A melon cmfsg gene mutant and a method for creating the same and an application thereof

Abstract

The application discloses a Cmfsg gene mutant of melon and a creation method and application thereof, and belongs to the technical field of plant genetic engineering. The mutant is obtained by CRISPR / Cas9 gene editing technology to direct mutation of the coding region or the promoter region of the Cmfsg gene of melon, the target point is recognized by a specific sgRNA primer, and is screened by specific primer PCR amplification and sequencing verification. The creation method comprises the following steps: constructing an editing vector of a target gene, introducing the wild-type queen melon by means of an agrobacterium-mediated genetic transformation technology, screening, regeneration and molecular identification to obtain the mutant. The Cmfsg gene has a regulation function on the fruit fur character and the fruit shape index of melon, can realize the direct and rapid creation of the appearance character of melon, overcomes the defects of long breeding period and low efficiency in traditional breeding, and the obtained mutant has stable genetic traits, and can be used as a parent or genetic resource for high-quality breeding of melon.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology, specifically, it relates to a muskmelon Cmfsg gene mutant and its creation method and application. Background Technology

[0002] Muskmelon (Cucumis melo L.) is an important horticultural crop with significant economic value and industrial scale. The fruit surface groove, as an important external structure of the muskmelon fruit, directly affects the fruit's marketability and is closely related to the integrity of the peel and post-harvest storage characteristics. The fruit shape index is a key indicator for measuring the appearance quality of muskmelon fruits, significantly influencing consumer preferences and market value. Therefore, both are core target traits for muskmelon quality breeding.

[0003] Existing research indicates that the genetic regulatory mechanism of the fruit surface groove trait in melons is not fully understood. Related studies have only progressed to the level of genetic mapping and association analysis of the trait, failing to verify the function of key regulatory genes using precise molecular biology methods. This has resulted in the inability to establish a direct causal relationship between genes and phenotypes. Furthermore, current technologies have not systematically explored the potential impact of these regulatory genes on other key agronomic traits such as fruit shape index, lacking analysis of pleiotropic gene functions, thus limiting their breeding application value. In addition, current marker-based breeding methods can only screen for existing trait types in naturally occurring populations, unable to actively create new germplasm with ideal fruit surface groove phenotypes and optimized fruit shape, severely restricting the efficiency and precision of melon appearance quality breeding.

[0004] Therefore, it is urgent to develop new gene targets and technical pathways to achieve precise improvement of the appearance traits of melon fruits and meet the industry's demand for high-quality melon germplasm. Summary of the Invention

[0005] The purpose of this application is to provide a muskmelon Cmfsg gene mutant, its creation method and application. The CRISPR / Cas9 system can be used to target and knock out the muskmelon Cmfsg gene, which not only directly verifies the core role of the gene in fruit surface groove formation, but also further reveals its significant regulatory effect on fruit shape index, providing a new gene target and technical path for the precise improvement of the appearance traits of muskmelon fruit.

[0006] To achieve the above objectives, this application provides a muskmelon Cmfsg gene mutant, obtained by directed mutation of the coding region sequence or promoter region sequence of the muskmelon Cmfsg gene using gene editing technology; wherein, the Cmfsg gene is MELO3C019694.2, the promoter region sequence is shown in SEQ ID NO.1, and the coding region sequence is shown in SEQ ID NO.2.

[0007] Furthermore, the editing target site of the Cmfsg gene coding region is targeted and recognized by the sgRNA corresponding to the primers shown in SEQ ID NO.4-SEQ ID NO.7; the editing target site of the Cmfsg gene promoter region is targeted and recognized by the sgRNA corresponding to the primers shown in SEQ ID NO.8-SEQ ID NO.11.

[0008] Furthermore, the mutant was obtained by extracting genomic DNA using the CTAB method, and then performing PCR amplification and sequencing verification using primers for identifying the coding regions shown in SEQ ID NO.12-SEQ ID NO.13 or primers for identifying the promoter regions shown in SEQ ID NO.14-SEQ ID NO.15.

