Use of MYC2a, MYC2b, COI1, AMY3, BAM1 and / or BAM5 genes in creating a cucumber male sterile line
By knocking out the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes using CRISPR/Cas9 gene editing technology, a male-sterile cucumber line was created. This solved the problems of high cost, low efficiency, and long breeding cycle of artificial emasculation in cucumber hybrid seed production, and achieved stable inheritance and high-efficiency breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANGZHOU UNIV
- Filing Date
- 2026-06-23
- Publication Date
- 2026-07-24
AI Technical Summary
The production of hybrid cucumber seeds is hampered by high costs and low efficiency of artificial emasculation, a scarcity of male-sterile varieties, and a long breeding cycle, resulting in insufficient seed purity and reduced market competitiveness.
Using CRISPR/Cas9 gene editing technology on the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes, the target genes were knocked out, creating a stable and heritable male-sterile cucumber line. The loss of gene function was achieved through Agrobacterium-mediated infection, avoiding artificial emasculation.
It significantly reduces seed production costs, improves seed purity and production efficiency, shortens the breeding cycle, and is suitable for large-scale hybrid seed production of cucumbers, solving the bottleneck problems of traditional breeding.
Smart Images
Figure CN122445709A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of plant genetic engineering and vegetable molecular breeding technology, specifically to the application of MYC2a, MYC2b, COI1, AMY3, BAM1 and / or BAM5 genes in the creation of male-sterile cucumber lines. Background Technology
[0002] Cucumber is one of the most widely cultivated and consumed vegetable crops in my country, holding a prominent industrial position. Yield increases and quality improvements heavily rely on the utilization of hybrid vigor. Currently, cucumber hybrid seed production still primarily relies on traditional manual emasculation and pollination, which faces a series of insurmountable bottlenecks in large-scale production: manual emasculation is costly, inefficient, time-consuming, and labor-intensive, severely squeezing profit margins as labor costs continue to rise; manual emasculation is prone to omissions and non-standard operations, directly leading to insufficient purity of hybrid seeds, affecting the quality and market competitiveness of commercial varieties; cucumber male-sterile germplasm resources are extremely scarce, with only a limited range of available sterility gene types and limited molecular marker and functional gene resources; traditional sterile line breeding cycles are extremely long, relying on multiple generations of backcrossing and transformation, typically taking 3-5 years to develop stable sterile lines, making it difficult to meet the demands of rapid breeding; some existing sterile materials are greatly affected by environmental factors such as temperature and light, resulting in unstable sterility and making them unsuitable for multi-regional and multi-seasonal production; research on the molecular mechanisms of cucumber male sterility is lagging, resulting in a shortage of key genes that can be used for gene editing and precision breeding, limiting the application of modern bio-breeding technologies.
[0003] Therefore, discovering key genes for male sterility in cucumbers and establishing efficient, stable, and rapid techniques for creating sterile lines are of great theoretical and practical value for reducing seed production costs, ensuring seed purity, and promoting the modernization and industrialization of cucumber hybrid breeding. Summary of the Invention
[0004] This invention addresses the problems of high cost, low efficiency, few male-sterile varieties, and long breeding cycles in existing cucumber hybridization breeding. It provides a set of cucumber genes that can be stably male-sterile through gene editing with large fragment deletions, and establishes a complete system for creating male-sterile lines.
[0005] Therefore, in a first aspect, the present invention provides the application of the MYC2a, MYC2b, COI1, AMY3, BAM1 and / or BAM5 genes in the creation of male-sterile cucumber lines, wherein the nucleotide sequences of the MYC2a, MYC2b, COI1, AMY3, BAM1 and BAM5 genes are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.
[0006] Furthermore, the application involves inducing stable, heritable male sterility in cucumbers by knocking out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes.
[0007] Furthermore, the application involves knocking out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes using CRISPR / Cas9 gene editing technology.
[0008] Furthermore, the application utilizes CRISPR / Cas9 gene editing technology. Knock out the COI1 gene; or Knock out the AMY3 gene; or Simultaneously knock out the MYC2a and MYC2b genes; or Simultaneously, the BAM1 and BAM5 genes were knocked out.
