CN112921051A

CN112921051A - Method for creating watermelon male sterile new germplasm by gene editing technology

Info

Publication number: CN112921051A
Application number: CN202110195588.0A
Authority: CN
Inventors: 魏春华; 张显; 张锐敏; 李佳悦; 李好; 张勇; 马建祥; 杨建强; 岳贞
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2021-06-08
Anticipated expiration: 2041-02-19
Also published as: CN112921051B

Abstract

The invention discloses a method for creating a novel male sterile germplasm of a watermelon by a gene editing technology, belonging to the technical field of plant genetic engineering. The invention firstly utilizes a CRISPR/Cas9 gene editing system to carry out gene editing and knockout on a gene ClATM1 or a conservative structural domain thereof, so that the gene is functionally deleted or mutated, thereby forming a recessive cell nucleus male sterility phenotype. Compared with the normal material, the male sterile line created by the method has no visible phenotypic change in other tissues such as female flowers, leaves, tendrils, stems, roots, growth vigor and the like except that the fertility of the male flowers is changed; if the created new male sterile germplasm is used for hybrid production or population improvement, the labor cost is greatly reduced, the breeding efficiency is improved, and the method has important production and application potentials.

Description

Method for creating watermelon male sterile new germplasm by gene editing technology

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a method for creating a watermelon male sterile new germplasm by a gene editing technology.

Background

Watermelon (Citrullus lanatus), belonging to the Cucurbitaceae (Cucurbitaceae) watermelon genus (Citrullus), is an important Cucurbitaceae crop planted widely in the world, and is a fruit-type economic crop, and is evaluated as the fifth largest fruit in the world due to the planting area and annual consumption. In production, watermelons have obvious hybrid vigor, and the conventional seed production process still adopts complex procedures such as manual bagging isolation, pollination and the like, so that the production cost is high, and the purity of seeds is difficult to completely ensure.

The male sterility of plants is a common phenomenon in nature, and is not only an important tool for researching the utilization of crop heterosis, but also an ideal material for researching the development function of plants. At present, 5 male sterility mutant genes of watermelon have been reported: smooth hairless male sterility gene (mgs); plant dwarf male gene (ms-dw), male sterile gene ms-1, male sterile gene ms-2 and male sterile gene ms-3. However, the above genes have not been finely positioned and cloned yet, and the application of the gene editing technology to the creation of male sterile germplasm is limited.

Therefore, the problem to be solved by those skilled in the art is to provide a method for creating a new male sterile germplasm of watermelon by using a gene editing technology.

Disclosure of Invention

In view of the above, the present invention provides a method for creating a new male sterile germplasm of a watermelon by using a gene editing technology.

In order to achieve the purpose, the invention adopts the following technical scheme:

the fine positioning of the sterile gene ClATM1 in the watermelon male sterile mutant Se18 is completed through the traditional gene positioning technology, and the gene is found to belong to a bHLH transcription factor family through sequence analysis and contains a conserved structural domain bHLH. Based on the gene, the invention creates the watermelon male sterile material by using the CRISPR/Cas9 technology, and further applies the watermelon male sterile material to the production process of watermelon hybrid seeds, thereby greatly improving the seed production efficiency and the purity of the hybrid seeds and reducing the production cost.

A method for creating a watermelon male sterile new germplasm by a gene editing technology comprises the following specific steps:

(1) designing a Target site for gene editing according to a CDS sequence of ClATM1 and the Target site, and designing two sets of editing sites, wherein each set comprises two Target sites, the first set comprises Target1 and Target2, and the second set comprises Target3 and Target 4;

a first set of:

target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3', respectively; SEQ ID No. 2;

target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3', respectively; SEQ ID No. 3;

the second set is as follows:

target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3', respectively; SEQ ID No. 4;

target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3', respectively; SEQ ID No. 5;

