Muskmelon salt-tolerant gene CmDUF1 and application thereof
By overexpressing the CmDUF1 gene in melon, the problem of insufficient salt tolerance in saline-alkali land cultivation was solved, which significantly improved the salt stress tolerance of melon and enhanced its growth performance.
Patent Information
- Application Number
- CN202510702618.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-26
AI Technical Summary
In the prior art, melons are insufficient in salt-tolerant in saline-alkali land cultivation, resulting in a decrease in yield and deterioration in quality, and the key genes and regulatory mechanisms in rootstocks that respond to salt stress and their regulatory mechanisms are unclear.
Through transcriptome analysis and gene function verification, it was found that the melon salt-tolerant gene CmDUF1 was overexpressed in melon through gene editing and overexpression techniques, significantly improving its tolerance to salt stress.
The tolerance of melon to salt stress was enhanced, which was manifested as greener leaves, higher chlorophyll content, more fresh and dry weight in the above ground and roots, significantly reduced MDA content and REC, improving the growth performance of melon under salt stress environment.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular breeding, and particularly relates to a melon salt-tolerance gene CmDUF1 and an application thereof. Background Art
[0002] China is a veritable major producer of greenhouse vegetables, occupying a pivotal position in the global greenhouse vegetable industry. Greenhouse vegetable cultivation offers many advantages, such as effectively resisting the adverse effects of the natural environment, ensuring year-round vegetable supply, and improving land utilization. However, greenhouse vegetable cultivation also faces several pressing challenges, with secondary soil salinization being the most prominent. Due to factors such as long-term, inappropriate irrigation and fertilization during greenhouse cultivation, as well as a lack of natural rainfall, salt accumulates in the soil, increasing soil solution concentration and triggering secondary salinization. This phenomenon is particularly common in areas with dense greenhouse vegetable cultivation and has become a key constraint to the sustainable development of the greenhouse vegetable industry. Grafting is a long-standing and widely used agronomic practice that plays a vital role in agricultural production. It involves grafting a bud or branch from one plant onto another, allowing them to grow together into a complete plant. This technique cleverly leverages the growth characteristics of different plants to achieve numerous beneficial effects.
[0003] Muskmelon (Cucumis melo) is a popular and important cash crop, not only for its sweet taste and rich nutrition, but also for its high economic value, making it a key component of agricultural production in many regions. Rich in nutrients such as vitamin C, vitamin A, B vitamins, minerals, and dietary fiber, it can be eaten fresh or processed into juice, preserved fruit, and other products, making it a popular choice among consumers. However, melon growth faces many challenges, particularly salt stress, which poses a serious threat to melon production. Saline-alkali soils are often a thorny issue in melon cultivation. Excessive salt content in saline-alkali soils can negatively impact melon growth, such as inhibiting root development and hindering nutrient absorption, leading to reduced yield and poor quality. However, grafting techniques and selecting salt-tolerant rootstocks can effectively improve the salt tolerance of melons. Salt-tolerant rootstocks can better maintain their physiological functions in high-salt environments. Their well-developed root systems absorb water and nutrients more efficiently, while also secreting beneficial substances that alleviate salt stress. When melon scions are combined with such rootstocks, the melon plants can leverage the rootstock's salt tolerance to grow better in saline-alkali soils. This not only increases the survival rate of melons in saline-alkali soils, but also significantly improves their yield and quality. This allows melons to be grown in saline-alkali soils that were previously difficult to cultivate, opening up new avenues for increasing agricultural production and income, and is of great practical significance.
[0004] However, the key genes and their regulatory mechanisms in melon rootstocks responding to salt stress are still unclear. Summary of the Invention
[0005] Through transcriptome analysis and gene function verification, the present invention discovered the melon salt-tolerance gene CmDUF1. The nucleotide sequence of its CDS is shown in SEQ ID NO. 1. It encodes a protein containing a DUF (Domain of Unknown Function) domain. In melon, CmDUF1 expression is closely related to salt tolerance and is significantly upregulated under salt stress conditions. Overexpression of this gene can significantly enhance the salt tolerance of grafted melons. This demonstrates that CmDUF1 is a key gene regulating salt tolerance in grafted melons and, through gene editing and overexpression techniques, validates its role in improving salt tolerance in melons.