[0009] A second aspect of the invention provides target sequences for CRISPR / Cas9 gene editing knockout of the MYC2a, MYC2b, COI1, AMY3, BAM1, and / or BAM5 genes. The target sequence of the MYC2a gene is selected from SEQ ID NO:7 and / or SEQ ID NO:8; The target sequence of the MYC2b gene is selected from SEQ ID NO:9 and / or SEQ ID NO:10; The target sequence of the COI1 gene is selected from SEQ ID NO:11 and / or SEQ ID NO:12; The target sequence of the AMY3 gene is selected from SEQ ID NO:13 and / or SEQ ID NO:14; The target sequence of the BAM1 gene is selected from SEQ ID NO:15 and / or SEQ ID NO:16; The target sequence of the BAM5 gene is selected from SEQ ID NO:17 and / or SEQ ID NO:18.
[0010] A third aspect of the present invention provides a method for creating a male-sterile cucumber line, the method comprising: Construct a recombinant vector based on the target sequence described above; The recombinant vector was used to infect explants of cucumber recipients via Agrobacterium-mediated infection. Positive transgenic seedlings were obtained through cultivation and resistance screening; The positive transgenic seedlings were sequenced and identified to obtain homozygous mutant lines with the target gene successfully knocked out; Phenotypic identification was performed on the homozygous mutant lines to screen for stable hereditary male-sterile cucumber lines.
[0011] Furthermore, the stamens of the cucumber male-sterile line are abnormally developed, the anthers do not dehisce, and the pollen is non-viable or completely sterile; the pistils of the cucumber male-sterile line are normally developed and can be pollinated and bear fruit normally.
[0012] In a fourth aspect, the present invention provides a recombinant vector for creating male-sterile lines of cucumber, the recombinant vector comprising the target sequence described above.
[0013] Furthermore, the method for constructing the recombinant vector includes: PCR amplification of DNA fragments containing the target sequence; The amplified fragment is ligated to the linearized vector; The ligation product was transformed into E. coli, and the recombinant vector was obtained through positive clone screening and sequencing verification.
[0014] In a fifth aspect, the present invention provides the application of the cucumber male-sterile line prepared by the method or the recombinant vector thereon in cucumber hybridization breeding and large-scale production of hybrid seeds.
[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention utilizes the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes combined with CRISPR / Cas9 gene editing technology to knock out target genes, thereby achieving gene function loss. This allows for the efficient creation of cucumber male-sterile lines with stable heritable traits. The resulting male-sterile lines exhibit abnormal stamen development, eliminating the need for artificial emasculation. This effectively solves the problems of high cost, low efficiency, scarcity of male-sterile varieties, and long breeding cycles associated with artificial emasculation in cucumber hybridization breeding. It significantly reduces hybrid seed production costs and improves seed purity, making it of significant application value in cucumber hybridization breeding and large-scale seed production. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.
[0017] Figure 1 This is a schematic diagram of the structure and target location of cucumber MYC2a, MYC2b, and COI1 genes provided in an embodiment of the present invention, wherein Figure A shows COI1. WT (Wild type) and coi1-1, coi1-2 The mutant gene structure and target location are shown in Figure B, which is MYC2a. WT(Wild type) and myc2a-1 , myc2a-3 The mutant gene structure and target location are shown in Figure C, which represents MYC2b. WT (Wild type) and myc2b- 1 , myc2b-3 mutant gene structure and target location; Figure 2 This is a schematic diagram of the gene structure and target location of AMY3, BAM1, and BAM5 provided in the embodiments of the present invention, wherein Figure D is AMY3. WT (Wild type) and amy3-1 , amy3-2 Mutant gene structure and target location, Figure E shows BAM1. WT (Wild type) and bam1-1 , bam1-2 Mutant gene structure and target location, Figure F shows BAM5. WT (Wild type) and bam5-1 , bam5-2 mutant gene structure and target location; Figure 3 This is a comparison diagram of the anther phenotypes of wild-type and mutant plants provided in an embodiment of the present invention. Figure A shows the wild-type and mutant anther phenotypes. coi1-1 Stereoscopic observation of the mutant and paraffin cross-section; Figure B shows WT (wild type) and... myc2a myc2b Stereoscopic observation of the mutant and paraffin cross-section, Figure C is WT (wild type). amy3-1, bam1-1, bam5-1, bam1 bam5 The mutant anthers were observed under a stereomicroscope on the day of flowering. Figure D shows the wild-type (WT). amy3-1, bam1-1, bam5-1, bam1 bam5 Paraffin cross-section of the mutant anther on the day of flowering; Figure 4 This is a comparison chart of pollen viability detection between wild-type and mutant strains provided in an embodiment of the present invention. Figure A shows the pollen viability of WT (wild-type) and mutant strains. coi1-1 Pollen germination in vitro on the day of flowering of the mutant; Figure B shows the results for WT (wild type) and... myc2a myc2b Pollen germination in vitro on the day of flowering of the mutant; Figure C shows the results for WT (wild type) and... amy3-1, bam1-1, bam5-1, bam1 bam5 Pollen germination in vitro on the day the mutant blooms. Detailed Implementation
[0018] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.
