Genome-wide analysis of BrbHLH gene family and functional identification of the involvement of BcbHLH57 in abiotic stress in wucai (Brassica campestris L.)

Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a subspecies of Brassica campestris, is a kind of cold-tolerant vegetable widely cultivated in the Yangtze-Huai River Basin of China. To breed new varieties of resistance to stress, it is necessary to study the tolerance of Resistance to different stresses. Transcriptomic data showed that exogenous application of 2, 4-epbrassinolide (EBR) significantly increased the bHLH genes expression in wucai at low temperature, and bHLH57 was up regulated significantly. BHLH (Basic helix-loop-helix) TFs (transcription factor) is a superfamily of TFs with complex functions and related to various plant life activities. In this study, a total of 239 bHLH genes were identified, and their amino acid physicochemical properties, chromosome location, gene structure, phylogeny and cis-acting elements were analyzed by bioinformatics. Phylogenetic analysis showed that bHLH gene families in Brassica rapa and Arabidopsis were divided into six groups, and the distribution of bHLH genes in Brassica rapa was uneven. The BrbHLHs were irregularly mapped in the cultivated B. rapa genome. The maximum number (40) of BrbHLH genes were mapped on Chr09. In addition, there were tandem repeats in some genes. The collinearity analysis showed that 152 AtbHLH genes and 239 BrbHLH protein genes formed 296 collinearity pairs, and the Ka/Ks ratio ofall duplicated BrbHLH gene pairs had a Ka/Ks ratio of < < 1, indicating that the bHLH family genes may have undergone strong purification and selection during the evolution process. Cis-acting elements analysis showed that the promoter region of bHLH family genes in Brassica rapa had more responsive elements related to light, hormone and abiotic stress. The BcbHLH57 overexpressing Arabidopsis thaliana lines were subjected to different stress treatments, and the seed germination rate, root growth, survival rate and various plant physiological indexes were determined. The results showed that the transgenic lines were more tolerant to salt, heat and drought stress. In this study, the response of BcbHLH57 gene to salt, heat and drought stress was reported for the first time, which provided a basis for further research on the function of BcbHLH57 in abiotic stress. BcbHLH57 overexpressing Arabidopsis thaliana lines showed strong tolerance to salt, heat and drought stress, which provided a basis for further research on the function of BcbHLH57 in abiotic stress.


Introduction
Plants encounter many environmental challenges during their lifespan, such as high or low temperature salinity, and drought, which lead to weaken the growth and development of plants Abdulbaki et al. 2022;Habibpourmehraban et al. 2022;Raza et al. 2022a, b Extended author information available on the last page of the article activities through complex defense mechanisms (Barnabás et al. 2008;Farooq et al. 2009;Zhu 2002). Plant stress response genes are regulated by transcription factors (TFs) through cis-acting elements in their promoter region, which makes plants respond quickly to environmental stresses (Goyal et al. 2018;Spitz and Furlong 2012;Wan et al. 2018;Yang et al. 2004).
Depending on the DNA-binding domain, TFs can be divided into four major categories: basic leucine zippe (bZIP), helix-turn-helix (HTH), zinc finger (ZnF), and basic helix-loop-helix (bHLH) (Lu et al. 2022). The bHLHs are characterized by a DNA-binding basic domain and an HLH domai consisting of two alpha helices separated by a loop of several amino acids (Webb et al. 2011). The bHLH domain contains two functional regions: one is HLH region at the C-terminal, and other is basic amino acid region at the N-terminal (Wang et al. 2015). In recent years, bHLH family genes have been identified and analyzed in different plants, such as orchardgras (Lu et al. 2022), grape (Wang et al. 2018b), Aquilegia (Zhang et al. 2023) The bHLH TF family is a transcription factor superfamily, which is related to the growth and development process, metabolic pathway regulation, hormone information transmission and response to stress of plants (Dong et al. 2021;Pireyre and Burow 2015;Sun et al. 2018). There are relatively few studies on bHLH TFs in Brassica campestris L. and wucai.
Wucai (Brassica campestris L. ssp.) is a variety of Chinese cabbage subspecies . As a biennial leafy vegetable, wucai was characterized by excellent quality, rich nutrition and strong cold resistance. It is widely distributed in the Jianghuai River Basin and is popular among consumers (Yuan et al. 2021a(Yuan et al. , 2021b. Abiotic stresses such as low temperature, high temperature, salinity and drought, threaten the growth, yield and commodity value of wucai (Yuan et al. , 2021cZou et al. 2016).
In this study, BcbHLH57 gene was cloned from "WS-1" and transgenic plants of Arabidopsis thaliana with BcbHLH57 overexpression were obtained. The response mechanism of bHLH57 under stress was verified by comparing the phenotypic, physiological and biochemical differences between the transgenic plants and wild-type plants after different stress treatments. The genomewide of bHLH TF family genes in Brassica rapa was systematically identified and mined by bioinformatics method. The physicochemical properties and conserved moieties of the protein sequences of bHLH family genes in Brassica rapa were analyzed. The chromosome mapping, exon-intron structure and cis-acting elements of the gene promoter region were analyzed, and phylogenetic trees were constructed with amino acid sequences of Arabidopsis thaliana. In this study, the functions of bHLH gene in Brassica rapa and BcbHLH57 gene in wucai were analyzed, which provided a theoretical basis for improving plant stress resistance.

