity. To explore the response of TGA TFs to drought stress, twenty-day-old seedlings were immersed in Hoagland nutrient solution containing 10% PEG6000 to simulate drought treatment, with a treatment time of 12 h and 24 h, respectively. Additionally, 0.1 g of B. napus leaf samples were collected at 0 h, 12 h, and 24 h under drought treatment, respectively, and put into a 2.0 centrifuge tube marked with the sample number. The leaves were collected at the designated time point and subjected to three replicate samplings, immediately flash-frozen in liquid nitrogen, and then stored at −80 °C for subsequent analysis.
The A. thaliana Col-0 and PAN mutant seeds were purchased from AraShare (https://www.arashare.cn/index/Product/index.html), with Col-0 serving as the wild-type control. The seeds were surface-sterilized by treating them with 70% ethanol for 5 min, followed by 3% sodium hypochlorite for 5 min, and then rinsed with sterile water 10 times. The sterilized seeds were germinated on 1/2MS medium. Simultaneously, PEG6000 was added to the 1/2MS medium at concentrations of 10%, 20%, and 30% to simulate drought conditions [60]. A. thaliana seeds were vernalized for 24 h at 4 °C and then the 1/2MS medium was placed vertically in a greenhouse. After three days, the germination rates of A. thaliana Col-0 and PAN mutants under different treatments were recorded. After 7 days of growth, the phenotypic characteristics of seedling root growth under drought conditions were documented.

4.2. Identification of Members of the TGA Gene Family in Brassica napus

To identify TGA genes in B. napus, we utilized 10 TGA gene sequences (Table S2) in A. thaliana, obtained from The Arabidopsis Information Resource (TAIR, http://arabidopsis.org (accessed on 10 March 2023)). The whole B. napus genome sequence was obtained from Ensemble Plants (https://plants.ensembl.org (accessed on 10 March 2023)) [61]. The AtTGA protein sequences were used as a query to implement a BLASTP search (E-value < 10 × 10−5) for the whole genome protein of B. napus to screen out the candidate TGA genes in B. napus. The BLAST search was completed in TBtools V2.096 [62]. Subsequently, the candidate proteins sequence were submitted to the SMART database (http://smart.embl.de/ (accessed on 20 March 2023)) and CDD database (https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 20 March 2023)) websites, respectively, to check whether the putative TGA genes contained entire bZIP and DOG1 domains to further discern the TGA gene in B. napus [63,64]. The theoretical isoelectric point (pI) and molecular weight (MW) of the retrieved TGA protein sequence were predicted by the online tool ExPASy (https://web.expasy.org/protparam/ (accessed on 10 April 2023)) [32,65]. Subcellular localization of all retrieved TGA protein sequences was determined using (https://www.genscript.com/wolf-psort.html (accessed on 10 April 2023)).

4.3. Chromosomal Location, Gene Structure, Conserved Motif, and Conserved Domain Analysis of TGA Gene Family in Brassica napus

To validate whether the putative 39 BnTGA genes can be classified into the same five groups as the TGA genes in A. thaliana, the maximum likelihood (ML) method was used to analyze the gene clustering of B. napus and A. thaliana TGA proteins, with 1000 bootstrap replicates.
The positional arrangement and dispersion of the TGA genes on the B. napus chromosome were obtained based on the B. napus GFF file and the candidate BnTGA gene, utilizing the Gene Location Visualize from GTF\GFF function within the TBtools V2.096 software; all parameters were set to their default values according to the program [62].
Based on the downloaded B. napus genome annotation files and the candidate TGA protein sequence, the BnTGAs structure, motif, and conserved domains were depicted employing the Gene Structure View tool in TBtools V2.096; all parameters were set to their default values according to the program [62,66].

4.4. Protein Interaction, Secondary and Tertiary Structural Analysis of TGA Proteins in Brassica napus

Based on the TGA gene ID and protein sequences of B. napus, OrthoVenn3 (https://orthovenn3.bioinfotoolkits.net/ (accessed on 16 May 2024)) was used to compare the homologous relationship between TGA genes in B. napus and those in A. thaliana. The STRING website (https://cn.string-db.org/ (accessed on 16 May 2024)) was used to establish a protein-protein interaction network, selecting a confidence score of 0.4 for the interactome and keeping the remaining parameters unchanged at their default values. The maximum number of interactors to be displayed was limited to 20. Finally, Cytoscape 3.10.2 was utilized to visualize the protein-protein interaction network.
The secondary structure prediction of BnTGA proteins was completed using the SOPMA application (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html (accessed on 16 May 2024)). To estimate and visualize the tertiary structures of the proteins, the Swiss-Model software (https://swissmodel.expasy.org/interactive (accessed on 17 May 2024)) was utilized [67,68].

