A method for creating a cucumber germplasm without tendril by site-directed mutagenesis

Abstract

The application discloses a method for creating a no-vine cucumber germplasm through site-directed mutation. The method is realized by mutating the coding gene of CsLFY protein in a vine cucumber variety; the CsLFY protein is a protein with an amino acid sequence shown in SEQ ID No: 3; the mutation is that the CsLFY gene shown in SEQ ID No: 1 is mutated into CsLFY / -1bp; the CsLFY / -1bp is a DNA molecule obtained by deleting the 281th nucleotide G from the 5' end of SEQ ID No: 1 and keeping other nucleotide sequences of SEQ ID No: 1 unchanged. The method can edit the coding gene of the CsLFY protein of the cucumber, and a no-vine cucumber is obtained. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a method for creating tendrilless cucumber germplasm through site-directed mutagenesis. Background Technology

[0002] Tendrils are unique locomotion organs of cucurbitaceous plants, enabling them to climb upwards. Cucumbers are cultivated vegetable crops, primarily managed through manual vine tying. Therefore, cucumbers do not require tendrils for climbing; furthermore, to reduce the biomass consumption of tendrils, they generally need to be removed manually, which is time-consuming and labor-intensive. Thus, developing tendril-free cucumber varieties has significant economic and practical importance for high-yield and labor-saving cucumber cultivation.

[0003] Currently, research on genes regulating plant tendril growth and development has made some progress. HOFER et al. found that HD-ZIPⅠ is a key regulator of pea tendril development; HD-ZIPⅠ inhibits leaf expansion, leading to the metamorphosis of tendrils. HD-ZIPⅠ belongs to a special subfamily of genes, suggesting that the evolution of tendrils in Vitex negundo (Papilionoideae) is related to the acquisition of the HD-ZIPⅠ gene. BOSS et al., based on a dwarf, tendril-free grape, discovered that grape tendrils are metamorphic organs of the inflorescence. Further research revealed that the dwarf, tendril-free grape is a gibberellin-insensitive mutation of VvGAI (encoding the DELLA protein), meaning that gibberellin can inhibit grape inflorescences and promote tendril development. Calonje et al. cloned two MADS-box genes, VFUL-L and VAP1, from grape, finding that they are expressed throughout tendril development and demonstrating that grape tendrils are allologous organs of the flower. OIZUMI et al. reported a tendril-free melon material, “ChibaTL.” “ChibaTL” is a spontaneous mutation of the melon variety 'Fuyukei1', a single-gene recessive mutation. Later, MIZUNO et al. obtained a heterozygote of “ChibaTL”, thus confirming that melon tendrils are a modified stem-leaf structure with lateral branching. Qi Chunzhang et al. collected a mutant of a South China-type white-skinned cucumber from Hubei Province, characterized by soft hairs on the stem and leaf surface, no tendrils, and no tubercles or thorns on the fruit surface. They found that the absence of tendrils was linked to the soft hairs and the absence of tubercles and thorns on the fruit. Wang et al. discovered a tendril-free cucumber mutant, ten, in more than 3000 cucumber germplasms. This mutant was caused by a mutation in the CsTEN gene of the TCP transcription factor family. CsTEN is specifically expressed in cucumber tendrils; rare mutations significantly reduce the transcriptional activation activity of CsTEN, causing tendrils to degenerate, lose thoracotropy, and develop into leaf shapes. CHEN et al. obtained a cucumber tendril-less mutant, td-1, through EMS mutagenesis screening. td-1 is a recessive mutation. It also exhibits diverse defective phenotypes, including dwarfing, reduced epidermal hairs, wrinkled leaves, and short roots. Map-based cloning and BSA sequencing identified the mutant gene as CsGCN5 (Csa6G527060), a missense mutation causing an aspartic acid mutation at position 82 to asparagine. Tissue expression analysis showed that CsGCN5 was highly expressed in cucumber roots, stems, leaves, and tendrils, consistent with the diverse defective phenotypes of td-1. Summary of the Invention

[0004] The purpose of this invention is to create tendril-free cucumber germplasm.

[0005] This invention first protects a method for cultivating tendrilless cucumbers, which is achieved by mutating the gene encoding the CsLFY protein (i.e., the CsLFY gene) in a tendril cucumber variety;

[0006] The CsLFY protein may be a1), a2), or a3):

[0007] a1) The amino acid sequence is that of the protein shown in SEQ ID No: 3;

[0008] a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2);

[0009] a3) A protein obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No: 3.

[0010] The proteins in a2) above are labeled as shown in Table 1.

