Genome‑wide identifcation and expression analysis of the growth-regulating factors under drought in Brassica juncea

doi:10.21203/rs.3.rs-4128359/v1

Growth-regulating factors (GRFs) are plant-specific transcription factors (TFs) involved in the regulation of plant growth, development, and abiotic stress processes. However, the functions of Brassica juncea (L.) Czern & Coss GRFs remain largely unknown. In this study, 34 BjGRF genes were identified in B. juncea. BjGRF members of the same subfamily were found to share a similar motif composition and gene structure. In total, 663 cis-acting element sites were found in the promoter regions of BjGRF genes, which were related to light response, hormone response, environmental stresses, and plant growth/development. Additionally, 48 pairs of segmental duplication genes were identified during gene duplication events, and no tandemly duplicated genes were identified. qRT-PCR analysis showed that the 34 BjGRF genes were primarily expressed in the roots, followed by the leaves. Furthermore, the 10 BjGRF genes were screened in response to drought stress, and the expression patterns of the genes were relatively consistent, with a maximum expression level at 3 or 24 h. This preliminary study clarifies the response of the BjGRF gene family to drought stress and provides ideas for further analyses of the biological functions of BjGRF genes.

Biological sciences/Biological techniques

Biological sciences/Genetics

Growth-regulating factors (GRFs) are plant-specific transcription factors (TFs) with highly conserved QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) domains at the N-terminal [1]. The QLQ domain is responsible for the SNH domain of GRF-interacting factor (GIF) binding, whereas the WRC domain plays a role in transcriptional regulation by combining with cis-elements of downstream genes, as well as DNA-binding motifs, namely, the zinc finger structure and nuclear localization signal region [1,2]. The C-end contains TQL (Thr, Gln, Leu), FFD (Phe, Phe, Asp), GGPL (Gly, Gly, Pro, Leu), and other structural domains [3,4]. Unlike the conserved N-terminal amino acid (aa) residues, the C-terminus is composed of variable aa residues and has a transcriptional activation function [5].

GRF TFs are subject to post-transcriptional regulation by miR396 and inhibit the expression of GRFs by degrading their encoded mRNAs or inhibiting their translation through complementary pairing with mRNAs [6,7]. Therefore, GRF-miR396 is involved in the regulation of plant growth, development, and abiotic stress tolerance through regulatory networks [8]. The expression of Sp-miR396a-5p in tomato is upregulated under salt and drought stress. After the heterologous expression of Sp-miR396a-5p, the expression of NtGRF1, NtGRF3, NtGRF7, and NtGRF8 in transgenic plants is downregulated [9]. The detection of physiological and biochemical indicators showed that the osmotic regulation ability of transgenic plants is enhanced but that the amount of reactive oxygen species decreases, indicating that the ability to resist drought, salt, and low-temperature stress is enhanced [9]. Additionally, overexpression of the AtGRF7 gene enhances resistance to osmotic and drought stress [8]. The atgrf7 mutant is more resistant to salt and drought stress than wild-type plants [10]. Sakuma et al. found that AtGRF7 can improve tolerance to salt and drought stress by inhibiting the expression of the dehydration response element-binding protein, DREB2A [11]. These results indicate that GRF TFs play a regulatory role in plant drought resistance responses.

The seasonal distribution of precipitation in Guizhou, China, is uneven, with more precipitation occurring in spring and summer and less in autumn and winter. During autumn and winter, rapeseed seedlings are prone to drought stress, which significantly affects production. Brassica juncea is a characteristic oilseed crop cultivated in Guizhou, with strong drought resistance. It can be planted in mountainous environments and is a rich resource for drought resistance genes. Therefore, exploring drought-resistant genes in B. juncea is important for the improvement of its varieties and germplasm resource innovation. The GRF family plays important roles in plant growth, development, and drought stress response. At present, GRF genes have been studied in Arabidopsis [2], rice (Oryza sativa) [12], Brassica napus [13], cotton (Gossypium hirsutum) [14], wheat (Triticum aestivum) [15], foxtail millet (Setaria italica) [16], and Brassica rapa [17]; however, no GRF gene has been reported in B. juncea. Here, we identified GRF family genes of B. juncea at the whole-genome level and comprehensively analyzed the physical and chemical characteristics, evolutionary relationships, homology, conserved motifs, gene structure, gene duplications, cis-elements, and expression patterns of BjGRF genes under drought stress at the seedling stage (four-leaf stage) to provide a scientific basis for further study of the potential function of BjGRF genes in the drought response and provide candidate genes for the drought-tolerant breeding of B. juncea.