4.5. Phylogenetic Analysis and Collinearity Analysis of TGA Gene Family in Brassica napus

To further understand the evolutionary relationships between BnTGA proteins and proteins from other plant species, including Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, and Camelina sativa, we constructed a phylogenetic tree. The TGA gene family in Arabidopsis lyrata, Arabidopsis halleri, and Camelina sativa, were identified according to the same method as B. napus. In addition, the TGA protein from Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, and Camelina sativa were obtained using the same methodology as in the case of B. napus. All TGA protein sequences in five species were multi-aligned through MUSCLE in MEGA (Version 11.0.13) [69]. Based on the JTT+G model, the phylogenetic tree was assembled using MEGA11 with default parameters, employing the maximum likelihood (ML) method with 1000 bootstrap replicates. The beautification of the evolutionary tree built using five species was completed by the Evolview online website (https://evolgenius.info//evolview-v2/#login (accessed on 26 April 2023)) [70]. The gene collinearity analysis of the BnTGA genes was carried out by MCScanX [71] to detect TGA gene replication events in B. napus with default parameters. In addition, six cruciferous plants (Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, Camelina sativa, Brassica juncea, and Brassica oleracea) were selected to construct a syntenic analysis map with B. napus.

4.6. Cis-Element Analysis in Promoters of TGA Gene Family in Brassica napus

The 2,000 bp upstream DNA sequences preceding the start codon (ATG) of the BnTGA genes were retrieved using TBtools V2.096. These sequences were then submitted to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 4 June 2023)) to identify and retrieve cis-regulatory elements (CREs) within the promoter region of BnTGA genes [66]. TBtools V2.096 was employed for visualizing the distribution data of the cis-elements associated with BnTGA genes.

4.7. Expression Profile Analyses of TGA Gene Family in Brassica napus

To investigate the expression of the BnTGA genes under drought, heat stresses, and under infection of Sclerotinia sclerotiorum, RNA-Seq data were acquired from the NCBI database (GSE156029 and GSE81545). GSE81545 was used to study the expression of BnTGAs after Sclerotinia sclerotiorum infection [72]. GSE156029 was used to study the expression of BnTGAs under drought and heat stress [73]. The generation of Advanced Heatmap Plots was carried out using the tools available on OmicStudio tools at https://www.omicstudio.cn (accessed on 28 June 2023).

4.8. RNA Extraction and Quantitative qRT-PCR Validation

Total RNA from B. napus was extracted with a FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). RNA was used to synthesize cDNA through the HiScript Ⅲ First Strand cDNA Synthesis kit (+gDNA wiper) (Vazyme, Nanjing, China). The process referred to the manufacturer’s instruction and the reverse transcription product was diluted as a template. The qRT-PCR was conducted using a ChamQ Universal SYBR® qPCR Master Mix (Vazyme, Q311, Nanjing, China), with three replicates per sample. Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 28 July 2023)) was used to design primers, and Actin gene was used as the internal reference gene. The results of the relative expression for target genes were calculated according to the 2−ΔΔCt method [74]. The quantitative data were compiled using Excel 2016 software, while statistical analysis and visualization were conducted using GraphPad Prism8.

5. Conclusions

In this study, a total of 39 BnTGA genes were discovered and categorized into five subgroups after an extensive analysis of the B. napus genome. By analyzing the gene structure, conserved motif, conserved domain, phylogenetic relationship, cis-element, and 3D structures of the BnTGA gene, the biological characteristics of BnTGA genes were comprehensively evaluated. The expression differences of 39 BnTGAs were assessed in response to Sclerotinia sclerotiorum infection, leading to the identification of the BnTGA4/6/11 genes as potential key players in the defense against Sclerotinia sclerotiorum. The qRT-PCR results demonstrated significant changes in the expression levels of three genes under drought stress: BnTGA14 and BnTGA17 exhibited a significant increase in expression, while BnTGA23 displayed a considerable decrease. Meanwhile, by subjecting different varieties of B. napus to drought treatment, it can be inferred from the results that the superior traits and stress tolerance of parental rapeseed can be transmitted to the offspring through hybridization. These findings lay a solid foundation for future investigations exploring the regulatory roles of the TGA gene family in the growth and disease resistance mechanisms of B. napus. Furthermore, they offer valuable guidance for the potential application of the BnTGA gene family in resistance breeding programs for B. napus.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25126355/s1.

