In this study, HMMER 3.3.2 software was used to perform a homology search (E-value threshold of 1e^-5) based on the Hidden Markov Model (HMM) of the CBL domain (PF04833) in the Pfam database (https://pfam.xfam.org/) (Mistry et al., 2021) for S. bicolor BTx623 and E048 protein sequences downloaded from the Phytozome v13 (https://phytozome-next.jgi.doe.gov/) (Goodstein et al., 2012) and SGMD databases (https://S.bicolor.genetics.ac.cn/SGMD, accessed on 5 May 2025) (Chen et al., 2025), respectively. The gene sequences obtained from the initial screening were further validated for conserved structural domains using NCBI Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to ensure that the identified sequences contained complete and typical CBL domains, thus accurately identifying members of the S. bicolor CBL gene family.

The coding sequences (CDS) of the genes were aligned with the corresponding genomic sequences and visualized using TBtools software (Chen et al., 2023) to show the exon-intron structure of the SbCBL genes. Then, the conserved motifs of S. bicolor CBL proteins were predicted using MEME Suite 5.5.3 (https://meme-suite.org/meme/tools/meme) (Bailey et al., 2009) software, setting the maximum number of motifs to 10, the motif length range from 6-50 amino acids, and other parameters as default. In addition, the conserved structural domains of the S. bicolor CBL gene family were predicted by NCBI-CDD. Finally, the gene structures, conserved motifs, and conserved structural domains of S. bicolor CBL gene family members were comprehensively analyzed using TBtools software (Chen et al., 2023).

The amino acid sequences of S. bicolor CBL gene family members and CBL protein sequences of A. thaliana, O. sativa, Z. mays, Solanum lycopersicum (S. lycopersicum) and Setaria italica (S. italica) were subjected to multiple sequence comparison using MEGA 7.0 software (Kumar et al., 2016). Neighbor-joining (NJ) was then used to construct the phylogenetic tree. In the parameter settings, the number of bootstrap tests was 1000, and the Poisson correction model was selected to calculate the genetic distance. Finally, the evolutionary tree was embellished by iTOL (https://itol.embl.de/) online website to show the evolutionary relationship between S. bicolor CBL gene family and members of this gene family in other plants, and to analyze the evolutionary pattern and classification of the gene family.

Tandem and genome-wide replication events of the S. bicolor CBL gene family were analyzed using MCScan X (Wang et al., 2012). The Simple Ka/Ks Calculator module in TBtools software was used to input the coding sequences (CDS), protein sequences, and immediately homologous gene pairs of the genes, respectively, and the Ka (non-synonymous substitution rate)/Ks (synonymous substitution rate) values between homologous genes were calculated to estimate the selection pressure. In addition, MCScan X (Wang et al., 2012) was applied to analyze the covariation events between S. bicolor and A. thaliana, S. lycopersicum, O. sativa, and S. italica CBL gene families.

Transcriptome data of different tissues of S. bicolor (including seedlings, leaves, roots, stems, inflorescences, and seeds, etc.) at different developmental stages were obtained from the S. bicolor Genome and Mutant Bank SGMD database (Chen et al., 2025). Then, we utilized Tbtools software to analyze gene expression data from various tissues, screening for CBL genes that exhibited high expression levels across different tissues and developmental stages. The data are shown in a heatmap with gene expression in different tissues with row-scaled transcriptome atlas (TPM values). Red and blue boxes indicate high and low expression levels of SbCBL genes.

S. bicolor was grown in a growth chamber at Yan’an University with 16.0 hours of light, temperature maintained at 25°C and 70% humidity. To determine the expression level of CBL gene after NaCl and PEG treatments, S. bicolor seedlings at the three-leaf-one-heart stage were selected and treated with 150 mM NaCl and 15% PEG, respectively, followed by collection of S. bicolor root samples 7 days. All experiments were performed in three biological replicates (Three biological replicates and three technical replicates per biological sample were performed.). Total RNA was isolated using the Plant Total RNA Kit from Beijing Zhuangmeng International BioGenetics Co. Ltd. and reverse transcription was performed using HiScript IV All-in-One Ultra RT SuperMix for qPCR from Novozymes. The RT-qPCR amplification reaction system consisted of 5 μL 2×SYBR, 3 μL ddH₂O, 1 μL cDNA template, and 0.5 μM forward and reverse primers in a total volume of 10 μL. The expression level of SbCBL gene was analyzed by the 2^-ΔΔCT method in response to different stress treatments (Xue et al., 2024), and SbACTIN was used as an internal reference gene to analyze the expression level of SbCBL gene under different stress treatments.