Acquisition of BjGRF family members and prediction of physical and chemical properties

Thirty-four BjGRF genes in the B. juncea genome were identified using two HMMER searches, and all contained QLQ and WRC domains. BjGRF01–BjGRF34 were named based on their position on chromosomes. The physicochemical properties of the family revealed that the length of aa varied significantly, ranging from 261 aa (BjGRF19) to 905 aa (BjGRF28). The isoelectric point ranged from 6.19 (BjGRF02) to 9.35 (BjGRF03), with an average value of 8.33, and 88.24% of BjGRF was basic protein. The predicted molecular weight ranged from 29.82 kDa (BjGRF19) to 102.90 kDa (BjGRF28), and the protein instability indices of BjGRF proteins were between 51.13 (BjGRF08) and 78.24 (BjGRF19), both of which were greater than 40, indicating that these proteins are unstable. The fatty acid index ranged from 43.65 (BjGRF01) to 78.78 (BjGRF22), the mean value of hydrophilicity (GRAVY) was between –1.07 (BjGRF31) and –0.45 (BjGRF22), and the GRAVY of all hydrophilic BjGRF proteins was negative, which may be attributed to the absence of hydrophobic residues. Subcellular localization prediction showed that 31 encoded BjGRF proteins were located in the nucleus, BjGRF04 was localized to the peroxisome, BjGRF25 was located to the cytoplasm, and BjGRF28 was located in the chloroplast (Table 1), indicating that BjGRF plays a major regulatory role as a transcription factor in the nucleus.

Table 1. Physicochemical properties of GRF family members

Gene	Gene ID	Number of amino acids (aa)	Molecular weight (kDa)	pI	Instability index (II)	Aliphatic index (AI)	GRAVY	Subcellular localization
BjGRF01	BjuA01g06510S	400	45.26	7.77	62.53	43.65	-1.03	Nucleus
BjGRF02	BjuA01g31170S	402	44.39	6.19	61.70	48.61	-0.77	Nucleus
BjGRF03	BjuA01g42700S	451	49.33	9.35	60.60	62.93	-0.62	Nucleus
BjGRF04	BjuA02g29680S	321	34.98	8.49	52.48	65.26	-0.61	Peroxisome
BjGRF05	BjuA03g03410S	443	48.31	9.30	60.55	59.01	-0.65	Nucleus
BjGRF06	BjuA03g11330S	419	45.74	6.68	53.08	53.82	-0.65	Nucleus
BjGRF07	BjuA03g25350S	369	41.98	8.63	60.48	47.07	-10.00	Nucleus
BjGRF08	BjuA03g34750S	518	55.46	9.02	51.13	57.86	-0.59	Nucleus
BjGRF09	BjuA03g41020S	368	40.37	8.86	76.23	52.55	-0.68	Nucleus
BjGRF10	BjuA04g05650S	535	60.65	8.43	59.53	72.71	-0.61	Nucleus
BjGRF11	BjuA04g23150S	390	42.81	7.75	61.80	49.90	-0.76	Nucleus
BjGRF12	BjuA04g28180S	437	49.13	9.34	59.95	61.28	-0.82	Nucleus
BjGRF13	BjuA05g07930S	388	42.73	8.25	73.02	54.10	-0.75	Nucleus
BjGRF14	BjuA05g30670S	387	43.72	8.22	60.87	46.38	-1.02	Nucleus
BjGRF15	BjuA07g05380S	400	45.82	8.92	62.88	71.90	-0.59	Nucleus
BjGRF16	BjuA07g18060S	527	59.10	8.98	55.86	71.40	-0.57	Nucleus
BjGRF17	BjuA09g20250S	382	42.68	6.70	70.59	49.84	-0.87	Nucleus
BjGRF18	BjuB01g08310S	423	48.06	8.57	65.26	50.05	-0.93	Nucleus
BjGRF19	BjuB01g22600S	261	29.82	8.76	78.24	60.11	-0.70	Nucleus
BjGRF20	BjuB01g43920S	380	41.91	7.31	65.49	50.42	-0.74	Nucleus
BjGRF21	BjuB01g49280S	427	47.98	9.05	56.47	64.78	-0.80	Nucleus
BjGRF22	BjuB02g26640S	386	43.49	9.04	59.60	78.78	-0.45	Nucleus
BjGRF23	BjuB02g69190S	427	46.75	6.79	57.58	50.52	-0.69	Nucleus
BjGRF24	BjuB02g75260S	474	51.63	8.95	51.90	59.87	-0.68	Nucleus
BjGRF25	BjuB05g34880S	365	40.43	8.01	57.63	63.01	-0.57	Cytoplasm
BjGRF26	BjuB05g51630S	418	46.06	6.56	58.16	47.66	-0.79	Nucleus
BjGRF27	BjuB05g61460S	476	52.21	9.23	62.49	63.91	-0.64	Nucleus
BjGRF28	BjuB06g00370S	905	102.91	9.18	52.05	74.53	-0.47	Chloroplast
BjGRF29	BjuB06g09050S	532	59.89	8.71	54.74	72.20	-0.56	Nucleus
BjGRF30	BjuB06g50190S	380	41.81	8.77	73.00	51.66	-0.73	Nucleus
BjGRF31	BjuB07g45940S	379	43.12	8.69	65.14	45.04	-1.07	Nucleus
BjGRF32	BjuB08g07310S	380	42.56	7.33	71.93	50.87	-0.83	Nucleus
BjGRF33	BjuB08g35300S	517	55.21	9.15	49.68	57.21	-0.61	Nucleus
BjGRF34	BjuB08g43680S	374	41.37	8.21	72.14	51.71	-0.74	Nucleus