plant material
This experiment was conducted in the breeding basement of Anhui Agricultural University, Hefei, China. The high-generation inbred line "WS-1" used in this study was a stable genetic material of Wucai. The seedlings were planted in a greenhouse at 25 ± 2 °C (day) and 18 ± 1.5 °C (night) with 70-75% relative humidity. When the seedlings grew to 4-5 leaves, the seedlings were treated with room temperature (NT) of 25/18 °C (day/night), low temperature (LT) of 8/3 °C (day/night) and high temperature (HT) of 40/30 °C (day/night). After 3 days of treatment, functional leaves were immediately frozen in liquid nitrogen and maintained at − 80 °C for further physiological and biochemical analyses.
In the experiment on the effects of LT and brassinosteroids (EBR) on plants, seedlings with uniform size at 5-6 leaf stage (45 d after planting) were selected and randomly divided into 2 groups for treatment. One group (n = 50) was sprayed with 0.1 μM EBR solution (the optimal concentration has been screened), and the other group (n = 50) was sprayed with the same amount of ddH 2 O. Two days after treatment, each group was subdivided into two groups, one group (n = 25) was NT control, the other group (n = 25) continued to grow under LT stress.
Arabidopsis (Columbia-0) and transgenic Arabidopsis were placed in a growth chamber of 25/15 °C (18 h day/6 h night) at a light intensity of 300 μmol·m-2·s-1 for 30 days, and the leaves were collected and quick-frozen. Salt stress and osmotic stress treated transgenic T3 and wild-type seeds in 1/2 Murashige and Skoog (MS) liquid medium supplemented with 150 mM NaCl and 250 mmol·L-1 mannitol, respectively. For heat stress, seedlings were placed in 1/2 ms liquid medium for 40 °C simulation. Seedlings under normal conditions were used as controls under all stress treatments.

Genome-wide identification of BrbHLH genes in Brassica rapa
Brassicaceae Database (Brassica rapa Genome Sequencing Project Consortium 2011) (http:// brass icadb. cn) and Arabidopsis thaliana database (http:// arabi dopsis. org) were used to download the whole genome sequence of Brassica rapa and Arabidopsis thaliana, CDS sequence and annotation file of the genome, and establish a database. According to the reported names of bHLH gene family members in Arabidopsis (Bailey et al. 2003), protein sequences of bHLH gene family members in Arabidopsis were extracted for identification of bHLH gene family members in Brassica rapa.

Complete genome identification and physicochemical properties analysis of BrbHLH gene family
BLAST sequence comparison was performed with protein sequences of bHLH gene family members of Arabidopsis thaliana in Brassicaceae Database (Edgar 2010) to screen the BrbHLH gene family members. Pfam, SMART and other databases were used to further identify candidate members. The NCBI-CDD database (https:// www. ncbi. nlm. nih. gov/ Struc ture/ bwrpsb/ bwrpsb. cgi) was used to predict conservative domain structure, finalized BrbHLH family members.
The physical and chemical properties of the BrbHLH gene family were analyzed using the online website Expasy-Protparam Tool (https:// web. expasy. org/ProtParam/). The subcellular localization of BrbHLH gene family members were forecast and analysis by the WoLF PSORT website (Horton et al. 2007) (https:// wolfp sort. hgc. jp/).

