Use of a melon cmSOS2 related gene in enhancing plant resistance to salt stress

Abstract

The application provides application of a melon CmSOS2 related gene in enhancing plant salt stress resistance, and relates to the technical field of plant genetic engineering. The melon CmSOS2 gene family is identified for the first time in a system, and it is found that the melon CmSOS2 related gene plays a key role in salt stress response. Experiments show that under salt stress conditions, overexpression of the above melon CmSOS2 related gene can significantly improve melon root growth, maintain root cell structure integrity, and effectively regulate ion homeostasis by increasing K+ content, reducing Na+ content and Na+ / K+ ratio in the root, thereby enhancing melon salt tolerance. The application also provides a q-PCR primer pair for detecting the expression level of the gene family and a method for enhancing the salt stress resistance of melon. The above gene and primer can provide important gene resources and identification tools for melon salt-tolerant molecular breeding and salt-tolerant cultivation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of a melon CmSOS2-related gene in enhancing plant salt stress resistance. Background Technology

[0002] Muskmelon (Cucumis melo L.) belongs to the Cucurbitaceae family and is an important economically valuable fruit and vegetable crop, widely cultivated due to its rich content of various vitamins and minerals. However, with the large-scale application of protected cultivation techniques, long-term high-density planting and unreasonable irrigation measures have led to the continuous accumulation of soil salts. Salt stress causes chlorosis in muskmelon leaves, reduces chlorophyll content, induces stomatal closure, and decreases net photosynthetic rate, thereby hindering water absorption. Excessive sodium... + and Cl - The accumulation of ions disrupts intracellular ion balance, inhibits the activity of key metabolic enzymes, further hinders the synthesis and accumulation of photosynthetic products, and ultimately affects melon growth and fruit development. Therefore, a deeper understanding of the molecular mechanisms by which melons respond to salt stress, especially the identification of salt tolerance-related signaling pathways and key genes, is of great significance for enhancing melon salt tolerance, ensuring stable production, and expanding cultivation in saline-alkali land.

[0003] Under high-salt conditions, when Na+ in the cytoplasm... + When the concentration rises to toxic levels, plants will activate Na+. + / H + The reverse transport system will transfer Na + They exit the cell or isolate themselves into the vacuoles, thereby maintaining lower cytoplasmic Na+ levels. + The process relies primarily on the synergistic action of salt-overly sensitive (SOS) proteins located on the plasma membrane and NHX family proteins located on the vacuolar membrane. Its regulatory center depends on the highly conserved SOS signaling pathway. In the signaling network of plant responses to salt stress, the SOS pathway is currently the most clearly understood and functionally defined ion homeostasis regulation system. This pathway is mediated by SOS1 (plasma membrane Na+). + / H + The cellular Na+ system is centered around three key proteins: SOS2 (serine / threonine protein kinase) and SOS3 (calcium receptor protein), and is finely regulated by cofactors such as SOS4 and SOS5, working together to maintain cellular Na+. + / K + Homeostasis. SOS2, as a key core kinase in this pathway, senses Ca2+. + It has the dual function of signaling and transducing downstream. Its C-terminus contains a FISL motif, enabling it to... +Upon signal activation, it can bind to SOS3 to form an SOS3-SOS2 complex, which subsequently phosphorylates and activates SOS1, promoting Na+ activation. + External discharge. Furthermore, SOS2 can enhance the vacuolar membrane H... + -ATPase and NHX-type Na + / H + The activity of antitransporters promotes Na+ transport. + It enters the vacuole through compartmentalization. In addition to regulating ion homeostasis, SOS2 can also activate antioxidant factors such as NDPK2, CAT2, and CAT3, enhancing the ability to scavenge reactive oxygen species (ROS), thereby reducing oxidative damage caused by salt stress.

[0004] Recent studies have shown that PKS5 in Arabidopsis thaliana negatively regulates the SOS signaling pathway. The transcriptional repressor SSN1 regulates the degradation of the SOS2-PIF4 complex through liquid-liquid phase separation, forming "saltbodies." The ESCRT-III component FYVE4 positively regulates salt tolerance by enhancing the SOS1-SOS2 interaction. SOS2 can also optimize salt stress response by activating AMT1;1 to maintain ammonium uptake. In crops, SOS2 in tomato (Solanum lycopersicum) regulates root Na+... + External discharge, xylem Na + Loading and vacuolar compartmentalization enhance salt tolerance. Furthermore, SOS homologs have been identified in species such as rice (Oryza sativa L.), wheat (Triticum aestivum L.), and sunflower (Helianthus annuus L.), indicating that this pathway is functionally conserved within the plant kingdom. However, compared to model plants and major food crops, research on the genomic characteristics, evolutionary patterns, and expression patterns of the SOS2 family in cucurbitaceous crops, particularly melons, remains weak. Therefore, investigating and exploring salt-tolerance-related genes in melons and applying them to the breeding of melons with salt tolerance is a problem to be solved in this field.