[0009] This application also provides a method for creating a muskmelon Cmfsg gene mutant, comprising the following steps: designing and constructing a CRISPR / Cas9 gene editing vector targeting the coding region or promoter region of the Cmfsg gene; and introducing the CRISPR / Cas9 gene editing vector into wild-type muskmelon using Agrobacterium-mediated genetic transformation technology to obtain mutant plants. The "creation" in this application refers to innovative preparation, involving the innovative construction of gene sequences, the discovery of functions or uses, and the innovation of preparation methods.

[0010] Furthermore, the construction process of the CRISPR / Cas9 gene editing vector includes:

[0011] sgRNAs were designed targeting the coding region or promoter region of the Cmfsg gene, and corresponding upstream and downstream primers were synthesized. The primer sequences for the coding region sgRNAs are shown in SEQ ID NO.4-SEQ ID NO.7, and the primer sequences for the promoter region sgRNAs are shown in SEQ ID NO.8-SEQ ID NO.11.

[0012] The upstream and downstream primers of the sgRNA were denatured and annealed to obtain double-stranded sgRNA.

[0013] The tandem vector was digested with BbsI enzyme, and the vector fragment was recovered and ligated with the double-stranded sgRNA to construct the tandem-sgRNA intermediate vector.

[0014] The tandem-sgRNA intermediate vector and the final vector pB7-CAS9-TPC were digested with SpeI and KpnI, ligated, and then identified by colony PCR and sequenced to obtain a positive CRISPR / Cas9 gene editing vector. The colony PCR was performed using the upstream and downstream primers corresponding to SEQ ID NO.4-SEQ ID NO.7 or SEQ ID NO.8-SEQ ID NO.11.

[0015] Furthermore, the conditions for the denaturation-annealing treatment are: reaction at 95°C for 5 minutes, followed by placement at room temperature for 20 minutes;

[0016] The enzyme digestion system for the tandem vector with BbsI is as follows: 1 μg DNA vector, 5 μL r-cut buffer (restriction endonuclease buffer), 1 μL Enlyme (enzyme), and sterile water to a final volume of 50 μL; the reaction conditions are 37℃ for 3 h and 65℃ for 20 min.

[0017] The ligation system for the recovered vector fragment and the double-stranded sgRNA is as follows: 5 μL of double-stranded sgRNA, 25 ng of digested vector, 1 μL of ligase T4 (T4 DNA ligase), 1 μL of 10X ligase buffer (T4 polynucleotide kinase reaction buffer), and sterile water to a final volume of 10 μL. The reaction conditions are 16°C for 12 h.

[0018] Furthermore, the Agrobacterium-mediated genetic transformation technology includes the following steps:

[0019] Queen melon seeds were used as explants, sterilized, and then cultured in the dark. OD was prepared simultaneously. 600 =0.4-0.5% Agrobacterium bacterial suspension; wherein, the Agrobacterium is the AGL1 strain with a positive CRISPR / Cas9 gene editing vector introduced;

[0020] The explants after dark culture were co-cultured with the Agrobacterium tumefaciens culture for 3 days;

[0021] The explants after co-culture were transferred to a selection medium for selection culture, and then subcultured after the clustered shoots grew.

[0022] Seedlings that have grown to a suitable height through subculture are transferred to a rooting medium to root and obtain regenerated plants;

[0023] Genomic DNA was extracted from the regenerated plants, and the Cmfsg gene-editing mutant was obtained through PCR amplification and sequencing verification.

[0024] Furthermore, the disinfection process includes, in sequence:

[0025] Soak in sterile water for 30 seconds;

[0026] Disinfect with 75% alcohol for 30 seconds;

[0027] Disinfect with 5% sodium hypochlorite for 13 minutes;

[0028] Rinse three times with sterile water for five minutes each time.

[0029] This application also provides an application of the Cmfsg gene mutant in melon breeding, including:

[0030] As a male or female parent, it is used for hybridization and breeding of melon varieties with specific fruit surface groove phenotype;

[0031] As a genetic resource, it is used to create melon germplasm with optimized fruit shape index.