Chromosome localization analysis of BrbHLH gene family
TBtools (Chen et al. 2020) and annotated files of Brassica rapa genome were used to locate BrbHLH family genes on corresponding chromosomes and visualize them.

Analysis of exon-intron and conserved motif of BrbHLH gene family
The online website MEME (Bailey et al. 2006) (http:// meme-suite. org/ tools/ meme) was used to predict the conserved motifs of the protein sequences of the BrbHLH gene family, and the number of searched motifs was set to 10. The schematic diagram of exon-intron and conserved motifs of the gene family was drawn using TBtools.

Cis-acting element analysis of BrbHLH gene family
The sequence of 2000 bp upstream of the start codon of family genes was extracted from the genome sequence of Brassica rapa and used as the analysis sequence of promoter region. The online wibesite PlantCARE (Lescot et al. 2002) (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ htmL/) was used to analyze promoter region. The critical cis-acting elements were screened and visualized by TBtools software.

Phylogenetic analysis and collinearity analysis of BrbHLH gene family
The reported protein sequences of bHLH gene family members in Arabidopsis thaliana were combined with those of BrbHLH gene family. ClustalW program (Kumar et al. 2016) in MEGA 7.0 was used for sequence alignment, and neighbor-joining was used to construct phylogenetic tree. Set the Bootstrap verification parameters to 1000 times, and set the Partial deletion parameter to 95.
Interspecific collinearity was analyzed by MCScanX program and mapped by TBtools. KaKs Calculator (Wang et al. 2010) was used to analyze the gene non-synonymous substitution (Ka) and synonymous sub-substitution (Ks) between the two species, and the values of Ks < 0 were discarded to avoid the calculation error.

Cloning and construction of overexpression vector of bHLH57 from wucai and generation of BcbHLH57-overexpressing Arabidopsis
Total RNA Extraction from the functional leaves of "WS-1" was carried out by MiniBEST Plant RNA Extraction Kit (TaKaRa) RNA Extraction Kit. The cDNA was obtained by referring to the instruction of PrimeScriptTM RT Reagent Kit (TaKaRa).
BrbHLH57 cDNA was cloned into the pMD19-T simple vector, and inserted into plasmid pCambia1305-35S-nFLAG-cMYC and infected the inflorescence of Arabidopsis thaliana. The seeds received were cultured in 50% hygromycin B medium, and according to Mendel's separation law, a 3:1 separation ratio of T2 generation plants would occur. Select the plants with good growth and continue to cultivate in the incubator for seed collection. Four weeks later, the copy number of BrbHLH57 of each transgenic line was detected by Quantitative Real-time PCR (qRT-PCR), and three overexpression positive transgenic lines were identified (OE#14, OE#26, OE#27). The total RNA was isolated from OE#14, OE#26, OE#27 using a total RNA kit (Takara Biomedical Technology Co., Beijing, China). Primer software v6.0 (Premier Biosoft International, Palo Alto, CA, USA) was used to design specific gene primers (Table S5). The gene encoding actin was used as the control. RT-qPCR was performed using SYBR GREEN Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China). Relative gene expression levels were calculated using the 2 −ΔΔCT method (Schmittgen et al. 2008).

Physiological index measurement of transgenic plants
Transgenic T3 and wild-type seeds were planted on ½ MS medium of 40 °C (heat stress), 150 mmol·L-1 NaCl (salt stress) and 250 mmol·L-1 mannitol (osmotic stress), respectively. Ordinary ½ MS medium was used as the control group for all stress treatments, and the germination rate within 7 days was calculated. The root conditions of transgenic plants and wildtype plants under different stress treatments were observed and measured after 14 days of growth.
Transgenic T3 and wild-type Arabidopsis thaliana seedlings were cultured for 20 days at high temperature 38/30 °C and room temperature (24/16 °C) for 3 days, and to observe the effect of high temperature stress on survival rate of different lines. Transgenic and wild-type Arabidopsis thaliana seedlings at 20 d of seedling age were sprayed with 150 mmol·L-1 NaCl for 7 d (water spraying as control) to observe the effects of salt stress on survival rate of different lines. Transgenic and wildtype Transgenic and wild-type Arabidopsis thaliana seedlings aged 3 weeks were treated with drought for 14 days, and normal seedlings cultured at the same time were used as controls. The survival rate of different lines after 3 days of drought and rehydration was calculated.
Relative electrical conductivity (REC) was measured according to the method described by Baziramakenga et al. with some modifications (Baziramakenga et al. 1995). The H 2 O 2 and O 2 − content, as well as the total antioxidant capacity, were measured using the Solarbio reagent kit (Cat #BC3595, 1290 and 1315, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Statistical analysis
The mean ± standard error of three biological replicates was selected for the data in this paper. SPSS 22.0 software (SPSS Institute Inc., USA) was used for statistical analysis, and the significance level was set at P = 0.05. Duncan's multiinterval test was used for mean separation test. Graphing was implemented using GraphPad Prism 6.01software (Mitteer et al. 2020) (GraphPad, USA).