Using HMMER 3.3.2 software based on the Hidden Markov Model of the CBL conservative domain (PF04833) in the Pfam database, we searched and validated S. bicolor protein sequences using NCBI-CDD. Finally, we identified ten CBL gene family members. These genes were named SbCBL1-SbCBL10 and SbECBL1-SbECBL10 based on their location on the chromosomes. The distribution of these 20 genes on the S. bicolor chromosomes was visualized using TBtools software, and the results are shown in Figure 1 . S. bicolor CBL gene family members were unevenly distributed across three chromosomes: five genes (SbCBL1-SbCBL5) were found on chromosome 1; four genes were found on chromosome 2; and one gene was found on chromosome 6. This uneven distribution pattern may be related to the evolution of gene families, chromosome structure, and gene function.

This study systematically analyzed the amino acid length, molecular weight, isoelectric point, instability index, and hydrophilicity of these members. The results showed that the lengths of the amino acid sequences encoded by the S. bicolor CBL gene family members differed significantly. For instance, SbCBL1 encodes 187 amino acids, whereas SbCBL7 encodes a protein consisting of 673 amino acids ( Figure 2A ). Additionally, the amino acid lengths of CBL homologous genes differed between the two S. bicolor varieties. For instance, SbCBL9 corresponds to SbECBL9, which has 446 and 686 amino acids, respectively. This difference in length may reflect the functional complexity of different members. In terms of molecular weight, members of the S. bicolor CBL gene family exhibited similar diversity. The results showed that family proteins have a wide range of molecular weights. For example, SbCBL1 has a molecular weight of 46.832 kDa, while SbCBL7 has a molecular weight as high as 74.62 kDa. There are also members with a predicted molecular weight of about 36.72 kDa ( Figure 2B ). This difference in molecular weight may be closely related to the structural and functional diversity of the proteins. Notably, 70% of the SbCBL gene family members in Sorghum bicolor exhibit an isoelectric point (pI) exceeding 7. This prevalence of basic pI values underscores the potential functional adaptations of these proteins for operating in alkaline cellular environments or interacting with negatively charged macromolecules ( Figure 2C ). Additionally, 70% of SbCBL proteins were predicted to be unstable (PI ≥ 40), while 30% were stable ( Figure 2D ). This result establishes a foundation for the subsequent in-depth study of this family’s protein functions. Through computational predictions of subcellular localization, all members of the SbCBL protein family exhibited exclusive targeting to the plasma membrane, indicating a highly conserved localization pattern. This striking uniformity in membrane association strongly suggests that SbCOBL proteins may play specialized roles in cell wall-plasma membrane interactions, potentially regulating cellulose deposition patterns or mediating mechanical stress responses at the cell surface.

To resolve the evolutionary classification of the S. bicolor CBL gene family, this study used MEGA 7.0 software to conduct a phylogenetic analysis of the amino acid sequences of 10 SbCBL and 10 SbECBL members. The results showed that the CBL proteins of the two S. bicolor varieties can be categorized into three subfamilies: Group I, Group II, and Group III ( Figure 3A ). Group I contains 12 members: SbCBL1, SbCBL2, SbCBL3, SbCBL4, SbCBL7, SbCBL8, SbECBL1, SbECBL2, SbECBL3, SbECBL4, SbECBL7, and SbECBL8. Group II includes SbCBL10 and SbECBL10, and Group III includes SbCBL5, SbCBL6, SbCBL9, SbECBL5, SbECBL6, and SbECBL9.