[0011] Table 1. Sequence of Labels

[0012] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0013] The protein in a3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0014] The proteins mentioned in a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0015] The gene encoding the protein in a3) above can be obtained by deleting one or more amino acid residues from the codons of the DNA sequence shown in SEQ ID No: 1 or SEQ ID No: 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0016] In the above method, the mutation can be to mutate the CsLFY gene shown in SEQ ID No: 1 to CsLFY / -1bp; CsLFY / -1bp is a DNA molecule obtained by deleting nucleotide G at position 281 from the 5' end of SEQ ID No: 1, while keeping the other nucleotide sequences of SEQ ID No: 1 unchanged.

[0017] In the above method, the gene encoding the CsLFY protein in the mutant tendril cucumber variety is introduced into the cucumber using a CRISPR / Cas9 system. The CRISPR / Cas9 system may include a recombinant expression vector that expresses a DNA molecule containing gRNA targeting the gene encoding the CsLFY protein.

[0018] In the above method, the target sequence of the gRNA may be as shown in SEQ ID No: 1, positions 274-298 from the 5' end.

[0019] In the above method, the recombinant expression vector can be the recombinant plasmid CRISPR / CAS9-CsLFY. The recombinant plasmid CRISPR / CAS9-CsLFY can be obtained by inserting the DNA double-stranded molecule shown in SEQ ID No: 4 into the recognition site of the restriction endonuclease Bsal in the CRISPR / CAS9 vector.

[0020] This invention also protects the use of the material encoding the CsLFY protein (i.e., the CsLFY gene) of any of the mutant tendril cucumber varieties in the cultivation of tendrilless cucumbers.

[0021] The CsLFY gene described above can be a DNA molecule of the following type: (b1) or (b2) or (b3) or (b4) or (b5):

[0022] (b1) A DNA molecule with a coding region as shown in SEQ ID NO:1;

[0023] (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1;

[0024] (b3) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:2;

[0025] (b4) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined by (b1) or (b2) or (b3) and encodes any of the CsLFY proteins described above;

[0026] (b5) A DNA molecule derived from cucumber and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA molecule defined in (b1), (b2), or (b3) and encoding any of the CsLFY proteins described above.

[0027] The stringent conditions were: hybridization in a solution of 2×SSC and 0.1% SDS at 68°C, followed by two washes of 5 min each, and then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by two washes of 15 min each.

[0028] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0029] Of these, SEQ ID NO:1 consists of 1161 nucleotides, SEQ ID NO:2 consists of 1948 nucleotides, and the nucleotides shown in SEQ ID NO:1 encode the amino acid sequence shown in SEQ ID NO:3.

[0030] Those skilled in the art can readily mutate the nucleotide sequence encoding any of the CsLFY proteins described above using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of any of the CsLFY proteins isolated according to this invention, as long as they encode any of the CsLFY proteins described above, are derived from and equivalent to the nucleotide sequence of this invention.

[0031] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence of the CsLFY protein shown in SEQ ID NO:3 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0032] In the above applications, the mutation can be the mutation of the CsLFY gene shown in SEQ ID No: 1 to CsLFY / -1bp; CsLFY / -1bp is a DNA molecule obtained by deleting nucleotide G at position 281 from the 5' end of SEQ ID No: 1, while keeping the other nucleotide sequences of SEQ ID No: 1 unchanged.

[0033] In the above applications, the gene encoding the CsLFY protein in the mutant tendril cucumber variety can be B1) or B2):

[0034] B1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the CsLFY protein;

[0035] B2) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B1).

[0036] In the above applications, the nucleic acid molecule described in B1) may be a DNA molecule expressing gRNA that targets the gene encoding the CsLFY protein or gRNA that targets the gene encoding the CsLFY protein.

[0037] In the above applications, the target sequence of the gRNA may be as shown in SEQ ID No: 1, positions 274-298 from the 5' end.

[0038] Experiments have shown that transforming cucumber inbred line 9930 with recombinant Agrobacterium containing the recombinant plasmid CRISPR / CAS9-CsLFY can edit the CsLFY gene. After editing the CsLFY gene using the CRISPR / Cas9 endonuclease, mutations occur. When both homologous chromosomes of the CsLFY gene are mutated (specifically, the CsLFY gene shown in SEQ ID No: 1 is mutated to CsLFY / -1bp; CsLFY / -1bp is a DNA molecule obtained by deleting nucleotide G at position 281 from the 5' end of SEQ ID No: 1, while keeping the other nucleotide sequences unchanged), the CsLFY protein activity is lost. This loss of CsLFY protein activity results in tendril-free cucumber germplasm. The method of this invention can achieve CsLFY gene editing in cucumber, obtaining tendril-free cucumbers. Therefore, the CsLFY protein can regulate cucumber tendrils. This invention has significant application value. Attached Figure Description

[0039] Figure 1 The positional relationship between the target, Oligo F, and Oligo R.

