Method for creating watermelon male sterility new germplasm through gene editing technology and application of watermelon male sterility new germplasm

Abstract

The invention discloses a method for creating a novel watermelon male sterility germplasm through a gene editing technology and application of the novel watermelon male sterility germplasm. The invention relates to the technical field of plant genetic engineering, in particular to a method for creating a novel watermelon male sterility germplasm through a gene editing technology and application of the novel watermelon male sterility germplasm. The protein or the expression substance for regulating and controlling the gene or the substance for regulating and controlling the activity or content of the protein can be applied to any one of the following applications: 1) regulating and controlling the male sterility of plants; 2) preparing a product for regulating plant male sterility; (3) cultivating plants with changed fertility; according to the present invention, the CRISPR / Cas9 gene editing system is adopted to perform gene editing knockout on the gene ClMYB33 so as to make the gene function be deleted or mutated so as to form the watermelon male sterility phenotype, and the male sterility material created by using the method can reduce the production cost of the watermelon hybrid and improve the seed production efficiency;

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a method for creating new watermelon male sterile germplasm by gene editing technology and application thereof. Background Art

[0002] watermelon( Citrullus lanatus ), a crop of the genus Citrullus in the family Cucurbitaceae, is a horticultural crop widely cultivated and eaten worldwide. Its planting area accounts for 7% of the world's vegetable crop planting area, and the global annual output exceeds 100 million tons. As one of the world's important watermelon producers and consumers, the development of the watermelon industry has played an important role in my country's agricultural development and in increasing farmers' income. In production, watermelon has obvious hybrid advantages, but it requires artificial emasculation and bagging during the hybridization process, which is labor-intensive and has high seed production costs. In addition, the stamens in the bisexual flowers lead to self-pollination of the female parent, making it difficult to obtain high-purity hybrids. Therefore, the creation of stable male sterile materials through gene editing can provide an efficient and cost-saving technical shortcut for the breeding of watermelon hybrids.

[0003] Male sterility is a natural phenomenon that results in the inability to pollinate normally due to abnormal development of male organs. Male sterility lines are an important tool for studying the utilization of hybrid vigor in crops and are also ideal materials for exploring plant developmental functions. At present, reported watermelon male sterility mutants include smooth hairless male sterility mutant (mgs); dwarf male sterility plant (ms-dw), male sterility lines ms-1, ms-2, ms-3, and male sterility dual-purpose line Se18. The cloning and functional verification of the male sterility gene ClATM1 in Se18 mutant material have been completed, but no relevant research has been conducted on male sterility genes in other mutants, and the regulatory mechanism of watermelon male sterility has not been fully resolved.

[0004] ClMYB33 is a transcription factor of the R2R3 MYB family. Studies in Arabidopsis have shown that MYB33 and MYB65 play an important role in tapetum development and their functions are complementary. In the myb33 myb65 double mutant, the loss of function of these two genes leads to anther development defects, indicating that they have a synergistic effect in tapetum development. The molecular mechanism of MYB33 is limited to the model plant Arabidopsis, and has not been reported in watermelon. It is of great significance to fully understand the molecular mechanism of regulating male flower development in watermelon, compare the conservation of ClMYB33 in different species, and mutate ClMYB33 in watermelon by combining gene editing technology.

[0005] Therefore, providing a method for creating new watermelon male sterile germplasm through gene editing technology is an urgent problem that technicians in this field need to solve. Summary of the invention

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

[0007] In order to solve the above problems, the present invention provides the use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in regulating plant male sterility.

[0008] The use of the protein or gene expression regulating substance provided by the present invention or the substance regulating the activity or content of the protein in any of the following: 1) Application in regulating plant male sterility; 2) Application in the preparation of products for regulating plant male sterility; 3) Application in breeding plants with altered fertility; 4) Use in the preparation of products for cultivating plants with altered fertility; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No. 2; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence of at least 75% identity with any of a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any protein defined in a1) to a3).

[0009] The name of the protein described in a1) above is ClMYB33.

[0010] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0011] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0012] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0013] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein ClMYB33 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein ClMYB33 isolated by the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein ClMYB33 and have the function of the protein ClMYB33.

[0014] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0015] Herein, 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 webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for a pair of amino acid sequences or nucleotide sequences to calculate the identity, then the value (%) of the identity can be obtained.

[0016] Herein, the 80% or greater identity may 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.

[0017] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0018] In the above application, the protein is derived from watermelon ( Citrullus lanatus ).

[0019] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene, wherein the gene encodes the protein ClMYB33.