[0006] Specifically, the present invention provides the use of the melon salt-tolerance gene CmDUF1, the protein encoded thereby, an expression cassette containing the gene, a recombinant vector, a transgenic cell line or a recombinant bacterium in any of the following applications:
[0007] A1) Regulating salt tolerance in melon;
[0008] A2) preparing a product for regulating salt tolerance of muskmelon;
[0009] A3) enhancing the tolerance of melon to salt stress;
[0010] A4) preparing a product for enhancing tolerance of melon to salt stress;
[0011] A5) Cultivate salt-tolerant melon varieties;
[0012] A6) preparing and cultivating salt-tolerant muskmelon varieties;
[0013] The nucleotide sequence of the melon salt-tolerance gene CmDUF1 is shown in SEQ ID NO.1.
[0014] Furthermore, overexpression of the CmDUF1 gene in melon improved tolerance to salt stress.
[0015] Furthermore, a CmDUF1 gene overexpression vector was constructed and introduced into recipient melons to obtain transgenic melon materials.
[0016] Furthermore, the obtained transgenic melon material showed stronger salt stress tolerance.
[0017] Furthermore, stronger salt stress tolerance is reflected in the fact that under the same salt concentration conditions, the leaves of the transgenic melon materials are greener, the chlorophyll content is higher, the fresh weight and dry weight of the aboveground parts and roots are also greater, and the MDA content and REC of the leaves and roots are significantly reduced.
[0018] The present invention also provides a method for enhancing the salt stress tolerance of melon, comprising enhancing, increasing or upregulating the expression level of the CmDUF1 gene in the melon or the function or activity of its protein, wherein the nucleotide sequence of the melon salt-tolerance gene CmDUF1 is shown in SEQ ID NO: 1.
[0019] Furthermore, a CmDUF1 gene overexpression vector was constructed and introduced into recipient melons to obtain transgenic melon materials.
[0020] Furthermore, the obtained transgenic melon material showed stronger salt stress tolerance.
[0021] Furthermore, stronger salt stress tolerance is reflected in the fact that under the same salt concentration conditions, the leaves of the transgenic melon materials are greener, the chlorophyll content is higher, the fresh weight and dry weight of the aboveground parts and roots are also greater, and the MDA content and REC of the leaves and roots are significantly reduced.
[0022] The present invention also provides a method for breeding salt-tolerant melon varieties, which comprises constructing a CmDUF1 gene overexpression vector and introducing it into a recipient melon to obtain a transgenic melon material. The nucleotide sequence of the melon salt-tolerant gene CmDUF1 is shown in SEQ ID NO.1.
[0023] Furthermore, the method comprises the following steps:
[0024] S1. Construction of CmDUF1 gene overexpression vector;
[0025] S2. Introducing the overexpression vector from step S1 into muskmelon plants through Agrobacterium-mediated method, and transplanting them after infection;
[0026] S3. After the seedlings survive, positive seedlings containing the overexpression vector are screened to obtain melon materials overexpressing CmDUF1.
[0027] Furthermore, the obtained transgenic melon material showed stronger salt stress tolerance.
[0028] Furthermore, stronger salt stress tolerance is reflected in the fact that under the same salt concentration conditions, the leaves of the transgenic melon materials are greener, the chlorophyll content is higher, the fresh weight and dry weight of the aboveground parts and roots are also greater, and the MDA content and REC of the leaves and roots are significantly reduced.
[0029] The present invention also provides a method for improving the salt tolerance of melon, comprising overexpressing the CmDUF1 gene in the melon and using the melon overexpressing the CmDUF1 gene as a rootstock for grafting, which can significantly improve the salt tolerance of the scion melon.
[0030] Furthermore, the nucleotide sequence of the CmDUF1 gene is shown in SEQ ID NO.1.
[0031] Furthermore, the expression level of the CmDUF1 gene in the root system of the rootstock was significantly increased, thereby achieving the effect of improving salt tolerance.
[0032] Furthermore, the method for overexpressing the CmDUF1 gene in melon comprises the following steps:
[0033] S1. Construction of CmDUF1 gene overexpression vector;
[0034] S2. Introducing the overexpression vector from step S1 into muskmelon plants through Agrobacterium-mediated method, and transplanting them after infection;
[0035] S3. After the seedlings survive, positive seedlings containing the overexpression vector are screened to obtain melon materials overexpressing CmDUF1.
[0036] The present invention also provides an application of the CmDUF1 gene in improving the salt tolerance of melons, including overexpressing the CmDUF1 gene in the melon and using the melon overexpressing the CmDUF1 gene as a rootstock for grafting, which can significantly improve the salt tolerance of the scion melon.