[0040] Figure 2 Sequencing results of six homozygous CsLFY mutant strains and mutation types of the cslfy gene and cslfy protein.

[0041] Figure 3 Phenotypic observation of 6 CsLFY homozygous mutant strains. Detailed Implementation

[0042] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] The cucumber inbred line 9930 is described in the following literature: Li Q, Li H1, Huang W, Xu Y, Zhou Q, Wang S, Ruan J, Huang S, Zhang Z (2019). A chromosome-scale genome assembly of cucumber (Cucumis sativus L.). Gigascience 8:giz072. It is publicly available from the Beijing Academy of Agricultural and Forestry Sciences (i.e., the applicant). This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes. Cucumber inbred line 9930 is a tendril cucumber variety.

[0045] The CRISPR / CAS9 vector construction kit is a product of Hangzhou Baige Biotechnology Co., Ltd., with product catalog number BGK03.

[0046] MS powder is a product of Phyto Technology Laboratories, catalog number M519.

[0047] The culture media involved in the following examples are as follows:

[0048] The solute and concentration of MS liquid medium were 4.43 g / L MS powder and 30 g / L sucrose, with water as the solvent and a pH of 5.7-5.8.

[0049] The solutes and their concentrations in the MS solid medium were 4.43 g / L MS powder, 30 g / L sucrose, and 2.5 g / L plant gel, with water as the solvent and a pH of 5.7-5.8.

[0050] The solutes and their concentrations in the differentiation medium were MS powder 4.43 g / L, sucrose 30 g / L, plant gel 2.5 g / L, 6-BA 0.5 mg / L and ABA 1 mg / L, with water as the solvent and a pH of 5.7-5.8.

[0051] The solutes and their concentrations in the resistance differentiation medium were: MS powder 4.43 g / L, sucrose 30 g / L, plant gel 2.5 g / L, 6-BA 0.5 mg / L, ABA 1 mg / L, kanamycin 25 mg / L and carbenicillin 500 mg / L, with water as the solvent and a pH of 5.7-5.8.

[0052] The solutes and their concentrations in the rooting medium were MS powder 4.43 g / L, sucrose 30 g / L and plant gel 2.5 g / L, with water as the solvent and a pH of 5.7-5.8.

[0053] The solute and concentration of 1 / 2 MS liquid medium were 2.22 g / L MS powder and 30 g / L sucrose, with water as the solvent and a pH of 5.7-5.8.

[0054] In this invention, the nucleotide sequence of the CsLFY gene in cucumber cDNA is shown in SEQ ID NO:1; the nucleotide sequence of the CsLFY gene in cucumber genomic DNA is shown in SEQ ID NO:2. The CsLFY gene encodes the CsLFY protein. The amino acid sequence of the CsLFY protein is shown in SEQ ID NO:3.

[0055] Example 1: Creation of Tendril-Free Cucumber Germplasm through Site-Directed Mutation

[0056] Target design was performed using the DNA sequence shown in SEQ ID No: 1 on the E-CRISPR website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html). In this embodiment, one target was selected for the experiment. The target sequence is: 5'-TTCCGCTGGGACCTTCTAGTGGGTG-3' (i.e., positions 274-298 from the 5' end of SEQ ID No: 1), corresponding to the CsLFY gene.

[0057] I. Construction of recombinant plasmid CRISPR / CAS9-CsLFY

[0058] 1. Design and synthesize Oligo F based on the target:

[0059] 5'-TGTGTTCCGCTGGGACCTTCTAGTGGGTG-3' and Oligo R:

[0060] 5'-AAACACCCACTAGAAGGTCCCAGCGGAA-3' (The positional relationship between the target, Oligo F, and Oligo R is as follows) Figure 1 (As shown).

[0061] 2. Dilute Oligo F and Oligo R to 10 μM with deionized water to obtain Oligo F dilution and Oligo R dilution, respectively.

[0062] 3. Preparation of the annealing reaction system. The annealing reaction system is 20 μL, consisting of 1 μL Oligo F diluent, 1 μL Oligo R diluent, and 18 μL BufferAneal (a component of the CRISPR / CAS9 vector construction kit).

[0063] 4. Take the reaction system prepared in step 3, anneal it, and obtain oligo dimer.

[0064] The annealing procedure is as follows: first 95℃ for 3 minutes, then cool down to 20℃ at a rate of 0.2℃ / s.