Phylogenetic tree of GRF protein

Phylogenetic analysis of GRF families of different species is helpful for exploring gene functions. Therefore, the full-length aa sequences of 35 B. napus, 16 B. rapa, and nine Arabidopsis GRFs were downloaded to construct a NJ phylogenetic tree based on the 34 identified BjGRF genes (Fig. 1). Ninety-four GRF TFs were clustered into four subfamilies (groups A–D), and 34 BjGRF family members were randomly distributed into four subfamilies. There were nine members in Group A, seven in Group B, six in Group C, and twelve in Group D. Additionally, the GRF family proteins in different plants were highly conserved, and they may have similar or identical gene functions.

Figure 1. Neighbor-joining (NJ) phylogenetic tree of GRF proteins. Circles represent GRF TFs in B. napus;squares represent GRF TFs in B. rapa; stars represent GRF TFs in B. juncea GRF TFs; and triangles represent GRF TFs in Arabidopsis thaliana.

Homology analysis between BjGRF gene family and the selected species

To study the evolutionary relationship of BjGRF family genes among species, the interspecific homology of B. juncea, Arabidopsis, and B. rapa GRF TFs was analyzed. As shown in Fig. 2, GRF genes were homologous between B. juncea, Arabidopsis (24), and B. rapa (27), indicating that the GRF gene family of B. juncea and B. rapa had a closer homologous evolutionary relationship and may have similar functions.

Figure 2. Homology between the BjGRF gene family and GRFs of Arabidopsis and B. juncea. Gray lines in the background indicate homology blocks for B. juncea and genomes of selected species, and the other color lines highlight the homology of BjGRF gene pairs.

Conserved motif and gene structure of GRFs

Conserved motifs help clarify the biological functions of GRF TFs. In this study, we found 15 conserved motifs in the 94 GRF of B. juncea, B. napus, B. rapa,and Arabidopsis (Figs. 3a and 3b), of which motif 1 constituted the WRC domain, motif 2 constituted the QLQ domain, motif 3 constituted the GGPL domain, and motif 5 constituted the FFD domain. WRC and QLQ were present in all GRF gene families (except Bra021521), indicating that the GRF homologous genes among different species of the cruciferous family were highly conserved. Additionally, all members of the Group D subfamily contained FFD. The GRF proteins of the same subfamily contained similar conserved motif types and sequences, and the protein structure of the same subfamily members was relatively conserved. In particular, BjGRF10, BjGRF16, BjGRF29, Bra019640, and Bnacnng50230D contained similar conserved motif types, which may have similar features.