[0005] Therefore, this invention is proposed to solve the above-mentioned technical problems. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide the application of members of the CmSOS2 gene family in regulating the salt stress resistance of plants (especially melons). This invention is the first to systematically identify the melon SOS2 gene family and discover that the SOS2 gene family plays a key role in the salt stress response. Experiments show that under salt stress conditions, overexpression of the CmSOS2 gene family significantly improves melon root growth, effectively maintains the integrity of root cell structure, and regulates ion homeostasis by increasing K⁺ content, decreasing Na⁺ content, and the Na⁺ / K⁺ ratio in roots, thereby enhancing the salt tolerance of melons.

[0007] The purpose of this invention is to provide a method for enhancing the salt stress resistance of melons by transforming biological materials containing salt stress resistance genes into recipient melon materials for use in salt-alkali resistant cultivation or salt-tolerant variety breeding of melons.

[0008] The present invention also aims to provide an application of q-PCR primers in the identification of salt stress resistance in melons. By detecting the expression level of the CmSOS2 gene family members of melons under salt stress conditions, the resistance of melon plants to salt stress can be rapidly and accurately identified.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides an application of the melon CmSOS2 gene family in enhancing plant salt stress resistance. The melon CmSOS2 gene family includes the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, and MELO3C007208 gene. The CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.1, the CDS nucleotide sequence of the MELO3C010334 gene is shown in SEQ ID NO.2, the CDS nucleotide sequence of the MELO3C011108 gene is shown in SEQ ID NO.3, and the CDS nucleotide sequence of the MELO3C007208 gene is shown in SEQ ID NO.4.

[0011] The present invention also provides an application of the protein encoded by the CmSOS2-related gene in melon in enhancing plant salt stress resistance.

[0012] Preferably, under salt stress conditions, the plant's resistance to salt stress is improved by overexpressing the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene.

[0013] Preferably, the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene can improve root growth and increase total root length and root surface area under salt stress conditions.

[0014] Preferably, the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene can regulate ion homeostasis under salt stress conditions, increasing K⁺ content in roots, decreasing Na⁺ content, and decreasing the Na⁺ / K⁺ ratio.

[0015] Preferably, the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene can maintain the structural integrity of root cells under salt stress conditions.

[0016] Preferably, the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene participates in stress response signaling pathways mediated by abscisic acid (ABA), salicylic acid (SA), or methyl jasmonate (MeJA).

[0017] This invention also provides an application of q-PCR primers in the identification of salt stress resistance in melons. The q-PCR primers include four primer pairs with sequences shown in SEQ ID NO. 5~12. The primer pairs are used to amplify the MELO3C006758, MELO3C010334, MELO3C011108 and MELO3C007208 genes, respectively.

[0018] This invention also provides a method for enhancing the salt stress resistance of melons, wherein biological material containing a salt stress resistance gene is transformed into recipient melon material, thereby overexpressing the salt stress resistance gene in the melon. The salt stress resistance gene is either the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene. The CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.1, the CDS nucleotide sequence of the MELO3C010334 gene is shown in SEQ ID NO.2, the CDS nucleotide sequence of the MELO3C011108 gene is shown in SEQ ID NO.3, and the CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.4.

[0019] Preferably, the biological material comprises any one of the following: A1, a recombinant expression vector containing the gene; A2, a recombinant bacterium containing the gene; A3, a recombinant bacterium containing the recombinant expression vector of A1; A4, a transgenic plant cell or transgenic plant tissue containing the gene; A5, a transgenic plant cell or transgenic plant tissue containing the recombinant expression vector of A1.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides the first systematic identification of the CmSOS2 gene family in melon and reveals the key role of its members in responding to salt stress. Experimental results show that under salt stress, overexpression of SOS2 gene family members significantly improves melon root growth, effectively maintains root cell structural integrity, and regulates ion homeostasis by increasing K⁺ content, decreasing Na⁺ content, and the Na⁺ / K⁺ ratio, thereby enhancing the salt tolerance of melon. Currently, there are no reports on the function of overexpressing the melon CmSOS2 gene in improving salt tolerance in melon. Furthermore, the q-PCR primers of this invention can rapidly and accurately detect the expression level of the melon CmSOS2 gene under salt stress, providing a reliable molecular detection tool for identifying the salt stress resistance of melon. Therefore, this invention provides important gene resources and technical means for the breeding of salt-tolerant melon varieties and salt-alkali tolerant cultivation, and has significant application value and promotion prospects. Attached Figure Description

[0022] Figure 1 This provides genome-wide information related to the SOS2 gene family in melon, including its chromosome distribution, conserved domains, and gene structure analysis diagrams.

[0023] Figure 2 Phylogenetic relationship and conserved motif composition analysis of SOS2 amino acid sequences in multiple species;

[0024] Figure 3 Diagram showing the sequence conservation, protein tertiary structure, and conserved motifs of the SOS2 gene family in melon.

[0025] Figure 4 Diagram showing interspecific collinearity, promoter cis-regulatory elements, and protein-protein interaction networks of the SOS2 gene family in melon;

[0026] Figure 5 A diagram illustrating the expression patterns of the SOS2 gene family in melon;

[0027] Figure 6 Functional analysis diagram of MELO3C007208 and MELO3C010334 genes in melon under salt stress. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments can be purchased through conventional commercial channels unless otherwise specified.