[0037] Furthermore, the nucleotide sequence of the CmDUF1 gene is shown in SEQ ID NO.1.
[0038] Furthermore, the expression level of the CmDUF1 gene in the root system of the rootstock was significantly increased, thereby achieving the effect of improving salt tolerance.
[0039] Beneficial Effects: The CmDUF1 gene provided by this invention has important agricultural value: through gene overexpression technology, the CmDUF1 gene can be introduced into other melon varieties to improve their salt tolerance, thereby enhancing melon growth under salt stress. In other words, this gene can be used for melon genetic improvement. In addition, the discovery of this gene provides new ideas and targets for salt tolerance research in other crops, and is expected to be applied in a wider range of agricultural fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0041] Figure 1 Effects of grafting on salt tolerance in melon. Phenotypes of self-grafted and grafted melons at 0, 1, 3, and 5 days after treatment with 200 mM NaCl (a). Chlorophyll content (b), shoot fresh weight (c), root fresh weight (d), shoot dry weight (e), root dry weight (f), leaf malondialdehyde (MDA) content (g), root malondialdehyde (MDA) content (h), leaf relative electrical conductivity (REC) (i), and root relative electrical conductivity (REC) (j) of self-grafted and grafted melons at 5 days after treatment with 200 mM NaCl. Different lowercase letters indicate significant differences between treatments (P < 0.05). ST2 / ST2: self-grafted melon with ST2 as rootstock and ST2 as scion. ST2 / XZM17: grafted melon with XZM17 as rootstock and ST2 as scion.
[0042] Figure 2 Principal component analysis (PCA) and differentially expressed genes (DGRs) analysis of root transcriptome data from self-grafted and grafted melons under salt stress. Principal component analysis (PCA) of root transcriptome data from self-grafted and grafted melons after 1 (a), 3 (b), and 5 (c) days of 200 mM NaCl treatment. (d) DGRs analysis of root transcriptome data from self-grafted and grafted melons after 1, 3, and 5 days of 200 mM NaCl treatment. ST2 / ST2: self-grafted melon with ST2 as rootstock and ST2 as scion. ST2 / XZM17: grafted melon with XZM17 as rootstock and ST2 as scion.
[0043] Figure 3 Figure 2. Venn diagram and heat map analysis of differentially expressed genes in the roots of self-grafted and grafted melons under salt stress. Venn diagram analysis of differentially expressed genes in the roots of self-grafted and grafted melons after 1 (a), 3 (b), and 5 (c) days of 200 mM NaCl treatment. Venn diagram analysis of differentially expressed genes in the roots of grafted melons compared to self-grafted melons after 1, 3, and 5 days of 200 mM NaCl treatment (d). Heat map analysis of 34 differentially expressed genes in the roots of grafted melons that responded to salt stress after 1, 3, and 5 days of 200 mM NaCl treatment (e). ST2 / ST2: Self-grafted melon with ST2 as the rootstock and ST2 as the scion. ST2 / XZM17: Grafted melon with XZM17 as the rootstock and ST2 as the scion.
[0044] Figure 4 Quantitative analysis of CmDUF1 gene expression in roots of self-grafted and grafted melons under salt stress. Real-time quantitative PCR (qPCR) analysis of CmDUF1 gene in roots of self-grafted and grafted melons after 1 day (a), 3 days (b), and 5 days (c) of 200 mM NaCl treatment. Different lowercase letters indicate significant differences between treatments (P < 0.05). ST2 / ST2: Self-grafted melon with ST2 as rootstock and ST2 as scion. ST2 / XZM17: Grafted melon with XZM17 as rootstock and ST2 as scion.
[0045] Figure 5 Effects of root knockout and overexpression of the CmDUF1 gene on salt tolerance in grafted melon. (a) CmDUF1 gene editing efficiency in ST2 / KODUF1 roots. (b) Effect of CmDUF1 overexpression in ST2 / OEDUF1 roots. Root gene-edited phenotypes (c), chlorophyll content (d), shoot (e) and root dry weight (f), malondialdehyde (MDA) content in leaves (g) and roots (h), and relative electrical conductivity (REC) in leaves (i) and roots (j) under salt stress. Different lowercase letters indicate significant differences between treatments (P < 0.05). ST2 / EV: grafted melon transformed with an empty vector in its roots; ST2 / KODUF1: grafted melon with CmDUF1 knockout in its roots; ST2 / OEDUF1: grafted melon with CmDUF1 overexpression in its roots. DETAILED DESCRIPTION
[0046] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.