[0019] In a first aspect, the present invention provides the application of the MYC2a, MYC2b, COI1, AMY3, BAM1 and / or BAM5 genes in the creation of male-sterile cucumber lines, wherein the nucleotide sequences of the MYC2a, MYC2b, COI1, AMY3, BAM1 and BAM5 genes are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.
[0020] In some embodiments, the above application induces stable and heritable male sterility in cucumbers by knocking out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes.
[0021] In some embodiments, the above application involves knocking out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes using CRISPR / Cas9 gene editing technology.
[0022] In some embodiments, the above applications are performed using CRISPR / Cas9 gene editing technology. Knock out the COI1 gene; or Knock out the AMY3 gene; or Simultaneously knock out the MYC2a and MYC2b genes; or Simultaneously, the BAM1 and BAM5 genes were knocked out.
[0023] This invention provides for the first time a set of key male sterility genes in cucumber, namely MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5, enriching the gene resources for male sterility in cucumber. By editing these genes through large fragment deletions, stable male sterility in cucumber can be achieved, with traits unaffected by the environment and complete sterility. The use of gene editing technology can rapidly create sterile lines, significantly shorten the breeding cycle, replace artificial emasculation, significantly reduce seed production costs, and improve the purity and production efficiency of hybrid seeds. It is suitable for large-scale hybrid seed production of cucumber and has important prospects for industrial application.
[0024] A second aspect of this invention provides target sequences for CRISPR / Cas9 gene editing and knockout of the MYC2a, MYC2b, COI1, AMY3, BAM1, and / or BAM5 genes. The target sequence of the MYC2a gene is selected from SEQ ID NO:7 and / or SEQ ID NO:8; The target sequence of the MYC2b gene is selected from SEQ ID NO:9 and / or SEQ ID NO:10; The target sequence of the COI1 gene is selected from SEQ ID NO:11 and / or SEQ ID NO:12; The target sequence of the AMY3 gene is selected from SEQ ID NO:13 and / or SEQ ID NO:14; The target sequence of the BAM1 gene is selected from SEQ ID NO:15 and / or SEQ ID NO:16; The target sequence of the BAM5 gene is selected from SEQ ID NO:17 and / or SEQ ID NO:18.
[0025] A third aspect of the present invention provides a method for creating a male-sterile cucumber line, comprising: Construct recombinant vectors based on target sequences; The recombinant vector was used to infect cucumber explants via Agrobacterium-mediated infection. Positive transgenic seedlings were obtained through cultivation and resistance screening; Sequencing and identification of positive transgenic seedlings yielded homozygous mutant lines with the target gene successfully knocked out; Phenotypic identification was performed on homozygous mutant lines to screen for stable hereditary male-sterile cucumber lines.
[0026] In some embodiments, the male-sterile cucumber line has abnormal stamen development, with anthers that do not dehisce and pollen that is non-viable or completely sterile; the pistil develops normally and can be pollinated and produce fruit normally.
[0027] A fourth aspect of the present invention provides a recombinant vector for creating male-sterile cucumber lines, comprising the aforementioned target sequence.