(3) construction of CRISPR/Cas9 editing vector

Carrying out PCR amplification by using an intermediate vector pCBC-DT1T2 as a template and primers of Target1F/Target2R and Target3F/Target4R respectively, and recovering a Target segment 1 and a Target segment 2 respectively;

carrying out enzyme digestion on the CRISPR/Cas9 vector pBSE402 by using restriction endonuclease BsaI-HF, and recovering the vector pBSE402 after enzyme digestion;

carrying out homologous recombination connection on the target fragment 1 and the target fragment 2 recovered in the step I and the vector pBSE402 subjected to enzyme digestion in the step II respectively to obtain a connection product 1 and a connection product 2;

fourthly, respectively transforming the ligation product 1 and the ligation product 2 into escherichia coli competence DH5 alpha, and extracting the recombinant plasmid 1 and the recombinant plasmid 2 after correct sequencing; respectively transforming to agrobacterium-infected cells EHA 105;

(3) genetic transformation of watermelon

After being verified to be correct, the gene is respectively used for watermelon genetic transformation to obtain gene editing plants atm1_1 and atm1_ 2.

Further, the created new male sterile germplasm of the watermelon is applied to hybrid production or population improvement.

The invention relates to a method for creating a novel male sterile germplasm of a watermelon by using a gene editing technology, which adopts CRISPR/Cas9 or other gene editing technologies to carry out fixed-point editing or knockout on a gene ClATM1 or a conservative domain sequence thereof, so that the function of the gene protein is deleted or mutated to generate a sterile phenotype of male flower without pollen. The application of the male sterile material created by the method in production can reduce the production cost of the watermelon hybrid and improve the purity and the seed production efficiency of the hybrid.

According to the technical scheme, compared with the prior art, the method for creating the novel watermelon male sterile germplasm by using the gene editing technology is disclosed, and the method firstly uses a CRISPR/Cas9 gene editing system to carry out gene editing and knockout on the gene ClATM1 or a conservative structural domain thereof, so that the gene is functionally deleted or mutated, and a recessive cell nucleus male sterile phenotype is formed. Compared with the normal material, the male sterile line created by the method has no visible phenotypic change in other tissues such as female flowers, leaves, tendrils, stems, roots, growth vigor and the like except that the fertility of the male flowers is changed; if the created new male sterile germplasm is used for hybrid production or population improvement, the labor cost is greatly reduced, the breeding efficiency is improved, and the method has important production and application potentials.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram showing the structure of ClATM1 gene of the present invention;

FIG. 2 is a vector map of pBSE402 according to the present invention;

FIG. 3 is a drawing showing a first set of sgRNA expression elements of recombinant plasmids of the present invention;

wherein U6-26p and U6-29p are promoters, U6-26t are terminators, gRNA-Sc is a gRNA framework, and Target1 and Target2 are Target sites;

FIG. 4 is a graph showing the comparison of YL of the present invention with atm1_1 and atm1_2 at 4 targets;

wherein, -represents a deletion; the red marked base is a PAM structure of a CRISPR/Cas9 system target;

FIG. 5 is a drawing of the male flower phenotype of the unedited and edited plants of the present invention;

where YL (unedited) male flowers are fertile, whereas edited plants are atm1_1 and atm1_2 male flowers are sterile.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 editing site selection of Gene ClATM1

Designing a Target site for gene editing according to a CDS sequence (SEQ ID NO.1) of ClATM1 and a Target site on-line design website CRISPR-P (http:// CRISPR. hzau.edu.cn/CRISPR2/news. php, V2), and designing two sets of editing sites (a first set: Target1 and Target 2; a second set: Target3 and Target4), wherein each set comprises two targets and four targets.

The structural schematic diagram of the ClATM1 gene is shown in figure 1. Among them, Target1, Target2 and Target3 are all in the non-bHLH conserved domain of gene ClATM1, while Target4 is in the conserved domain (light green). Two sets of Target points are designed, wherein each set comprises 2 targets, and the first set comprises Target1 and Target 2; the second set contains Target3 and Target 4.