The composition and number of gene introns/exons are important for studying gene function. This study analyzed the gene structure of the B. juncea, B. napus, B. rapa, and Arabidopsis GRF family (Fig. 3c) and found significant differences in the number of exons among different GRF genes, with BjGRF having 2–12 exons, B. napus GRF containing 2–10 exons, B. rapa GRF having 3–10 exons, and Arabidopsis GRF containing 3–6 exons. In 64.7% of BjGRF genes, 38.57% (17) of B. napus GRF genes, 62.5% (10) of B. rapa GRF genes, and 55.56% (5) of B. rapa GRF genes, there were four exons. BjGRF28, Bnacnng50230D, Bra019640,and AtGRF8 had the highest number of exons in B. juncea, B. napus, B. rapa,and Arabidopsis, containing 12, 10, 10, and 6 exons, respectively. Furthermore, a higher number of exons was found in BjGRF10, BjGRF16,and BjGRF29 genes, indicating that the alternatively spliced forms were more complex. The gene structure of some BjGRFs was more complex than that of GRF genes of B. napus, B. rapa and Arabidopsis.

Figure 3. Phylogenetic relationship, conserved motif, and gene structure of GRFs. (a) Phylogenetic tree of the GRF family in B. juncea, B. napus, B. rapa,and Arabidopsis. (b) Conserved motif of the GRF family in B. juncea, B. napus, B. rapa,and Arabidopsis, with different colors of rods representing different motifs. (c) Structural analysis of the GRF family gene in B. juncea, B. napus, B. rapa,and Arabidopsis, with yellow bars representing UTRs, green bars representing exons, and black lines representing introns.

Chromosomal localization and duplication of BjGRF genes

To clarify the expansion characteristics of BjGRFs, gene duplication in the BjGRF family was analyzed (Fig. 4). In total, 48 pairs of duplicated genes were detected in the BjGRF family, all of which were segmental duplications and randomly mapped to 14 chromosomes (except A06, A08, B03, and B04). Additionally, no tandem duplication events were detected, suggesting that segmental duplications were the main driving force for the expansion and evolution of BjGRF family members and played a major role in the evolution of BjGRF genes.

Figure 4. Analysis of duplicated genes of the GRF family of B. juncea. Gray lines in the background represent synchronous blocks within the B. juncea genome; red lines indicate segmental duplication BjGRF gene pairs.

Prediction of cis-acting elements of BjGRF genes

Using the PlantCARE website to predict and analyze the cis-acting elements in the 1.5 kb region upstream of the start codon of BjGRF genes was conducive to exploring the potential biological functions and regulatory mechanisms of BjGRF genes (Fig. 5). In total, 663 cis-acting elements were found in BjGRF genes, which were divided into four categories (Fig. 5b, Supplementary Table S1): light-response elements (291 sites), hormone signaling (192 sites), growth and development (36 sites), and environmental stress responses (144 sites). Light-response elements were present in all BjGRF genes, indicating that BjGRF participated in photo-response regulation. Among the environmental stress-related elements, anoxic induction (GC motif and ARE), drought induction (MBS), and low-temperature response elements were found, suggesting that the BjGRF family played a role in the response to stress. Additionally, five hormone-related elements, namely, abscisic acid (ABA) cis-acting element (ABRE), methyl jasmonate (MeJA) cis-acting element (TGACG-motif and CGTCA-motif), gibberellin (GA) response element (GARE-motif, P-box and TATC-box), auxin-response element (TGA-box and TGA-element), and salicylic acid (SA) response element (SARE and TCA-element), were found in most BjGRF genes. Circadian elements, meristem-related elements (CAT-box), endosperm expression (GCN4_motif and AACA_motif), and cell cycle regulation (MSA-like) were development-related elements found in BjGRF genes. In summary, the BjGRF genes contain a large number of elements related to hormone responses and environmental stress, suggesting that they respond to adverse environmental effects by regulating different hormone pathways and responding to stress.

Figure 5. Prediction analysis of the BjGRF genes promoter. (a) Distribution of BjGRF genes in the 1.5 kb promoter region. (b) Number of light-responsive cis-elements, hormone-responsive cis-elements, stress-responsive cis-elements, and plant growth–related cis-elements in BjGRF genes. (c) Number of different hormone (MeJA, GA, ABA, auxin, and SA)-responsive cis-elements in BjGRF genes. (d) Number of environmental stress (anaerobic, drought, defense, stress, wound, and low temperature)-related cis-elements upstream of BjGRF genes. (e) Number of plant growth–related cis-elements in BjGRF genes.