Comprehensive characterization of BrbHLH genes in Brassica rapa genome
To identify bHLH family genes in rapeseed, 152 AtbHLH protein sequences were used as queries for a BlastP search against the Brassica rapa genome. As a result, a sum of 239 BrbHLH genes was identified with the BrbHLH domain (Table 1). Henceforward, these genes are labeled as "Brb-HLH1-BrbHLH239." The physical and chemical properties of the BrbHLHs are shown in Table 1. Briefly, the gene length varied from 183 bp (BrbHLH8) to 2,109 bp (BrbHLH230), and the amino acid length varied from 60 (BrbHLH8) to 702 (BrbHLH230) amino acids. The anticipated molecular weights (MW) of the 239 BrbHLH proteins increased from 6.51 kDa (BrbHLH8) to 76.94 kDa (Brb-HLH230), the isoelectric points (PI) extended from 4.59 (BrbHLH185) to 11.86 (BrbHLH216), the instability coefficients of BrbHLH gene family proteins ranged from 26.61 (BrbHLH233) to 81.53(BrbHLH103), and the GRAVY ranged from − 4.44 (BrbHLH82) to − 0.04 (BrbHLH82), indicating that most of the BrbHLHs is hydrophilic. The transformations in MW and PI are primarily due to the elevated content of necessary amino acids and post-translational alterations. The in silico subcellular localization discovered that 210 BrbHLH proteins were situated on the nucleus, 16 AhAPX proteins on chloroplast, four BrbHLH proteins on cytoplasm, three BrbHLH proteins on peroxisome, two BrbHLH proteins on plasma membrane, one BrbHLH protein on cytoskeleton, one BrbHLH protein on golgi apparatus, and one BrbHLH protein on mitochondrion (Table 1).
In this study, a total of 239 BrbHLH genes were discovered in the B. rapa genome (Table 1). These genes were irregularly mapped in the cultivated B. rapa genome. The maximum number (40) of BrbHLH genes were mapped on Chr09, followed by Chr03/Chr05 with 27 genes on each chromosome ( Fig. 1). Only 13 BrbHLH genes were located on chr10 (Fig. 1). In addition, there were tandem repeats in some genes, indicating that there were homologous genes in the BrbHLH gene family, which increased the gene diversity.

Evolutionary analysis of BrbHLH gene family
To unreveal the evolutionary relationship between the Brb-HLHs and AtbHLHs genes, an unrooted phylogenetic tree was built by a multiple sequence alignment of the prophesied bHLH protein sequences from B. rapa and A. thaliana (Fig. 2). Based on the standard classification of Arabidopsis BrbHLH genes, the 391 bHLHs genes from two plant species were distributed into six main classes (Group1-6) ( Fig. 2). Further, Group1 had the largest number of 165 members, followed by Group3 with 92 gene members. Only seven genes of BrbHLH family and nine genes of AtbHLH family were divided into Group 6, suggesting that members of this group may have experienced more gene loss during the evolution process. In a nutshell, bHLHs grouping into the same sub-class may retain corresponding functions.

Collinearity analysis of BrbHLH gene family
Collinearity analysis was carried out to review the evolutionary association of the bHLH genes between B. rapa and A. thaliana ( Fig. 3 and Table S1). The results discovered a strong orthologous of bHLH genes among these two species (Fig. 3). Collinearity analysis showed that 152 AtbHLH genes and 239 BrbHLH genes formed 296 collinearity pairs (Fig. 3). There were 47, 36 and 35 collinear relationships on chromosome A09, A05 and A01 of B. rapa, respectively. There are only 18 collinear pairs on the chromosome A10. The Ka/Ks ratio is considered as a huge diagnostic marker in evaluating the sequence evolution in terms of selection pressures and duplication types (Hurst et al. 2002). Thus, to understand the evolutionary story of the BrbHLHs, the Ka, Ks, and Ka/Ks ratio was revealed (Table S1). The dataset unveiled that all duplicated BrbHLH gene pairs had a Ka/Ks ratio of < < 1 (Table S1) It indicates that the bHLH family genes may have undergone strong purification and selection during evolution, and were generally conserved in structure. It is speculated that their functions and functions are relatively similar.