A first set of:

target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3', respectively; SEQ ID No. 2;

target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3', respectively; SEQ ID No. 3;

the second set is as follows:

target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3', respectively; SEQ ID No. 4;

target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3', respectively; SEQ ID No. 5;

example 2CRISPR/Cas9 editing vector construction

(1) PCR amplification

Synthesizing the adapter primers according to the two sets of target sequences, which comprises the following steps:

a first set of:

target1 sequence primers:

Target1F：5’-TCGAAGTAGTGATTGCTGTTCAGGGAACTGTTCCGTTTTAGAGCTAGAAATAGC-3’；SEQ ID NO.6；

target2 sequence primers:

Target2R：5’-TTCTAGCTCTAAAACCTACGCCGTCTCCCACTACCAATCTCTTAGTCGACTCTAC-3’；SEQ ID NO.7；

the second set is as follows:

target3 sequence primers:

Target3F：5’-TCGAAGTAGTGATTGGCCGGAGTTTATGAAGACGGTTTTAGAGCTAGAAATAGC-3’；SEQ ID NO.8；

target4 sequence primers:

Target4R：5’-TTCTAGCTCTAAAACTCACTTCTCTTAAAAGCTCCAATCTCTTAGTCGACTCTAC-3’；SEQ ID NO.9；

underlined is the target sequence.

The intermediate vector pCBC-DT1T2 is used as a template, and high fidelity enzyme PrimeStar Max Premix (TaKaRa) is used for PCR amplification, wherein the amplification system is as follows: PrimeStarMax Premix (2X) 25. mu.L, template 2.5. mu.L (100 ng/. mu.L), upstream and downstream primers 2.5. mu.L each, ddH₂O17.5. mu.L. The PCR reaction program is: 10s at 98 ℃, 5s at 58 ℃, 15s at 72 ℃ and 38 cycles; 5min at 72 ℃. And (3) carrying out electrophoresis detection on the PCR products (the sizes of the fragments of the two sets of editing sites are 626bp), and respectively carrying out gel recovery to obtain a target fragment 1 and a target fragment 2.

(2) Vector cleavage and ligation

The CRISPR/Cas9 vector pBSE402 (vector map is shown in figure 2) is cut by using restriction enzyme BsaI-HF (NEW ENGLAND BioLabs) in the following way: pBSE 4022. mu.g, CutSmart buffer 5. mu.L, BsaI-HF 1. mu.L, ddH₂And O is supplemented to 50 mu L. Enzyme digestion is carried out for 2h at 37 ℃, and gel recovery is carried out after electrophoresis detection.

And connecting the PCR product with a vector: and respectively carrying out homologous recombination and connection on products (the target fragment 1 and the target fragment 2) amplified by the PCR and the digested vector pBSE402 to obtain a connection product 1 and a connection product 2. The connecting system is as follows: vector to insert molar ratio was about 1: 2; 5 × 4 μ L of reaction buffer; NovoRec Plus recombinase 1 μ L; ddH₂The content of O is filled to 20 mu L. Ligation was carried out at 50 ℃ for 10 min.

(3) Transformation of recombinant plasmids

Mu.l of each of the ligation product 1 and ligation product 2 was heat-shock transformed into E.coli competent DH 5. alpha. and spread on LB solid plates containing 50mg/L kanamycin, and cultured overnight at 37 ℃. And carrying out positive detection on the colony by using primers U626-IDF and U629-IDR, and carrying out bacterial liquid sequencing by using a primer EDIT.

Wherein, the specific primer sequences of U626-IDF and U629-IDR are as follows:

U626-IDF：5’-TGTCCCAGGATTAGAATGATTAGGC-3’；SEQ ID NO.10；

U629-IDR：5’-AGCCCTCTTCTTTCGATCCATCAAC-3’；SEQ ID NO.11；

wherein, the specific primer sequence of the EDIT is as follows:

EDIT：5’-GGGAATCTGAAAGAAGAGAAGCAG-3’；SEQ ID NO.12。

after the sequencing is correct, two sets of recombinant plasmids (recombinant plasmid 1 and recombinant plasmid 2) are respectively extracted and respectively transformed into agrobacterium-infected competent cells EHA105, and PCR verification (by using primers U626-IDF and U629-IDR) is correct and then used for watermelon genetic transformation.

Wherein, the first set of recombinant plasmid skeleton is shown in figure 3; the second set of recombinant plasmid scaffolds replaced Target1, Target2 of the first set of recombinant plasmid scaffolds with Target3 and Target4, respectively.