Expression of BjGRF genesin different tissue parts at the seedling stage

To investigate the function of BjGRF genes at the seedling stage of B. juncea, we used qRT-PCR to analyze the expression of 34 BjGRF genes in different tissues (Fig. 6, Supplementary Table S2). There were differences in the expression of BjGRF genes in different tissues, with 19 BjGRF genes having the highest expression levels in the roots and 11 BjGRF genes having the highest expression levels in the leaves. BjGRF10, BjGRF12, BjGRF15,and BjGRF22 showed the highest expression levels in the stems.

Figure 6. Expression of BjGRF genes in different tissue parts (root, stem, and leaf) at the B. juncea seedling stage. The expression data of 34 BjGRF genes in different tissues were stored in Supplementary Table S2.Significant differences between the data are indicated by lowercase letters.

Expression of BjGRF genes under simulated drought stress

Ten BjGRF genes were selected for qRT-PCR based on the expression of BjGRF genes in different tissues and their cis-element related to drought stress (Fig. 7, Supplementary Table S3). BjGRF03, BjGRF09, BjGRF16, and BjGRF32 genes had similar expression patterns, presenting an increase-decrease-increase trend, and the expression reached a peak at 3 h of drought stress. Moreover, the expression level of BjGRF03 was the highest among the 10 BjGRF genes at 3 h of drought stress, which was 17.56 times higher than that of the control (0 h). However, the expression of BjGRF06, BjGRF23, BjGRF26, BjGRF29, and BjGRF34 was the highest at 24 h. In particular, the expression trends of BjGRF26 and BjGRF34 were similar, suggesting that they had similar or identical functions. These studies revealed that BjGRF genes responded to drought treatment and that they may participate in the response to drought stress.

Figure 7. Expression of BjGRF genes under drought stress at the four-leaf stage. The qRT-PCR data was stored in Supplementary Table S3. Significant differences between the data are indicated by lowercase letters.

In recent years, owing to the continuous changes in global climate, many studies have focused on understanding how crops can resist drought stress and improve their resistance mechanisms [18]. Plants undergo changes in their morphological structure, gene expression, and metabolic processes after drought, which may even lead to the termination of photosynthesis and disruption of metabolism, thereby affecting crop yield and quality [19-21]. When plants perceive drought signals, they produce Ca²⁺, phosphatidylinositol, and other secondary messenger substances while increasing the concentration of intracellular calcium ions and initiating the regulatory network of the protein phosphorylation pathway [22, 23]. Finally, the target protein directly participates in cell protection or regulates the expression of related stress genes through TFs to improve stress resistance in plants [24, 25]. Therefore, TFs play an important role in drought stress response. According to the sequence and DNA-binding characteristics of TFs that respond to drought stress, they can be divided into different families, such as the GRF, ERF, MYB, and WRKY families [26].

The GRF gene family is a group of plant-specific TFs that play important roles in various aspects of plant growth, development, signal transduction, and plant defense responses [27]. Since the identification of the first GRF gene in O. sativa [28], an increasing number of GRF genes has been identified in multiple species, and they have been shown to affect plant growth, development, and stress responses [8, 29-32]. With the publication of the genome sequence of B. juncea, the identification of the BjGRF gene family has become possible [33]. In this study, 34 BjGRF genes were identified in the whole genome of B. juncea, and they were named BjGRF01–BjGRF34 based on their positions on the chromosomes, all of which contained highly conserved QLQ and WRC domains. Analysis of the physical and chemical properties showed that the number of aa and molecular weight of BjGRF (except BjGRF28) proteins were not significantly different, indicating that members of the BjGRF family might have similar functions. Gene structure analysis showed that 64.7% of BjGRF genes contained four exons, indicating that the structure of BjGRF genes was relatively evolutionarily conserved; however, the number of exons in BjGRF10, BjGRF16, BjGRP28, and BjGRF29 was large, and other BjGRF genes had fewer introns. Research suggests that the addition or deletion of exons or introns may lead to differences in gene structure and function, resulting in the generation of new genes [34-36]. Therefore, we speculated that the intron of BjGRF was lost during evolution, which may have altered gene function. In line with previous studies, we also found that the number of introns is related to gene expression, and when the number of introns in genes is large, it can quickly respond to various adverse factors.

