A method for creating new watermelon male sterile germplasm through gene editing technology and application thereof

Abstract

The application discloses a method for creating new watermelon male sterile germplasm through a gene editing technology and application thereof. The application relates to the technical field of plant genetic engineering, in particular to a method for creating new watermelon male sterile germplasm through a gene editing technology and application thereof. The protein or expression material of a regulation gene or a material for regulating the activity or content of the protein can be applied to any one of the following: 1) regulating plant male sterility; 2) preparing a product for regulating plant male sterility; 3) cultivating a plant with changed fertility; 4) preparing a product for cultivating a plant with changed fertility; and 5) plant breeding. A CRISPR / Cas9 gene editing system is used to perform gene editing knockout on a gene ClMYB33 so that the gene function is lost or mutated, thereby forming a watermelon male sterile phenotype. The male sterile material created by the method can reduce the production cost of a watermelon hybrid and improve seed production efficiency.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a method for creating new male-sterile germplasm of watermelon through gene editing technology and its application. Background Technology

[0002] watermelon( Citrullus lanatus Watermelon, belonging to the genus *Citrullus* of the family Cucurbitaceae, is a widely cultivated and consumed horticultural crop worldwide, accounting for 7% of the world's vegetable crop planting area, with a global annual output exceeding 100 million tons. As one of the world's major watermelon producers and consumers, China's watermelon industry has played a significant role in the country's agricultural development and increasing farmers' income. While watermelons exhibit significant hybrid vigor, the hybridization process requires manual emasculation and bagging, resulting in a large workload and high seed production costs. Furthermore, the stamens in hermaphroditic flowers lead to self-pollination of the female parent, making it difficult to obtain high-purity hybrids. Therefore, creating stable male-sterile materials through gene editing can provide an efficient and cost-effective technological shortcut for watermelon hybrid breeding.

[0003] Male sterility is a natural phenomenon in plants where abnormal development of male organs prevents normal pollination. Male-sterile lines are important tools for studying the utilization of heterosis in crops and ideal materials for exploring plant developmental functions. Currently reported male-sterile mutants in watermelon include the smooth, hairless male-sterile mutant (mgs); the dwarf male-sterile plant (ms-dw); male-sterile lines ms-1, ms-2, and ms-3; and the dual-purpose male-sterile line Se18. The male-sterile gene ClATM1 has been cloned and its function verified in the Se18 mutant material. Research on male-sterile genes in other mutants has not been conducted, and the regulatory mechanism of male sterility in watermelon is not fully understood.

[0004] ClMYB33 is an R2R3 MYB family transcription factor. Studies in Arabidopsis thaliana have shown that the MYB33 and MYB65 genes play important roles in tapetum development, and their functions are complementary. In the myb33 myb65 double mutant, the loss of function of these two genes leads to pollen sac development defects, indicating a synergistic effect in tapetum development. Molecular mechanism studies of MYB33 have been limited to the model plant Arabidopsis thaliana, and have not yet been reported in watermelon. A thorough understanding of the molecular mechanisms regulating male flower development in watermelon, comparing the conservation of ClMYB33 in different species, and combining gene editing technology to mutate ClMYB33 in watermelon are of great significance.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a method for creating a new watermelon male sterile germplasm through a gene editing technology. SUMMARY

[0006] The main problem to be solved by the present application is how to change the fertility of watermelon and obtain a watermelon male sterile germplasm.

[0007] In order to solve the above problems, the present application provides an application of a protein or an expression material of a regulatory gene or a material for regulating the activity or content of the protein in regulating plant male sterility.

[0008] The application of the protein or the expression material of the regulatory gene or the material for regulating the activity or content of the protein provided by the present application is in any one of the following:

[0009] 1) in regulating plant male sterility;

[0010] 2) in preparing a product for regulating plant male sterility;

[0011] 3) in cultivating plants with changed fertility;

[0012] 4) in preparing a product for cultivating plants with changed fertility;

[0013] 5) in plant breeding;

[0014] The protein is any one of the following proteins:

[0015] a1) a protein with an amino acid sequence of SEQ ID No. 2;

[0016] a2) a protein with an amino acid sequence shown in SEQ ID No. 2 after substitution and / or deletion and / or addition of one or more amino acid residues and having the same function;

[0017] a3) a protein with an amino acid sequence defined in any one of a1) or a2) having more than 75% identity and having the same function;

[0018] a4) a fusion protein obtained by connecting a terminal tag to the protein defined in any one of a1) to a3).