The agrobacterium transformation procedure is as follows:

adding 1 mu l of recombinant plasmid into agrobacterium EHA105 competent cells, standing on ice for 5min in sequence, quickly freezing in liquid nitrogen for 5min, carrying out water bath at 37 ℃ for 5min, finally carrying out water bath for 5min, adding 400 mu l of LB liquid culture medium without antibiotics into each tube for resuscitation, transferring the tubes to 200rpm, and carrying out shaking culture on a shaking table at 28 ℃ for 2-3 hours. And (3) taking 100 mu l of recovered bacterial liquid, plating the recovered bacterial liquid on an LB solid culture medium containing antibiotics (Kan), airing at room temperature, and after the bacterial liquid is fully absorbed, inversely placing the culture dish in an incubator at 28 ℃ for culturing for 2-3 d.

Example 3 genetic transformation of watermelon

Taking plump watermelon germplasm 'YL' seeds, soaking the seeds in distilled water at 50-55 ℃ for about 30min, and peeling off the seeds. And (3) disinfecting the peeled kernels with 75% alcohol for about 30s in an ultra-clean workbench, cleaning the kernels with sterile water twice, soaking and disinfecting the kernels with 3% sodium hypochlorite for 15min, cleaning the kernels with sterile water for 5-7 times, airing the kernels, paving the kernels in a BM solid culture medium (Agar 4.43g/L), and culturing the kernels in the dark at 25 ℃ for 3 d.

Taking out the seed after germination, cutting off two ends of the cotyledon, and dividing the remaining cotyledon part into 8 pieces for infection. During the period, EHA105 with correct PCR verification is singly selected into LB liquid culture medium containing 50mg/L kanamycin and 20mg/L rifampicin, and the bacterial liquid concentration is shaken to OD₆₀₀When the concentration was 0.8, the bacterial suspension was resuspended in MS culture medium (MS519(PhytoTech Labs)4.43g/L, sucrose 30g/L, 6-BA 1.5mg/L) to obtain OD at the final concentration₆₀₀0.2. The cut cotyledons are soaked in the bacterial liquid heavy suspension for 15min, taken out and dried, and then co-cultured in a co-culture medium CM (MS5194.43g/L, sucrose 30G/L; G3251(PhytoTech Labs) 3G/L; 6-BA 1.5mg/L) padded with filter paper, and cultured in the dark at 25 ℃ for 3 d.

After the co-culture for 3 days, the cotyledon blocks are taken out and washed with sterile water to remove the redundant agrobacterium liquid on the surface (about 5-7 times) until the sterile water is clear, and the cotyledon blocks are taken out and dried, and then placed on a recovery culture medium RM (MS5194.43g/L, 30G/L of sucrose; G32513G/L; 6-BA 1.5mg/L, 200mg/L of Timentin (Scientific Research Special)) for recovery culture and cultured at 28 ℃.

After 5-7 days of recovery culture, the cotyledons of the recovery culture were transferred to selection medium SM (MS5194.43g/L, sucrose 30G/L; G3251(PhytoTech Labs)3G/L, 1.5 mg/L6-BA, 200mg/L Timentin, 1.4mg/L Basta) for selection culture, subcultured at 28 ℃ for 3-4 weeks, and subcultured every 7 days. Explants with distinct shoots were then transferred to shoot elongation medium (MS524(PhytoTech Labs)4.43g/L, sucrose 30g/L, G32513 g/L, inositol 1g/L, SH organic solution 500. mu.L/L, NAA 0.01mg/L, 6-BA 0.1mg/L, Timentin 200mg/L, Basta 1.4.4 mg/L) at 28 ℃, 8h/d dark, 16h/d light, 8000Lx light intensity. The selected shoots were excised (note that the incisions did not contain callus), transferred to rooting medium (MS5194.43g/L, sucrose 30G/L, 6-BA 1.5mg/L, G32513G/L, 0.5mg/L IBA and 200mg/L Timentin) for rooting culture, and cultured at 28 ℃ until rooting. The preparation method of the SH organic solution comprises the following steps: 5g nicotinic acid, 5g VB₁、0.5g VB₆And the volume is up to 500 ml.