Author Contributions

Conceptualization, Y.D., L.X. and M.X.; methodology, Y.D., Z.X. and L.X.; formal analysis, Y.D., Z.X. and M.X.; software, Y.D., Z.X., Y.W. and X.Z.; validation, L.X., Z.X., Y.W. and Z.Z.; writing—original draft preparation, Y.D. and Z.X.; writing—review and editing, Y.D., H.L., Y.W. and X.Z.; visualization, Y.D., H.L., X.Z. and Z.Z.; supervision, L.X. and M.X.; project administration, L.X., Y.W. and M.X.; funding acquisition, L.X. and M.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Agriculture and Rural Affairs Department of Zhejiang Province (2023SNJF005), Science and Technology Department of Zhejiang Province (2022C02034), Science and Technology Program of Sichuan Province (2022NZZJ0018,2021YFYZ0018), Sichuan rapeseed innovation team (sccxtd), Fundamental Research Funds of Zhejiang Sci-Tech University (23042097-Y), and China Scholarship Council (CSC).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available on request from the authors.

Acknowledgments

Thanks are due to college of Life Sciences and Medicine, Zhejiang Sci-Tech University, Institute of Agriculture, The University of Western Australia, Leshan Academy of Agricultural Sciences. We also thank Rui Sun from the Agricultural Experiment Station of Zhejiang University for his assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of TGA genes on Brassica napus chromosome. The chromosome size is expressed by length. The variation in color represents the density of the region, where blue indicates low, yellow indicates medium, and red indicates high density. The blue lines on the chromosome represent the physical location of the BnTGA genes. The scale on the left is displayed as megabases (Mb).
Figure 1. Distribution of TGA genes on Brassica napus chromosome. The chromosome size is expressed by length. The variation in color represents the density of the region, where blue indicates low, yellow indicates medium, and red indicates high density. The blue lines on the chromosome represent the physical location of the BnTGA genes. The scale on the left is displayed as megabases (Mb).
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Figure 2. Phylogenetic relationship, motif, and domain of the TGA genes in Brassica napus and Arabidopsis thaliana. (a) The phylogenetic tree of the TGA protein sequence in B. napus and A. thaliana was constructed by the maximum likelihood (ML) method. The different groups were labeled from I to V. (b) Conserved motifs in BnTGAs. Various colors represented different motifs. (c) Comparison of conserved domain among AtTGAs and BnTGAs.
Figure 2. Phylogenetic relationship, motif, and domain of the TGA genes in Brassica napus and Arabidopsis thaliana. (a) The phylogenetic tree of the TGA protein sequence in B. napus and A. thaliana was constructed by the maximum likelihood (ML) method. The different groups were labeled from I to V. (b) Conserved motifs in BnTGAs. Various colors represented different motifs. (c) Comparison of conserved domain among AtTGAs and BnTGAs.
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Figure 3. Thirty-nine TGA gene structures. The green region stands for the CDS, the yellow region for the UTR, and the gray lines refer to exon regions. The ruler at the bottom indicates the length of the sequences.
Figure 3. Thirty-nine TGA gene structures. The green region stands for the CDS, the yellow region for the UTR, and the gray lines refer to exon regions. The ruler at the bottom indicates the length of the sequences.
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Figure 4. The prediction of the protein interaction network in Arabidopsis thaliana. Red represents TGA proteins, while green represents other proteins.
Figure 4. The prediction of the protein interaction network in Arabidopsis thaliana. Red represents TGA proteins, while green represents other proteins.
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Figure 5. Evolutionary relationship analysis of TGA proteins. Based on the alignment of the TGA domain, TGA amino acid sequences from the Brassica napus (Bn), Arabidopsis thaliana (AT), Arabidopsis lyrata (AIy), Arabidopsis halleri (Arh), and Camelina sativa (Csa) were used to construct a phylogenetic tree, with the maximum likelihood (ML) method and 1000 bootstrap replicates. Light green, light blue, light pink, light coral, and orange represent Group I, Group II, Group III, Group IV, and Group V, respectively. Bootstrap values are symbolized with a circular pattern, with less than or equal to 40 in gray, 41 to 80 in yellow, and 81 to 100 in red.
Figure 5. Evolutionary relationship analysis of TGA proteins. Based on the alignment of the TGA domain, TGA amino acid sequences from the Brassica napus (Bn), Arabidopsis thaliana (AT), Arabidopsis lyrata (AIy), Arabidopsis halleri (Arh), and Camelina sativa (Csa) were used to construct a phylogenetic tree, with the maximum likelihood (ML) method and 1000 bootstrap replicates. Light green, light blue, light pink, light coral, and orange represent Group I, Group II, Group III, Group IV, and Group V, respectively. Bootstrap values are symbolized with a circular pattern, with less than or equal to 40 in gray, 41 to 80 in yellow, and 81 to 100 in red.