[0019] The protein in a1) is named as ClMYB33.

[0020] In order to facilitate the purification or detection of the protein in a1), a tag protein can be connected to the amino terminal end or carboxyl terminal end of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0021] The above-mentioned protein can be artificially synthesized, or a gene encoding the protein can be synthesized first and then expressed biologically to obtain the protein.

[0022] The tag protein includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0023] A person of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein ClMYB33 of the present application by using known methods, such as a method of directed evolution or a method of point mutation. Those nucleotides artificially modified to have 75% or more identity with the nucleotide sequence of the protein ClMYB33 isolated in the present application are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application, as long as they encode the protein ClMYB33 and have the function of the protein ClMYB33.

[0024] The 75% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0025] In the present application, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity of a pair of amino acid sequences or nucleotide sequences can be calculated by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search in Advanced BLAST 2.1, and then the value of the identity (%) can be obtained.

[0026] In the present application, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0027] In the present context, the more than 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0028] In the above use, the protein is derived from watermelon (Citrullus lanatus). Citrullus lanatus

[0029] In the present context, the substance that modulates the activity and / or content of the protein can be a substance that modulates the expression of a gene that encodes the protein ClMYB33.

[0030] In the above, the substance that modulates the expression of a gene can be a substance that performs at least one of the following six types of modulation:

[0031] 1) modulation at the transcriptional level of the gene;

[0032] 2) modulation after the transcription of the gene (i.e. modulation of the splicing or processing of the primary transcript of the gene);

[0033] 3) modulation of the RNA transport of the gene (i.e. modulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm);

[0034] 4) modulation of the translation of the gene;

[0035] 5) modulation of the mRNA degradation of the gene;

[0036] 6) modulation of the post-translational of the gene (i.e. modulation of the activity of the protein translated from the gene).

[0037] In the present application, the modulation can be up-regulation or enhancement or increase. The modulation can also be down-regulation or weakening or decrease.

[0038] In the present context, the enhancement, increase or up-regulation of the expression of the gene that encodes the protein as described above in a recipient plant, or / and the enhancement, increase or up-regulation of the activity and / or content of the gene that encodes the protein as described above is achieved by introducing the gene that encodes the protein as described above into the recipient plant.

[0039] In the present context, the modulation of the expression of the gene that encodes the protein can be inhibition or decrease or down-regulation of the expression of the gene. The inhibition or decrease or down-regulation of the expression of the gene can be achieved by gene knockout or gene silencing.

[0040] In the above use, the substance that modulates the expression of a gene or the substance that modulates the activity or content of the protein can be a biological material that is associated with the protein as described above, and the biological material can be any one of the following:

[0041] ​c1) a nucleic acid molecule encoding a protein as described hereinbefore;

[0042] c2) an expression cassette comprising the nucleic acid molecule of c1);

[0043] c3) a recombinant vector comprising the nucleic acid molecule of c1), or a recombinant vector comprising the expression cassette of c2);

[0044] c4) a recombinant microorganism comprising the nucleic acid molecule of c1), or a recombinant microorganism comprising the expression cassette of c2), or a recombinant microorganism comprising the recombinant vector of c3);

[0045] c5) a transgenic plant cell line comprising the nucleic acid molecule of c1), or a transgenic plant cell line comprising the expression cassette of c2);

[0046] c6) a transgenic plant tissue comprising the nucleic acid molecule of c1), or a transgenic plant tissue comprising the expression cassette of c2);

[0047] c7) a transgenic plant organ comprising the nucleic acid molecule of c1), or a transgenic plant organ comprising the expression cassette of c2);

[0048] e1) a nucleic acid molecule inhibiting or reducing or silencing the expression of a gene encoding a protein as described hereinbefore;

[0049] e2) an expression cassette comprising the nucleic acid molecule of e1);

[0050] e3) a recombinant vector comprising the nucleic acid molecule of e1), or a recombinant vector comprising the expression cassette of e2);

[0051] e4) a recombinant microorganism comprising the nucleic acid molecule of e1), or a recombinant microorganism comprising the expression cassette of e2), or a recombinant microorganism comprising the recombinant vector of e3);

[0052] e5) a transgenic plant cell line comprising the nucleic acid molecule of e1), or a transgenic plant cell line comprising the expression cassette of e2);

[0053] e6) a transgenic plant tissue comprising the nucleic acid molecule of e1), or a transgenic plant tissue comprising the expression cassette of e2);

[0054] e7) a transgenic plant organ comprising the nucleic acid molecule of e1), or a transgenic plant organ comprising the expression cassette of e2).