[0028] In some embodiments, the method for constructing the recombinant vector includes: PCR amplification of DNA fragments containing the target sequence; The amplified fragment is ligated to the linearized vector; The ligation product was transformed into E. coli, and the recombinant vector was obtained through positive clone screening and sequencing verification.
[0029] A fifth aspect of this invention provides the application of the prepared cucumber male-sterile line or recombinant vector in cucumber hybridization breeding and large-scale production of hybrid seeds.
[0030] Example 1: Obtaining the nucleotide sequence of the target gene Cucumber leaves were used as material. Total RNA was extracted from cucumber using the Trizol method, and cDNA of cucumber genes was synthesized using a reverse transcription kit. The coding region sequences of MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 were amplified using gene-specific primers. The sequences were detected by agarose gel electrophoresis, and the target fragments were recovered using a recovery kit. The fragments were then ligated into vectors and sequenced. The full-length coding region sequences of cucumber MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 were obtained as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.
[0031] Example 2 Target Design Using gene editing target design software, CRISPR / Cas9 targets were designed for conserved regions and functional domains of various genes. Two or more targets were designed within the conserved regions of each gene's functional domain, with a target length of 20 bp and a PAM (NGG) sequence at the 3' end. This ensures that editing results in large-scale deletions in the gene's coding region, thereby inactivating the target protein. After design, the targets were validated for specificity to avoid off-target effects.
[0032] The sequence of the first target fragment of the MYC2a gene is shown in SEQ ID NO:7, and the sequence of the second target fragment is shown in SEQ ID NO:8; The sequence of the first target fragment of the MYC2b gene is shown in SEQ ID NO:9, and the sequence of the second target fragment is shown in SEQ ID NO:10. The sequence of the first target fragment of the COI1 gene is shown in SEQ ID NO:11, and the sequence of the second target fragment is shown in SEQ ID NO:12; The sequence of the first target fragment of the AMY3 gene is shown in SEQ ID NO:13, and the sequence of the second target fragment is shown in SEQ ID NO:14; The sequence of the first target fragment of the BAM1 gene is shown in SEQ ID NO:15, and the sequence of the second target fragment is shown in SEQ ID NO:16; The sequence of the first target fragment of the BAM5 gene is shown in SEQ ID NO:17, and the sequence of the second target fragment is shown in SEQ ID NO:18.
[0033] Example 3: Preparation of the carrier 1. The vector backbone CsPTX041 was digested with restriction endonucleases. The digestion system (50 μL) consisted of: 10 μL vector backbone DNA, 1 μL restriction endonuclease, 5 μL 10× digestion buffer, and 34 μL sterile deionized water. The mixture was incubated at 37°C for 3–4 h. The digestion products were detected by 1% agarose gel electrophoresis. The linearized vector backbone was recovered, purified, and used for later use.
[0034] 2. Using a homologous recombination kit, the recovered target fragment was ligated to the linearized vector backbone via homologous recombination. The ligation system (20 μL) consisted of: X μL of linearized vector backbone, Y μL of target fragment, 4 μL of 5× homologous recombinase buffer, 1 μL of homologous recombinase, and sterile deionized water to a final volume of 20 μL. The mixture was incubated at 37°C for 30 min and immediately placed on ice for 5 min to cool.
[0035] The amount of linearized vector backbone and the amount of target fragment used are calculated using the formula for X / Y.
[0036] The optimal amount of linearized vector backbone used in the recombinant reaction system is 0.03 pmol, and the optimal amount of target fragment is 0.06 pmol (molar ratio of linearized vector backbone to target fragment is 1:2). The DNA mass corresponding to these molar amounts can be roughly calculated using the following formula: Optimal linearized vector backbone usage amount = [0.02 × number of linearized vector backbone base pairs] ng (0.03 pmol) Optimal amount of target fragment used = [0.04 × number of base pairs of target fragment] ng (0.06 pmol) For example, when cloning a 2kb target fragment into a 5kb linearized vector backbone, the optimal amount of linearized vector backbone should be 0.02 × 5000 = 100 ng; the optimal amount of target fragment should be 0.04 × 2000 = 80 ng.
[0037] 3. Transform the recombinant ligation product into E. coli DH5α competent cells. The specific steps are as follows: (1) Take 10 μL of the ligation product and add it to 50 μL of DH5α competent cells. Mix gently and incubate on ice for 30 min.