[0047] Example 1 Identification of the melon salt-tolerance gene CmDUF1
[0048] 1.1 Cultivation and salt treatment of grafted melon seedlings
[0049] The melon varieties used in the experiment, 'Shoutian 2' ('ST2') and 'Xizhoumi 17' ('XZM17'), were purchased from Hebei Liertian Seed Co., Ltd. First, the melon seeds were sterilized in 55°C pure water for 15 minutes, followed by soaking at 25°C for 6 hours. The seeds were then placed in a Petri dish (10 cm diameter) lined with three layers of moistened filter paper and placed in a 28°C incubator for germination. After germination, the seeds were sown on a sponge soaked in 1 / 2 Hoagland nutrient solution. When the cotyledons were flat, the melon seedlings were transplanted into a hydroponic cup (90 mm diameter, 57 mm bottom diameter, 135 mm height) and filled with 400 mL of 1 / 2 modified Hoagland nutrient solution. The nutrient solution consisted of 1mM MgSO4·7H2O, 4mM CaCl2, 60mM KNO3, 0.5mM Ca(H2PO4)2·H2O, and 74.93mg / L of solid trace elements (DZPM0059) from Coolaber. After preparation, the pH of the nutrient solution was adjusted to 6.5 using 1M KOH. Melon seedling cultivation conditions were: daytime temperature of 30°C, nighttime temperature of 18°C, and light intensity of 250μmol·m -2 ·s -1 , photoperiod 14h. When the melon seedlings grew to 2 leaves and 1 heart, a nutrient solution without salt was used as the control group, and a nutrient solution supplemented with 200mM NaCl was set as the salt treatment group. Each treatment was repeated 3 times, with 6 plants in each repeat. 5 days after salt treatment, phenotypic photos were taken, and samples were taken to measure growth indicators and damage indicators (malondialdehyde (MDA) content and relative electrical conductivity (REC)). According to the above-mentioned self-rooted melon seedling cultivation method, melon rootstocks (salt-sensitive variety "ST2" and salt-tolerant variety "XZM17") and scion "ST2" were raised. When the rootstock emerged from the soil, germination of the scion began. After the cotyledons of the scion flattened, grafting was performed using the cutting method. After the graft survived, its cultivation conditions were the same as those for self-rooted melon seedlings. When the grafted melons grew to 2 leaves and 1 heart, salt treatment was performed, using the same salt treatment method as for self-rooted melon seedlings. On days 1, 3, and 5 after salt treatment, random samples were taken for phenotypic photography and determination of morphological and physiological parameters. Root samples were also stored at -80°C for subsequent transcriptome sequencing analysis.
[0050] 1.2 Determination of chlorophyll content and biomass
[0051] After 5 days of 200 mM NaCl treatment, the SPAD values of the first true leaf of self-rooted and grafted melon seedlings were measured using a SPAD-502 chlorophyll meter. Biomass was then measured. The fresh weight of the shoots and roots was measured, followed by drying in a 105°C oven for 15 minutes and at 72°C for 72 hours. Finally, the dry weight was measured.
[0052] 1.3 Determination of malondialdehyde (MDA) content and relative conductivity (REC)
[0053] After 5 days of treatment with 200 mM NaCl, malondialdehyde (MDA) content and relative electrical conductivity (REC) were measured in leaves and roots of native and grafted melon seedlings (Niu et al., 2018). MDA content was determined by weighing 0.1 g of fresh leaves or roots, adding 5 mL of 5% trichloroacetic acid, grinding, and centrifuging at 10,000 rpm for 20 minutes. 2 mL of the supernatant was mixed with 2 mL of 0.67% thiobarbituric acid solution and heated in a 100°C water bath for 15 minutes. A solution containing all reagents was used as a blank control, and the absorbance of the samples was measured at 450 nm, 532 nm, and 600 nm. MDA content was calculated using the formula: C (nmol / g) = (6.45(A532 - A600) - 0.56A450) * 5 / 0.1. Relative conductivity (REC): Weigh 0.1 g of plant leaves or roots, rinse three times with distilled water, blot dry with filter paper, place in a 15 mL centrifuge tube, add 5 mL of pure water, and apply vacuum (0.8 MPa) for 20 minutes. Measure the conductivity (R1) of the extract using a conductivity meter. Heat the sample in a boiling water bath for 30 minutes, cool to room temperature, shake well, and measure the conductivity (R2) of the extract again. Relative conductivity = R1 / R2 × 100% (Niu et al. 2018).