[0055] In the above uses, the nucleic acid molecule of c1) can be a DNA molecule as defined in any one of the following,

[0056] d1) the nucleotide sequence is a DNA molecule as defined in SEQ ID No. 1 ;

[0057] d2) The coding sequence is the DNA molecule shown in SEQ ID No. 3;

[0058] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above;

[0059] d4) A DNA molecule that hybridizes under strict conditions with a nucleotide sequence defined by d1) or d2) and encodes the protein described above.

[0060] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0061] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector pBSE406.

[0062] Construct a plant expression vector containing existing plant expression vectors. ClMYB33 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0063] use ClMYB33 When constructing recombinant plant expression vectors, any type of enhancing promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0064] In order to facilitate the identification and screening of the transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants, including but not limited to, enzymes or luminescent compounds that can produce color changes (GUS genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes), etc. For the safety of transgenic plants, no selective marker genes are added, and the transformed plants are directly screened under stress.

[0065] The gene or gene fragment provided by the present application is introduced into plant cells or recipient plants by using any vector that can guide the expression of foreign genes in plants, so that transgenic cell lines and transgenic plants with altered fertility can be obtained. ClMYB33 The expression vector carrying the ClMYB33 gene can be transformed into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. conventional biological methods, and the transformed plant tissues are cultivated into plants.

[0066] Alternatively, the expression cassette of e2) is an expression cassette having a DNA molecule shown in SEQ ID No. 6.

[0067] In the above application, the nucleotides 305-589 of SEQ ID No. 6 of the expression cassette sequence are promoters for initiating the transcription of sgRNA genes, the sgRNA1 gene is located at positions 17-36 of SEQ ID No. 6 of the sequence listing, the sgRNA2 gene is located at positions 590-609 of SEQ ID No. 6 of the sequence listing, and the nucleotides 113-304 are terminators for terminating the transcription of sgRNA genes.

[0068] The present application also provides a method for improving plant male sterility, which comprises the step M of inhibiting or reducing or silencing the activity and / or content of the protein described above in the plant of interest, or / and, inhibiting or reducing or silencing the expression amount of the gene encoding the protein described above, to improve the plant male sterility.

[0069] The present application also provides a method for reducing plant male sterility, which comprises the step P of enhancing, increasing or up-regulating the activity and / or content of the protein described above in the plant of interest, or / and, enhancing, increasing or up-regulating the expression amount of the gene encoding the protein described above, to reduce the plant male sterility.

[0070] In the above method, the reducing the expression amount and / or activity of the gene encoding the protein ClMYB33 in the plant of interest can be: reducing or inactivating the activity of the gene encoding the protein ClMYB33 in the genome of the plant of interest by using gene mutation, gene knockout, gene editing or gene knockdown technology.

[0071] The gene knockout refers to the phenomenon of inactivating a specific target gene by gene editing technology. The gene knockout inactivates a specific target gene by changing the DNA sequence.

[0072] The present application provides a method for breeding a male sterile plant, comprising inhibiting or reducing or silencing the expression of the gene encoding the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the plant of interest, or / and inhibiting or reducing or silencing the activity and / or content of the gene encoding the above-mentioned protein, to obtain a male sterile plant.

[0073] In an embodiment of the present application, the breeding method for breeding a male sterile plant comprises the following steps:

[0074] (1) constructing a recombinant expression vector for inhibiting or reducing or silencing the gene encoding the above-mentioned protein;

[0075] (2) transforming the recombinant expression vector constructed in step (1) into a recipient plant to obtain a male sterile plant.

[0076] In the present application, the purpose of plant breeding can include breeding a male sterile plant.

[0077] In the present application, the specific performance of the male sterile plant can be: anther does not dehisce, pollen is not released, resulting in male sterility.

[0078] In the present application, the plant can be as follows:

[0079] G1) a monocotyledonous plant or a dicotyledonous plant;

[0080] G2) a Cucurbitaceae plant;

[0081] G3) a Cucurbitaceae plant;

[0082] G4) Citrullus;

[0083] G5) watermelon.