[0065] 5. Ligate the oligo dimer obtained in step 4 with the CRISPR / CAS9 vector (a component in the CRISPR / CAS9 vector construction kit) to obtain the recombinant plasmid CRISPR / CAS9-CsLFY.

[0066] The ligation system consisted of 10 μL of oligo dimer, 2 μL of CRISPR / CAS9 vector, 0.5 μL of restriction endonuclease BsaⅠ (Anza, IVGN0366), 2.5 μL of T4 ligase (Anza, IVGN2104), 1 μL of Anza buffer (included with T4 ligase), and ddH2O.

[0067] The connection procedure is: let it stand at room temperature for 1 hour.

[0068] The recombinant plasmid CRISPR / CAS9-CsLFY was sequenced. Sequencing results showed that the recombinant plasmid CRISPR / CAS9-CsLFY was obtained by inserting the DNA double-stranded molecule shown in SEQ ID No: 4 into the recognition site of the restriction endonuclease Bsal in the CRISPR / CAS9 vector.

[0069] II. Obtaining the CsLFY homozygous mutant

[0070] Because cucumbers are diploid plants, when Cas9 begins to edit specific genes, both alleles on the two homologous chromosomes within the same cell can be edited, producing the same or different types of mutations. Therefore, two alleles in a plant are considered two gene editing events. A homozygous mutant is one where the CsLFY gene on both homologous chromosomes of the plant has the same mutation. A biallelic mutant is one where the CsLFY gene on both homologous chromosomes of the plant has been mutated, but in different forms. A heterozygous mutant is one where the CsLFY gene on one of the two homologous chromosomes of the plant has been mutated, while the CsLFY gene on the other homologous chromosome has not been mutated. A wild-type is one where the CsLFY gene on neither of the two homologous chromosomes of the plant has been mutated.

[0071] 1. Preparation of Agrobacterium infection solution

[0072] (1) The recombinant plasmid CRISPR / CAS9-CsLFY was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.

[0073] (2) A single clone of recombinant Agrobacterium was inoculated into 2 ml of YEB liquid medium containing 50 mg / L kanamycin and 70 mg / L Rif, and cultured overnight at 28°C with shaking at 200 rpm / min to obtain culture solution 1. 2 ml of culture solution 1 was inoculated into 50 ml of YEB liquid medium containing 50 mg / L kanamycin and 70 mg / L Rif, and cultured at 28°C with shaking at 200 rpm / min to obtain OD. 600nm 2. The culture solution is 0.6-0.8.

[0074] (3) Take the culture medium obtained in step (2), centrifuge at 5000 rpm for 5 minutes, and collect the bacterial cells; wash the bacterial cells with 1 / 2 MS liquid medium, and then dilute with 1 / 2 MS liquid medium to obtain OD. 600nm The Agrobacterium infection solution is approximately 0.2.

[0075] 2. Recombinant Agrobacterium was transformed into cucumber inbred line 9930 (hereinafter referred to as cucumber). After differentiation and rooting, T0 generation transgenic cucumbers were obtained. The specific steps are as follows:

[0076] (1) Select plump and intact cucumber seeds, peel them, disinfect them in 70% (v / v) ethanol aqueous solution for 30s, then sterilize them in 2.0% sodium hypochlorite solution for 15min, rinse them several times with sterile water, and finally sow them on MS solid medium and culture them at 28℃ for 2-3 days until the cotyledons break open.

[0077] (2) After completing step (1), take the cucumber seed, cut off the growing point and hypocotyl, cut off the upper half (1 / 2-1 / 3) of both cotyledons, and leave the lower half as the explant.

[0078] (3) After completing step (2), infect the explants with the Agrobacterium infection solution prepared in step 1 for 15 min; after the infection is completed, use sterile filter paper to absorb the excess Agrobacterium infection solution, and inoculate the explants with their backs facing down on the differentiation medium and incubate them in the dark at 28°C for 2 days.

[0079] (4) After completing step (3), place the explants in the resistance differentiation medium and culture them at 28°C with alternating light and dark conditions (16 hours of light / 8 hours of darkness; light intensity is about 2000 lx) for 15-20 days to obtain resistant shoots about 1.0-1.5 cm long.

[0080] (5) After completing step (4), cut off the resistant buds and place them in a rooting medium containing 100 mg / L kanamycin. Induce rooting by alternating light and dark culture at 28°C (16 hours of light / 8 hours of darkness; light intensity of approximately 2000 lx). After the root system has developed well, transplant the cucumber plants into flowerpots filled with sterile soil and cover them with plastic wrap to retain moisture. Cultivate them in an artificial climate chamber for 1 week. Then, move them to a greenhouse and allow them to adapt for 3-5 days. After that, manage them as usual to obtain the T0 generation of transgenic cucumbers.

