Genome-wide characterization of the MBF1 gene family and its expression pattern in different tissues and stresses in Medicago

doi:10.21203/rs.3.rs-2594095/v1

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Genome-wide characterization of the MBF1 gene family and its expression pattern in different tissues and stresses in Medicago

https://doi.org/10.21203/rs.3.rs-2594095/v1

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Background

Multiprotein bridging factor 1 are transcription factors that play critical roles in plant life cycle and in plant tolerance to environmental stresses. Medicago sativa is an important perennial legume forage grass, whereas the potential information in the MBF1 genes associated with stress resistance remains poorly understood.

Results

Three MBF1 genes were identified from each of the M. truncatula and M. sativa genomes. Multiple sequence alignment analysis showed that all these members contain conserved MBF1 and HTH domains. The MBF1 genes showed similar exon-intron organizations, and similar architectures in their conserved motifs. A number of cis-acting elements associated with drought, MeJA and light stress were identified in their promoter regions. In addition, these MBF1 genes were shown in genechip and transcriptome data to exhibit divergent expression patterns in various tissues or in response to drought and salt treatments. In particular, qRT-PCR results showed that the expression of MtMBF1b and MtMBF1c were significantly induced by NaCl treatment, indicating that they are likely to play a role in salt stress response.

Conclusions

Our comprehensive analysis provides valuable information for elucidating the evolutionary process of MBF1 genes and their expression patterns in different tissues and under four stresses. This work will facilitate the application of MBF1 genes in molecular breeding of highly resistant alfalfa.

Medicago truncatula

Medicago sativa

MBF1 genes

Abiotic stresses

Expression profiling

Environmental stresses such as drought, salinity, high temperature, and low temperature constrain plant growth and development, often leading to physiological and morphological changes and even death. [1]. In response, plants have evolved complex and efficient information transfer systems to cope with these environmental stresses during their life cycle. [2]. Plants receive environmental signals through these stress-inducible factors in vivo, and subsequently important transcription factors (TFs) are activated to mediate appropriate plant responses, including bZIP, MYB, bHLH, WAKY, ERF, NAC, MBF1, and etc. [3]. Multiprotein bridging factor 1 (MBF1), as one of the crucial transcriptional coactivators, and plays a critical role in gene expression and regulation during stress responses [4].

MBF1 family members with a fexible N-terminal region and highly conserved helix-turn-helix (HTH) domain in its C-terminal [4]. Binding to activated proteins is the function performed by the N-terminus, while the C-terminus plays a role in protein folding and dimer formation of MBF1. [4]. MBF1 was identified as a non-DNA binding transcription co-factor and the general factor TATA box Binding Protein (TBP), which is essential for transcription initiation. MBF1 members continually shifts its the locations in the cytoplasm and nucleus to conduct its functions [5]. The discovery in yeast demonstrates a direct interaction of yeast MBF1 with the basic leucine zipper (bZIP) family of yeast transcription factors, GCN4, forming the GCN4-MBF1-TBP complex [6]. Each member of this structure exhibits sophisticated linkages in plant resistance to biotic and abiotic stresses.

In Arabidopsis, there are three MBF1 genes, wrer found in plant bodies without tissue-specific expression, such as root, stem, leaf, flower, fruit, seed and anther organs [7], but some specifc expression abundances were found among the three different MBF1 genes during plant development [8, 9]. AtMBF1a overexpression was found to be more tolerant to salt stress and pathogen infection after displaying a phenotype of glucose hypersensitivity [10]. Overexpression of AtMBF1c not only increases seed number in Arabidopsis and soybean [11, 12], but also MBF1c rapidly accumulates and interacts with trehalosephosphate synthase in response to heat stress. [12]. Ectopic expression in grapes in Arabidopsis improved drought tolerance [13], and the ectopic expression of MBF1 gene in wheat improved heat tolerance in rice [14]. The MBF1 gene appears to function as a negative regulator in the cold stress response in pepper. [7]. Despite the indispensable role of MBF1 genes in plant growth and development, there is little information on Medicago, except for some reports describing MBF1 genes of several species.