[0032] In summary, the beneficial technical effects of this application are as follows:

[0033] 1. This application is the first to use CRISPR / Cas9 gene editing technology to understand the regulatory role of the Cmfsg gene on the groove trait of melon fruit surface, which solves the core shortcoming of existing technologies that can only perform genetic localization of the gene and cannot clarify the causal relationship between gene function and phenotype, and provides direct evidence for the study of the regulatory mechanism of melon fruit surface traits.

[0034] 2. This application reveals for the first time the dual regulatory function of the Cmfsg gene, confirming that it not only regulates the formation of fruit surface grooves, but also significantly reduces the fruit shape index (making the fruit closer to round / flat round from oblong), breaking through the single understanding of the gene's function and enhancing the gene's comprehensive application value in multi-trait improvement.

[0035] 3. This application realizes the targeted creation of key appearance traits such as fruit surface grooves and fruit shape in melons, freeing them from the dependence on natural variation in traditional breeding, thereby significantly shortening the breeding cycle and improving breeding efficiency, and further providing a technical path for the rapid cultivation of new high-quality melon germplasm.

[0036] 4. This application utilizes the targeting capabilities of the CRISPR / Cas9 system to avoid the linkage burden problem of traditional hybridization breeding. The mutant traits obtained are stably inherited, providing key technical support for the precise improvement of melon appearance and quality, and can help the industrial application of breeding. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is an information diagram of the tandem carrier proposed in the embodiments of this application.

[0039] Figure 2 This is an information diagram of the final carrier pB7-CAS9-TPC proposed in the embodiments of this application.

[0040] Figure 3 These are the identification results of the Cmfsg knockout mutants proposed in this application, among which cmfsg-1, cmfsg-2, cmfsg-3, cmfsg-4, cmfsg-5 and cmfsgp-1 are Cmfsg knockout mutants.

[0041] Figure 4 The fruits of wild-type melon (WT) and Cmfsg gene knockout mutants (cmfsg-1 and cmfsgp-1) at different stages are presented in this application.

[0042] Figure 5 This is a statistical comparison chart of the fruit shape index of Cmfsg knockout mutant and wild-type melon proposed in this application. Detailed Implementation

[0043] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0044] This application provides a muskmelon Cmfsg gene mutant, its creation method, and its application. The muskmelon Cmfsg gene is MELO3C019694.2, its promoter sequence is shown in SEQ ID NO.1, its nucleotide sequence is shown in SEQ ID NO.2, and its encoded protein amino acid sequence is shown in SEQ ID NO.3. The muskmelon material involved in this application is the Queen variety.

[0045] Among them, SEQ ID NO.1:

[0046]

[0047] SEQ ID NO.2:

[0048]

[0049] SEQ ID NO.3:

[0050] MEFMQKREVELQSHNNYLRAQIAEHERIQQQQQQQQTNMMQRATYESVGGQYDDENRSTYGAVGALMDSDSHYAPQDHLTALQL.

[0051] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0052] Example 1

[0053] This embodiment describes the construction of a CRISPR / Cas9 gene editing vector targeting the Cmfsg gene (coding / promoter region) in melon, providing a tool vector for subsequent Agrobacterium-mediated genetic transformation. The vector contains sgRNA and Cas9 nuclease expression elements that specifically recognize the Cmfsg gene, enabling targeted cleavage of the target gene in recipient cells. Specifically, two target sites were selected each in the promoter and coding region of the Cmfsg gene, which is associated with the fruit groove trait in melon, to construct gRNA expression cassettes. These target gRNA expression cassettes were then ligated into a knockout vector to obtain the Cmfsg knockout vector.

[0054] Specifically, the following steps are included:

[0055] (1) Target site design and primer synthesis

[0056] Log in to the website bioinfogp.cnb.csic.es / tools / breakingcas / ?gset=melon, enter the promoter and CDS sequence of the Cmfsg gene, and select a target with high score and high specificity.