[0020] In the above, the substance regulating gene expression may be a substance that performs at least one of the following six types of regulation: 1) Regulation at the transcriptional level of the gene; 2) regulation after the transcription of the gene (i.e., regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm); 4) Regulation of the translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0021] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be down-regulation, attenuation or reduction.

[0022] Herein, the enhancement, increase or upregulation of the expression level of the gene encoding the protein mentioned above in the recipient plant, or / and the enhancement, increase or upregulation of the activity and / or content of the gene encoding the above protein is achieved by introducing the gene encoding the above protein into the recipient plant.

[0023] Herein, the regulation of the expression of the coding gene of the protein may be inhibition, reduction or down-regulation of the coding gene expression. Inhibition, reduction or down-regulation of the coding gene expression may be achieved by gene knockout or gene silencing.

[0024] In the above application, the substance regulating gene expression or the substance regulating the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following: c1) a nucleic acid molecule encoding the protein described above; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce or silence the expression of the protein encoding gene mentioned above; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0025] In the above application, the nucleic acid molecule in c1) can be any of the following DNA molecules, d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No.1; d2) the coding sequence is the DNA molecule shown in SEQ ID No.3; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence specified in d1) or d2) and encodes the protein described above; d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0026] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0027] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, phages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors. Specifically, the vector may be pBSE406.

[0028] Use existing plant expression vectors to construct ClMYB33 The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as but not limited to Agrobacterium crown gall induction (Ti) plasmid genes (such as carmine synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0029] use ClMYB33 When the gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter and corn ubiquitin promoter, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0030] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0031] Any vector capable of directing the expression of foreign genes in plants is used to express the ClMYB33Genes or gene fragments are introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered fertility. Expression vectors carrying the ClMYB33 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, and the transformed plant tissues can be cultivated into plants.

[0032] Optionally, the expression cassette described in e2) is an expression cassette having a DNA molecule shown in SEQ ID No.6.

[0033] In the above application, nucleotides 305-589 of SEQ ID No.6 of the expression cassette sequence in e2) are promoters for initiating transcription of the sgRNA gene, the sgRNA1 gene is located at positions 17-36 of SEQ ID No.6 of the sequence list, the sgRNA2 gene is located at positions 590-609 of SEQ ID No.6 of the sequence list, and nucleotides 113-304 are terminators for terminating transcription of the sgRNA gene.

[0034] The present invention also provides a method for improving plant male sterility, which comprises step M, wherein step M is to inhibit, reduce or silence the activity and / or content of the protein mentioned above in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein mentioned above, so as to improve the male sterility of the plant.

[0035] The present invention also provides a method for reducing plant male sterility, which comprises step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein mentioned above to reduce the male sterility of the plant.

[0036] In the above method, reducing the expression level and / or activity of the gene encoding the protein ClMYB33 in the target plant can be: using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the gene encoding the protein ClMYB33 in the genome of the target plant.

[0037] The gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by changing the DNA sequence.

[0038] The present invention provides a method for cultivating male sterile plants, comprising inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, so as to obtain male sterile plants.

[0039] In one embodiment of the present invention, the breeding method for cultivating male sterile plants comprises the following steps: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein mentioned above; (2) The recombinant expression vector constructed in step (1) is transferred into a recipient plant to obtain a male sterile plant.

[0040] In the present invention, the purpose of plant breeding may include cultivating male sterile plants.

[0041] In the present invention, the specific manifestation of male sterility may be: anthers do not dehisce and pollen does not release, resulting in male sterility.

[0042] In the present invention, the plant may be as follows: G1) monocots or dicots; G2) Cucurbitales; G3) Cucurbitaceae; G4) Citrullus; G5) Watermelon.

[0043] The present invention discloses a method for creating a new watermelon male sterile germplasm by gene editing technology. The method uses the CRISPR / Cas9 gene editing system for the first time to perform gene editing and knockout on the gene ClMYB33, so that the gene function is lost or mutated, thereby forming a male sterile phenotype. Compared with normal materials, the watermelon male sterile line created by the method of the present invention has no visible phenotypic changes in other tissues such as female flowers, leaves, tendrils, stems, roots, growth potential, etc., except that the fertility of male flowers has changed; if the created new male sterile germplasm is used for hybrid production or population improvement, it will greatly reduce labor costs and improve breeding and seed production efficiency, and has important production application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 for ClMYB33 Schematic diagram of gene structure and target location; the gene structure of ClMYB33 includes 3 exons and 2 introns, Target1 is in the first exon (red box), and Target2 is in the second exon (red box).

[0045] Figure 2 This is the pBSE406 vector map.

