A method for creating diploid seedless watermelon by gene editing ClCKI1

Abstract

The invention discloses a method for creating diploid seedless watermelon by gene editing ClCKI1, and belongs to the technical field of plant genetic engineering breeding. The invention discloses a method for creating diploid seedless watermelon by gene editing ClCKI1, which uses CRISPR / Cas9 technology for the first time to perform targeted editing on the ClCKI1 gene of watermelon, so that its gene function is lost, female gametes are sterile, self-pollination fruits swell normally, double fertilization cannot form zygotes, seeds are aborted, and only a small amount of white-shelled seeds are produced, thereby creating diploid seedless watermelon. Compared with wild-type materials, the edited diploid seedless watermelon plants created by the method of the invention have no obvious phenotypic changes in the vegetative growth stage and male gametes, except for the sterility of female gametes. The method has the advantages of being simple and easy to operate, high efficiency, low cost and short cycle.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering breeding, and more specifically to a method for creating diploid seedless watermelon by gene editing ClCKI1. Background Art

[0002] Watermelon (Citrullus lanatus), whose ripe fruit is rich in sugar, organic acid, vitamins and minerals, has important nutritional and edible value, and is an important horticultural crop that is widely cultivated and has high economic value all over the world. Seedless watermelon is favored by consumers and producers because of its seedless and convenient consumption, high sweetness, rich nutritional quality, strong disease resistance and storage resistance. In recent years, the research on seedless watermelon has mainly focused on the hybridization of diploid and tetraploid watermelons to obtain triploid seedless, hormone seedless and chromosome translocation seedless. Traditional triploid seedless watermelon is obtained by hybridization of tetraploid and diploid. It is difficult to obtain tetraploid mother parent during the breeding process, and the formation of triploid seeds is difficult, the seed quantity is small, the seed coat is thick, the seed germination rate is low and the seedling rate is low, resulting in high seed production costs. The breeding process of hormone seedless and chromosome translocation seedless is complicated, time-consuming and labor-intensive, with low fruit setting rate, poor seedless and commercial properties of the fruit (Zheng Xiaoying et al., 2005). The long breeding cycle and high breeding cost have greatly restricted the promotion and development of seedless watermelon. In recent years, technical research on seedless watermelon at the molecular biology level is in its initial stage. With the application of genetic engineering in the field of breeding, the reproductive development genes of watermelon can be modified to prevent double fertilization from failing to form seeds normally, making it possible to create seedless watermelon at the diploid level.

[0003] The life cycle of angiosperms undergoes an alternation from the diploid sporophyte generation to the haploid gametophyte generation, which is achieved through gametophyte development, double fertilization to form a zygote, and embryonic development. Female gametophyte development is of great significance to plant sexual reproduction and crop yield. Cytokinin histidine protein kinase (CKI1) is a constitutive activator of cytokinin signaling, a key determinant of the central cell of angiosperms, a precursor of the endosperm, and plays an important role in the development of female gametes. CKI1 is stably expressed from the FG1 (female gametophyte 1) stage of embryo sac development to the FG7 stage of mature embryo sac, with high expression concentrated in the central cell of the mature embryo sac stage and low expression in the egg cell and synergid. The functional traits of CKI1 have not been reported in watermelon.

[0004] CRISPR / Cas9-mediated plant genome editing technology can modify the excellent traits of crops at specific sites, thereby achieving precise breeding of crops and improving breeding efficiency. Therefore, providing a method for creating diploid seedless watermelon by gene editing ClCKI1 is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a method for creating diploid seedless watermelon by gene editing ClCKI1, and uses CRISPR / Cas9 to edit the watermelon histidine kinase protein ClCKI1 (Cla97C02G040700) to create diploid horizontal seedless watermelon, shorten the seedless watermelon breeding cycle, reduce breeding costs, and accelerate the breeding process.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A dual-target knockout vector, comprising dual targets of gRNA1 and gRNA2, or gRNA3 and gRNA4;

[0008] The sequences of gRNA1, gRNA2, gRNA3 and gRNA4 are as follows:

[0009] gRNA1: 5'-AATTTTGCTAAACTTCTCA-3'; SEQ ID NO.3;

[0010] gRNA2: 5'-CATTGGTCAGTAGCAACAC-3'; SEQ ID NO.4;

[0011] gRNA3: 5'-TTCTTCGAACTCAAATCTA-3'; SEQ ID NO.5;

[0012] gRNA4: 5'-TCCACCACGGGAATATAGT-3'; SEQ ID NO. 6.