[0030] To further elucidate the function of the CmSOS2 family of genes in melon and promote the application of the SOS2 gene family in melon salt tolerance research and molecular breeding, this invention systematically identified and analyzed the SOS2 family. Bioinformatics analysis was conducted on its gene structure, conserved motifs, physicochemical properties of encoded proteins, chromosome distribution, phylogenetic relationships, and promoter cis-regulatory elements. Using public databases and transcriptome expression data, combined with real-time quantitative PCR (qRT-PCR), the tissue-specific expression pattern of the melon CmSOS2 gene and its response pattern under salt stress and treatment with various hormones (ABA, SA, MeJA) were analyzed. Genes with significant expression responses were screened, and their overexpression vectors were constructed. Functional verification was performed using a melon hairy root transformation system. By comparing the phenotypes, root structure, ion content (Na⁺, K⁺), and physiological indicators of overexpressing plants and controls under salt stress, the function of the CmSOS2 gene in regulating ion homeostasis and enhancing melon salt tolerance was systematically evaluated. This invention provides a comprehensive analysis of the molecular characteristics and expression regulation mechanisms of the SOS2 gene family in melon, offering important genetic resources and theoretical basis for a deeper understanding of the salt-tolerant molecular network in melon and for breeding new salt-tolerant melon varieties.

[0031] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0032] Example 1

[0033] Identification of the CmSOS2 gene family and analysis of its protein physicochemical properties

[0034] The whole genome sequence, gene annotation files, and protein sequences of melon were obtained from the CuGenDB database (http: / / cucurbitgenomics.org / ). The Hidden Markov Model (HMM) spectrum of the NAF domain (PF03822) was downloaded from the Pfam database (http: / / pfam.xfam.org). Using the local HMMER software, the sequence was analyzed with an E-value threshold of 1e. -15Candidate proteins containing the SOS2 domain were screened, and 20 melon CmSOS2 genes (named MELO3C) were preliminarily identified. The conserved domains were further validated using the SMART online software (https: / / smart.embl.de / ). The amino acid count, molecular weight, and theoretical isoelectric point of the proteins were analyzed using the ExPASyProtParam online software (https: / / web.expasy.org / protparam / ), and subcellular localization was predicted using Cell-PLoc2.0 (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ) (as shown in Table 1). Gene structure was predicted using the SoftBerry platform (http: / / www.softberry.com / ).

[0035] The results showed that these proteins had amino acid lengths ranging from 365 to 497 and molecular weights (Mw) varying from 41.02 to 56.11 kDa, exhibiting considerable variability. The theoretical isoelectric point (pI) ranged from 5.60 to 9.25, indicating that these proteins may exhibit different charge states under different pH conditions. The instability index (Ii) values ​​ranged from 28.96 to 48.85. Among them, MELO3C026055 (Ii=28.96) was predicted to be highly stable, while MELO3C010334 (Ii=48.85) was classified as unstable. The aliphatic index was generally high (77.29–93.99), indicating good thermal stability of the SOS2 protein family. All members exhibited negative average hydrophobicity coefficients (GRAVY), ranging from -0.54 to -0.16, confirming their hydrophilicity and suggesting potential involvement in cytoplasmic and membrane-related signal transduction processes.

[0036] Subcellular localization prediction revealed that SOS2 proteins are widely distributed across multiple compartments, including the cytoplasm (MELO3C002661, MELO3C011108), chloroplasts (MELO3C002766, MELO3C007208), nucleus (MELO3C005987, MELO3C006758), endoplasmic reticulum (MELO3C010234), plasma membrane (MELO3C006483), and cytoskeleton (MELO3C026055). This diverse subcellular distribution suggests that the SOS2 gene family may be involved in regulating various biological processes, such as environmental sensing, ion homeostasis, and stress response. In summary, the SOS2 gene family in melon exhibits significant diversity in terms of Mw, pI, and subcellular localization.

[0037] Table 1. Physiological and biochemical characteristics of SOS2 family members identified in the melon genome.

[0038]

[0039] The CDS sequence of the MELO3C006758 gene is shown in SEQ ID NO.1:

[0040]

[0041] The CDS sequence of the MELO3C010334 gene is shown in SEQ ID NO.2:

[0042]

[0043] The CDS sequence of the MELO3C011108 gene is shown in SEQ ID NO.3:

[0044]

[0045] The CDS sequence of the MELO3C007208 gene is shown in SEQ ID NO.4:

[0046]

[0047] Example 2

[0048] Chromosomal localization analysis and structural analysis

[0049] Based on the CmSOS2 gene family information obtained in Example 1 and the mustard genome annotation information, the chromosomal location of the CmSOS2 gene was determined using the melon genome database. The MG2C software (https: / / qiaoyundeng.github.io / ) was used to draw a chromosomal mapping map of the melon CmSOS2 family members. The chromosomal locations are shown below. Figure 1 As shown in Figure A (scale bar is 5 Mb), a phylogenetic tree was constructed using the NJ method (neighbor-joining method) based on the amino acid sequences of this family members to clarify their evolutionary grouping. Furthermore, gene structure was analyzed using the SoftBerry platform (http: / / www.softberry.com / ), and the results are as follows: Figure 1 B (The figure shows, from left to right, the phylogenetic tree, conserved domains, and gene structure of the melon CmSOS2 gene family. Green represents the pkinase domain, yellow represents the NAF domain, and red represents the APH domain. The gray box represents the UTR region, the cyan box represents the exon, and the black line represents the intron.)