[0046] Figure 3It is a recombinant plasmid expression element, in which U6-26p and U6-29p are promoters, U6-26t and U6-29t are terminators, gRNA-Sc is the gRNA skeleton, and is an element in the intermediate vector pCBC-DT1T2; Target1 and Target2 are target sites.

[0047] Figure 4 The comparison results of ZZJM (WT) and clmyb33 at two target sites of the present invention are shown; wherein, - indicates deletion; the Target sequence and PAM sequence are marked with underscores and bold letters, respectively.

[0048] Figure 5 This is the male flower phenotype of clmyb33. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed 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 all be obtained from commercial channels.

[0051] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0052] 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 public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0053] The pBSE406 in the following examples has been described in: Chen YY, Wang ZP, Ni HW, et al. CRISPR / Cas9-mediated baseediting system efficiently generates gain-of-function mutations in Arabidopsis[J]. Sci China Life Sci, 2017, 60(5):520-523. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0054] The watermelon germplasm material 'ZZJM' in the following example has been recorded 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 public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0055] Embodiment 1, ClMYB33 Obtaining gene knockout mutants 1. Editing site design of ClMYB33 (Cla97C08G151350) The target sites for gene editing were designed according to the genomic DNA sequence of ClMYB33 (SEQ ID No. 1) and the target site online design website CRISPR P (http: / / crispr.hzau.edu.cn / CRISPR2 / news.php, V2). A total of two sets of editing sites, Target1 and Target2, were designed. ClMYB33 Schematic diagram of gene structure Figure 1 .

[0056] In the genome DNA of watermelon cultivar 97103 (http: / / cucurbitgenomics.org / organism / 21), the genome sequence encoding the ClMYB33 protein is shown in SEQ ID No.1 in the sequence list, and the encoded amino acid sequence is shown in SEQ ID No.2 in the sequence list. The CDS encoding the ClMYB33 protein is shown in SEQ ID No.3 in the sequence list. ClMYB33 The first exon of the gene, Target2 is in the second exon of the gene. Exons are represented by capital letters, and introns are represented by lowercase letters.

[0057] Target-1 sequence: 5'-ATAGTGGAGGGAATGCTAG-3' (SEQ ID No. 4); Target-2 sequence: 5'-GAATACTCGAATAAAGAGG-3' (SEQ ID No. 5).

[0058] 2. Obtaining the target fragment PCR amplification was performed using the intermediate vector pCBC-DT1T2 as a template and Target2-BsF / Target2-F0 / Target1-R0 / Target1-BsR (containing Target1 and Target2, respectively) as primers using Vazyme P505 high-fidelity enzyme (PhantaMax Super-Fidelity DNA Polymerase).

[0059] Amplification system (50μL): 2xPhantaMax Buffer 25μL, dNTP Mix (10mM) 1μL, Target2-BsF / Target1-BsR primers (10μM) 2μL each, Target2-F0 / Target1-R0 (0.5μM) 2μL each, Phanta Max Super-Fidelity DNA Polymerase 1μL, template DNA 2μL, ddH2O 13μL.

[0060] PCR reaction program: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 55℃ for 15s, extension at 72℃ for 30s, 35 cycles; complete extension at 72℃ for 5min. The target fragment (Target-1)-(gRNA-Sc)-(U6-26t)-(U6-29p)-(Target-2) (SEQ ID No.6) was recovered by 1% agarose gel electrophoresis.

[0061] The primer sequences of Target2-BsF, Target2-F0, Target1-R0, and Target1-BsR are as follows: Target2-BsF: 5'-ATATATGGTCTCGATTG GAATACTCGAATAAAGAGG GTT-3'; Target2-F0: 5'-TG GAATACTCGAATAAAGAGGGTTTTAGAGCTAGAAATAGC-3'; Target1-R0: 5'-AAC CTAGCATTCCCTCCACTAT CAATCTCTTAGTCGACTCTAC-3′; Target1-BsR: 5'-ATTATTGGTCTCGAAAC CTAGCATTCCCTCCACTAT C-3'.

[0062] 3. Construction of CRISPR / Cas9 editing vector The CRISPR / Cas9 vector pBSE406 (vector map, see Figure 2 ) Enzyme digestion and recombination ligation. Enzyme digestion and recombination system (15μL): 10xBSA 1.5μL, pBSE406 (1μg / μl) 2μL, BsaI-HF (NEB) 1μL, T4 Ligase (NEB) 1μL, 10xNEB T4 Buffer 1.5μL, target fragment 2μL, ddH2O 6μL. Reaction procedure: 37℃ 5 h, 50℃ 5min, 80℃ 10min, transform DH5α competent cells.