Alfalfa (Medicago sativa) is a perennial legume plant, which is one of the important forage crops. Alfalfa is rich in protein, minerals, vitamins and other nutrients, which is the optimal forage for improving the quality of livestock products [15]. Nevertheless, some unfavorable environmental factors limit the growth of plants; therefore, it is urgent to explore key genes to improve the resistance of alfalfa and thus increase the yield [16]. The improvement of yield and quality of tetraploid alfalfa is limited by its genetic complexity and intolerance to environmental stresses. Fortunately, Medicago truncatula, the model legume, is evolutionarily related to alfalfa. The genetic advantages and genomic resources of Medicago truncatula as a model plant facilitate the study of alfalfa. In this study, we identified MBF1 genes from both Medicago, and analyzed their multiple sequence alignment, phylogenetic relationship, gene structure, protein motifs, and cis-acting elements. Furthermore, the expression profiles of MBF1s of M. truncatula and M. sativa in response to various abiotic stresses were analyzed. Our comparative studies on MBF1 genes will provide a basis for future investigation on abiotic stress responses medicated by MBF1s, and will facilitate the functional characterization of individual MBF1s genes in responses to stresses and developmental signals.

Identification of MBF1 genes in the M. truncatula and M. sativa genome

Based on domain confirmation and homology search, a total of four putative MBF1s (MtMBF1a.1, MtMBF1a.2, MtMBF1b, MtMBF1c) from M. truncatula, and two putative MBF1s (MsMBF1a and MsMBF1c) from M. sativa, were identified, respectively (Table 1). The molecular property analysis revealed that most MBF1 proteins were between 110aa and 150aa, which is similar to the MBF1 profile of tomato [26]. Differently, the MtMBF1c and MsMBF1a proteins are shorter in length, 56aa and 74aa, respectively. The predicted pI values ranged from 8.6 (MtMBF1b) to 10.24 (MsMBF1c). The predicted molecular weights ranged from 6.18 kDa (MtMBF1c) to 15.6 kDa (MtMBF1a.1). The corresponding homologous MBF1 genes of M. sativa and M. truncatula were identified in parallel by sequence alignment. The predicted subcellular locations suggested that two proteins (Mt/MsMBF1c) were located in the nuclear, MtMBF1a.2 were located in the cytoplasmic, and the remaining three proteins (MtMBF1a.1, MtMBF1b and MsMBF1a) were located in extracellular.

Table 1
Properties of the predicted MBF1 proteins in *M. truncatula* and *M.sativa*
Gene number	Gene Name	TIGR Locus	Start site	End site	Homologous Gene	pI	MW(kDa)	Protein Length	Subcellular Localization
MtMBF1-1	MtMBF1a.1	MtrunA17_Chr2g0320211	41480478	41484655	MsMBF1a	9.95	15.35	141	Extracellular
MtMBF1-2	MtMBF1a.2	MtrunA17_Chr4g0041301	38998265	39003781	MsMBF1a	9.91	15.6	143	Cytoplasmic
MtMBF1-3	MtMBF1b	MtrunA17_Chr4g0057871	50949726	50950391	MsMBF1a	8.6	13.75	121	Extracellular
MtMBF1-4	MtMBF1c	MtrunA17_Chr6g0485281	39789414	39792731	MsMBF1c	9.9	6.18	56	Nuclear
MsMBF1-1	MsMBF1a	MsG0480021982.01.T01	66798008	66798612	MtMBF1a.1	9.45	7.8	74	Extracellular
MsMBF1-2	MsMBF1c	MsG0680035636.01.T01	109794752	109795081	MtMBF1c	10.24	11.87	110	Nuclear

Multiple sequence alignment, phylogenetic analysis and classification of MBF1 genes in Medicago

Sequence alignments of the multiple amino acids between MtMBF1s and MsMBF1s indicated that the sequences are highly conserved (Fig. 1): all the MBFs containing MBF1 domain in N-terminal (MtMBF1b was an incomplete domain) and HTH domain was present in the C-terminal (except MsMBF1a).