Exon-intron and conserved motif analysis of BrbHLH family genes
The exon-intron arrangements and conserved motifs of the BrbHLH genes were analyzed to get insights into the advancement of the BrbHLH family genes in Brassica rapa genome (Fig. 4). The conservative motifs of BrbHLH gene family were analyzed by the website MEME (Fig. 4). A total of 10 conserved motifs were predicted. It was found that the distribution of these motifs was more similar among genes that were closely related. Analysis of the exon-intron structure of BrbHLH gene family showed that the number of exons and introns was different among different gene clusters, and the closer the genetic relationship was, the more similar the gene structure was. It is speculated that closely related genes may have similar functions.

Cis-acting element analysis of BrbHLH genes
To better understand the regulatory role of BrbHLH genes toward Brassica rapa growth and development, and tolerance to abiotic stress and phytohormones treatment, cis-regulatory elements in the promoter of BrbHLH were explored (Fig. 5). The complete dataset of cis-elements is presented in Supplementary Table 3. Mainly, three abiotic stress-responsive (low temperature, light, defense and stress) elements and hormone related (abscisic acid, salicylic acid, MeJA, gibberellin, auxin) element were detected. These elements consist of G-box, ABRE, TCT-motif, Box 4, GT1-motif, etc. (Table S4). These results suggest that these genes may actively respond to light, hormone regulation, resist stress, and play a key role in promoting normal plant growth and development (Fig. 5, Table S4), indicating their defensive role against stress conditions.

Identification of functional candidate genes for low temperature tolerance of wucai
Exogenous application of EBR (Mitteer et al. 2020) can significantly improve the low temperature tolerance of "WS-1" under low temperature stress. Figure 6A shows the clustering heat map analysis of transcription factors in the transcriptomic data of "WS-1" treated with EBR at low temperature (Fig. 6A). Cluster analysis was performed on the DEGs related to the bHLH family genes of "WS-1" (Fig. 6B). The results showed that the expression level of bHLH57 gene in LT + EBR group was up-regulated 228 times compared with LT group (Table S2). In addition, the relative expression level of BcbHLH57 gene was significantly upregulated by high and low temperature treatment of "WS-1" (Fig. 6D).
The principal component analysis (PCA) showed that the two groups had good repeatability (Fig. 6E, F). It was speculated that BcbHLH57 gene might be involved in mediating EBR to enhance the low temperature tolerance of "WS-1".

Cloning of BcbHLH57 gene and construction of overexpression vector of wucai
The cDNA template was obtained by reverse transcription of the RNA of "WS-1", and the primer fragment of BrbHLH57 gene was designed (Table S5), and the coding sequence of BcbHLH57 gene with a length of 936 bp was finally amplified (Fig. 7). DNAMAN 6.0 software was used for sequence comparison, and it was found that the similarity of bHLH57 gene coding sequences between wucai and Brassica campestris was as high as 98.93%, with only a few base substituents, indicating a very close genetic relationship (Fig. 7). The 35S: pCambia1305-BcbHLH57 expression vector was constructed and introduced into Arabidopsis thaliana by inflorescence infection (Fig. 8A). Three T3 transgenic lines (OE14, OE26 and OE27) with high relative expression levels were obtained after screening, and their relative expression levels were 62.83, 44.43 and 11.37 times that of WT, respectively (Fig. 8B).

Effects of different stresses on seed germination of transgenic BcbHLH57 plants
The seeds of three T3 transgenic lines and wild type of Arabidopsis thaliana were subjected to high temperature (40 °C), osmotic stress (250 mmol L-1D-mannitol) and salt stress (150 mmol·L-1NaCl), respectively, (Fig. 9). In the control (normal treatment) group, the germination rates of ◂ transgenic and wild-type seeds showed roughly the same trend within 7 days (Fig. 9A, E). The germination rate of the three transgenic lines was significantly higher than that of the wild type in the treatment of high temperature stress, salt stress and osmotic stress ( Fig. 9B-D, F-H).