[0057] Specific sgRNA primers were designed for the coding region sequence SEQ ID NO.2, as shown below:

[0058] fsg-sgRNA1-F:5'-attgGCAAACGAACATGATGCAAA-3' (SEQ ID NO.4);

[0059] fsg-sgRNA1-R:5'-aaacTTTGCATCATGTTCGTTTGC-3' (SEQ ID NO.5);

[0060] fsg-sgRNA2-F:5'-attgAGGGCAACATATGAGAGCGT-3' (SEQ ID NO.6);

[0061] fsg-sgRNA2-R:5'-aaacACGCTCTCATATGTTGCCCT-3' (SEQ ID NO.7); where uppercase letters represent the core sequence and lowercase letters represent the vector homologous arms.

[0062] An sgRNA was designed targeting the promoter region sequence SEQ ID NO.1, as shown below:

[0063] fsgp-sgRNA1-F:5′-ATTGCTACGTTGTAATAGCCTCAC-3′ (SEQ ID NO.8);

[0064] fsgp-sgRNA1-R:5′-AAACGTGAGGCTATTACAACGTAG-3′ (SEQ ID NO.9);

[0065] fsgp-sgRNA2-F:5′-ATTGCTGACTCCCAATCAGCCTCG-3′ (SEQ ID NO.10);

[0066] fsgp-sgRNA2-R:5′-AAACCGAGGCTGATTGGGAGTCAG-3′ (SEQ ID NO. 11).

[0067] (2) Primer denaturation-annealing treatment

[0068] Add 1 μL of sgRNA F (100 μM) and 1 μL of sgRNA R (100 μM) to 48 μL of ddH2O, react at 95°C for 5 minutes (denaturation), and then incubate at room temperature for 20 minutes (annealing). Here, sgRNA F refers to fsg-sgRNA1-F, fsg-sgRNA2-F, fsgp-sgRNA1-F, and fsgp-sgRNA2-F from step (1), and sgRNA R refers to fsg-sgRNA1-R, fsg-sgRNA2-R, fsgp-sgRNA1-R, and fsgp-sgRNA2-R from step (1). The primers for single-stranded sgRNA are denatured and annealed to form double-stranded sgRNA (containing BbsI restriction sites and sticky ends for easy ligation to a vector).

[0069] (3) Vector digestion and ligation

[0070] Tandem was digested with BbsI enzyme. Figure 1 The vector (shown) generates sticky ends that match the double-stranded sgRNA. The double-stranded sgRNA is then inserted into the tandem vector using T4 ligase to construct the tandem-sgRNA intermediate vector (two types: coding region targeted and promoter region targeted).

[0071] The enzyme digestion system is shown in Table 1, and the ligation system is shown in Table 2.

[0072] Table 1 Enzyme digestion system

[0073]

[0074] Table 2 Connection System

[0075]

[0076] (4) Enzyme digestion and ligation of intermediate and final vectors, and identification

[0077] The intermediate vector and the final vector pB7-CAS9-TPC obtained by double digestion with SpeI and KpnI (3) were ligated. Figure 2 (As shown in Table 1). The enzyme digestion system is shown in Table 1. The sgRNA expression cassette was transformed into the final vector containing the Cas9 gene. Colony PCR and sequencing were performed using primers sgRNA1-F and sgRNA2-R to verify the correctness of the vector construction. Then, Agrobacterium was transformed to obtain engineered Agrobacterium that can be used for transformation.

[0078] The reaction conditions for colony PCR are as follows:

[0079] 1) Pre-denaturation: 94℃, 3 min;

[0080] 2) Denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 45 seconds, cycled 30 times;

[0081] 3) After 30 cycles, extend for 10 minutes.

[0082] The PCR products were detected by 1% agarose gel electrophoresis, and the size of the amplified fragments was determined by comparison with the DL 2000plues DNA Marker. Bacterial cultures with a correct band of 500 bp were used to extract plasmids using a plasmid extraction kit (Vazyme, 7E1713L4) and sent for sequencing. The correctly sequenced plasmids were transformed into Agrobacterium AGL1 (Vazyme Biosciences, AC1020S), and then identified by colony PCR. Positive clones were preserved in 25% glycerol to obtain the correctly edited Agrobacterium AGL1 engineered strain. The correctly edited vector refers to a positive CRISPR / Cas9 editing vector (coding region targeted, promoter region targeted).