To further elucidate the phylogenetic position of the S. bicolor CBL genes, this study integrated CBL protein sequences from S. bicolor, A. thaliana, S. lycopersicum, O. sativa, S. italica and Z. mays for a multiple comparison analysis ( Figure 3B ). Phylogenetic trees constructed using the neighbor-joining method showed that the CBL genes of all species could be divided into three significant branches ( Figure 4B ). Group I mainly included six SbCBLs, six SbECBLs, five AtCBLs, ten SlCBLs, seven OsCBLs, five ZmCBLs, and six SiCBLs. Group II mainly consisted of three SbCBLs, three SbECBLs, five AtCBLs, five SlCBLs, three OsCBLs, three ZmCBLs, and two SiCBLs. Group III mainly consisted of one SbCBL, one SbECBL, one AtCBL, two SlCBLs, one OsCBL, one ZmCBL, and one SiCBL. The results of the phylogenetic analysis showed that the S. bicolor CBL gene family has evolved to be related to CBL genes from other plants while maintaining its own specificity. ZmBk2L3, a COBRA family protein, functions in the regulation of cell wall dynamics and carbohydrate partitioning (Julius et al., 2021). Phylogenetic analysis revealed that its clustering with SbCBL1/2 and SbECBL1/2 suggests involvement in cell membrane-associated signaling or cell wall modification via similar mechanisms. ZmBk2 maintains the flexibility of plant organs by modulating the lignin-cellulose interaction pattern (Sindhu et al., 2007). Additionally, phylogenetic analyses revealed that ZmBk2 clusters with SbECBL4 and SbCBL4, indicating that the protein may perform similar functions via conserved molecular mechanisms.

A gene structure and conserved Motif analysis of S. bicolor CBL genes revealed significant structural and functional differences. Gene structure revealed notable variations in exon-intron structure among different subgroups. Specifically, members of subgroup I have three to seven exons and exhibit structural diversity. Members of subgroup II, such as SbCBL10 and SbECBL10, have six and four exons, respectively. Notably, subgroup III members have a more concise gene structure with only one or two exons. This diversity in gene structure may be closely related to functional differentiation and the evolutionary history of genes ( Figures 4A, B ). Different exon-intron structures may lead to variations in gene transcription and translation processes, which may affect gene function and expression regulation.

Conserved motif analysis performed by MEME Suite 5.5.3 identified a total of 10 characteristic motifs (Motif1-Motif10). Systematic analysis revealed that all motifs were intact in subgroup I, suggesting that these core elements may collectively maintain the basal biological functions of CBL proteins. Subgroup II exhibited a distinct motif deletion pattern: SbCOBL10 lacked Motif9, and SbECOBL10 lacked Motifs 1 and 9 ( Figure 4C ). Members of the third subgroup contained only Motifs 1, 2, 3, and 4, which were distributed in a manner that may be related to the functional differentiation of gene family members. The distribution of motifs 1, 2, 3, and 4 is specific and may be related to the functional differentiation of gene family members ( Figure 4C ). Analysis of gene structure and conserved motifs revealed that S. bicolor CBL gene family members are structurally conserved yet diverse. These conserved structural features may ensure the gene family’s basic function, while diverse structures provide the basis for the genes’ functional differentiation and evolution, enabling different gene members to play unique roles in S. bicolor growth, development, and environmental adaptation.

To reveal the expansion mechanism of the S. bicolor CBL gene family and its evolutionary constraints, this study systematically analyzed the types of replication events and selection pressures that characterize its members. As shown in the Figure 5 , tandem duplication (TD) events drove the clustered distribution of CBL3 and CBL4. Meanwhile, Whole Genome Duplication (WGD) events contributed to the generation of two paralogous gene pairs: CBL3/CBL7 and CBL5/CBL9.

Further analysis of the ratio of non-synonymous to synonymous substitution rates (Ka/Ks) revealed that the Ka/Ks values of these replication events were significantly less than one (CBL3/CBL7: Ka/Ks = 0.1; CBL5/CBL9: Ka/Ks = 0.27), suggesting that these genes underwent strong purifying selection ( Supplementary Table 2 ). This indicates that these genes experienced strong purifying selection during evolution.

To elucidate the evolutionary trajectory of the CBL gene family in S. bicolor, this study performed whole-genome synteny analysis using MCScan X between S. bicolor and representative species, including the dicot model plant A. thaliana, monocot crops O. sativa and S. italica, as well as the solanaceous crop S. lycopersicum. A total of 3 (S. bicolor–A. thaliana), 10 (S. bicolor–S. lycopersicum), 10 (S. bicolor–O. sativa), and 10 (S. bicolor–S. italica) CBL homologous gene pairs were identified ( Figure 6 ). Notably, O. sativa, S. italica and S. bicolor, as closely related species within the Gramineae family, exhibited significantly more syntenic gene pairs than A. thaliana, indicating that the CBL gene family retained higher genomic structural conservation after monocot–dicot divergence. Further analysis revealed “one-to-many” homologous relationships between certain S. bicolor CBL genes and multiple species. For instance, SbCBL3 showed synteny with both SlCBL1 and SlCBL1 in S. lycopersicum. Similarly, SbCBL3 corresponded to three homologs in S. italica (SiCBL2 and SiCBL7) and two in O. sativa (OsCBL1 and OsCBL6).