Effects of different stresses on root growth of transgenic plants with BcbHLH57 gene
After 14 days of different stress treatments, 15 transgenic plants and wild-type plants were randomly selected for the measurement of taproot length, and the mean value and standard error were calculated. As shown in Fig. 10, the root length of transgenic plants in each stress treatment group was significantly larger than that of the wild type after 14 days of treatments.

Effects of different stresses on seedling survival rate of transgenic plants with BcbHLH57 gene
After 38/30 °C heated for 3 days and 150 mmol·L-1 NaCl spraying for 7 days, the survival rate of seedlings of the wild-type and three T3 transgenic lines were significantly higher than that of wild-types, while there was no significant difference between the transgenic and wild-type seedlings of the control group (normal treatment) (Fig. 11). The seedlings of 3-week-old wild type and transgenic Arabidopsis thaliana were rewatered for 3 days after 14 days of drought treatment (14 days without watering), and the survival of each line was observed. After 7 days of drought stress, the leaves of wild-type plants curled, withered and wilted with a significant dehydration phenomenon. There was no obvious dehydration in all transgenic lines, which was similar to the control group. After 14 days of drought stress, the dehydration of wild-type plants was more significant, and most of them died. All transgenic lines had obvious dehydration, but the survival rate was higher than that of wild-type plants. After rehydration for 3 days, the leaves of all transgenic lines turned green and the survival rate was significantly higher than that of the wild type. The survival rate of OE#14 and OE#27 transgenic plants was more than 80% after rehydration for 3 days, while the survival rate of wild-type plants was less than 10%, which was obviously weaker than other transgenic lines.

Effects of different stresses on physiological indexes of transgenic plants with BcbHLH57 gene
In addition, the physiological characteristics including the accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2.-), Relative conductivity (REL), and total antioxidant capacity (T-AOC) also reflected the stress tolerance.
We detected these indices in the wild type and three lines of transgenic Arabidopsis over-expressing BcbHLH57 (OE14, OE26 and OE27) (Fig. 12). Compared with the wild-type, the contents of REL, H2O2 and O2.-of the transgenic plants under heat, salt and drought stresses decreased, while the T-AOC increased, while there was no significant difference among the plants under normal treatment (Fig. 12). This indicated that compared with wild-type plants, transgenic plants had greater reactive oxygen species scavenging ability, thereby maintaining cell homeostasis. These results further indicated that overexpression of bHLH57 improved the tolerance of transgenic Arabidopsis to heat, salt and drought stresses.