[0013] Furthermore, a method for creating diploid seedless watermelon by gene editing ClCKI1 is provided, wherein the specific steps are as follows:

[0014] 1) Design the target gRNA1, gRNA2, gRNA3, and gRNA4 according to the ClCKI1 CDS sequence;

[0015] 2) Construction of ClCKI1 CRISPR / Cas9 dual-target knockout vector; gRNA1 and gRNA2, gRNA3 and gRNA4 were combined with dual targets respectively;

[0016] (1) Using restriction endonuclease BsaI-HF to digest the CRISPR / Cas9 vector pBSE402, and recovering the digested vector pBSE402;

[0017] (2) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using primers Target1-1F / Target1-2R and Target1-3F / Target1-4R to recover target fragment 1 and target fragment 2, respectively;

[0018] (3) homologously recombining the vector pBSE402 digested in step (1) with the target fragment recovered in step (2) to transform DH5α competent cells;

[0019] (4) shaking the colonies obtained in step (3) and extracting the recombinant plasmids, sequencing and comparing them, and transforming the recombinant plasmids with correct sequencing into Agrobacterium EHA105 competent cells;

[0020] (5) performing PCR detection on the bacterial colonies obtained in step (4), and performing genetic transformation of watermelon after verification;

[0021] 3) Genetic transformation of watermelon is carried out through infection, co-cultivation, recovery culture, selection culture, bud elongation culture and rooting culture stages to obtain transgenic plants;

[0022] 4) Performing gene editing detection on the transgenic plants obtained in step 3), and transplanting the gene-edited plants to a test field to observe the seedless phenotype.

[0023] Furthermore, the ClCKI1 CDS sequence is shown in SEQ ID NO.2.

[0024] Further, the sequences of the primers Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows:

[0025] Target1-1F:

[0026] 5'-TCGAAGTAGTGATTG TGAGAAGTTTAGCAAAATT GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.7;

[0027] Target1-2R:

[0028] 5'-TTCTAGCTCTAAAAC GTGTTGCTACTGACCAATG CAATCTCTTAGTCGACTCTAC-3';SEQ IDNO.8;

[0029] Target1-3F:

[0030] 5'-TCGAAGTAGTGATTG TTCTTCGAACTCAAATCTA GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.9;

[0031] Target1-4R:

[0032] 5'-TTCTAGCTCTAAAAC ACTATATTCCCGTGGTGGA CAATCTCTTAGTCGACTCTAC-3'; SEQ ID NO. 10.

[0033] Furthermore, the dual-target knockout vector or the method described is used in seedless watermelon breeding.

[0034] The material selected for genetic transformation of watermelon in the present invention is the wild variety 'YL'.

[0035] The gene editing is achieved through the CRISPR / Cas9 system. The vegetative growth and male gametes of the Clcki1 mutant are not affected, but the female gametes are sterile. The gene editing method of the present invention can shorten the breeding cycle of seedless watermelons, save breeding costs, accelerate the breeding process of seedless watermelons, and enrich the germplasm resources of seedless watermelons.

[0036] The present invention uses CRISPR / Cas9 technology to knock out the ClCKI1 gene in watermelon, thereby causing the gene to be functionally deficient. The mutant exhibits female gamete sterility, fertilization failure, seed abortion, and only a small amount of white-shelled seeds are produced, thereby achieving seedless watermelon at the diploid level. The diploid seedless watermelon created by CRISPR / Cas9 in the present invention effectively shortens the breeding cycle of seedless watermelon and provides a theoretical basis for the study of seedless watermelon at the molecular biology level.

[0037] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses a method for creating diploid seedless watermelon by gene editing ClCKI1. For the first time, CRISPR / Cas9 technology is used to target and edit the watermelon ClCKI1 gene, so that its gene function is lost, the female gamete is sterile, fertilization fails, and only a small amount of white shell seeds can be formed, thereby creating a diploid seedless watermelon. Compared with the wild-type material, the edited diploid seedless watermelon plant created by the method of the present invention has no obvious phenotypic changes in male gametes and vegetative growth stages except for female gamete sterility. The method has the advantages of simple operation, high efficiency, low cost and short cycle. Creating diploid seedless watermelon by gene editing ClCKI1 shortens the breeding cycle, saves breeding costs, and enriches seedless watermelon breeding resources, which is of great significance for accelerating the breeding of seedless watermelon varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0039] Figure 1 The accompanying drawing is a schematic diagram of the structure of the ClCKI1 gene of the present invention; black boxes represent exons, black lines represent introns, and the target sites of the ClCKI1 gene are marked by arrows;