[0054] 1.4 Transcriptome analysis
[0055] Transcriptome sequencing was performed on the roots of self-grafted and grafted melon seedlings after treatment with 200 mM NaCl for 1, 3, and 5 days. RNA sequencing was performed by Beijing Qingke Biotechnology Co., Ltd. Genomic data were obtained from the Cucurbit Genomics Database (http: / / cucurbitgenomics.org / ) and aligned using Melon DHL92genome v4. Quality control analysis of the sequencing data revealed that Q20 values for all samples were above 85% and Q30 values were above 80%. Gene expression was calculated using transcripts per million (TPM), and differentially expressed genes were identified and counted using DESeq2 software (Love et al., 2014). Differentially expressed genes were identified using a Log2 (Foldchange value) > 1.0 and a P < 0.05 (Tilak et al., 2023).
[0056] result:
[0057] 1.1 Grafting improves the salt tolerance of melon
[0058] To investigate the effects of grafting on salt tolerance in melons, this study constructed two grafting combinations (ST2 / ST2 and ST2 / XZM17) using the salt-sensitive cultivar 'ST2' and the salt-tolerant cultivar 'XZM17' as rootstocks, respectively, with the salt-sensitive cultivar 'ST2' as the scion. Salt treatment with 200 mM NaCl was then applied. Results showed that, under control conditions, ST2 / XZM17 exhibited superior growth compared to ST2 / ST2 after five days of treatment, with greener leaves, higher chlorophyll content, and greater fresh and dry weights of both the shoots and roots. Under salt treatment, ST2 / XZM17 exhibited superior phenotypes compared to ST2 / ST2, with leaf SPAD values increased by 70.8%, shoot and root fresh weights increased by 86.3% and 143.4%, and shoot and root dry weights increased by 61.7% and 126.9%, respectively. ST2 / XZM17 also suffered less damage, with leaf and root MDA contents decreased by 51.4% and 45.1%, respectively, and leaf and root REC decreased by 44.1% and 36.1%, respectively. Furthermore, under salt stress, compared to the control, leaf SPAD values decreased by 53.3% and 32.1%, shoot fresh weight decreased by 61.3% and 60.5%, root fresh weight decreased by 72.8% and 60.5%, shoot dry weight decreased by 43.8% and 38.1%, and root dry weight decreased by 70.4% and 55.5%, respectively. At the same time, compared with ST2 / ST2, ST2 / XZM17 was less damaged. Compared with the control, under salt stress, the MDA content and REC in the leaves and roots of ST2 / ST2 and ST2 / XZM17 were significantly reduced ( Figure 1 In conclusion, grafting salt-tolerant melon as rootstock can significantly improve the salt tolerance of salt-sensitive melon.
[0059] 1.2 CmDUF1 in the rootstock root system is a key gene for improving salt tolerance in melon grafting
[0060] Given the key role of the root system of the rootstock in determining the salt tolerance of grafted melons, this study further explored the key genes that improve the salt tolerance of melons by grafting. To this end, transcriptome sequencing was performed on the roots of the root system after 1, 3 and 5 days of salt treatment. First, principal component analysis (PCA) was performed on the transcriptome data. The results showed that the three samples of each treatment were all within the 90% confidence ellipse, indicating that the transcriptome data were accurate and reliable. Subsequently, the number of differentially expressed genes was analyzed, and it was found that the number of differentially expressed genes in the roots of the "ST2" rootstock was 5766, 5553 and 7623, respectively, and the number of differentially expressed genes in the roots of the "XZM17" rootstock was 3767, 3557 and 6097, respectively, after 1, 3 and 5 days of salt treatment. Figure 2). Further analysis of these differentially expressed genes using a Venn diagram revealed that 881, 998, and 1,101 genes responded only to salt stress in the "XZM17" root system, but not in the "ST2" root system, at 1, 3, and 5 days of salt treatment, respectively. Taking the intersection of these genes, it was found that only 34 genes responded to salt stress in the "XZM17" root system, but not in the "ST2" root system, at 1, 3, and 5 days of salt treatment. A heat map analysis of the expression levels of these 34 genes showed that MELO3C022991 had the highest expression level, and under salt stress, the expression level in the "XZM17" root system was significantly higher than that in the "ST2" root system. The protein sequence of this gene was compared with that of Arabidopsis thaliana, and it was found to belong to the DUF family, so it was named CmDUF1( Figure 3 Further quantitative polymerase chain reaction (qPCR) results showed that under control conditions, there was no significant difference in the expression of CmDUF1 in the roots of "XZM17" and "ST2". However, after salt treatment for 1, 3, and 5 days, the expression of CmDUF1 in the roots of "XZM17" increased significantly by 3.7, 3.5, and 3.0 times, respectively, compared with "ST2". Figure 4 In summary, CmDUF1 was identified as the key gene for improving salt tolerance of melon by grafting, and its nucleotide sequence is shown in SEQ ID NO.1.