[0084] The application discloses a method for creating a new watermelon male sterile germplasm through a gene editing technology, which first uses a CRISPR / Cas9 gene editing system to perform gene editing knockout on a gene ClMYB33, so that the gene function is lost or mutated, and thus a male sterile phenotype is formed. Compared with normal materials, the watermelon male sterile line created by the method has no visible phenotype change in other organizations such as female flowers, leaves, tendrils, stems, roots and growth potential, except that the fertility of male flowers is changed; if the created male sterile new germplasm is used for hybrid production or population improvement, the artificial cost can be greatly reduced, the breeding and seed production efficiency can be improved, and the method has important production and application potential. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 For ClMYB33 The gene structure and target position schematic diagram; the gene structure of ClMYB33 includes three exons and two intron structures, Target1 is in the first exon (red box), and Target2 is in the second exon (red box).

[0086] Figure 2 It is a pBSE406 vector map.

[0087] Figure 3 It is a recombinant plasmid expression element, wherein U6-26p and U6-29p are promoters, U6-26t and U6-29t are terminators, gRNA-Sc is a gRNA skeleton, and is an element in the intermediate vector pCBC-DT1T2; Target1 and Target2 are target sites.

[0088] Figure 4 It is an alignment result of ZZJM (WT) and clmyb33 at two target sites in the application; wherein '-', Target sequence and PAM sequence are respectively indicated by underlining and letter bolding.

[0089] Figure 5 It is a clmyb33 male flower phenotype. DETAILED DESCRIPTION

[0090] The application will be further described in detail in combination with specific embodiments, and the examples given are only for illustrating the application, rather than limiting the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0091] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0092] The quantitative tests in the following examples, unless otherwise specified, are all set up with three repeated experiments, and the results are taken as the average value.

[0093] The pCBC-DT1T2 in the following examples has been described in: Tian, S., Jiang, L., Gao, Q. et al. Efficient CRISPR / Cas9-based gene knockout in watermelon. Plant Cell Rep 36, 399-406 (2017). https: / / doi.org / 10.1007 / s00299-016-2089-5. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0094] The pBSE406 in the following examples has been described in: Chen YY, Wang ZP, Ni HW, et al. CRISPR / Cas9-mediated base editing system efficiently generates gain-of-function mutations in Arabidopsis [J]. Sci China Life Sci, 2017, 60(5):520-523. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0095] The watermelon germplasm material 'ZZJM' in the following examples has been described in: Ren et al., Evolutionary gain of oligosaccharide hydrolysis and sugar transport enhanced carbohydrate partitioning in sweet watermelon fruits, THE PLANT CELL 2021: 33: 1554-1573. The biological material can be obtained from the applicant, which is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0096] Example 1, ClMYB33Obtaining of gene knockout mutants

[0097] 1. Editing site design of ClMYB33 (Cla97C08G151350)

[0098] According to the genomic DNA sequence (SEQ ID No. 1) of ClMYB33 and the online target site design website CRISPR P (http: / / crispr.hzau.edu.cn / CRISPR2 / news.php, V2), the target site for gene editing was designed, and two sets of editing sites Target1 and Target2 were designed. ClMYB33 The schematic diagram of gene structure is shown in Figure 1 .

[0099] In the genome (http: / / cucurbitgenomics.org / organism / 21) of watermelon cultivar 97103, the genomic sequence encoding ClMYB33 protein is shown in SEQ ID No. 1 of the sequence listing, and the protein encoding the amino acid sequence is shown in SEQ ID No. 2 of the sequence listing. The CDS encoding ClMYB33 protein is shown in SEQ ID No. 3 of the sequence listing. Among them, the target point Target1 is in the first exon of the gene, and Target2 is in the second exon of the gene. The exon is represented by capital letters, and the intron is represented by lowercase letters. ClMYB33 The schematic diagram of gene structure is shown in

[0100] Target-1 sequence: 5'-ATAGTGGAGGGAATGCTAG-3' (SEQ ID No. 4);

[0101] Target-2 sequence: 5'-GAATACTCGAATAAAGAGG-3' (SEQ ID No. 5).

[0102] 2. Obtaining of target fragment

[0103] Using the intermediate vector pCBC-DT1T2 as the template, and Target2-BsF / Target2-F0 / Target1-R0 / Target1-BsR (containing Target1 and Target2, respectively) as the primers, PCR amplification was performed using Vazyme P505 high-fidelity enzyme (PhantaMax Super-Fidelity DNA Polymerase).