Gene duplications are the main driving force of genome and genetic evolution [37]. Related studies have shown that gene duplication not only increases the number of GRF genes but is also a way to produce new genes, which supports plant adaptation to various adverse environments [38]. In this study, a total of 48 pairs of duplicated genes were detected, all of which were segmental duplications, indicating that segmental duplication was the main mechanism for increasing the number of genes in the family. Segmental duplication has been reported to effectively promote the amplification of GRF gene family members in Arabidopsis and strawberries, and no tandem duplication has been found in this gene family in either species [27, 39]. The results of the present study are consistent with those of previous studies on Arabidopsis and strawberry families, suggesting that the GRF family can increase the number of genes and produce new genes through segmental duplication in different plants.

In this study, the prediction of cis-acting elements revealed that 19 BjGRF genes in this family contained 1–2 drought response elements (MBS) and ABRE. ABA is a key hormone involved in plant responses to drought stress. Under drought conditions, plants perceive external stimuli, and the transcription and protein levels of ABA synthase are upregulated upon receiving drought signals, leading to an increase in endogenous ABA content [40]. Additionally, the responses to drought stress and resistance to drought requiring gene regulation can be divided into two types according to the mode of action of drought stress genes: functional genes that have protective effects on plants and directly participate in improving the drought resistance ability of plants and regulatory genes that regulate signal transduction and gene expression. TFs, as regulatory genes, participate in the response to drought stress, whereas GRFs, as plant-specific TFs, play a role in plant growth and abiotic stress by coordinating the stress response and defense signals [4, 32, 41]. Regulatory functions have also been reported in response to drought stress. Du et al. found that the expression levels of MtGRF2 and MtGRF8 in alfalfa were higher under drought stress [42]. In wheat, the expression of the TaGRF21 gene is significantly upregulated under drought stress [15]. In this study, qRT-PCR showed that BjGRF had a similar expression trend after drought treatment, especially BjGRF03, BjGRF09, BjGRF16, and BjGRF32, which reached their maximum expression after 3 h of stress, indicating that these four genes are responsive to drought stress and may play regulatory roles in drought stress. The expression level of BjGRF03 was the highest among the 10 BjGRFs genes after 3 h, which was 17.56 times higher than that of the control (0 h), indicating that it had a strong response to drought stress and could be used as a candidate gene related to drought resistance. In conclusion, this study provides new genetic resources for further exploration of the role of BjGRF gene family members in drought stress tolerance, as well as a theoretical basis for breeding drought-tolerant rapeseed varieties.

In this study, 34 BjGRF genes were found in B. juncea, which were divided into four subfamilies exhibiting similar conserved motifs and gene structures. Furthermore, 48 pairs of segmental duplications were found in B. juncea via collinearity analysis. The BjGRF promoter region contains cis-acting elements related to light response, hormone response, environmental stress response, and growth/development. We also detected the expression of 34 BjGRF genes in the seedling stage (roots, stems, and leaves) of B. juncea, as well as the expression patterns of 10 BjGRF genes under drought conditions. We found that the expression patterns of BjGRF genes were similar under drought stress and that they may be involved in the regulation of drought stress. In general, our study lays a biological foundation for the future discovery of the functions of BjGRF genes in cruciferous plants.

Materials and treatments

B. juncea seeds used in this experiment were provided by the Oil Research Institute of Guizhou Province, Guizhou Academy of Agricultural Sciences. Whole seeds were selected and planted in soil (substrate:soil = 3:1) until the four-leaf stage, after which the roots, stems, and leaves were collected. Plants were subjected to a drought simulation treatment with 20% PEG 6000, and the leaves were collected at 0, 3, 6, 12, and 24 h. All plant samples were immediately frozen in liquid nitrogen and then stored in a deep freezer at –80 ℃ for the next experiment.