[0040] Figure 2 The accompanying drawing is a schematic diagram of the recombinant construction of the pBSE402 CRISPR / Cas9 vector of the present invention;

[0041] Among them, A: dual targets of gRNA1 and gRNA2; B: dual targets of gRNA3 and gRNA4; expression frames pro35S:zCAS9, pro35S:GFP, pro35S:Basta belong to the original vector pBSE402;

[0042] Figure 3 The accompanying drawing is a schematic diagram of the Clcki1 CRISPR / Cas9 mutation types of the present invention;

[0043] Among them, WT is the wild-type 'YL' sequence, Clcki1 is the 'YL' mutant sequence; the gRNA target sequence and PAM sequence are underlined and bolded, respectively; the missing bases of the mutant are indicated by black dotted lines;

[0044] Figure 4 The accompanying drawing shows the phenotype observation of the Clcki1 seedless fruit of the present invention;

[0045] Among them, a: self-pollinated mature fruit of wild type WT; b: mature fruit of Clcki1-1 mutant; c: cross-section of self-pollinated mature fruit of wild type WT; d: cross-section of mature fruit of Clcki1-1 mutant; e: number of seeds in self-pollinated mature fruit of wild type WT; f: number of seeds in mature fruit of Clcki1-1 mutant. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Example 1 Analysis of ClCKI1 (Cla97C02G040700) gene structure and design of editing sites

[0048] The BlastClCKI1 gene in the watermelon genome database of the Cucurbitaceae website (http: / / cucurbitgenomics.org / ) was obtained, with gene ID ClCKI1: Cla97C02G040700; and its gene sequence was analyzed.

[0049] The watermelon ClCKI1 gene sequence is shown in SEQ ID NO.1, and the sequence analysis is shown in Figure 1 As shown, the ClCKI1 gene has five exons. According to the CDS sequence of ClCKI1 (Cla97C02G040700) SEQ ID NO.2 (YL), the target was designed on the CRISPR direct website (http: / / crispr.dbcls.jp / ), including 4 targets, ClCKI1 target gRNA1, gRNA2, gRNA3, gRNA4; gRNA1 and gRNA3 target on the first exon, gRNA2 and gRNA4 target on the second exon.

[0050] Watermelon ClCKI1 gene sequence:

[0051]

[0052] ClCKI1 CDS sequence:

[0053] ATGTATGTGTCCACACCA AATTTTGCTAAACTTCTCA

[0054] The ClCKI1 target sequence is as follows:

[0055] gRNA1: 5'-AATTTTGCTAAACTTCTCA-3'; SEQ ID NO.3;

[0056] gRNA2: 5'-CATTGGTCAGTAGCAACAC-3'; SEQ ID NO.4;

[0057] gRNA3: 5'-TTCTTCGAACTCAAATCTA-3'; SEQ ID NO.5;

[0058] gRNA4: 5'-TCCACCACGGGAATATAGT-3'; SEQ ID NO. 6.

[0059] Example 2 Construction of ClCKI1 CRISPR / Cas9 dual-target knockout vector

[0060] Among them, ClCKI1 gRNA1 and gRNA2, gRNA3 and gRNA4 respectively combine dual targets. The main expression elements of the recombinant plasmid are shown in Figure 2 .

[0061] The CRISPR / Cas9 vector pBSE402 was digested with restriction endonuclease BsaI-HF (NEW ENGLAND BioLabs) and the digested vector pBSE402 was recovered. Enzyme digestion system (30 μL): 10x CutSmart buffer 3 μL, pBSE402 (1-2 μg) 2 μL, BsaI-HF 1 μL, ddH2O 24 μL.

[0062] Using the intermediate vector pCBC-DT1T2 as a template, Target1-1F / Target1-2R (containing gRNA1 and gRNA2, respectively) and Target1-3F / Target1-4R (containing gRNA3 and gRNA4, respectively) as primers, PCR amplification was performed using Vazyme P505 high-fidelity enzyme (PhantaMax Super-Fidelity DNA Polymerase).