[0038] (2) Heat shock in a 42℃ water bath for 45 seconds, then immediately ice bath for 2 minutes, avoiding shaking.
[0039] (3) Add 450 μL of antibiotic-free LB liquid medium to the centrifuge tube and culture at 37°C and 200 r / min for 1 h to restore cell activity.
[0040] (4) Spread 100 μL of bacterial culture onto LB solid medium containing Kan resistance and incubate upside down at 37°C for 12-16 h to obtain single colonies. Pick single colonies and inoculate them into LB liquid medium containing Kan resistance. Incubate at 37°C with shaking at 200 r / min for 8-10 h. Perform bacterial culture PCR identification. Further extract plasmids from PCR-positive clones and perform sequencing verification to confirm that the target site is correctly inserted into the vector backbone and that there are no sequence mutations. Successfully construct the recombinant vectors of each gene and store the plasmids at -20°C for later use.
[0041] Example 4: Agrobacterium-mediated transformation and cucumber genetic transformation 1. The recombinant vector was transformed into Agrobacterium EHA105 by electroporation, and positive Agrobacterium strains were obtained by PCR identification; 2. Infect cucumber plants with positive Agrobacterium strains. The specific steps are as follows: Soak the required seeds in water at 55℃-65℃ for 4-6 hours. Remove the seed coat with tweezers. Wash the seeds with 75% alcohol for 30 seconds, then wash them with 30% sodium hypochlorite solution (84 disinfectant: distilled water = 1:1) for 10-15 minutes. Inoculate the washed seeds onto the inoculation medium and culture in the dark at 25℃ for 2 days, until the two cotyledons are slightly open. Infect the explants with Agrobacterium for 10-15 minutes, then place the infected explants in a co-culture medium and co-culture in the dark for 2 days. Transfer the co-cultured explants to the differentiation medium and culture them under light and constant temperature for about 15-28 days until the callus redifferentiates and grows adventitious shoots. Transfer the selected explants that have grown in the differentiation medium to the differentiation medium and subculture for about 15 days. For explants that have grown well in the subculture, cut off the leaf parts, leaving only the callus and adventitious shoots, and place them in a new differentiation medium to continue culturing for about 15 days. When the adventitious buds grow to 2-3 cm, cut them off from the base of the root and transfer them to a rooting medium to induce rooting. Once the adventitious roots of the seedlings are growing well, remove the transformed seedlings, wash off the culture medium from the roots, and transplant them into plastic containers filled with vermiculite.
[0042] 3. The nutrient systems for each culture medium are shown in Tables 1, 2, 3, and 4.
[0043] Table 1. Inoculation medium (pH=5.6-5.8)
[0044] Table 2. Co-culture medium (pH=5.4)
[0045] Table 3 Differentiation medium (pH=5.6-5.8)
[0046] Table 4 Rooting medium (pH=5.6-5.8)
[0047] Example 5: Obtaining positive transgenic seedlings Genomic DNA was extracted from regenerated plants, and PCR amplification was performed using vector-specific primers and gene-specific primers. Positive transgenic seedlings were obtained by electrophoresis detection and screening.
[0048] Example 6 Sequencing Identification Using the genomic DNA of positive transgenic seedlings as a template, the target gene fragment was amplified, recovered, and sequenced. The fragment was compared with the wild-type sequence to identify the large-fragment deletion mutation type and obtain homozygous deletion mutant lines.
[0049] Example 7: Combined application of genes Mutant 1: coi1 Mutants; Mutant 2: amy3 Mutants; Combinatorial mutant 1: myc2a myc2b Double-gene mutant; Combinatorial mutant 2: bam1 bam5 Double-gene mutant.
[0050] Due to functional redundancy, combined mutants 1 and 2 were cross-pollinated in the T0 generation to form a double-mutant mutant. The gene structures and target sites of the wild-type and mutants are as follows: Figure 1 and Figure 2 As shown.