[0050] The results showed that these CmSOS2 genes were unevenly distributed across nine chromosomes (Chr2, Chr3, Chr4, Chr5, Chr6, Chr7, Chr8, Chr11, and Chr12). Notably, Chr8 contained the most SOS2 genes, while Chr2 and Chr3 each carried only 1-2 genes. The absence of SOS2 genes on Chr1, Chr9, or Chr10 indicates uneven evolutionary expansion or loss of this gene family within the melon genome. Phylogenetic analysis based on amino acid sequences and using the NJ method divided the CmSOS2 gene family into four distinct branches. All members contain a typical pkinase domain, crucial for kinase activity and participation in phosphorylation cascades. Most members also possess a NAF domain, known to mediate interactions with SOS3-like calcium receptors and play a key role in responses to abiotic stresses such as salt stress. In addition, several genes (MELO3C014269 and MELO3C007208) contain APH domains, suggesting possible functional specialization in protein-protein interaction networks or specific stress adaptations. Gene structure analysis shows that members within the same phylogenetic branch are highly conserved in exon-intron organization. Figure 1(B) For example, genes in branch I (MELO3C026055 and MELO3C026873) exhibit similar exon numbers and lengths, indicating conservation of transcriptional and translational regulation. In contrast, significant structural differences were observed between branches. MELO3C021231 and MELO3C005987 contain more exons and complex intron configurations, which may reflect functional diversity associated with tissue-specific expression or fine stress response regulation.

[0051] Example 3

[0052] Phylogenetic and protein sequence alignment analysis of the SOS2 gene in different plants

[0053] Protein sequences annotated as SOS2 were downloaded from the Arabidopsis (https: / / www.arabidopsis.org / ), cucumber (http: / / cucurbitgenomics.org / ), watermelon (http: / / cucurbitgenomics.org / ), and melon (http: / / cucurbitgenomics.org / ) genome databases. Multiple sequence alignment was performed using MUSCLE in MEGA11, and a phylogenetic tree was constructed using neighbor-joining (NJ) with 1000 replicates. The phylogenetic tree was visualized and annotated using the iTOL online tool (https: / / itol.embl.de / ). Figure 2 Left panel). Use the MEME suite (https: / / meme-suite.org / meme / ) to identify conserved motifs in the SOS2 protein sequence, with the number of motifs set to 15 ( Figure 2 The right panel shows motifs 1-15, each color-coded according to the legend. To investigate the sequence conservation of the melon CmSOS2 gene family, protein secondary structures were aligned using ESPript 3.0 software (https: / / espript.ibcp.fr / ). The three-dimensional structure of the SOS2 protein was obtained in PDB format from the AlphaFold database (https: / / alphafold.ebi.ac.uk / ) and visualized using PyMOL software. Figure 3 ).

[0054] The results showed that the phylogenetic tree was divided into seven distinct branches (I to VII). Notably, most branches contained SOS2 homologs from multiple species. Specifically, branch II included MELO3C004428 and MELO3C010234 from melon, AT4G30960 from Arabidopsis thaliana, and CICG02G017100 and CICG06G012300 from watermelon, indicating strong evolutionary conservation of these genes in their core functions. A total of 15 conserved motifs (motifs 1-15, indicated by boxes of different colors) were identified. The schematic diagram visually illustrates the presence, order, and relative position of these conserved motifs in each gene. The motif composition and distribution pattern showed that all genes in branch I contained core motifs, such as motifs 1, 2, 3, and 5. These motifs are associated with pkinase domains (involved in phosphorylation and downstream target regulation) and NAF regulatory domains (mediating interactions with SOS3-like calcium receptors). This suggests functional conservation of this branch in processes such as ion homeostasis and salt stress response. Most SOS2 proteins contain more than 10 conserved motifs, which are mainly concentrated in the central region of the sequence. This distribution is consistent with the concentrated organization of SOS2 protein functional domains and further supports the reliability of motif analysis.