[0061] Example 2 Verification of CmDUF1 gene function
[0062] 2.1 Creation of root knockout and CmDUF1 overexpressing grafted melon materials
[0063] Using "XZM17" melon as the rootstock and "ST2" melon as the scion, grafted melons were constructed with no transformation vector, CmDUF1 gene knockout, and overexpression in the root system. The CmDUF1 (MELO3C022991) overexpression vector and CRISPR / Cas9 vector were constructed according to the method of Geng et al. (An efficient root transformation system for CRISPR / Cas9-based analyses of shoot–root communication in cucurbit crops, 2022). Both vectors are constructed using pBSE403. Construction of the overexpression vector: After digestion with XabI and SacI, the CDS sequence of CmDUF1 was inserted into the pBSE403 vector. Construction of the CRISPR / Cas9 vector: After digestion with BsaI, the sgRNA (AAAAGTTGCAGTGATTTGA) was inserted into the pBSE403 vector. These vectors were used to transform Escherichia coli 5α. Subsequently, Agrobacterium K599 was transformed with the pBSE403 empty plasmid, the pBSE403 plasmid containing the CmDUF1 sgRNA, and the pBSE403 plasmid containing the CmDUF1 CDS according to the K599 manufacturer's instructions (Weidi, Shanghai). Transformed Agrobacterium K599 was used to infect melon plants, which were transplanted 4 days after infection. After the seedlings emerged, roots without red fluorescence were removed every 5 days, while red fluorescent roots were retained. Grafting was performed when the seedlings had one leaf and one heart. After grafting, the plants were protected from light and maintained at 100% humidity for 3 days, followed by gradual ventilation and exposure to light. Before salt treatment, red fluorescent roots of knockout plants were sequenced by Hi-TOM sequencing to determine gene editing efficiency, and red fluorescent roots of overexpressing plants were analyzed by quantitative real-time PCR (qRT-PCR) to verify overexpression. After identification, grafted melon seedlings with CmDUF1 gene knockout or overexpression in their roots were obtained. The seedlings were then transplanted into hydroponic cups for cultivation. When the grafted melon seedlings reached two leaves and one heart, a control group was treated with a nutrient solution containing no salt, and a salt-treated group with a nutrient solution supplemented with 200 mM NaCl. Each treatment was replicated three times, with six plants per replicate. On days 1, 3, and 5 after salt treatment, random root samples were taken for morphological and physiological measurements, and root samples were stored at -80°C.