[0104] Amplification system (50 μL): 2x PhantaMax Buffer 25 μL, dNTP Mix (10 mM) 1 μL, Target2-BsF / Target1-BsR primer (10 μM) 2 μL each, Target2-F0 / Target1-R0 (0.5 μM) 2 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 μL, template DNA 2 μL, ddH2O 13 μL.

[0105] PCR reaction program: pre-denaturation 95 °C for 3 min; denaturation 95 °C for 15 s, annealing 55 °C for 15 s, extension 72 °C for 30 s, 35 cycles; complete extension 72 °C for 5 min. The target fragment (Target-1)-(gRNA-Sc)-(U6-26t)-(U6-29p)-(Target-2) (SEQ ID No. 6) was recovered by 1% agarose gel electrophoresis.

[0106] Target2-BsF, Target2-F0, Target1-R0, Target1-BsR primer sequences are as follows:

[0107] Target2-BsF: 5'-ATATATGGTCTCGATTG GAATACTCGAATAAAGAGG GTT-3';

[0108] Target2-F0: 5'-TG GAATACTCGAATAAAGAGG GTTTTAGAGCTAGAAATAGC-3';

[0109] Target1-R0: 5'-AAC CTAGCATTCCCTCCACTAT CAATCTCTTAGTCGACTCTAC-3';

[0110] Target1-BsR: 5'-ATTATTGGTCTCGAAAC CTAGCATTCCCTCCACTAT C-3'.

[0111] 3. CRISPR / Cas9 editing vector construction

[0112] The CRISPR / Cas9 vector pBSE406 (vector map see Fig. 1) was digested with restriction endonuclease Bsal-HF (NEB) and T4 Ligase (NEB) to obtain the vector backbone pBSE406-BsaI and the gRNA expression cassette pBSE406-gRNA. Figure 2) enzyme digestion and recombination. Enzyme digestion and recombination system (15 μL): 10xBSA 1.5 μL, pBSE406 (1 μg / μl) 2 μL, BsaI-HF (NEB) 1 μL, T4 Ligase (NEB) 1 μL, 10xNEB T4 Buffer 1.5 μL, target fragment 2 μL, ddH2O 6 μL. Reaction procedure: 37 °C for 5 h, 50 °C for 5 min, 80 °C for 10 min, transform DH5α competent cells.

[0113] The transformed E. coli was subjected to colony PCR detection using identification primers U626-F and U629-R, and the primer sequences were as follows:

[0114] U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3';

[0115] U629-IDR: 5'-AGCCCTCTTCTTTCGATCCATCAAC-3'.

[0116] After the correct band size was identified, the bacteria were inoculated and shaken, and after sequencing was completed, sequence alignment was performed to obtain the recombinant plasmid pBSE406-sgRNA1-sgRNA2 (expression elements of the recombinant plasmid are shown in SEQ ID No. 6). Figure 3 ).

[0117] The structure of pBSE406-sgRNA1-sgRNA2 is described as follows: the small fragment between the restriction endonuclease BsaI-HF recognition site and the T4 Ligase recognition site of the starting vector pBSE406 is replaced with the DNA molecule of SEQ ID No. 6, and the other sequences of the starting vector remain unchanged to obtain the recombinant vector.

[0118] The recombinant plasmid pBSE406-sgRNA1-sgRNA2 with correct sequencing was transformed into Agrobacterium EHA105 competent cells. PCR detection was performed on Agrobacterium EHA105 using primers U626-IDF and U626-IDR to obtain Agrobacterium positive clone EHA105 / pBSE406-sgRNA1-sgRNA2.

[0119] 4. Watermelon genetic transformation

[0120] Seeding: 50 seeds of the laboratory watermelon germplasm material 'ZZJM' were soaked in a 55 °C water bath for 30 min, and the seed coat was removed. The peeled seeds were sterilized with 75% alcohol for 1 min, soaked in 3% sodium hypochlorite for 15 min, and then washed with sterile water for 5 times. The seeds were placed on a breeding medium (BM, breeding medium; H2O, Agar 6 g / L) in a clean bench and cultured at 25 °C in the dark for about 3 d.

[0121] Inoculation: The single colony of Agrobacterium EHA105 / pBSE406-sgRNA1-sgRNA2 which was verified by colony PCR was inoculated into LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin. When the concentration of the bacterial solution was shaken to OD 600 0.6-0.8, the bacterial bodies were collected by centrifugation at 5000 rpm for 5 min, and then resuspended in MS liquid medium (MS+30% sucrose+6-BA) to make the final concentration of OD 600 =0.4.