[0055] Multiple sequence alignment was performed on the identified SOS2 protein amino acid sequence. Figure 3 A represents a multiple sequence alignment of amino acids from members of the CmSOS2 family. The alignment includes several amino acid sequences encoding the CmSOS2 protein. The black shaded areas represent highly conserved residues. Predicted secondary structural elements, including β1 to β8 (β-sheets), α1 to α7 (α-helices), and η1 to η3 (η-helices), are marked above the alignment results, reflecting the conserved sequences and structural motifs among family members. The results show a large number of highly conserved amino acid residues (black shaded areas) among different SOS2 members, with particularly high consistency observed in secondary structural elements such as α-helices (α1-α7), β-sheets (β1-β8), and η-helices (η1-η3). In Arabidopsis thaliana, SOS2 (AT5G35410) is known to interact with the calcium receptor protein SOS3 under salt stress, forming a complex that phosphorylates and activates the plasma membrane Na+ / H+ antiporter SOS1, thereby driving sodium efflux to maintain cellular ion homeostasis. To further analyze the structural basis of this function, the known functional homolog AT5G35410 in Arabidopsis thaliana was selected as a reference, and a three-dimensional structural model based on homology was performed with that of melon MELO3C010334. Figure 3B shows the tertiary structure modeling of the SOS2 protein. Simulated structures of the Arabidopsis reference protein AT5G35410 (blue) and the melon CmSOS2 protein MELO3C010334 (pink) are displayed, with the N-terminus (N-ter) and C-terminus (C-ter) labeled. The results indicate that the two proteins exhibit high spatial conformational similarity, both employing typical protein kinase domain folding, including an N-terminal (N-ter) catalytic core and a C-terminal (C-ter) regulatory region. This structural similarity demonstrates the evolutionary structural conservation of the SOS2 protein and further supports the potential functional conservation between melon SOS2 and Arabidopsis SOS2. Furthermore, conserved motifs within the SOS2 family were predicted using MEME software, and further analysis was performed using Sequencelogo. Figure 3 C). Four core conserved motifs (motifs 1-4) were identified. The height of each amino acid residue represents its degree of conservation at that position, and color distinguishes residue types. The E value on the right highlights the statistical significance of the motifs, reflecting their core sequence attributes and functional importance within the family. Motif 1 contains conserved residues such as "HTTCGTPNVY," corresponding to the ATP-binding and catalytic phosphorylation region within the pkinase domain. Based on these findings, we hypothesize that the melon CmSOS2 gene may play an important biological role in response to salt stress.

[0056] Example 4

[0057] Collinearity, promoter cis-elements, and protein-protein interaction network analysis of the CmSOS2 gene family in melon

[0058] To investigate the evolutionary conservation of the CmSOS2 gene family in melon, systematic collinearity analysis was performed on melon, Arabidopsis thaliana, watermelon, and cucumber using the JCVI software package. Figure 4A shows the collinearity analysis results of the SOS2 gene among melon, Arabidopsis thaliana, watermelon, and cucumber. The chromosomes of melon (Cm01 to Cm12), Arabidopsis thaliana (At01 to At05), watermelon (Cl01 to Cl11), and cucumber (Cs01 to Cs07) are displayed. Colored lines connect collinear SOS2 homologous gene pairs across species, demonstrating the evolutionary conservation and genomic collinearity of this gene family. Furthermore, the collinearity analysis indicates that the CmSOS2 gene is located within a widely conserved genomic block in these four species. This finding supports the conservation of their core functions in plant salt stress responses. Further calculations showed that the Ka / Ks ratios (see Table 2) were all significantly less than 1, indicating that the CmSOS2 gene underwent intense purifying selection during evolution, resulting in a high degree of functional conservation. Although the homology of this gene is high within the Cucurbitaceae family (melon, watermelon, and cucumber), none of the compared Ka / Ks values ​​showed significant signs of positive selection. This also means that the functional stability of this gene family has been maintained, and large-scale adaptive differentiation has not occurred.

[0059] Table 2

[0060]

[0061] Potential cis-regulatory elements in the 2000bp upstream promoter region of the CmSOS2 gene were predicted using the PlantCare database (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ), and element distribution heatmaps were generated using TBtools-II. Figure 4B shows the distribution of cis-regulatory elements in the upstream 2000bp promoter region of the melon CmSOS2 gene. Each row represents the functional category of the cis-regulatory elements, including light response (G-Box, AE-box), hormone response (ABRE corresponds to ABA, TGA-element corresponds to SA, GARE-motif corresponds to GA), stress response (e.g., MBS, LTR), and development regulation (e.g., C-box, A-box, AE-box, GT1-motif, TCC-motif). Each column corresponds to a single CmSOS2 gene. The color intensity and numerical value of the cells represent the abundance of each element type in the promoter region. The figure shows significant differences in the cis-regulatory element composition among different CmSOS2 genes. For example, MELO3C004428 is highly enriched with ABRE elements, suggesting a potential role in ABA-mediated responses. In contrast, MELO3C002661 and MELO3C006758 contain a large number of light-responsive elements, suggesting their potential involvement in photoperiod or photosynthetic-related growth regulation. This diversity of cis-elements provides a transcriptional regulatory basis for the functional differentiation of the SOS2 gene in various biological processes such as growth, development, and stress response.

[0062] Predict the protein-protein interaction network of the CmSOS2 protein using the STRING database (https: / / cn.string-db.org / ). Figure 4 C represents the protein-protein interaction network diagram, further revealing the potential interaction network involving melon SOS2 proteins. Nodes prefixed with MELO3C represent members of the melon SOS2 family, while nodes prefixed with A0A indicate proteins predicted by the database to potentially interact. Edges represent predicted functional associations. In the constructed network, core SOS2 proteins such as MELO3C014269, MELO3C010334, and MELO3C021231 serve as hub nodes. The functional diversity of promoter cis-elements and the complexity of the protein interaction network further support their potential involvement in multifunctional regulation, including abiotic stress responses and growth and development.