[0083] Table 3 Bacterial PCR System

[0084]

[0085] Example 2

[0086] This embodiment is used to introduce the editing vector constructed in Example 1 into melon explants via Agrobacterium-mediated transformation, and to screen for positive mutant plants (T0 generation) with the Cmfsg gene directed to be edited.

[0087] Specifically, the following steps are included:

[0088] (1) Preparation of explants and bacterial culture

[0089] Seeds of the melon variety Queen were used as explants. The melon seeds were soaked in sterile water for 30 seconds, disinfected with 75% alcohol for 30 seconds, and then sterilized with 5% sodium hypochlorite for 13 minutes. The seeds were rinsed with sterile water for 5 minutes and repeated 3 times. The seeds were placed at room temperature in the dark for 12-20 hours. After peeling off the inner seed coat, the seeds were placed on MS medium (MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, pH=5.8) and incubated in the dark at 28°C for one day.

[0090] Simultaneously, the Agrobacterium AGL1 engineered bacteria obtained in Example 1 were shaken at 28°C and 200 r / min until OD was reached. 600 After reaching 0.5-0.6, add it to 1 / 2 MS infection solution (MS 2.2 g / L, sucrose 30 g / L, AS 0.2 mM) and shake at 28℃ and 200 r / min for 12 h until OD reaches 0.5-0.6. 600 =0.4-0.5, to obtain Agrobacterium infection solution containing editing vector.

[0091] (2) Infection and co-cultivation

[0092] Remove the tail 1 / 3 and about 5 mm of the radicle from the seed. Cut the remaining part in half from the middle (leaving 4 usable parts for each seed). Place the cut seeds in the invasion staining solution and vacuum-treat them 10 times with a syringe. Dry the seeds on filter paper and place them in a co-culture medium (MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, CuSO4·5H2O 1 mg / L, MES 0.6 g / L, 6-BA (cytokinin) 0.5 mg / L, IAA (auxin) 0.1 mg / L, AS (acetylsyleugenol) 0.2 mM, pH=5.87) at 26℃ in a 3-day cycle of 16 h light + 8 h dark.

[0093] Through infection and co-culture, Agrobacterium can be brought into full contact with explants. During co-culture, Agrobacterium can transfer sgRNA and Cas9 gene into explant cells.

[0094] (3) Screening and cultivation

[0095] Explants were transferred from co-culture medium to selection medium for selection culture. Untransformed explants were eliminated by using glufosinate (a carrier resistance marker) in the selection medium, while resistant explants that successfully integrated T-DNA (sgRNA, Cas9 gene) were retained. This induced the differentiation of clustered shoots, resulting in melon clustered shoots with glufosinate resistance.

[0096] The screening medium consisted of: MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, CuSO4·5H2O 1 mg / L, MES 0.6 g / L, 6-BA 0.5 mg / L, IAA 0.1 mg / L, Cef (cephalosporin) 250 mg / L, Tim (termethin, ticarcillin) 150 mg / L, and glufosinate 4 mg / L.

[0097] (4) Subgeneration

[0098] Three weeks later, the explants sprouted clusters of shoots and were transferred to a new selection medium. Subculture was then performed every three weeks by removing vitrified and etiolated portions. When the stems reached 1-2 cm in length, they were cut and placed in rooting medium (MS 4.4 g / L, sucrose 30 g / L, agar 8 g / L, Tim 150 mg / L) to induce rooting. Once complete regenerated plants (T0 generation) were obtained, DNA could be extracted from leaves for identification as the seedlings matured.

[0099] (5) Genomic DNA was extracted from regenerated plants using the CTAB method, and specific primers were used to detect whether the Cmfsg gene had been edited, and positive mutants were screened.

[0100] The specific steps for extracting genomic DNA from regenerated plants are as follows:

[0101] 1) Take an appropriate amount of plant leaves into a 2mL centrifuge tube, put 2 3mm steel balls into each tube, add 500μL of 2% CTAB buffer, and grind them in a grinder (grinding conditions: 60Hz, 90s).