Taking out the regenerated seedling from the culture bottle when the regenerated seedling grows to 4-5 true leaves, slowly washing the culture medium at the root with clear water, transplanting the seedling into a substrate which is sterilized at high temperature and high pressure in advance, watering the substrate thoroughly, then carrying out heat preservation and moisture preservation culture, covering a plug tray with water drops after 3-4 days, and gradually uncovering and hardening the seedling.

Example 4 detection of transgenic plants of watermelon

DNA of watermelon regenerated seedlings with GFP fluorescence (GFP carried on the vector pBSE 402) was extracted by the CTAB method, and the steps were as follows: quickly grinding a small part of tender leaves in liquid nitrogen into powder, and putting the powder into a centrifugal tube of 1.5 ml; adding preheated 800 μ l CTAB extraction buffer solution, and water bathing at 65 deg.C for 30 min; adding equal volume of chloroform isoamyl alcohol, wherein the volume ratio of chloroform to isoamyl alcohol is 24: 1, centrifuging at 8000r/min for 10min after uniformly mixing; transferring the supernatant into a new centrifugal tube, and adding 2-3 volumes of isopropanol, and slightly mixing the mixture by turning upside down; centrifuging at 10000r/min for 10 min; pouring out the supernatant, washing the precipitate twice with 75% ethanol, draining the rest liquid, drying for 3min, and adding 100 μ l ddH₂O (containing 0.1% RNase) was dissolved and stored at 4 ℃ for further use.

And respectively carrying out PCR amplification on the sequences of the two sets of four target sites by using the extracted DNAs as templates and using primers ATM1-CRJC-F and ATM1-CRJC-R, wherein a positive control is a recombinant plasmid, and a negative control is non-transgenic plant DNA.

Wherein the specific primer sequences of the ATM1-CRJC-F and the ATM1-CRJC-R are as follows:

ATM1-CRJC-F：5’-CCATGCCCACTGCCTATACT-3’；SEQ ID NO.13；

ATM1-CRJC-R：5’-CGCCGGCGACATGGTGAAGA-3’；SEQ ID NO.14。

the amplification system is as follows: 2 XTaq PCR StarMix with loading Dye 10. mu.L, template 1. mu.L, primers 1. mu.L each, ddH₂O7. mu.L. The PCR reaction program is: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s, 58 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃. PCR products were recovered according to the size of the band, and TA cloning (pClone007 Versatile Simple Vector Kit, TSINGKE) was performed, and after positive detection of PCR of bacterial liquid (using primer ATM1-CRJC-F, ATM1-CRJC-R), single clones were selected and sent for testing and editing confirmation.

Wherein, the Target sequencing result corresponding to Target1 in atm1_1 is as follows:

5’-CTTCAGGGAACTGTTCC-3’；SEQ ID NO.15；

the Target sequencing results in atm1_1 corresponding to Target2 are as follows:

5’-GTAGTGGGAGACTAG-3’；SEQ ID NO.16；

the Target sequencing results in atm1_2 corresponding to Target3 are as follows:

5’-GCCGGAGTTTATGAAGACG-3’；SEQ ID NO.17；

the Target sequencing results in atm1_2 corresponding to Target4 are as follows:

5’-GAGCTTTTAAGAGATGA-3’；SEQ ID NO.18；

the comparison result is shown in figure 4, a gene editing plant atm1_1 is obtained by the first set of Target points, 2bp and 4bp (-representation) are respectively deleted in Target point regions of Target1 and Target 2; a second set of targets obtained a gene editing plant atm1_2, which lacked 2bp (-representation) in the Target region of Target 4. Both editing plants atm1_1 and atm1_2 are homozygous edits at the target.

Example 5 phenotypic Observation of transgenic plants of watermelon

The edited plants atm1_1 and atm1_2 were planted in sunlight greenhouse, managed normally, and after the male flower opened, the phenotype was observed, and the result is shown in FIG. 5. Figure 5 results show that: compared with an unedited plant YL, the overall shape, petals and stamens of the two plants of atm1_1 and atm1_2 are smaller, and the stamens have no pollen shed and are expressed as complete sterility.