[0122] Immersion: When the embryo radicles were about 1 cm long, the cotyledons were cut at both ends, and the explants were cut into small pieces for immersion. The cut cotyledons were mixed evenly in a 20 mL syringe containing 10 mL of resuspended bacterial solution, and then immersed under negative pressure for 15 min. The explants were taken out and dried on sterile filter paper, and then transferred to co-culture medium (CM, co-culture medium; MS+30% sucrose+plant gel+6-BA) padded with filter paper for co-culture, and cultured in the dark at 28°C for 3 d. MS was purchased from Phytotech Company, and 6-BA was purchased from Yuanye Company.

[0123] Recovery culture: After 3 d of co-culture, the cotyledon pieces were transferred to recovery medium (RM, recovery medium; MS+30% sucrose+plant gel+6-BA+Timentin) and cultured at 28°C under the conditions of light intensity 20000 lux and light duration 16 h / d for 7 d.

[0124] Selection culture: After the recovery culture, the explants were transferred to selection medium (SM, selective medium; MS+30% sucrose+agar+6-BA+Timentin+Basta) for selection culture, and subcultured at 28°C for 3-4 weeks, with subculture every 12 d.

[0125] Seedling elongation culture: The explants with obvious bud points carrying the GFP label were transferred to seedling elongation medium (SE, seedling elongation; MS+30% sucrose+agar+6-BA+Timentin) for culture.

[0126] Rooting culture: The selected buds were transferred to MS medium containing IAA and Timentin for rooting culture, and cultured at 28°C until rooting.

[0127] Transplanting: When the regenerated seedlings were rooted and grew to 4-5 true leaves, they were taken out of the culture bottle, the root medium was carefully removed, and they were transplanted into a flowerpot with a substrate: vermiculite ratio of 1:3, and then cultured in normal management after watering and keeping warm and moist.

[0128] The MS medium used in the experiment is PhytoTech M519.

[0129] Finally, positive ClMYB33 Gene editing strain.

[0130] 5、 ClMYB33 Transgenic plant editing detection

[0131] Sampling of pBSE406-sgRNA1-sgRNA2 vector containing GFP fluorescence watermelon ClMYB33 Gene editing strain regeneration seedlings, and genomic DNA was extracted by CTAB method.

[0132] Specific steps: take a small part of the tender leaves of watermelon in liquid nitrogen and grind into powder, put it in a 1.5ml centrifuge tube; add 800μL preheated CTAB extraction buffer, 65℃ water bath for 30min; add equal volume of chloroform isoamyl alcohol (the volume ratio of chloroform and isoamyl alcohol is 24:1), mix well and centrifuge at 8000r / min for 10min; transfer the supernatant to a new 1.5mL centrifuge tube, add 2 / 3 volume of isopropanol, mix gently and precipitate in-20℃ refrigerator for 1h; centrifuge at 10000r / min for 10min; discard the supernatant, wash the precipitate with 75% ethanol twice, dry the remaining liquid after pouring off, dissolve with 100μL ddH2O (containing 0.1% RNAase) at room temperature, and store at 4℃ for standby.

[0133] Using the extracted genomic DNA as the template, the sequence containing two target sites was amplified by PCR with primers ClMYB33-IDF1 / ClMYB33-IDR1 and ClMYB33-IDF2 / ClMYB33-IDR2.

[0134] Among them, the specific primer sequences of ClMYB33-IDF1 / ClMYB33-IDR1 and ClMYB33-IDF2 / ClMYB33-IDR2 are as follows:

[0135] ClMYB33-IDF1: 5'-GGCATCCGAAAAATGAGATTG-3';

[0136] ClMYB33-IDR1: 5'-GTGCCCATTTGTTTCCCAT-3';

[0137] ClMYB33-IDF2: 5'-ACCTTTCGTGCTTTGCTGTAG-3';

[0138] ClMYB33-IDR2: 5'-CATCGGGACCAGGAAATAA-3'.

[0139] Amplification system (50 μL) and reaction procedure: 2 × T5 SuperMIX 25 μL, primer (10 μM) 2 μL each, template DNA 1 μL, ddH2O 20 μL.