To analyze the transcriptional and regulatory features of the S. bicolor CBL gene family, this study systematically analyzed the cis-acting elements in the upstream promoter region (2,000 bp prior to the transcriptional start site) of the gene using the PlantCARE database. The results showed that the CBL gene promoter region contained abundant regulatory elements, mainly categorized into four groups: hormone response, abiotic stress response, light signaling regulation, and growth and development ( Figure 7 ; Supplementary Table 3 ). All members carried abscisic acid (ABA) response elements (ABRE, ACGTG) and methyl jasmonate (MeJA) response elements (TGACG motif, TGACG), indicating that the S. bicolor CBL gene family may be involved in regulating adversity acclimatization through ABA and MeJA signaling pathways. Additionally, some members contained cis-regulatory elements related to growth hormones, salicylic acid, and gibberellin, implying that these members may be involved in multiple stress responses through hormone crosstalk. Abiotic stress response elements included the drought response element MBS (CAACTG), the low temperature response element LTR (CCGAAA), the anaerobic-induced element ARE (AAACCA), and the mechanical damage response element. Members of subgroup III (e.g., SbCBL7/9) showed a notably high frequency of the low-temperature response element LTR (CCGAAA) in the promoter region. This density was significantly higher than that of the other subgroups. This suggests that subgroup III may enhance cold hardiness by activating low-temperature acclimation pathways. Meanwhile, subgroups II and III specifically carried the mechanical damage response element, the WUN motif (AAATTACCT), which may respond to physical stress by regulating the cell wall.

We analyzed the expression patterns of 10 S. bicolor CBL gene family members based on transcriptome data of different S. bicolor tissues (e.g., roots, stems, leaves, flowers, and seeds) at different developmental stages in the SGMD database ( Supplementary Table 4 ). The data are presented in a heatmap illustrating gene expression across various tissues, utilizing a row-scaled transcriptome atlas based on TPM (Transcripts Per Million) values. In this visualization, red boxes signify high expression levels of SbCBL genes, while blue boxes indicate low expression levels. As illustrated in Figure 8 , various CBL genes exhibited significant expression variations across different S. bicolor tissues and developmental stages. In root tissues, the expression levels of SbCBL1, SbCBL5 and SbCBL9 were relatively high, suggesting that these genes play important roles in root growth and development. The normal development of roots is crucial for plant growth and survival because they are an important organ for water and nutrient uptake in plants. These genes may promote root growth and development by regulating cell wall synthesis and modification in root cells, as well as affecting cell elongation and differentiation. In stem tissues, SbCBL2, SbCBL4 and SbCBL8 exhibited high expression levels, suggesting their involvement in stem elongation and thickening processes. Stem growth and development play a key role in supporting the plant and transporting materials. These genes may regulate the arrangement and deposition of cellulose microfilaments in stems, enhancing their mechanical strength to support the plant during growth. SbCBL6, SbCBL7 and SbCBL10 are expressed at significantly higher levels in inflorescence tissues than in other tissues, suggesting that they play important roles in flower development and reproduction.

The expression level of SbCBL3 was significantly higher in the seed grain than in other tissues, suggesting that this gene plays an important role in seed grain development. Because material accumulation and maturation directly affect S. bicolor yield and quality, SbCBL3 may influence seed grain size, shape, and starch accumulation by regulating cell wall synthesis and modification, ultimately affecting S. bicolor yield and quality. Gene expression pattern analysis revealed that members of the S. bicolor CBL gene family exhibit distinct expression patterns in various tissues and developmental stages. This differential expression may be closely related to the functional differentiation of these genes. These gene family members play unique roles in different stages of S. bicolor growth and development, as well as in different tissues. They are involved in plant growth, development, and reproduction by regulating cell wall synthesis and modification.