[0063] Example 5

[0064] Expression analysis of CmSOS2 in different tissues

[0065] Expression data of the CmSOS2 gene in melon at different tissues and developmental stages (including roots, stems, leaves, flowers, and fruits) were obtained from the MelonetDB database (https: / / melonet-db.dna.affrc.go.jp / ap / top). Based on publicly available transcriptome data, tissue-specific expression heatmaps were generated. Figure 5A is a heatmap showing the expression of members of the SOS2 gene family in melon at different tissues and developmental stages, including callus, seeds at different post-germination days, various parts of seedlings, stems, leaves, flowers, ovaries, fruits at different post-flowering days (DAF), and post-harvest storage. The color gradient from blue to red represents the gene expression values ​​after log10 transformation, illustrating the expression specificity of family members at different tissues and developmental stages. Figure 5 A schematic diagram illustrating the expression levels of the B melon gene MELO3C010334 in different tissues and developmental stages. In vegetative organs, MELO3C010334 and MELO3C021231 showed the highest expression levels in roots and hypocotyls (FPKM>15), indicating their potential roles in ion uptake and transport. MELO3C007208 was specifically highly expressed in leaves, possibly related to calcium signaling in chloroplasts. In reproductive organs, MELO3C006758 was significantly upregulated during early ovary development at day 0 post-flowering (DAF), while MELO3C026055 remained highly expressed during fruit ripening (29-50 DAF), suggesting functional specialization of this family in floral organ differentiation and fruit quality formation. Developmental dynamics analysis showed the most significant expression changes from seed germination to seedling stage, with 11 genes upregulated more than 2-fold. Among them, MELO3C014269 showed the highest induction (4.3-fold). These spatiotemporal expression patterns are consistent with the distribution of tissue-specific cis-elements (CAT-box, A-box) in promoters, reflecting the decisive role of transcriptional regulation in functional partitions.

[0066] Example 6

[0067] 1. Plant materials and experimental treatment

[0068] The experiment used the thick-skinned melon variety 'Yuyuan 28', bred by the Horticulture Research Institute of Henan Academy of Agricultural Sciences. Plump and uniform seeds were selected, soaked, and germinated until the radicle emerged before sowing in seedling trays. At the one-leaf-one-heart stage, seedlings were transplanted into 6cm × 6cm seedling pots filled with a sterilized peat moss substrate mixture and cultured in a light incubator under the following conditions: light intensity 30,000 lx, photoperiod 14h light / 10h dark, temperature 28°C / 18°C, and relative humidity 60%. When the seedlings reached the three-leaf stage, the following abiotic stress treatment was applied: to apply salt stress, the plants were irrigated with NaCl solutions prepared with deionized water at concentrations of 100, 200, and 300 mM. The solution was irrigated until it seeped from the bottom of the pot, ensuring uniform and acute stress was applied to the entire root zone. For hormone treatment, 1 mM salicylic acid (SA), 100 μM abscisic acid (ABA), and 100 μM methyl jasmonate (MeJA) solutions were used. Root and third true leaf samples were collected at 0, 12, 24, and 48 hours after treatment, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent RNA extraction.

[0069] 2. qRT-PCR analysis of the CmSOS2 gene under abiotic stress

[0070] To investigate the abiotic stress response of the CmSOS2 family, four representative genes (MELO3C006758, MELO3C010334, MELO3C011108, and MELO3C007208) were selected for in-depth analysis based on their phylogenetic distribution across different branches, promoter cis-elements, and preliminary stress response expression patterns. Root tissues from different treatments were ground in liquid nitrogen, and total RNA was extracted using a plant total RNA extraction kit (Vazyme Biotech Co., Ltd., China). RNA integrity was verified by agarose gel electrophoresis. cDNA was synthesized using a reverse transcription kit (Vazyme) with 1 μg of total RNA. Primers were designed using Primer 5.0, with amplicon lengths between 150-300 bp (Table 3). CmActin7 was used as an internal control gene. qRT-PCR was performed using SYBR qPCR MasterMix (Vazyme). A 20 μL reaction mixture contained 10 μL MasterMix, 0.5 μL each of forward and reverse primers, 2 μL cDNA, and 7 μL ddH2O. The reaction program was: 95°C pre-denaturation for 30 s; 95°C for 10 s, 60°C for 30 s, for a total of 40 cycles; melting phase: 95°C for 15 s, 60°C for 60 s, 95°C for 15 s. 2 -ΔΔCt The method calculated relative gene expression levels. Three biological replicates were performed, with three technical replicates per replicate. GraphPadPrism 8.0 was used for data analysis and graphing; results are shown below. Figure 5 C Figure 5 D、 Figure 5 E and Figure 5 F.