[0063] The transformed E. coli was tested by colony PCR using identification primers U626-F and U629-R. The primer sequences are as follows: U626-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3'; U629-IDR: 5'-AGCCCTCTTCTTCGATCCATCAAC-3'.

[0064] After the band size was confirmed to be correct, the bacteria were inoculated and shaken. After sequencing, the sequence was aligned to obtain the recombinant plasmid pBSE406-sgRNA1-sgRNA2 (the recombinant plasmid expression element is shown in Figure 3 ).

[0065] The structure of pBSE406-sgRNA1-sgRNA2 is described as follows: a recombinant vector is obtained by replacing the small fragment between the restriction endonuclease BsaI-HF recognition site and the T4 Ligase recognition site of the starting vector pBSE406 with a DNA molecule of SEQ ID No. 6, and keeping the other sequences of the starting vector unchanged.

[0066] The correctly sequenced recombinant plasmid pBSE406-sgRNA1-sgRNA2 was transformed into Agrobacterium EHA105 competent cells. PCR detection of Agrobacterium EHA105 was performed using primers U626-IDF and U626-IDR to obtain Agrobacterium positive clones EHA105 / pBSE406-sgRNA1-sgRNA2.

[0067] 4. Genetic transformation of watermelon Sowing: Take 50 seeds of the laboratory watermelon germplasm material 'ZZJM', soak them in a 55℃ water bath for 30 minutes, and then peel off the seed shells. In the clean bench, disinfect the peeled seed kernels with 75% alcohol for 1 minute, soak them in 3% sodium hypochlorite for 15 minutes, and then wash them with sterile water for 5 times. Spread the seed kernels on the sowing medium (BM, breeding medium; H2O, Agar 6g / L), and culture them in the dark at 25℃ for about 3 days.

[0068] Inoculation: Pick the single colony of Agrobacterium EHA105 / pBSE406-sgRNA1-sgRNA2 verified by colony PCR into LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and shake the bacterial solution until the concentration reaches OD 600 When the OD was 0.6-0.8, the cells were collected by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in MS liquid medium (MS + 30% sucrose + 6-BA) to a final concentration of OD 600 =0.4.

[0069] Dip: When the radicle of the seed kernel grows to about 1 cm, cut off the two ends of the cotyledon and cut the explant into small pieces for dipping. Mix the cut cotyledon evenly in a 20mL syringe containing 10mL of resuspended bacterial solution, and then dip under negative pressure for 15 minutes. Take out the explant and dry it on sterile filter paper, transfer it to co-culture medium (CM; MS+30% sucrose+plant gel+6-BA) padded with filter paper for co-cultivation, and culture it in the dark at 28℃ for 3 days; MS was purchased from Phytotech and 6-BA was purchased from Yuanye Company.

[0070] Recovery culture: After 3 days of co-cultivation, the cotyledon blocks were transferred to recovery medium (RM; MS + 30% sucrose + phytagel + 6-BA + timentin) and cultured for 7 days at 28°C, with a light intensity of 20,000 lux and a light duration of 16 h / d.

[0071] Selective culture: After the recovery culture is completed, the explants are transferred to selective medium (SM; MS + 30% sucrose + agar + 6-BA + Timentin + Basta) for selective culture, and subcultured at 28°C for 3-4 weeks, with subculture every 12 days.

[0072] Shoot elongation culture: The explants with obvious buds and GFP tags were transferred to the shoot elongation medium (SE; MS + 30% sucrose + agar + 6-BA + timentin) for culture.

[0073] Rooting culture: The selected buds were transferred to MS medium containing IAA and timentin for rooting culture and cultured at 28°C until roots formed.

[0074] Transplanting: When the regenerated seedlings take root and grow 4-5 true leaves, take them out of the culture bottle, carefully remove the culture medium at the roots, and transplant them into a flowerpot with a substrate: vermiculite ratio of 1:3. Water thoroughly to keep warm and moisturize, and manage normally after cultivation.

[0075] The MS medium used in the experiment was PhytoTech M519.

[0076] Finally got positive ClMYB33 Gene-edited strains.

[0077] 5. ClMYB33 Transgenic plant editing detection Sampling GFP fluorescent watermelon containing pBSE406-sgRNA1-sgRNA2 vector ClMYB33 The gene-edited strains were regenerated and genomic DNA was extracted using the CTAB method.