Discussion
There are many TF families involved in physiological processes, abiotic and biological stress responses in plants (Liu et al. 2021), such as AP2/EREBP (Song et al. 2005), MYB (Ambawat et al. 2013;Pireyre and Burow 2015), WRKY (Chen et al. 2012), NAC (Olsen et al. 2005). The bHLH family is the second largest TF family in plants (Feller et al. 2011), and has been identified and studied in many plants, such as Arabidopsis ) (147 family members), apple (Mao et al. 2017) (188 family members), rice (Li et al. 2007) (167 family members), Solanum potato (Wang et al. 2018a, b) (124 family members), tomato (Sun et al. 2015) (159 family members), and maize ) (208 family members). As the tripling event and gene loss event of the whole genome of Brassica (Lüthje and Martinez-Cortes 2018), the number of BrbHLH TFs should be about 300 in theory, about 2-3 times of the number of Arabidopsis thaliana members. While only 239 members were identified, indicating that a large number of gene loss occurred in the Brassica genome during the evolution process. The equal number of 239 BrbHLH TFs and 234 Bob-HLH TFs (Shan et al. 2019) indicates that Brassica species are consistent in the evolutionary process, in line with the theory of "U's Triangle" . The results of chromosomal localization analysis showed that BrbHLHs was unevenly distributed on the chromosome of Brassica rapa and there were tandem repeat gene pairs, which was consistent with the report in potato. Tandem duplication contributed to gene amplification, suggesting it promoted the amplification of bHLHs in Brassica rapa .
We constructed a phylogenetic tree of BrbHLH in Brassica rapa and AtbHLH in Arabidopsis thaliana. Brb-HLH and AtbHLH genes can be divided into five groups. Group1-group5 were evenly distributed, and the numbers of BrbHLHs were about 1.5 times that of AtbHLHs. However, the numbers of BrbHLH and AtbHLH genes in Group6 were similar and the least, which may be due to the occurrence of gene loss in the process of evolution. In addition, colinear analysis revealed a large number of colinear gene pairs between Brassica rapa and Arabidopsis thaliana, indicating a high degree of homology between the two species, which was also related to their belonging to the cruciferous family. The result of Ka/Ks showed that these collinear genes may have undergone strong purification selection, and their structures are relatively conserved, suggesting that their functions and functions are similar.
We analyzed the structure of BrbHLHs gene and predicted ten conserved motifs. It was found that the distribution of these motifs was more similar among genes that were closely related. The number of exons and introns was different among different gene clusters, and the closer the genetic relationship was, the more similar the gene structure was. It is speculated that closely related genes may have similar functions. Diversity of gene structure leads to diversity of gene functions and plays a role in plant growth and development and stress resistance ). Analysis of cis-acting elements in the promoter region of BrbHLH genes in Brassica rapa showed that most genes were involved in light, low temperature stress response, and hormone regulation. These genes may be involved in plant photomorphogenesis, regulation of hormone metabolism pathways and abiotic stress tolerance, which is consistent with the results of ZmbHLHs in maize .
AtbHLH9 (AtPIF4) can promote stem elongation to adapt to heat stress by regulating plant auxin levels (Franklin et al. 2011). AtbHLH17 (AIB) participates in the regulation of ABA signal and enhances drought resistance of Arabidopsis thaliana (Li et al. 2007). OsbHLH035 can enhance the resilience of rice seedlings under salt stress . Transcriptomic analysis showed that bHLH57 gene was significantly upregulated by EBR at low temperature. QRT-PCR analysis showed that Bcb-HLH57 was up-regulated at low and high temperatures at 24 h. These results suggest that bHLH57 mediate the physiological regulation process of hormone regulation against abiotic stress in wucai. BHLH57 was an important gene involved in seed germination and is involved in ABA mediate regulation of seed dormancy (Tian et al. 2020). BHLH57 positively regulate seed dormancy by inducing the expression of genes encoding ABA biological synthase (NCED6 and NCED9) (Alam et al. 2019). BHLH57 can also interact with ODR1 protein to inhibit the expression of NCED6 and NCED9 in the nucleus and promote seed germination (Alam et al. 2019). Overexpression of soybean bHLH57 gene can significantly upregulate downstream iron absorption genes, resulting in higher iron content in plants and enhanced tolerance to iron deficiency. The sequence of bHLH57 gene was compared with bHLH57 gene of Brassica rapa. The results were highly similar, indicating that the genetic relationship between bHLH57 gene of Brassica campestris and Brassica rapa was very close, but there were some base substitutions. The comparison of the coding sequences of BrbHLH57 and BcbHLH57 showed that the genetic relationship between BrbHLH57 and BcbHLH57 was very close, while there were some base substitutions. It is speculated that genetic mutations occurred during the evolution of Brassica campestris resulting in the emergence of wucai. The substitution of these bases may cause the functional difference of bHLH57 gene between Brassica campestris and wucai.
In this study, the overexpression vector 35S: PCAM-biA1305-BcbHLH57 was cloned and constructed using the high-generation inbred line "WS-1" as the template. The vector was successfully introduced into Arabidopsis thaliana and T3 transgenic lines with high expression were obtained. Different stress treatments were carried out on transgenic lines, and phenotypes related to stress tolerance were measured. It was found that high temperature, salt and osmotic stress affected seed germination rate and root growth of wild type (Col-0) more than transgenic lines. It was speculated that the expression of BcbHLH57 gene could improve the tolerance of plants to high temperature, salt and osmotic stress during seed germination and rooting stage, which was consistent with previous reports that bHLH57 gene was involved in seed germination (Hao et al. 2021;Liu et al. 2020). In this study, physiological indices of BcbHLH57 OE lines of transgenic Arabidopsis thaliana were determined after high temperature, salt and drought stress. The results showed that the over-expression of BcbHLH57 gene significantly reduced the relative conductivity, H 2 O 2 and superoxide anion contents of Arabidopsis thaliana plants, and increased the total antioxidant capacity of plants, suggesting that bHLH57 gene can respond to abiotic stresses. These results suggest that overexpression of BcbHLH57 gene can remove ROS in vivo, thus maintaining the  stress. This study provides theoretical reference for the functional study of BcbHLH57 gene and the high-quality breeding of wucai.  need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.