Sequence-based phylogenetic analysis among M. truncatula, M. sativa, Arabidopsis, O. sativa, Z. mays, V. unguiculata and B. rapa showed that these proteins were grouped into 3 distinct clades (a, b, c, Fig. 2). MBF1a and MBF1b were detected to have a closer relationship, which suggested that they might conduct more similar functions. However, MBF1c was displayed to be a relatively independent group (Fig. 2), which indicated that it might play some specifc roles related to the development process in plants.

Analyses of conserved motif and gene structure

In order to understand the structural characteristics of MBF1 genes of Medicago, the motifs and exons/introns structure analyses of the MBF1 family genes from the Medicago were conducted and the results are consistent with the phylogenetic analyses (Fig. 3). Ten preserved motifs were detected in these MBF1 proteins. Motif 1 and motif 2 were detected in all members’ MBF1 domain, where motif1 is MBF1 domain and motif 2 is HTH domain (Fig. 3B). Therefore, the similar motif distribution of the MBF1 proteins in Medicago may promote to the prediction of the functions of MBF1s.

The gene structures of exon numbers ranged from one to four, and the intron numbers ranged from zero to three for two species (Fig. 3C). MtMBF1a. 2 and MtMBF1c contain more UTR sequences, accounting for 55% and 46% of the gene sequences respectively, while MtMBF1b and MsMBF1c do not contain UTR, and differences in exon or intron length were also observed between orthologous members.

Analysis of chromosome location and collinearity of MBF1 genes

The chromosome location data of each MBF1 gene were download from the Phytozome database, based on which these MBF1 gene were anlysised by TBtools and mapped on their corresponding chromosomes (Fig. 4A, B). As a result, the distribution of MBF1 genes were not even in either M. truncatula or M. sativa. MtMBF1 were distributed on chromosome 2, 4, 6, and two genes (MtMBF1a.2 and MtMBF1b) are distributed on chromosome 4 (Fig. 4A). MsMBF1a and MsMBF1c were distributed on chromosome 4 and 6 respectively. (Fig. 4B). In addition, there were no segmental duplication and tandem duplication events in both M. truncatula and M. sativa.

Furthermore, three comparative syntenic maps of M. sativa and M. truncatula associated with the representative plant species Arabidopsis were constructed to illustrate the evolution relationship of MBF1 genes family (Fig. 4C). Two pairs of genes are covalently related between M. sativa and M. truncatula. MtMBF1a.2 was collinear with MsMBF1a, and MtMBF1c was collinear with MsMBF1c. Moreover, to better understand the evolutionary selection pressure during the formation of MBF1 gene families, the non-synonymous/synonymous substitution (Ka/Ks) values of MBF1 gene pairs were analyzed between M. sativa and M. truncatula. Generally, Ka/Ks > 1, Ka/Ks = 1, and Ka/Ks < 1 indicate positive selection, neutral evolution, and purifying selection, respectively [27]. The ratios of two (MtMBF1a.2-MsMBF1a, MtMBF1c-MsMBF1c) homologous pairs were even smaller than 0.2, suggesting that these genes have undergone purifying selection after segmental and whole genome duplications.

Analysis of cis-acting element of MBF1 genes

To further determine the molecular functions and expression pattern of the MBF1 family, we focused on hormones, abiotic stress and growth & development related cis-acting elements (Fig. 5 and Addition file 1). In phytohormone pathways, most of the MBF1 genes were detected to have more cis-element involved in Methyl jasmonate (MeJA) and ethylene responsiveness (Fig. 5A). In addition, a series of cis-acting elements were identifed with abiotic stress. All MBF1 genes were mainly related to drought and light responsiveness. Combining hormone and abiotic stress cis-elements, it was concluded that MBF1 gene family had strong response to external environmental stimuli. Furthermore, MBF1 gene family contains a large number of promoter enhancement regions and transcription initiation elements, which may play a key role in the rapid response of plants to external stimuli. Notably, an exclusive cis-element associated with circadian control was obtained in MtMBF1a.2 and MsMBF1a, suggests that MBF1 genes may play a role in regulating plant cyclic rhythms.