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Figure 6. Gene duplication events of BnTGAs and synteny analyses of TGA genes among different plants. (a) Genome-wide gene syntenic analysis of Brassica napus. The gray lines represent the syntenic relationships of each gene in B. napus, while the red lines represent the replication events of the BnTGA genes. (b) Syntenic pairs of TGA genes between B. napus and other species (including Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, Brassica juncea, Brassica oleracea, and Camelina sativa). Gray lines in the background indicate the collinear blocks within B. napus and other plant genomes, while the syntenic TGA gene pairs are linked with red lines.
Figure 6. Gene duplication events of BnTGAs and synteny analyses of TGA genes among different plants. (a) Genome-wide gene syntenic analysis of Brassica napus. The gray lines represent the syntenic relationships of each gene in B. napus, while the red lines represent the replication events of the BnTGA genes. (b) Syntenic pairs of TGA genes between B. napus and other species (including Arabidopsis thaliana, Arabidopsis lyrata, Arabidopsis halleri, Brassica juncea, Brassica oleracea, and Camelina sativa). Gray lines in the background indicate the collinear blocks within B. napus and other plant genomes, while the syntenic TGA gene pairs are linked with red lines.
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Figure 7. (a) Phylogenetic tree of the TGA protein sequence in Brassica napus. (b) Cis-acting element prediction of the BnTGA gene family in B. napus. Different colors represent the different cis-acting elements.
Figure 7. (a) Phylogenetic tree of the TGA protein sequence in Brassica napus. (b) Cis-acting element prediction of the BnTGA gene family in B. napus. Different colors represent the different cis-acting elements.
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Figure 8. Expression analysis of TGA genes in Brassica napus leaves under biotic and abiotic stress. (a) Expression levels of TGA genes in B. napus leaves under drought and heat stress. (b) Expression levels of TGA genes in both susceptible (Westar) and tolerant (ZY821) genotypes of B. napus leaves infected with Sclerotinia sclerotiorum.
Figure 8. Expression analysis of TGA genes in Brassica napus leaves under biotic and abiotic stress. (a) Expression levels of TGA genes in B. napus leaves under drought and heat stress. (b) Expression levels of TGA genes in both susceptible (Westar) and tolerant (ZY821) genotypes of B. napus leaves infected with Sclerotinia sclerotiorum.
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Figure 9. Schematic diagram of Brassica napus cross-breeding in this study.
Figure 9. Schematic diagram of Brassica napus cross-breeding in this study.
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Figure 10. Analysis of BnTGA genes’ expression levels in Brassica napus under drought stress. (a) Relative expression levels of three selected BnTGA genes in eight experimental materials, at different time points, were measured by qRT-PCR in response to drought stress conditions. The expression levels were normalized to that of Actin. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, no significant difference). (b) Summary of qRT-PCR expression of three BnTGA genes treated by drought in different varieties of B. napus.
Figure 10. Analysis of BnTGA genes’ expression levels in Brassica napus under drought stress. (a) Relative expression levels of three selected BnTGA genes in eight experimental materials, at different time points, were measured by qRT-PCR in response to drought stress conditions. The expression levels were normalized to that of Actin. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, no significant difference). (b) Summary of qRT-PCR expression of three BnTGA genes treated by drought in different varieties of B. napus.
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Duan, Y.; Xu, Z.; Liu, H.; Wang, Y.; Zou, X.; Zhang, Z.; Xu, L.; Xu, M. Genome-Wide Identification of the TGA Gene Family and Expression Analysis under Drought Stress in Brassica napus L. Int. J. Mol. Sci. 2024, 25, 6355. https://doi.org/10.3390/ijms25126355

AMA Style

Duan Y, Xu Z, Liu H, Wang Y, Zou X, Zhang Z, Xu L, Xu M. Genome-Wide Identification of the TGA Gene Family and Expression Analysis under Drought Stress in Brassica napus L. International Journal of Molecular Sciences. 2024; 25(12):6355. https://doi.org/10.3390/ijms25126355

Chicago/Turabian Style

Duan, Yi, Zishu Xu, Hui Liu, Yanhui Wang, Xudong Zou, Zhi Zhang, Ling Xu, and Mingchao Xu. 2024. "Genome-Wide Identification of the TGA Gene Family and Expression Analysis under Drought Stress in Brassica napus L." International Journal of Molecular Sciences 25, no. 12: 6355. https://doi.org/10.3390/ijms25126355

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