Identification of BjGRF family genes

To identify GRF genes in the whole genome of B. juncea, the hidden Markov model files of WRC (PF08879) and QLQ (PF08880) domains were downloaded from the Pfam database (http://pfam.xfam.org/) [43]. All BjGRF proteins in the B. juncea protein database were searched using HMMER software (version 3.0; http://hmmer.org/) with the E-value of < e^-5. A specific hidden Markov model of B. juncea was constructed and searched again in the B. juncea protein database, with an E-value threshold of < 10^-3. The protein sequences of candidate genes were submitted to the Pfam and NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/) databases to verify the WRC and QLQ domains and screen reliable BjGRF candidate genes. The physicochemical properties of proteins, including the number of aa, molecular weight (kDa), theoretical isoelectric point, instability index, aliphatic index, and mean value of hydrophilicity (GRAVY), were predicted using ProtParam (https://web.expasy.org/protparam/) [44]. The subcellular localization was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/).

Phylogenetic and homology analysis of GRF family members

Phylogenetic relationships were analyzed using full-length aa sequences of GRF TFs in Arabidopsis [13], B. rapa[17], B. napus [13], and B. juncea. A phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0, with 1,000 bootstrap replications [45], and visualized using the online software Evolview (https://evolgenius.info/evolview-v2/) [46]. The genome annotation file (GFF3) and CDS sequences of Arabidopsis and B. rapa were downloaded from the Ensembl Plants database, and the homology of the GRF gene between BjGRFand Arabidopsis and B. rapa was determined using MCScanX software [47].

Conserved motif and gene structure of BjGRFs

The CDS and DNA sequences of BjGRF genes were submitted to the Gene Structure Display Server (http://gsds.gao-lab.org/) website to determine the gene structure [48]. The conserved motif of BjGRF protein was predicted using the online program MEME (https://meme-suite.org/meme/) with the following parameters: maximum number of motifs 20; motif width, between 6 and 100 aa. The conserved motif was visualized using TBtools software [49].

Prediction of cis-acting elements ofthe BjGRF gene promoter and gene duplications

The 1.5 kb sequence upstream of the BjGRF gene start code was extracted using Perl language and submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify the cis-acting elements in BjGRF genes [50]. Segmental duplications and tandem repeat genes of BjGRF family members were obtained using MCScanX software (http://chibba.pgml.uga. edu/mcscan2/) [47], and segmental duplications and tandem repeat genes between BjGRF genes were analyzed using Circos software [51].

Expression pattern analysis

A SteadyPure Plant RNA Extraction Kit (Accurate biotechnology, China) was used to extract total RNA from the roots, stems, and leaves of B. juncea seedlings. RNA was also extracted from the leaves after PEG 6000 treatment, and the concentration and quality of RNA were determined using a micro-ultraviolet spectrophotometer. The cDNAs were synthesized using an EvoM-MLV reverse transcription premix kit (Accurate biotechnology, China) and stored at –20 °C. The ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, China) was used for qRT-PCR, with BjUBQ9 as the internal reference gene, according to the manufacturer’s protocol. The procedure of qRT-PCR was as follows: 95 °C for 5 min at the pre-denaturation stage; 40 cycles of 95 °C for 15 s and 60 °C for 30 s at the PCR stage; and 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s at the melt curve stage, with three repeats for each treatment. The 2^−ΔΔCt [52] method was used to calculate the relative expression of genes. Data collation, analysis, and visualization were performed using Excel 2010, and one-way analysis of variance was performed using SPSS 22.0 software. The LSD method was used for multiple ratios (P < 0.05). The primers used for qRT-PCR are listed in Supplementary Table S4.

Acknowledgements

We thank for lab members for their assistance in this study. We would like to thank Editage (www.editage.cn) for English language editing the English text of a draft of this manuscript.

Author contributions

Y.Z. conceived and designed the study, performed the experiments and wrote the manuscript. F.L., L.W. and B.Y. helped with bioinformatics analysis and created the figures. H.J., R.T. and S.L. helped revamp the manuscript. H.X. and C.Z. guided the experiments and revised the manuscript. All authors have read and approved the final version of this manuscript.

Competing interests

The authors declare no conflict of interest.

Data Availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Ethics statement

The collection of specimens conformed to the requirement of international ethics, which did not incur any damage to the environment and the species itself. The process and purpose of this experimental research were in line with the rules and regulations of our institute. There are no ethical issues or specific permissions are required.

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No competing interests reported.

Genome‑wide identifcation and expression analysis of the growth-regulating factors under drought in Brassica juncea

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Abstract

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Discussion

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