To explore the expression pattern of the CBL gene in S. bicolor under drought and salt stress, we selected a CBL gene with high expression levels in the roots and performed RT-qPCR analysis. As shown in Figure 9 , the expression levels of several CBL genes changed significantly under NaCl and PEG treatment. Under NaCl treatment, the expression level of SbCBL4 increased, while the expression levels of SbCBL3, SbCBL5, SbCBL7, SbCBL8 and SbCBL9 decreased. Conversely, PEG treatment up-regulated the expression of genes such as SbCBL4 and SbCBL8, while down-regulating the expression of SbCBL1, SbCBL2, SbCBL3, SbCBL5 and SbCBL9. Notably, the SbCBL3, SbCBL5 and SbCBL9 genes were down-regulated following both drought and salt stress treatments. These results suggest that the CBL gene in S. bicolor plays a role in the S. bicolor response to salt and drought stress.

In this study, 10 members of the CBL gene family were identified from the S. bicolor BTx623 and E048 genomes, respectively ( Figure 1 ). Gene structure analysis showed that the number of exons of these members ranged from 1-7, and there were differences in exon lengths and intron phases ( Figure 3 ). Through conserved motif analysis, we identified 10 conserved motifs, of which Motif1, Motif2, Motif3 and Motif4 were distributed among all members, indicating that these motifs are important for maintaining the basic structure and function of CBL proteins ( Figure 3 ). The structural features of genes are usually closely related to their functions (Sajjad et al., 2023). The diversity in exon-intron structure of S. bicolor CBL gene family members implies that they may be functionally differentiated. For example, SbCBL2, SbCBL3, SbCBL4, SbCBL7 and SbCBL8, which have a higher number of exons, may encode proteins with more complex structures and functions, which are involved in the regulation of multiple processes in S. bicolor growth and development, whereas SbCBL5, SbCBL6 and SbCBL9, which have a relatively low number of exons, may encode proteins that are simpler. This phenomenon has been observed in other species (Sajjad et al., 2023). Based on gene structure and conserved motif analyses combined with existing research reports, it is hypothesized that members of the S. bicolor CBL gene family are involved in several aspects of S. bicolor growth and development. However, these functional speculations require further experimental validation. Future studies could knock out or overexpress members of the S. bicolor CBL gene family using gene editing techniques, such as CRISPR-Cas9, to observe their effects on growth and development, cell wall structure, and related physiological processes. Simultaneously, proteomics and biochemical methods will be used to thoroughly study the interaction mechanism between CBL proteins and other molecules and clarify their specific functions and pathways of action within the cell.

In this study, we identified a total of 10 CBL genes in S. bicolor, which aligns closely with the number of genes in this family reported in other monocotyledonous plants. For instance, O. sativa has 11 CBL genes (Li et al., 2003), Z. mays has 9 (Brady et al., 2007). In contrast, among dicotyledonous plants, A. thaliana possesses 12 CBL genes (Roudier et al., 2002), S. lycopersicum has 17 (Cao et al., 2012), P. trichocarpa 14 (Sajjad et al., 2023), and G. hirsutum has 39. This indicates a significant variation in the number of CBL family genes across different plant species.This suggests that the CBL gene family is highly conserved across monocotyledonous. Phylogenetic tree analysis revealed that the CBL genes of S. bicolor are interspersed with those of O. sativa and Z. mays, forming several small sub-branches ( Figure 4 ). This indicates that S. bicolor is evolutionarily related to O. sativa and Z. mays in the CBL gene family and may share some functions with them. For instance, SbCOBL5, SbCOBL9, SbECOBL5, and SbECOBL9 clustered with OsBC1L1 and OsBC1L8, as well as ZmBK2L1 and ZmBK2L8, forming a sub-branch with some maize CBL genes ( Figure 4 ).

By comparing the conserved motifs of CBL genes in different species, it was found that several species have Motif1, Motif2, Motif3, and Motif4 ( Figure 3 ). These motifs are found in CBL proteins from S. bicolor, A. thaliana, O. sativa and Z. mays. This suggests that they play important roles in the basic functions of the CBL gene family. They may be involved in the interaction of CBL proteins with other molecules or in maintaining the proteins’ structural stability. A comparative analysis of the CBL gene family with CBL gene families of other species reveals the evolutionary conservation and specificity of the S. bicolor CBL gene family. This conservation is reflected in similarities with other plants in terms of gene structure, conserved motifs, and evolutionary relationships. These similarities provide important clues for a deeper understanding of the CBL gene family’s basic functions.