Expression profiles of MBF1 genes in different tissues and validation by qRT-PCR

We investigated the expression patterns of MBF1s in various tissues of M. truncatula with genechip dataset from the MtGEA web server, including roots, stems, petioles, leaves, flowers, buds, pods, seeds and seeds coat (Fig. 6A, Additional file 2). MtMBF1a.1 and MtMBF1a.2 showed relatively high expression level in these tissues, MtMBF1b and MtMBF1c were expressed at relatively low level in different tissues (Fig. 6A). As for M. sativa, six tissues of different type were analyzed based on transcriptome data, including roots, elonged-stems, pre-elonged-stems, leaves, flowers and nodules (Fig. 6C, Additional file 3). Among them, there was no significant difference in the expression of MsMBF1a in different tissues, but the expression of MsMBF1c in leaves and flowers was significantly higher than that in roots, stems and nodules (Fig. 6C).

To understand the potential function of the Medicago MBF1 genes, the expression pattern were examined using qRT-PCR in different organs, including the roots, stems, leaves and flowers (Fig. 6B, D, Additional file 2, 3). All of the MBF1 genes were detected in these four tissues (Fig. 6B, D). Remarkably, the expression levels of all genes in leaves were significantly higher than those in other tissues, followed by flowers in M. truncatula and roots in M. sativa.

Expression profile of MBF1 gene in Medicago under stress

Expression profiles of MBF1 genes from M. truncatula were initially analyzed based on the genechip data, including samples from roots and shoots under drought treatment, and roots under vitro culture salinity and under hydroponic salinity (Additional File 2). As for M. sativa, transcriptome data for samples from roots and shoots under NaCl and drought treatment were also analyzed (Additional File 3).

Under NaCl conditions, the expression changes of four genes showed the similar trend under two different NaCl treatments (Fig. 7A). Among them, MtMBF1a.1 and MtMBF1a.2 showed relatively high expression level, MtMBF1b and MtMBF1c were expressed at relatively low level (Fig. 7A), this is consistent with the expression of genes in tissues (Fig. 6A). MtMBF1a showed an increasing and then decreasing trend under vitro culture salinity conditions, while it showed a decreasing and then increasing trend under hydroponics salinity conditions. Notably, under drought conditions, the expression trends of these genes were consistent in both roots and stems, and both gradually increased with increasing stress time. (Fig. 7B). Contrary to roots drought treatment, the expression of MtMBF1a.2 and MtMBF1b showed a downward trend after rewatering in shoots drought treatment, (Fig. 7B). This may be related to the special response mechanism of MBF1 genes to abiotic stress. In M. sativa, the expression of both members (MsMBF1a and MsMBF1c) showed an increasing trend under NaCl and drought stress treatments, with MsMBF1a showing a decrease in expression at 24 h of drought.

Validation of the expression profile of stress-responsive MBF1 gene by qRT-PCR

In order to verify the expression profiles of MBF1 genes from genechip data and microarray data, seedlings were treated with NaCl, mannitol, hot and cold at 1h, 3h, 6h, 12h, 24h, 48h and used for qRT-PCR analysis (Fig. 8, Additional File 4). As expected, most of the MBF1 genes responded to different stress treatments. For example, MtMBF1b tended to increase and then decrease under all stress treatments and peaked at 3-6h (Fig. 8). The expression level of MtMBF1a.1 decreased obviously under NaCl treatment, which was consistent with the genechip data (Fig. 7). The expression level of MtMBF1c increased significantly under NaCl treatment, but remained unchanged under drought stress (Fig. 8). In the face of heat stress, MtMBF1b was induced during the early stage, the other genes have no obvious changes. Notably, MsMBF1a and MsMBF1c showed extremely low levels of expression at all times in response to NaCl and drought treatments.