[0071] Table 3 Gene-specific primer sequences used for quantitative real-time expression analysis

[0072]

[0073] Figure 5 C Figure 5 D、 Figure 5 E and Figure 5F shows the relative expression levels of MELO3C006758 (sequence shown in SEQ ID NO.1), MELO3C010334 (sequence shown in SEQ ID NO.2), MELO3C021108 (sequence shown in SEQ ID NO.3), and MELO3C007208 (sequence shown in SEQ ID NO.4) at different time points (0, 12, 24, and 48 hours) after salt stress treatment, salicylic acid, abscisic acid, and methyl jasmonate treatment. Different lowercase letters above the bars indicate significant differences (P < 0.05) determined by Duncan's multiple comparison test after one-way ANOVA.

[0074] Depend on Figure 5 As shown in Figure C, salt stress rapidly induced the expression of several SOS2 genes. MELO3C010334 exhibited the most significant response, increasing 3.0-fold at 12 hours (P<0.01) and peaking at 24 hours (4.0-fold). MELO3C006758 was upregulated 2.0-fold at 12 hours but significantly decreased at 24 hours. MELO3C007208 showed a sharp increase of 6.0-fold at 12 hours. In contrast, MELO3C011108 did not show significant changes throughout the treatment (P>0.05), indicating functional differentiation of SOS2 genes in the salt stress response.

[0075] Depend on Figure 5 As shown in D, under SA treatment, MELO3C010334 was significantly upregulated by 2.0 times at 12 hours (P<0.05), but rapidly decreased at 24 hours, exhibiting a pattern of rapid activation and decay. MELO3C007208 was also upregulated by 0.9 times at 12 hours (P<0.05), indicating that it may be involved in SA signal transduction.

[0076] Depend on Figure 5 E indicates that ABA treatment can sustainably activate some SOS2 genes. MELO3C006758 reached a 5.0-fold expression peak at 12 hours (P<0.01) and remained at a high level (2.0-fold) at 24 hours. MELO3C010334 was upregulated by 1.5-fold at 12 hours (P<0.05), suggesting that they may play a role in ABA signal transduction.

[0077] Depend on Figure 5As shown in F, both MELO3C010334 and MELO3C007208 exhibited a delayed response to MeJA, peaking at 24 hours with upregulation of 4.0-fold and 3.0-fold, respectively (P<0.01), suggesting their involvement in JA-mediated stress responses. In summary, MELO3C010334 and MELO3C007208 showed significant responses to multiple treatments (including salt, SA, ABA, and MeJA), with particularly strong upregulation under salt stress. This indicates that these two genes may play a crucial role in melon's response to abiotic stress.

[0078] Example 7

[0079] Cloning of CmSOS2 family genes in melon, construction and functional verification of expression vectors.

[0080] 1. CmSOS2 gene cloning

[0081] Based on expression analysis, MELO3C007208 and MELO3C010334 were selected. Homologous recombination primers were designed using PrimerPremier5.0 software based on the complete coding sequences of the two genes (Table 4).

[0082] Table 4. Specific primer sequences for gene cloning

[0083]

[0084] PCR amplification was performed using polymerase with cDNA as a template. The PCR reaction system and conditions were as follows: μL each of forward and reverse primers, μL of polymerase, μL of template cDNA, μL of ddH2O, and a total reaction volume of [missing information]. The PCR reaction was performed according to the following program: 94℃ for 2 min; 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 2 min, for 32 cycles; 72℃ for 7 min, and then isothermal at 4℃. The PCR product was detected by 1% agarose gel electrophoresis. After confirming the result was correct, the PCR product was recovered from the gel using a DNA recovery kit to obtain the target gene fragment.

[0085] 2. Construction of recombinant carriers

[0086] The coding sequences of these two genes were successfully cloned into the pCambia1300-YFP vector after digestion and ligation with KpnI and ScaI restriction endonucleases, thus constructing the recombinant overexpression vectors MELO3C007208-YFP and MELO3C010334-YFP. Figure 6 A).