[0081] 3. Using genomic DNA from T0 generation transgenic cucumber leaves as templates, PCR amplification was performed using primer pairs F (5'-CCTCGAGAAATGGTGGGAGG-3') and R (5'-AGGGGAGAGCCAAACAATCC-3') (reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min), yielding the corresponding PCR amplification products. The PCR amplification products were then sequenced. The sequencing results were compared with the CsLFY gene Cas9 target sequence (SEQ ID No: 2, positions 43 to 491 from the 5' end), and mutation types were identified.

[0082] 4. Self-pollinate the heterozygous mutants obtained in step 3. The resulting seeds are T1 generation seeds, and the plants grown from the T1 generation seeds are T1 generation plants.

[0083] 5. Using genomic DNA from leaves of the T1 generation plants as templates, PCR amplification was performed using primer pairs consisting of primer F: 5'-CCTCGAGAAATGGTGGGAGG-3' and primer R: 5'-AGGGGAGAGCCAAACAATCC-3' (reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min), yielding the corresponding PCR amplification products. The PCR amplification products were then sequenced. The sequencing results were compared with the Cas9 target sequence of the CsLFY gene (SEQ ID No: 2, positions 43 to 491 from the 5' end), and the mutation type was identified.

[0084] The results showed that six homozygous CsLFY mutant strains were obtained, named cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6, respectively. The mutation types of the cslfy gene and cslfy protein in cucumber inbred lines 9930, cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6 are described in [reference needed]. Figure 2(9930 is a partial sequence of cucumber inbred line 9930, and cslfy1-6 are 6 homozygous mutant strains of CsLFY). The CsLFY gene on both homologous chromosomes of cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6 all have the same mutation, specifically, a deletion of one nucleotide "G" in the CsLFY gene on both homologous chromosomes (i.e., deletion at position 281 from the 5' end of SEQ ID No: 1), which causes a frameshift, premature termination of the encoded protein, and loss of function of the CsLFY protein.

[0085] III. Phenotypic observation of CsLFY homozygous mutant strains

[0086] When the CsLFY homozygous mutants (cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6) and cucumber inbred line 9930 all grow to the stage of 4 leaves and 1 heart, observe whether they have tendril phenotype.

[0087] See results Figure 3 (CK-1 and CK-2 are both cucumber inbred lines 9930). The results showed that cucumber inbred line 9930 exhibited a tendril phenotype, while cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6 did not, and their tendrils appeared leaf-like. That is, cslfy-1, cslfy-2, cslfy-3, cslfy-4, cslfy-5, and cslfy-6 were all tendrilless cucumber mutants.

[0088] Therefore, it can be seen that the present invention can create tendrilless cucumber germplasm through site-directed mutagenesis.

[0089] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for breeding a cucumber without tendril, by mutating a CsLFY gene shown in SEQ ID No: 1 in a cucumber variety with tendril; The mutation is to delete CsLFY The gene mutation is CsLFY / -1bp ; the mutation is to delete CsLFY / -1bp is a DNA molecule obtained by deleting the nucleotide G at position 281 from the 5' end of SEQ ID No: 1, while keeping other nucleotide sequences of SEQ ID No: 1 unchanged.

2. The method of claim 1, wherein: The CsLFY gene in the mutant cucumber variety with tendril is achieved by introducing a CRISPR / Cas9 system into the cucumber; The CRISPR / Cas9 system includes a recombinant expression vector of a DNA molecule expressing a gRNA targeting CsLFY a gene of interest.

3. The method of claim 2, wherein: The target sequence of the gRNA is shown in SEQ ID No: 1 from the 5' end of the 274-298th position. 4.Use of a substance of a CsLFY gene shown in SEQ ID No: 1 in a mutant cucumber variety in breeding a cucumber without tendril; The mutation is to delete CsLFY The gene mutation is CsLFY / -1bp ; the mutation is to delete CsLFY / -1bp is a DNA molecule obtained by deleting the nucleotide G at position 281 from the 5' end of SEQ ID No: 1, while keeping other nucleotide sequences of SEQ ID No: 1 unchanged.

5. Use according to claim 4, characterized in that: The substance of the CsLFY gene in the mutant cucumber variety with tendril is a DNA molecule expressing a gRNA targeting the CsLFY gene or a gRNA targeting the CsLFY gene.

6. Use according to claim 5, characterized in that: The target sequence of the gRNA is shown in SEQ ID No: 1 from the 5' end of the 274-298th position.

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