Multiprotein Bridging Factor 1 (MBF1) is a plant superfamily with a long history of documented [5]. The MBF1 gene has been studied mainly in the model plant Arabidopsis, where it plays a role in the regulation of plant growth, development and stress response [10, 28]. After the discovery of four MBF1 genes in tomato by Sanchez Ballesta et al. information on the MBF1 family has been reported in several species [29], but a genome-wide identification and characterization of MBF1 genes in Medicago is still vacant. In the present study, we conducted an integrated investigation on the MBF1s, and a total of 4 MBF1 members from M. truncatula and 2 MBF1 members from M. sativa were identified (Table 1).

The results of multiple sequence matching showed that almost all Medicago MBF1 members contained MBF1 domains and HTH domains (Fig. 1), they have highly conserved amino acid sequences, which is consistent with tomato [29]. Compared with Arabidopsis, Medicago has at least two different MBF1 genes, of which MBF1a and MBF1c are essential members (Fig. 2). Phylogenetic analysis showed that MBF1a and MBF1b were closely related, while MBF1c was relatively independent (Fig. 2). Interestingly, the CDS length of the MBF1c gene was always longer than that of the MBF1a and MBF1b genes (Table 1). However, the MsMBF1a gene structure was significantly shorter than MtMBF1a (Fig. 3), which may be due to incomplete genome splicing. In addition, the gene structure with few or no introns may enhance the ability to respond to external stresses through rapid transcription and translation. [30].

Gene duplication and differentiation play a crucial role in family expansion and evolution [31]. We found no duplication events were found in M. truncatula or M. sativa, however, there is a collinear relationship between species (MtMBF1a.2-MsMBF1a and MtMBF1c-MsMBF1c) (Fig. 4). Previous studies suggested that collinear might provide some similar genetic functions and expression patterns [32]. Thus, the detailed functions and the essential members of these MBF1 genes could be revealed in further studies.

Previous studies have reported that MBF1 genes play a roles in plant growth and development [33]. In the present study, we identified the tissue specific expression patterns of all MBF1 genes in Medicago in a variety of tissues, and the results showed that MBF1 gene was mainly expressed in leaves (Fig. 6), so finding MBF1 key functional genes may be of significance for alfalfa yield improvement. High-salinity or drought soil is the most serious abiotic stress [34], while heat and cold extremes also limit plant reproduction [35]. It is urgent to improve ambient pressure tolerance of alfalfa to increase yield. Genechip data and transcriptome data under NaCl and drought treatments suggested that the expressions of almost six genes were highly induced in Medicago (Fig. 7), suggesting that these genes may be involved in plant responses to abiotic stresses. Subsequently, the expression pattern of all six genes was verified under NaCl, mannitol, heat and cold treatments in M. truncatula and M. sativa by qRT-PCR analyses (Fig. 8), and the results suggested that MtMBF1b, c genes are involved in NaCl induction, and MtMBF1a.2 and MtMBF1b genes are involved in hot induction. In the phylogenetic analysis, MBF1 genes were divided into three branches, which is consistent with Arabidopsis and maize [10]. Overexpressing the AtMBF1a gene in Arabidopsis confers increased resistance under the infection by B. cinerea [10]. The closely related genes of MtMBF1a.1, 2 and MsMBF1a may also involved in fending off pathogenic attack. Moreover, The ectopic expression of AtMBF1c did not infuence the response to cold stress in Arabidopsis seedlings [11]. The homologous genes MtMBF1c and MsMBF1c exhibited normal expression under cold stresses, suggesting that these two genes may not play a key role in response to cold stress.

In this study, six MBF1 genes on a genome-wide scale in M. sativa and M. truncatula were identified. Amino acid sequence, motif composition and conserved gene structure showed high similarity in these MBF1 genes. Moreover, phylogenetic analysis and covariance of MBF1 genes in different species reveal evolutionary patterns among them and predict their functions in response to complex environments. In addition, the expression profiles of MBF1 genes in different tissues and treatments of Medicago were analyzed and validated by qPCR and revealed that MtMBF1a.2, MtMBF1b, MsMBF1a and MsMBF1c gene pairs showed the same expression pattern in Medicago, and they may play an important role in response to drought stress. This study compares the MBF1 family of M. truncatula and M. sativa to provide new ideas for understanding the evolutionary relationship, which also provides a comprehensive information basis for better characterizing the role of the MBF1 family in plant stress resistance.