[0063] The primer sequences of Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows:

[0064] Target1-1F:

[0065] 5'-TCGAAGTAGTGATTG TGAGAAGTTTAGCAAAATT GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.7;

[0066] Target1-2R:

[0067] 5'-TTCTAGCTCTAAAAC GTGTTGCTACTGACCAATG CAATCTCTTAGTCGACTCTAC-3';SEQ IDNO.8;

[0068] Target1-3F:

[0069] 5'-TCGAAGTAGTGATTG TTCTTCGAACTCAAATCTA GTTTTAGAGCTAGAAATAGC-3';SEQ IDNO.9;

[0070] Target1-4R:

[0071] 5'-TTCTAGCTCTAAAAC ACTATATTCCCGTGGTGGA CAATCTCTTAGTCGACTCTAC-3'; SEQ ID NO. 10.

[0072] Amplification system (50μL): 2x Phanta Max Buffer 25μL, dNTP Mix (10mM) 1μL, upstream and downstream primers (10μM) 2μL each, Phanta Max Super-Fidelity DNA Polymerase 1μL, template DNA 2μL, ddH2O 17μL. PCR reaction program: pre-denaturation 95℃3min; denaturation 95℃15s, annealing 58℃15s, extension 72℃30s, 35 cycles; complete extension 72℃5min. Agarose gel electrophoresis was used to detect the recovery of target fragment 1 (with Target1-1F / Target1-2R as primers) and target fragment 2 (with Target1-3F / Target1-4R as primers).

[0073] The digested vectors were connected with the recovered target fragments 1 and 2 by homologous recombination using Vazyme C112ClonExpress II One Step Cloning Kit. Ligation reaction system (10 μL): 1 μL of linearized vector, 2 μL of target fragment, 2 μL of 5x CE II Buffer, 1 μL of Exnase II, 4 μL of ddH2O, reacted at 37°C for 30 min, and transformed into DH5α competent cells respectively. The transformation method was carried out according to the DH5α competent cell instruction manual of Shanghai Weidi Biotechnology Co., Ltd.

[0074] Primers U626-IDF and U629-IDR were used for colony PCR detection, and the primer sequences were as follows:

[0075] U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3'; SEQ ID NO.11;

[0076] U629-IDR: 5'-AGCCCTCTCTCTTTCGATCCATCAAC-3'; SEQ ID NO. 12.

[0077] The recombinant plasmids were extracted by shaking the bacteria, sequenced and compared, and the recombinant plasmids with correct sequencing were transformed into Agrobacterium EHA105 competent cells. The transformation method was carried out according to the instructions for Agrobacterium EHA105 competent cells of Shanghai Weidi Biotechnology Co., Ltd.

[0078] The primers U626-IDF and U629-IDR were used to perform PCR detection on Agrobacterium to verify the correct colonies.

[0079] Example 3 Watermelon Genetic Transformation

[0080] Sowing: Take full seeds of watermelon material 'YL', soak them in a 55℃ waterbath for 30 minutes, peel off the seed shells. Enter the clean bench, disinfect the peeled seed kernels with 75% alcohol for 30 seconds, soak and disinfect in 3% sodium hypochlorite for 15 minutes, wash with sterile water 5 times, then dry on sterile filter paper, spread on sowing medium (BM, breeding medium; H2O, Agar 6.64g / L), and culture in the dark at 28℃ for 2 days.

[0081] Dip infection: After the seeds germinate, cut off the two ends of the cotyledons, and divide the remaining explants into 8 equal pieces in a cross shape for dip infection. At the same time, pick the EHA105 single colony verified by PCR into LB liquid culture medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and shake the bacterial solution until the OD 600 =0.8, resuspend the bacterial solution in MS culture medium (M519 4.43 g / L, sucrose 30 g / L, 6-BA 1.5 mg / L) to a final concentration of OD 600 =0.2. The cut cotyledons were inoculated with a 20 mL syringe containing the resuspended bacterial solution under vacuum for 15 min, the explants were taken out and dried on sterile filter paper, and then spread on co-culture medium (CM; M519 4.43 g / L, sucrose 30 g / L, G3251 3 g / L, 6-BA 1.5 mg / L) padded with filter paper for co-culture, and cultured in the dark at 28°C for 3 days; M519 and G3251 were purchased from Phytotech, and 6-BA was purchased from Yuanye Company.