[0140] PCR reaction procedure: pre-denaturation 98℃ 3 min; denaturation 98℃ 10 s, annealing 58℃ 10 s, extension 72℃ 15 s, 35 cycles; complete extension 72℃ 5 min. 1% agarose gel electrophoresis was used to detect the band size. The remaining PCR product was sent for sequencing, and the sequencing primers were ClMYB33-IDR1 / ClMYB33-IDR2.

[0141] The alignment results of the ClMYB33 mutant target editing are shown in Figure 4 , wherein the ClMYB33 is deleted by 3 bp at the Target1 target and deleted by 2 bp at the Target2 target.

[0142] Compared with the wild type (ZZJM), the mutant clmyb33 has ClMYB33 gene mutations in both of the two homologous chromosomes ClMYB33 gene mutations are as follows: ClMYB33 In the gene, “5'-ATAGTGGAGGGAATGCTAGCGG-3' (corresponding to positions 77-98 of SEQ ID No. 1, positions 77-98 of SEQ ID No. 3)” is mutated to “5'-ATAGTGGAGGGACTAGCGG-3'”, with deletion of three nucleotides; “5'-GAATACTCGAATAAAGAGGCGG-3' (corresponding to positions 1748-1769 of SEQ ID No. 1, positions 396-417 of SEQ ID No. 3)” is mutated to “5'-GAATACTCGAATAAAGGCGG-3'”, with deletion of two nucleotides; the deletion of the above nucleotides causes a frame shift, resulting in changes in the ClMYB33 amino acid and premature termination of translation, leading to loss of function of the ClMYB33 protein.

[0143] Example 2, ClMYB33 Phenotype observation of gene edited plants

[0144] The edited plants clmyb33-1 were planted in the greenhouse of Yanqing base in the spring of 2024, and were normally managed. After the male flowers opened, their phenotypes were observed.

[0145] Compared with the wild type, clmyb33-1 there was no obvious difference in plant morphology and female flower development between the mutant and the wild type. The mutant female flowers could be normally pollinated. In the reproductive growth stage, the mutant male anthers were smaller, and the anthers did not dehisce in the later stage, with no pollen production Figure 5 .

[0146] In summary, the present application provides a method for creating new watermelon male sterile germplasm through gene editing technology, by editing the male sterile regulatory gene ClMYB33 , a completely male sterile line can be quickly obtained, which has important application potential in watermelon hybrid advantage utilization and population improvement.

[0147] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented within a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

Claims

1. Use of a substance for regulating expression of a gene or a substance for regulating protein content in any one of the following: 1) use in improving male sterility of a plant; 2) use in preparing a product for improving male sterility of a plant; 3) use in breeding a male sterile plant; 4) use in preparing a product for breeding a male sterile plant; the protein is a protein with an amino acid sequence of SEQ ID No. 2; the gene encodes the protein; the substance for regulating expression of a gene or the substance for regulating protein content is a biological material related to the protein, and the biological material is any one of the following: e1) a nucleic acid molecule for inhibiting expression of the gene encoding the protein; e2) an expression cassette containing the nucleic acid molecule of e1); e3) a recombinant vector containing the nucleic acid molecule of e1) or containing the expression cassette of e2); e4) a recombinant microorganism containing the nucleic acid molecule of e1) or containing the expression cassette of e2) or containing the recombinant vector of e3); e5) a transgenic plant cell line containing the nucleic acid molecule of e1) or containing the expression cassette of e2); e6) a transgenic plant tissue containing the nucleic acid molecule of e1) or containing the expression cassette of e2); e7) a transgenic plant organ containing the nucleic acid molecule of e1) or containing the expression cassette of e2); the plant is watermelon.

2. A method of increasing male sterility in a plant, comprising: The method comprises a step M of inhibiting the content of the protein of claim 1 or inhibiting the expression amount of the gene encoding the protein of claim 1 in a plant of interest to improve male sterility of the plant; the plant is watermelon.

3. A breeding method for breeding a male sterile plant, characterized by: The method comprises a step M of inhibiting the content of the protein of claim 1 or inhibiting the expression amount of the gene encoding the protein of claim 1 in a plant of interest to improve male sterility of the plant; 4. The method of claim 3, wherein: the plant is watermelon. The method comprises the following steps: (1) constructing a recombinant expression vector for inhibiting the gene encoding the protein of claim 1; (2) introducing the recombinant expression vector constructed in step (1) into a recipient plant to obtain a male sterile plant.

Images