[0064] 2.2 Effects of root knockout and overexpression of CmDUF1 on the phenotype of grafted melon under salt stress
[0065] To further investigate the effects of the CmDUF1 gene on salt tolerance in grafted melons, this study transformed empty vectors, CmDUF1 gene editing vectors, and CmDUF1 gene overexpression vectors into rootstock roots. The salt-sensitive variety "ST2" was then used as the scion for grafting. This resulted in grafted melons with empty vectors (ST2 / EV), CmDUF1 gene-edited grafted melons (ST2 / KODUF1), and CmDUF1 gene-overexpressed grafted melons (ST2 / OEDUF1) transformed into rootstocks. The CmDUF1 gene editing efficiency in the ST2 / KODUF1 root system was detected using HiTom technology, and the result was 72.1% ( Figure 5 (a)). The overexpression effect of CmDUF1 in the roots of ST2 / OEDUF1 was detected by qPCR. Compared with ST2 / EV, the expression level of CmDUF1 in the roots of ST2 / OEDUF1 was upregulated by 17.3 times ( Figure 5 (b)). Figure 5 As shown in (c)-(j), after salt treatment of these grafted melons, it was found that under normal conditions, ST2 / KODUF1 plants were smaller and had significantly lower aboveground and root dry weights than ST2 / EV, while the opposite was true for ST2 / OEDUF1. Under salt treatment, ST2 / KODUF1 had a worse phenotype than ST2 / EV, with leaf SPAD values reduced by 21.6% ( Figure 5(d)), the aboveground dry weight and root dry weight decreased by 39.6% and 50.9%, respectively, indicating a higher degree of damage, while the malondialdehyde (MDA) content in leaves and roots increased by 41.8% and 95.2%, and the relative electrical conductivity (REC) in leaves and roots increased by 45.8% and 55.2%, respectively; while the phenotype of ST2 / OEDUF1 was better, with the SPAD value increased by 29.6%, the aboveground dry weight and root dry weight increased by 35.6% and 171.3%, respectively, and the degree of damage was lower, with the MDA content in leaves and roots decreased by 44.5% and 56.6%, respectively, and the REC in leaves and roots decreased by 51.8% and 29.6%, respectively. Furthermore, compared with the control, under salt treatment, the leaf SPAD values of ST2 / EV, ST2 / KODUF1, and ST2 / OEDUF1 decreased by 50.9%, 59.4%, and 35.3%, respectively. The shoot dry weight decreased by 55.7%, 64.1%, and 49.0%, respectively, and the root dry weight decreased by 79.9%, 82.8%, and 64.9%, respectively. Leaf MDA content increased by 1.9-, 2.1-, and 0.6-fold, respectively, and root MDA content increased by 2.8-, 5.2-, and 1.3-fold, respectively. Leaf REC increased by 1.9-, 2.4-, and 1.1-fold, respectively, and root REC increased by 1.2-, 1.9-, and 0.8-fold, respectively. These results indicate that root knockout of CmDUF1 reduces salt tolerance in grafted melon, while overexpression has the opposite effect, suggesting that CmDUF1 positively regulates salt tolerance in grafted melon. This gene has broad application prospects in melon genetic improvement.
[0066] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.
Claims
1. Use of the melon salt-tolerance gene CmDUF1, the protein encoded thereby, an expression cassette containing the gene, a recombinant vector, a transgenic cell line, or a recombinant bacterium in any of the following applications: A1) Regulating salt tolerance in melon; A2) preparing a product for regulating salt tolerance of muskmelon; A3) enhancing the tolerance of melon to salt stress; A4) preparing a product for enhancing tolerance of melon to salt stress; A5) Cultivate salt-tolerant melon varieties; A6) preparing and cultivating salt-tolerant muskmelon varieties; The nucleotide sequence of the melon salt-tolerance gene CmDUF1 is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that Overexpression of the CmDUF1 gene in melon improves tolerance to salt stress.
3. The use according to claim 2, characterized in that A CmDUF1 gene overexpression vector was constructed and introduced into recipient melons to obtain transgenic melon materials.
4. The use according to claim 3, characterized in that The obtained transgenic melon materials showed stronger salt stress tolerance.
5. A method for enhancing the tolerance of melon to salt stress, characterized in that: The method comprises enhancing, increasing or up-regulating the expression level of the CmDUF1 gene in melon or the function or activity of its protein, wherein the nucleotide sequence of the CmDUF1 gene is shown as SEQ ID NO.
1.
6. The method according to claim 5, characterized in that A CmDUF1 gene overexpression vector was constructed and introduced into recipient melons to obtain transgenic melon materials.
7. The method according to any one of claim 6, characterized in that: The obtained transgenic melon materials showed stronger salt stress tolerance.
8. A method for cultivating salt-tolerant muskmelon varieties, characterized in that: A CmDUF1 gene overexpression vector was constructed and introduced into a recipient melon to obtain a transgenic melon material. The nucleotide sequence of the CmDUF1 gene is shown in SEQ ID NO.
1.
9. The method according to claim 8, characterized in that The method comprises the following steps: S1. Construction of CmDUF1 gene overexpression vector; S2. Introducing the overexpression vector from step S1 into muskmelon plants through Agrobacterium-mediated method, and transplanting them after infection; S3. After the seedlings survive, positive seedlings containing the overexpression vector are screened to obtain melon materials overexpressing CmDUF1.
10. The method according to any one of claims 8-9, characterized in that: The obtained melon materials showed stronger tolerance to salt stress.