[0102] 2) Place in a centrifuge at 6000 rpm for a brief 5 seconds;

[0103] 3) Add 500 μL of chloroform, invert fifty times to mix, and centrifuge at 12000 r / min for 5 min;

[0104] 4) Transfer the supernatant to a new tube, add an equal volume of isopropanol, invert the tube fifty times to mix, centrifuge at 12000 r / min for 5 min and remove the supernatant.

[0105] 5) Add 500 μL of 70% ethanol, centrifuge at 12000 r / min for 5 min, remove the supernatant, and repeat twice;

[0106] 6) After air-drying at room temperature, add an appropriate amount of ddH2O to dissolve completely. Use a spectrophotometer to detect the concentration and quality of the DNA until the concentration is 100ng / μL-200ng / μL, thus obtaining qualified melon genomic DNA.

[0107] The specific steps for screening positive mutants are as follows:

[0108] Primers fsg-F, fsg-R and fsgP-F, fsgP-R were designed based on the sequences flanking the Cmfsg target site. PCR amplification and sequencing identification were performed using primers specific to the coding and promoter regions, respectively (PCR amplification systems are shown in Table 3). Results are shown below. Figure 3 As shown, editing the Cmfsg coding region yielded five mutants, with cmfsg-1 and cmfsg-5 being insertion mutations. ， cmfsg-2, cmfsg-3, and cmfsg-4 are base mutations and deletion mutations. ， One mutant was obtained by editing the promoter region, namely a base mutation at gRNA1.

[0109] Primers for coding region identification:

[0110] fsg-F:5'-GCAGAACACGAGAGAATAC-3' (SEQ ID NO. 12);

[0111] fsg-R:5'-GAGCATAATGGCTGTCTGAA-3' (SEQ ID NO. 13).

[0112] Promoter region identification primers:

[0113] fsgP-F:5'-GTGTTTGGTTGGAGCAATGTCA-3' (SEQ ID NO. 14);

[0114] fsgP-R:5'-TTCCAAGAAAGTGAGCGGGG-3' (SEQ ID NO. 15).

[0115] Example 3

[0116] This embodiment directly demonstrates the direct causal relationship between Cmfsg gene editing and the formation of fruit surface grooves and a decrease in fruit shape index in melons by comparing the fruit phenotypes of the edited mutant and the wild type, while also quantifying phenotypic differences. Specifically, it includes the following steps:

[0117] The T1 generation mutant obtained by self-pollination of the mutant and the wild-type (Queen Melon variety) control plant were planted in the same greenhouse at the same time under the same management conditions. Phenotypic observation and data measurement were carried out from 0 to 40 days of fruit ripening.

[0118] (1) Characteristics of fruit surface grooves:

[0119] like Figure 4 As shown, from the day of flowering, the fruits of the cmfsg-1 and cmfsgp-1 mutants clearly showed grooves on the fruit surface and were rounder than the wild type. By 40 days, the fruits of all Cmfsg gene (coding region and promoter region) edited mutants showed clear longitudinal grooves on the fruit surface, while the WT fruit surface was smooth without any grooves. This indicates that the disruption of Cmfsg gene function is the direct cause of the induction of fruit surface groove formation.

[0120] (2) Fruit shape index:

[0121] The longitudinal and transverse diameters of mature fruits were measured, and the fruit shape index (longitudinal diameter / transverse diameter) was calculated. The results showed ( Figure 5 Compared to the wild type (WT, fruit shape index: 1.807±0.083), the fruit shape index of the Cmfsg coding region mutant was significantly reduced to 1.437±0.041, and the promoter region mutant's fruit shape index was further reduced to 1.267±0.060. This indicates that compared to the wild type, the mutant plants have a lower fruit shape index, with the promoter knockout mutant (Cmfsgp) having the lowest fruit shape index. Statistical analysis showed that the difference between the mutants and WT was extremely significant. This suggests that the Cmfsg gene plays an important regulatory role in both regulating fruit surface groove formation and overall fruit shape.

[0122] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of this application.