[0051] Example 8 Phenotypic Identification 1. Observation of anther phenotype During the flowering period, flower buds and open flowers were collected to observe the anther dehiscence of wild-type and mutant strains. For example... Figure 3 As shown, under a stereomicroscope, the wild-type anthers dehisced normally, opening and everting along the longitudinal sutures, with numerous pollen grains on the surface. In contrast, the mutant anthers had smooth surfaces, abnormal dehiscence, and no pollen release. Paraffin section observation revealed that the wild-type anther walls curled outwards, the septa ruptured, and no pollen in the anther cavity. The mutant anthers, however, did not show significant epidermal shrinkage, the pores remained connected by the septa, and pollen remained trapped within the anther cavity. In summary, the mutant exhibited anther dehiscence and significantly abnormal stamen development.
[0052] 2. Pollen viability testing Pollen viability was detected using an in vitro germination method. Figure 4 As shown, the wild type has a large number of pollen and a germination rate as high as 98%; the mutant strain has very few or no pollen, a pollen germination rate as low as 5% or no germination, and the pollen is completely sterile, thus it is identified as a male-sterile strain.
[0053] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.
Claims
1. The application of MYC2a, MYC2b, COI1, AMY3, BAM1 and / or BAM5 genes in the creation of male-sterile cucumber lines, characterized in that, The nucleotide sequences of the MYC2a, MYC2b, COI1, AMY3, BAM1 and BAM5 genes are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.
2. The application according to claim 1, characterized in that, The application involves inducing stable, heritable male sterility in cucumbers by knocking out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes.
3. The application according to claim 2, characterized in that, The application involves using CRISPR / Cas9 gene editing technology to knock out one or more of the MYC2a, MYC2b, COI1, AMY3, BAM1, and BAM5 genes.
4. The application according to claim 3, characterized in that, The application utilizes CRISPR / Cas9 gene editing technology. Knock out the COI1 gene; or Knock out the AMY3 gene; or Simultaneously knock out the MYC2a and MYC2b genes; or Simultaneously, the BAM1 and BAM5 genes were knocked out.
5. The target sequences for CRISPR / Cas9 gene editing knockout of the MYC2a, MYC2b, COI1, AMY3, BAM1, and / or BAM5 genes, characterized in that... The target sequence of the MYC2a gene is selected from SEQ ID NO:7 and / or SEQ ID NO:8; The target sequence of the MYC2b gene is selected from SEQ ID NO:9 and / or SEQ ID NO:10; The target sequence of the COI1 gene is selected from SEQ ID NO:11 and / or SEQ ID NO:12; The target sequence of the AMY3 gene is selected from SEQ ID NO:13 and / or SEQ ID NO:14; The target sequence of the BAM1 gene is selected from SEQ ID NO:15 and / or SEQ ID NO:16; The target sequence of the BAM5 gene is selected from SEQ ID NO:17 and / or SEQ ID NO:
18.
6. A method for creating male-sterile cucumber lines, characterized in that, The method includes: Constructing a recombinant vector from the target sequence according to claim 5; The recombinant vector was used to infect explants of cucumber recipients via Agrobacterium-mediated infection. Positive transgenic seedlings were obtained through cultivation and resistance screening; The positive transgenic seedlings were sequenced and identified to obtain homozygous mutant lines with the target gene successfully knocked out; Phenotypic identification was performed on the homozygous mutant lines to screen for stable hereditary male-sterile cucumber lines.
7. The method according to claim 6, characterized in that, The stamens of the cucumber male-sterile line are abnormally developed, the anthers do not dehisce, and the pollen is non-viable or completely sterile; the pistils of the cucumber male-sterile line are normally developed and can be pollinated and produce fruit normally.
8. A recombinant vector for creating male-sterile lines of cucumber, characterized in that, The recombinant vector includes the target sequence as described in claim 5.
9. The recombinant vector according to claim 8, characterized in that, The method for constructing the recombinant vector includes: PCR amplification of DNA fragments containing the target sequence; The amplified fragment is ligated to the linearized vector; The ligation product was transformed into E. coli, and the recombinant vector was obtained through positive clone screening and sequencing verification.
10. The application of the cucumber male-sterile line prepared by the method of claim 6 or 7 or the recombinant vector of claim 8 or 9 in cucumber hybridization breeding and large-scale production of hybrid seeds.