Identification of MBF1 genes in the M. truncatula and M. sativa genome

Genomic data of M. truncatula and M. sativa were downloaded from (https://figshare.com/articles/dataset/Medicago_sativa_genome_and_annotation_files/12623960) (accessed on 12 October 2022) and (https://www.jcvi.org/research/medicago-truncatula-genome-database) (accessed on 12 January 2022) website, respectively. The first step in determining the MBF1 (MBF1 domain (PF08523) and HTH domain (PF01381)) family is to download Hidden Markov Model (HMM) profiles from Pfam protein family database (https://pfam.xfam.org/, accessed on 16 October 2022). Moreover, the MBF1 gene sequence of Arabidopsis thaliana was obtained from the TAIR website (https://www.arabidopsis.org/) (accessed on 17 October 2022). In order to further screen the MBF1 genes, submit the proposed MBF1 sequence to InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search) (accessed on 18 October 2022), CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) (accessed on 18 October 2022), Pfam (https://pfam.xfam.org/) (accessed on 18 October 2022), and SMART (http://smart.embl-heidelberg.de/) (accessed on 18 October 2022). Finally, 4 MtMBF1 and 2 MsMBF1 genes were identified and located in the corresponding positions of chromosomes. Correspondingly, the molecular weight (MW) and isoelectric point (pI) of the deduced amino acid sequences were predicted with the Expert Protein Analysis System (ExPASy) on the proteomics server (http://www.expasy.ch/tools/protparam.html) (accessed on 20 October 2022). The subcellular localization was predicted using the Softberry Home Page (http://www.softberry.com/) (accessed on 20 October 2022).

Analyses on sequence and structures of the Medicago MBF1 genes

MEME program (http://meme-suite.org/tools/meme) (accessed on 25 October 2022) with the motif number of MBF1 set as 10 and the width range of 10 to 200 amino acids (aa) to identify conservative motifs. Subsequently, jalview software was used for sequence alignment. Visualization of exon-intron positions and conserved motifs by TBtools software [17].

Phylogenetic analysis and classification ofMBF1genes

MBF1 proteins from seven plant species (M. truncatula, M. sativa, Arabidopsis, O. sativa, Z. mays, V. unguiculata and B. rapa) were used in a multiple alignment in ClustalW [18]. Phylogenetic trees were constructed by the neighbor-joining method using MEGA-X software with a bootstrap of 1000 replicates. Meanwhile, subfamily clustering on the phylogenetic tree of other species was determined based on Arabidopsis member clustering. Subsequently, EvolView (https://evolgenius.info/evolview-v2/) (accessed on 1 November 2022) was used to view the phylogenetic tree.

Analyses of chromosome location and collinearity ofMBF1genes

The chromosomal localization of all MBF1s of M. truncatula and M. sativa were obtained from the corresponding website. Multiple collinear Scan toolkit (TBtools) was used to analyze the gene duplication events with default parameters [19]. All the MBF1 genes were similarly mapped to eight M. truncatula and M. sativa chromosomes, respectively [17]. To demonstrate Intraspecific and interspecific covariance among M. truncatula, M. sativa and a representative model plant (Arabidopsis), the syntenic maps were constructed using the Dual Systeny Plotter software (TBtools). The simple Ka/Ks calculator software is used to calculate non synchronous (Ka) and synchronous (Ks) values of MBF1 gene pairs (TBtools) [17].

Analyses of cis-acting elements and location ofMBF1genes inMedicago

TBtools software was used to identify the promoter sequence of MBF1 gene (1.5 kb upstream of the translation initiation site), and online program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 1 November 2022) was used to predict the cis-elements in the promoter region [20]. TBtools was used to visualize the cis-acting elements of all MBF1 genes of Medicago.