Gene expression pattern analysis revealed that members of the S. bicolor CBL gene family exhibit distinct expression patterns in various tissues and developmental stages ( Figure 8 ). Root growth and development is a complex process involving cell division, elongation, and differentiation, and CBL genes may affect cell morphogenesis and physiological functions by regulating cell wall synthesis and modification in root cells (Shao et al., 2020). In A. thaliana and O. sativa, it has been shown that CBL genes regulate the initiation and growth of root hairs (Roudier et al., 2005; Ben et al., 2015; Li et al., 2022). In root tissues, the high expression of SbCBL1, SbCBL5 and SbCBL9 may regulate root growth and development ( Figure 8 ). For instance, these genes may regulate the orientation of cellulose microfilaments, affecting root cell elongation and differentiation. The highly expressed CBL genes in S. bicolor root tissues may have similar functions and play an important role in regulating root growth and development.

SbCBL2, SbCBL4, and SbCBL8 are highly expressed in stem tissues ( Figure 8 ). The mechanical strength of stems is crucial for upright growth and material transportation, as they are an important support structure in plants (Zhao et al., 2020; Zhang et al., 2022). SbCBL2, SbCBL4, and SbCBL8 may enhance cell wall strength and toughness by regulating cellulose microfilament arrangement and deposition in the stem, thus promoting elongation and thickening. Phylogenetic analyses confirmed this hypothesis, showing that ZmBk2 maintains plant organ flexibility by regulating the interaction between lignin and cellulose (Sindhu et al., 2007). ZmBk2 clusters with SbECBL4 and SbCBL4, suggesting that it may perform similar functions through conserved molecular mechanisms. Different SbCBL genes showed differential expression patterns under drought and salt stress conditions ( Figure 9 ). Specifically, NaCl treatment significantly increased the expression level of SbCBL4 while decreasing the expression of SbCBL3, SbCBL5, SbCBL7, SbCBL8 and SbCBL9. Conversely PEG treatment increased the expression of SbCBL4 and SbCBL8 while decreasing the expression of SbCBL1, SbCBL2, SbCBL3, SbCBL5, and SbCBL9. Analysis of the cis-acting elements of the SbCBL promoters revealed that SbCBL5, SbCBL7, SbCBL8 and SbCBL9 contain regulatory elements related to drought response. These results suggest that the SbCBL gene family plays a critical role in sorghum’s response to drought and salt stress. This role has also been confirmed in other plants, such as rice and cotton (Sun et al., 2022; Fu et al., 2024). This finding is consistent with previous research indicating that CBL genes play important roles in drought and salt tolerance in other species. These findings provide important clues for understanding the molecular regulatory mechanisms of S. bicolor growth and development and potential gene targets for genetic improvement and molecular breeding of S. bicolor. Future studies can explore these genes’ specific mechanisms during S. bicolor growth and development through gene function validation experiments. This will provide stronger theoretical support for improving S. bicolor varieties and agricultural production.

In this study, a more comprehensive bioinformatics analysis of the CBL gene family of S. bicolor was conducted. However, certain limitations still exist. First, regarding gene function validation, this study only analyzed gene structure, evolution, and expression patterns using bioinformatics methods. Thus, experimental validation of gene functions has not yet been carried out. Although potential gene functions were hypothesized based on gene structure and expression patterns, these hypotheses must be verified using techniques such as gene editing and transgenesis. Second, due to limitations in experimental conditions and technical capabilities, this study only used transcriptomic data for gene expression analysis, lacking protein-level validation. It is important to note that gene expression ultimately reflects protein expression; however, transcriptome data only reflects changes in the transcription level of genes, which does not accurately reflect protein expression. Future research can be carried out in the following directions: 1. To verify gene function, use CRISPR-Cas9 and other gene-editing technologies to construct S. bicolor CBL gene family knockout mutants and overexpression plants. Through phenotyping and analyzing physiological and biochemical indexes, study the specific functions and mechanisms of the genes in S. bicolor growth, development, and response to adverse stress. 2. Combine proteome data with transcriptome data to study protein expression. Using proteomics technology, we will analyze protein expression in different S. bicolor tissues and developmental stages. This will allow us to verify the relationship between gene and protein expression and further clarify gene function.3. We will study the interactions between members of the CBL gene family and other genes. Then, we will construct a gene regulatory network to help us understand the molecular regulatory mechanisms of S. bicolor growth, development, and response to adversity.