[0087] 3. Transformation of hairy roots in melon

[0088] The constructed target fusion protein expression vector and control vector were introduced into *Agrobacterium tumefaciens* strain K599 competent cells via freeze-thaw method. Transformants were plated on LB agar containing appropriate antibiotics and incubated upside down at 28°C for 36 h. Single colonies were picked, multiplied in liquid medium, and the cells were harvested. Then, 1 μL of 0.5% acetylsyleugenone was added to the bacterial suspension and vortexed for 30 s. Uniform 5-day-old melon seedlings were selected, and the taproots were aseptically removed on a clean bench. The cut ends were immediately immersed in *Agrobacterium* suspension with an OD600 of 0.8. The inoculated seedlings were carefully transplanted into sterile vermiculite substrate and co-cultured at 30°C in the dark for 2 days, keeping the substrate moist to maintain bacterial activity. After co-culture, the seedlings were transferred to 1 / 2 Hoagland nutrient solution for hydroponics. Subsequently, the seedlings were treated with 200 mM NaCl for 15 days for functional verification. The phenotypic results are as follows: Figure 6 B (Phenotype of melon plants after salt stress treatment: growth status of plants transformed with pCam1300-YFP (empty vector), MELO3C007208-YFP and MELO3C010334-YFP after 15 days of treatment with 200mM NaCl; scale bar = 5cm) and Figure 6 C (Cross-section observation of root tissue. Melon root tissue section after salt stress treatment; scale bar = 25 μm) shows that, as can be seen from the figure, compared with plants carrying the empty vector or overexpressing MELO3C007208-YFP, melon plants overexpressing MELO3C010334-YFP showed significantly better root growth. Root tissue section analysis revealed that under salt stress, root cells in plants carrying the empty vector or overexpressing MELO3C007208-YFP were disordered, while root cells in plants overexpressing MELO3C010334-YFP showed intact structure and no obvious damage. Further quantitative analysis indicated that… Figure 6 D (total root length after 15 days of salt stress) Figure 6 E (total root surface area after 15 days of salt stress): After salt stress, the total root length and total root surface area of ​​plants overexpressing MELO3C010334-YFP were significantly higher than those of the empty vector control and plants overexpressing MELO3C007208-YFP (P<0.01). Relative chlorophyll content (SPAD value) measurements showed no significant difference among the three groups. Figure 6 F). Compared with the empty vector, plants K overexpressing MELO3C007208-YFP... + The content did not change significantly, while Na + The content increased by an average of about 3.3%, Na + / K + The ratio increased by an average of approximately 3.4%. In contrast, plants overexpressing MELO3C010334-YFP showed a decrease in K... +The content increased by 33.7%-71.1%, Na + The content decreased by 21.0%-37.2%, Na + / K + The ratio decreased significantly by 44.7%-62.9%, showing a clear trend towards improved ion stability. (See...) Figure 6 G (determination of Na⁺ content in roots), 6H (determination of K⁺ content in roots), and 6I (statistical analysis of Na⁺ / K⁺ ratio in roots). Data are expressed as mean ± standard deviation. ** indicates extremely significant difference compared with the empty vector group (n=6, P<0.01)). Comprehensive analysis shows that overexpression of MELO3C010334 significantly improved root growth under salt stress and effectively regulated Na⁺ content in roots. + and K + This balance enhances the salt tolerance of melons. These results directly confirm that MELO3C010334 plays a key positive regulatory role in the response of melons to salt stress.

[0089] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of a melon CmSOS2-related gene in enhancing plant salt stress resistance, characterized in that, The melon CmSOS2-related genes include the MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, and MELO3C007208 gene. The CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.1, the CDS nucleotide sequence of the MELO3C010334 gene is shown in SEQ ID NO.2, the CDS nucleotide sequence of the MELO3C011108 gene is shown in SEQ ID NO.3, and the CDS nucleotide sequence of the MELO3C007208 gene is shown in SEQ ID NO.

4.

2. Application of a protein encoded by a melon CmSOS2-related gene in enhancing plant salt stress resistance.

3. The application according to claim 1, characterized in that, Under salt stress conditions, overexpression of the MELO3C006758, MELO3C010334, MELO3C011108, or MELO3C007208 genes can enhance plant resistance to salt stress.

4. The application according to claim 1, characterized in that, The MELO3C006758, MELO3C010334, MELO3C011108, or MELO3C007208 genes can improve root growth and increase total root length and root surface area under salt stress conditions.

5. The application according to claim 1, characterized in that, The MELO3C006758, MELO3C010334, MELO3C011108, or MELO3C007208 genes can regulate ion homeostasis under salt stress, increasing K⁺ content in roots and decreasing Na⁺ content and Na⁺ / K⁺ ratio.

6. The application according to claim 1, characterized in that, The MELO3C006758, MELO3C010334, MELO3C011108, or MELO3C007208 genes can maintain the structural integrity of root cells under salt stress conditions.

7. The application according to claim 1, characterized in that, The MELO3C006758 gene, MELO3C010334 gene, MELO3C011108 gene, or MELO3C007208 gene are involved in stress response signaling pathways mediated by abscisic acid, salicylic acid, or methyl jasmonate.

8. The application of a q-PCR primer in the identification of salt stress resistance in melon, characterized in that, The q-PCR primers include four primer pairs with sequences shown in SEQ ID NO.5~12. These primer pairs are used to amplify the MELO3C006758, MELO3C010334, MELO3C011108, and MELO3C007208 genes, respectively.

9. A method for enhancing the salt stress resistance of melons, characterized in that, Biological materials containing salt-stress resistance genes were transformed into recipient melon materials, thereby overexpressing the salt-stress resistance genes in the melons. The salt-stress resistance genes were MELO3C006758, MELO3C010334, MELO3C011108, or MELO3C007208 genes. The CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.1, the CDS nucleotide sequence of the MELO3C010334 gene is shown in SEQ ID NO.2, the CDS nucleotide sequence of the MELO3C011108 gene is shown in SEQ ID NO.3, and the CDS nucleotide sequence of the MELO3C006758 gene is shown in SEQ ID NO.

4.

10. The method according to claim 9, characterized in that, The biological material includes any one of the following: A1, a recombinant expression vector containing the gene; A2, a recombinant bacterium containing the gene; A3, a recombinant bacterium containing the recombinant expression vector of A1; A4, a transgenic plant cell or transgenic plant tissue containing the gene; A5, a transgenic plant cell or transgenic plant tissue containing the recombinant expression vector of A1.

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