[0123] Finally, it should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0124] This application uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A muskmelon Cmfsg gene mutant, characterized in that, The mutant is either the coding region mutant cmfsg-1 or the promoter region mutant cmfsgp-1, obtained by targeted mutation of a specific target in the coding region or a specific target in the promoter region of the melon Cmfsg gene using CRISPR / Cas9 gene editing technology. The Cmfsg gene is MELO3C019694.2, and its promoter region sequence is shown in SEQ ID NO.1, and its coding region sequence is shown in SEQ ID NO.

2. The cmfsg-1 is obtained by inserting a C base mutation between positions 4041 and 4042 of the coding region sequence of the Cmfsg gene in SEQ ID NO.2; the cmfsgp-1 is obtained by replacing positions 996, 1000, and 1014 of the promoter region sequence of the Cmfsg gene with A bases, C bases, and G bases, respectively.

2. A method for creating the muskmelon Cmfsg gene mutant of claim 1, characterized in that, Includes the following steps: Design and construct CRISPR / Cas9 gene editing vectors that target specific targets in the coding region or promoter region of the Cmfsg gene. The CRISPR / Cas9 gene editing vector was introduced into wild-type melon using Agrobacterium-mediated genetic transformation technology to obtain mutant plants; The construction process of the CRISPR / Cas9 gene editing vector includes: sgRNAs were designed targeting specific sites in the coding region or promoter region of the Cmfsg gene, and corresponding upstream and downstream primers were synthesized. The primer sequences for the coding region sgRNAs are shown in SEQ ID NO.4-SEQ ID NO.5, and the primer sequences for the promoter region sgRNAs are shown in SEQ ID NO.8-SEQ ID NO.

9. The upstream and downstream primers of the sgRNA were denatured and annealed to obtain double-stranded sgRNA. The tandem vector was digested with BbsI and BsaI in sequence. After recovering the vector fragment, it was sequentially ligated with the double-stranded sgRNA to construct the tandem-sgRNA intermediate vector. The tandem-sgRNA intermediate vector and the final vector pB7-CAS9-TPC were digested with SpeI and KpnI, ligated, and then identified by colony PCR and sequenced to obtain a positive CRISPR / Cas9 gene editing vector. The colony PCR was performed using the upstream and downstream primers corresponding to SEQ ID NO.4-SEQ ID NO.5 or SEQ ID NO.8-SEQ ID NO.

9.

3. The method according to claim 2, characterized in that, The conditions for the denaturation-annealing treatment are: reaction at 95℃ for 5 min, followed by placement at room temperature for 20 min. The enzyme digestion system for the tandem vector using BbsI and BsaI is as follows: 1 μg DNA vector, 5 μL r-cut buffer, 1 μL Enlyme, and sterile water to a final volume of 50 μL; the reaction conditions are 37℃ for 3 h and 65℃ for 20 min. The ligation system for the recovered vector fragment and the double-stranded sgRNA was as follows: 5 μL of double-stranded sgRNA, 25 ng of enzyme-digested vector, 1 μL of ligase T4, 1 μL of 10X ligase buffer, and sterile water to a final volume of 10 μL. The reaction conditions were 16°C for 12 h.

4. The method according to claim 2, characterized in that, The Agrobacterium-mediated genetic transformation technology includes the following steps: Queen melon seeds were used as explants, sterilized, and then cultured in the dark. OD was prepared simultaneously. 600 =0.4-0.5% Agrobacterium bacterial suspension; wherein, the Agrobacterium is the AGL1 strain with a positive CRISPR / Cas9 gene editing vector introduced; The explants after dark culture were co-cultured with the Agrobacterium tumefaciens culture for 3 days; The explants after co-culture were transferred to a selection medium for selection culture, and then subcultured after the clustered shoots grew. Seedlings that have grown to a suitable height through subculture are transferred to a rooting medium to root and obtain regenerated plants; Genomic DNA was extracted from the regenerated plants, and the Cmfsg gene-editing mutant was obtained through PCR amplification and sequencing verification.

5. The method according to claim 4, characterized in that, The disinfection process includes, in sequence: Soak in sterile water for 30 seconds; Disinfect with 75% alcohol for 30 seconds; Disinfect with 5% sodium hypochlorite for 13 minutes; Rinse three times with sterile water for five minutes each time.

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