Analysis of expression level ofMBF1genes

Genechip data from M. truncatula were downloaded for roots and shoots and for the MtMBF1 gene under drought and salt stress conditions. The expression levels of MtMBF1 genes in different tissues were also analyzed. Amazing HeatMap software was used to generate the heatmap [17]. The original transcriptome data from M. sativa under NaCl and mannitol treatments at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h (SRR7160314-15, 22–23, 25–49, 51–52, 56–57) were downloaded from the NCBI database [21]. Then, the data were converted to fastq files using the SRA-Toolkit v2.9 [22]. Raw reads were trimmed using Trimmomatic-0.39 [23]. Gene expression level was determined by mapping cleaned reads to the corresponding alfalfa reference genomes using the StringTie v2.1.3 package and the log2(FPKM) values [24].

Plant Materials And Treatments

The M. sativa (Zhongmu No.1, College of Grassland Science, Qingdao Agricultural University, Qingdao, China) and M. truncatula (cv. Jemalong A17, College of Grassland Science, Qingdao Agricultural University, Qingdao, China) plants used in this study was stored at the Qingdao Agricultural University. The roots, stems, leaves, flowers, pods (15-day-old pods) and seeds (15-day-old seeds) of mature plants were collected for RNA extraction and qRT-PCR analysis. Meanwhile, the expression patterns of MBF1 genes were also investigated under different stress treatments (15% minnitol, 300mM NaCl, 45 ^◦C heat and 4 ^◦C cold) [25].

Analysis Of Gene Expression By Qrt-pcr

Total RNAs were extracted by using Eastep® Super total RNA Extraction kit (Promega, Shanghai, China) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using Trans® Script One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) per manufacturer’s recommendations. qRT-PCRs were carried out using a 2×RealStar Green Fast Mixture (GeneStar, Shanghai, China) on ABI 7500 real-time Detection System (Applied Biosystems, USA). The housekeeping gene actin-related protein 4A gene was used as an internal control. The reaction was carried out as follows: 94 ^◦C for 30 s, followed by 40 cycles of 5s at 94 ^◦C and 34s at 60 ^◦C. The relative expression levels of the genes were determined with the comparative 2^−ΔΔCt method. The primer sequences used in this study were shown in Table S1.

Availability of data and materials：

MsMBF1: (http://www.medicagohapmap.org/downloads/cadl)

MtMBF1: (https://www.jcvi.org/research/medicago-truncatula-genome-database)

Arabidopsis, Oryza sativa, Zea mays, Vigna unguiculata and Brassica rapa: (https://www.uniprot.org/)

MBF1 proteins in M. truncatula and M.sativa: Table 1

Gene expression data: Additional file 4

Primer information: Table S1

Funding: This study was supported by the National Nature Science Foundation of China (U1906201), Shandong Forage Research System (SDAIT-23-01), First Class Grassland Science Discipline Program of Shandong Province (1619002), China.

Authors' contributions: Y.Z. Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft. X.Z. Formal analysis. S.Y. Formal analysis. Y.W. and H.W. Resources. J.L. Supervision. G.Y. Conceptualization, Funding acquisition, Project administration, Visualization, Writing - review & editing.

Competing interests: All the authors declared no competing interests.

Acknowledgements: Not applicable.

Ethics approval and consent to participate: Not applicable.

Consent for publication: Not applicable.

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Genome-wide characterization of the MBF1 gene family and its expression pattern in different tissues and stresses in Medicago

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Abstract

Background

Results

Conclusions

Figures

Introduction

Results

Identification of MBF1 genes in the M. truncatula and M. sativa genome

Multiple sequence alignment, phylogenetic analysis and classification of MBF1 genes in Medicago

Analyses of conserved motif and gene structure

Analysis of chromosome location and collinearity of MBF1 genes

Analysis of cis-acting element of MBF1 genes

Expression profiles of MBF1 genes in different tissues and validation by qRT-PCR

Expression profile of MBF1 gene in Medicago under stress

Validation of the expression profile of stress-responsive MBF1 gene by qRT-PCR

Discussion

Conclusion

Materials And Methods

Plant Materials And Treatments

Analysis Of Gene Expression By Qrt-pcr

Declarations

References

Additional Declarations

Supplementary Files

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