[0082] Recovery culture: After 3 days of co-cultivation, the cotyledon blocks were transferred to recovery medium (RM; M5194.43 g / L, sucrose 30 g / L, G3251 3 g / L, 6-BA 1.5 mg / L, 200 mg / LTimentin) and cultured for 7 days at 28°C, with a light intensity of 20,000 lux and a light duration of 16 h / d.

[0083] Selective culture: After the recovery culture is completed, the explants are transferred to selective medium (SM; M519 4.43 g / L, sucrose 30 g / L, Agar 6.4 g / L, 6-BA 1.5 mg / L, 1.4 mg / L Basta and 200 mg / LTimentin) for selective culture, and subcultured at 28°C for 3-4 weeks, with subculture every 12 days.

[0084] Shoot elongation culture: The explants with obvious buds carrying GFP tags were transferred to shoot elongation medium (SE; M519 4.43 g / L, sucrose 30 g / L, Agar 6.4 g / L, 6-BA 0.5 mg / L and Timentin 200 mg / L) for culture.

[0085] Rooting culture: Create new incisions on the selected buds and transfer them to MS medium containing 0.5 mg / L IAA and 200 mg / L Timentin for rooting culture. Culture at 28°C until roots form.

[0086] Transplanting: When the regenerated seedlings take root and grow 4-5 true leaves, take them out of the culture bottle, rinse with clean water to remove the culture medium on the roots, transplant them into a flowerpot with a substrate: vermiculite ratio of 2:1, cover with plastic wrap to keep warm and moist for 3-4 days, gradually remove the film to harden the seedlings, and move them into the field for normal management.

[0087] Example 4 Editing detection of watermelon Clcki1 transgenic plants

[0088] CTAB method was used to extract DNA from watermelon regeneration seedlings (containing different target sites) carrying Clcki1 GFP tags. Cut young leaves and place them in a 2ml centrifuge tube containing steel balls, precool them in liquid nitrogen, and grind them into powder by rapid shaking; add 800μL CTAB extraction buffer preheated at 65℃, and place them in a 65℃ water bath for 30min, turning them over every 10min; add an equal volume of 24:1 (chloroform: isoamyl alcohol) solution, mix them upside down, and centrifuge them at 8000r / min for 10min; transfer the supernatant to a 1.5ml centrifuge tube, add an equal volume of isopropanol, and mix them gently upside down; centrifuge them at 10000r / min for 10min, discard the supernatant, rinse the precipitate twice with 75% ethanol, pour it out and absorb the remaining liquid, and dissolve it with 100μL ddH2O (containing 0.1% RNase) after drying, and store it at 4℃ for later use.

[0089] Using the DNA of watermelon regenerated seedlings with Clcki1 carrying GFP tag as template, the sequences containing the two target sites of ClCKI1 were amplified by PCR using primers Crispr Clcki1 detection primer F and Crispr Clcki1 detection primer R, respectively, and the negative control was non-transgenic plant DNA. Detection primer Crispr ClCKI1 detection primer F was sent to test.

[0090] The sequences of Crispr Clcki1 detection primer F and Crispr Clcki1 detection primer R are as follows:

[0091] Crispr Clcki1 detection primer F: 5'-TTCCCATGATCTCCTTATGAACAA-3'; SEQ ID NO.13;

[0092] Crispr Clcki1 detection primer R: 5'-CAATGGAGACCACATGGAATCAT-3'; SEQ ID NO.14.

[0093] Amplification system (25 μL) and reaction procedure: 2x T5 SuperMIX 12.5 μL, upstream and downstream primers (10 μM) 1 μL each, template DNA 1 μL, ddH2O 9.5 μL. PCR reaction procedure: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 15 s, 35 cycles; complete extension at 72°C for 5 min. Detection by agarose gel electrophoresis.

[0094] The results of target editing comparison of Clcki1 mutants are shown in Figure 3Among them, Clcki1-1 lacked 2bp at the gRNA1 target site, Clcki1-2 lacked 2bp at the gRNA3 target site, and Clcki1-3 lacked 22bp at the gRNA3 target site.

[0095] Example 5 Phenotypic observation of Clcki1 transgenic plants The phenotypic observation is described using Clcki1-1 as an example.

[0096] The edited plants Clcki1-1, ClClcki1-2 and Clcki1-3 were planted in the Caoxinzhuang experimental field of Northwest Agriculture and Forestry University and managed normally to observe the phenotype.

[0097] The edited plants Clcki1-1, Clcki1-2 and Clcki1-3 had normal vegetative growth and male gamete development, but sterile female gametes during the reproductive stage.