In conclusion, the invention provides a method for creating a novel watermelon male sterile germplasm by using a gene editing technology, a recessive nucleus male complete abortion line can be quickly obtained by editing a sequence or a conserved structural domain bHLH of a male sterility regulatory gene ClATM1, and the method has important application potential in utilization of watermelon heterosis and population improvement.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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cttcagtttc aacaggcgcc cgctggaggt tttgataaca gtaatatcaa ccctgatttt 300

ggtcaagaag taacttctga ttccaaccgt atggtgtgtc ttgaccaatc cgattgggtt 360

gggactcaaa ttcaagaaat ggggtttaat caccaccacc atcaccagat tcaatctcaa 420

gatcaccatc atcatccgca ccagcagcag ttttcagatt ccgccatgcc cactgcctat 480

actcaagctc ccgacctctt gaacttctta aacatgccgg cccctgccag atgccccaac 540

aactcttcca tctccttctc caatcaccat acctcaccca tgggaggctt tcttggagac 600

cttcccgccg gagacgctgg caactcatcg tcgacttcgc tttcaataca ctacgatcct 660

ctgtttcacc tgaatcttcc gccgcagcca ccgctgttca gggaactgtt ccactctctt 720

cctcatggat atggtatacc ggcggcgagt tccagaggcc gaggaggtag tttgttcccg 780

gaaggggaga taatggagac agaaggaact gccggagttt atgaagacgg ggatggaagc 840

ggtgttttgg agttcagtag agatatggcg gattgtgttg ggaaaggaag aaatgggaaa 900

atgactaaac attttaccac tgaacgccaa agaagagttc aactgaatga gaaatataat 960

gctctcaaga gtttggttcc tattcctact aagaatgata gggcatcagt tgtgggagac 1020

gccataaatt acatccaaga gcttttaaga gaagtgaagg aactgaaact gctggtggag 1080

aagaagagat gcagcagaga gaggagcaag aggcacagga cggcggagga attagaaggg 1140

ggcggcgcgt gggatgttga aagcacaaat gcaaaggcag gtggtgtagt gggagacggc 1200

gtagaggatc aaagctacaa tttgagaagc tcatggctgc agagaaagac aaaagatact 1260

gaagttgatg tgagaattgt tgatgatgaa gtaaccataa agcttgtgca gcgtaaactc 1320

aactgcttgt tgcttgtctc taaattgctt gacgatcttc agcttgatct tcaccatgtc 1380

gccggcggcc acatcggcga ttactacagc ttcttgttca ataccaagat atatgaaggt 1440

tcatcagtgt atgcaagtgc catagccaac aaggttatgg aggcagtgga cagacaatac 1500

aacaacacca ccacatccaa tacctattaa 1530

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ctgttcaggg aactgttcc 19

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ccatgcccac tgcctatact 20

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cgccggcgac atggtgaaga 20

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cttcagggaa ctgttcc 17

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gtagtgggag actag 15

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gagcttttaa gagatga 17

Claims

1. A method for creating a watermelon male sterile new germplasm by a gene editing technology is characterized by comprising the following specific steps:

a first set of:

target1 sequence: 5'-CTGTTCAGGGAACTGTTCC-3', respectively; SEQ ID No. 2;

target2 sequence: 5'-GTAGTGGGAGACGGCGTAG-3', respectively; SEQ ID No. 3;

the second set is as follows:

target3 sequence: 5'-GCCGGAGTTTATGAAGACG-3', respectively; SEQ ID No. 4;

target4 sequence: 5'-GAGCTTTTAAGAGAAGTGA-3', respectively; SEQ ID No. 5;

(2) construction of CRISPR/Cas9 editing vector

(3) genetic transformation of watermelon

2. The method for creating the male sterile new germplasm of watermelon by using the gene editing technology as claimed in claim 1, wherein the created male sterile new germplasm of watermelon is applied to hybrid production or population improvement.