[0098] Since the female gametes of Clcki1-1 were aborted and the male gametes were normal, the female flowers of Clcki1-1 were self-fertilized on the day they opened. The fertilization during self-pollination was abnormal, and the fruit swelled normally. There were a few white-shelled seeds in the mature watermelon fruits of Clcki1-1. Figure 4 .

[0099] In summary, the present invention provides a method for creating diploid seedless watermelon by gene editing ClCKI1, which targets and knocks out watermelon ClCKI1 to make its gene function lack, female gametes sterile, self-pollination fruits expand normally, but cannot be fertilized normally to form zygotes, and mature fruits present a seedless phenotype, thereby creating seedless watermelon at the diploid level and accelerating the creation process of diploid seedless watermelon. The diploid seedless watermelon created by CRISPR / Cas9 in the present invention effectively shortens the breeding cycle of seedless watermelon, saves breeding costs, promotes the breeding of seedless watermelon varieties, and provides a theoretical basis for the study of seedless watermelon at the molecular biology level.

[0100] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A dual-target knockout vector, characterized in that: Including dual targets of gRNA1 and gRNA2, or gRNA3 and gRNA4; The sequences of gRNA1, gRNA2, gRNA3 and gRNA4 are as follows: gRNA1: 5'-AATTTTGCTAAACTTCTCA-3'; SEQ ID NO.3; gRNA2: 5'-CATTGGTCAGTAGCAACAC-3'; SEQ ID NO.4; gRNA3: 5'-TTCTTCGAACTCAAATCTA-3'; SEQ ID NO.5; gRNA4: 5'-TCCACCACGGGAATATAGT-3'; SEQ ID NO.

6.

2. A method for creating diploid seedless watermelon by gene editing ClCKI1, characterized in that: The specific steps are as follows: 1) Designing the target gRNA1, gRNA2, gRNA3, and gRNA4 described in claim 1 according to the ClCKI1 CDS sequence; 2) Construction of ClCKI1 CRISPR / Cas9 dual-target knockout vector; gRNA1 and gRNA2, gRNA3 and gRNA4 were combined with dual targets respectively; (1) Using restriction endonuclease BsaI-HF to digest the CRISPR / Cas9 vector pBSE402, and recovering the digested vector pBSE402; (2) Using the intermediate vector pCBC-DT1T2 as a template, PCR amplification was performed using primers Target1-1F / Target1-2R and Target1-3F / Target1-4R to recover target fragment 1 and target fragment 2, respectively; (3) homologously recombining the vector pBSE402 digested in step (1) with the target fragment recovered in step (2) to transform DH5α competent cells; (4) shaking the colonies obtained in step (3) and extracting the recombinant plasmids, sequencing and comparing them, and transforming the recombinant plasmids with correct sequencing into Agrobacterium EHA105 competent cells; (5) performing PCR detection on the bacterial colonies obtained in step (4), and performing genetic transformation of watermelon after verification; 3) Genetic transformation of watermelon is carried out through infection, co-cultivation, recovery culture, selection culture, bud elongation culture and rooting culture stages to obtain transgenic plants; 4) performing gene editing detection on the transgenic plants obtained in step 3), and transplanting the gene-edited plants to a test field to observe the seedless phenotype; The sequences of the primers Target1-1F / Target1-2R and Target1-3F / Target1-4R are as follows: Target1-1F: 5’-TCGAAGTAGTGATTG TGAGAAGTTTAGCAAAATT GTTTTAGAGCT AGAAATAGC-3’;SEQ IDNO.7; Target1-2R: 5′-TTCTAGCTCTAAAAC GTGTTGCTACTGACCAATG CAATCTCTTAGT CGACTCTAC-3':SEQ IDNO.8; Target1-3F: 5'-TCGAAGTAGTGATTG TTCTTCGAACTCAAATCTA GTTTTAGAGCTA GAAATAGC-3';SEQ ID NO.9; Target1-4R: 5′-TTCTAGCTCTAAAAC ACTATATTCCCGTGGTGGA CAATCTCTTAGT CGACTCTAC-3';SEQ IDNO.10。 3. The method for creating diploid seedless watermelon by gene editing ClCKI1 according to claim 2, characterized in that: The ClCKI1 CDS sequence is shown in SEQ ID NO.

2.

4. Use of the dual-target knockout vector according to claim 1 or the method according to any one of claims 2 to 3 in seedless watermelon breeding.

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