[0078] Specific steps: Take a small amount of young watermelon leaves and quickly grind them into powder in liquid nitrogen, and put them in a 1.5ml centrifuge tube; add preheated 800μL CTAB extraction buffer and place in a 65℃ water bath for 30min; add an equal volume of chloroform and isopropanol (the volume ratio of chloroform to isopropanol 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 by turning upside down and place in a -20℃ refrigerator to precipitate for 1h; centrifuge at 10000r / min for 10min; discard the supernatant, rinse the precipitate twice with 75% volume ethanol, pour it out and absorb the remaining liquid, dry it at room temperature, dissolve it with 100μL ddH2O (containing 0.1% RNase), and store it at 4℃ for later use.

[0079] Using the extracted genomic DNA as a template, the sequence containing the two target sites was amplified by PCR using primers ClMYB33-IDF1 / ClMYB33-IDR1 and ClMYB33-IDF2 / ClMYB33-IDR2.

[0080] Among them, the specific primer sequences of ClMYB33-IDF1 / ClMYB33-IDR1 and ClMYB33-IDF2 / ClMYB33-IDR2 are as follows: ClMYB33-IDF1: 5'-GGCATCCGAAAAATGAGATTG-3'; ClMYB33-IDR1: 5'-GTGCCCATTTGTTTCCCAT-3'; ClMYB33-IDF2: 5'-ACCTTTCGTGCTTTGCTGTAG-3'; ClMYB33-IDR2: 5'-CATCGGGACCAGGAAATAA-3'.

[0081] Amplification system (50 μL) and reaction procedure: 2×T5 SuperMIX 25 μL, primers (10 μM) 2 μL each, template DNA 1 μL, ddH2O 20 μL.

[0082] PCR reaction program: pre-denaturation at 98℃ for 3min; denaturation at 98℃ for 10s, annealing at 58℃ for 10s, extension at 72℃ for 15s, 35 cycles; complete extension at 72℃ for 5min. Band size was detected by 1% agarose gel electrophoresis. The remaining PCR product was sequenced with ClMYB33-IDR1 / ClMYB33-IDR2 as the sequencing primer.

[0083] The results of target editing comparison of ClMYB33 mutants are shown in Figure 4 , where ClMYB33 lacks 3bp at the Target1 target site and 2bp at the Target2 target site.

[0084] Compared with the wild type (ZZJM), the mutant clmyb33 ClMYB33 Gene, 2 homologous chromosomes ClMYB33 The following gene mutations occurred: ClMYB33The "5'-ATAGTGGAGGGAATGCTAGCGG-3' (corresponding to positions 77-98 of SEQ ID No.1 and positions 77-98 of SEQ ID No.3)" in the gene mutated to "5'-ATAGTGGAGGGACTAGCGG-3'", with a deletion of three nucleotides; the "5'-GAATACTCGAATAAAGAGGCGG-3' (corresponding to positions 1748-1769 of SEQ ID No.1 and positions 396-417 of SEQ ID No.3)" mutated to "5'-GAATACTCGAATAAAGGCGG-3'", with a deletion of two nucleotides; the deletion of the above nucleotides caused a frameshift, resulting in changes in the ClMYB33 amino acids and premature termination of translation, leading to the loss of ClMYB33 protein function.

[0085] Embodiment 2, ClMYB33 Phenotypic observation of gene-edited plants Will edit the plant clmyb33-1 It will be planted together with wild-type watermelons in the greenhouse of the Yanqing base in the spring of 2024 and managed normally. Its phenotype will be observed after the male flowers bloom.

[0086] Compared with the wild type, clmyb33-1 There were no significant differences in plant morphology and female flower development. The mutant female flowers could be pollinated normally. During the reproductive growth stage, the mutant male flower anthers were smaller, and the anthers did not crack in the later stage, and no pollen was produced ( Figure 5 ).

[0087] In summary, the present invention provides a method for creating new watermelon male sterile germplasm by gene editing technology, by regulating male sterility genes ClMYB33 By editing, male-complete sterile lines can be quickly obtained, which has important application potential in the utilization of watermelon hybrid vigor and population improvement.

[0088] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in any of the following: 1) Application in regulating plant male sterility; 2) Application in the preparation of products for regulating plant male sterility; 3) Application in breeding plants with altered fertility; 4) Use in the preparation of products for cultivating plants with altered fertility; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No. 2; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein having an amino acid sequence identity of more than 80% with that specified in a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any one of the proteins defined in a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from watermelon.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown below: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No.1; d2) the coding sequence is the DNA molecule shown in SEQ ID No.3; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described in claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for improving plant male sterility, characterized in that: The method comprises step M, wherein step M is to inhibit, reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit, reduce or silence the expression level of the gene encoding the protein described in claim 1 or 2, so as to improve the male sterility of the plant.

6. A method for reducing plant male sterility, characterized in that: The method comprises step P, wherein step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described in claim 1 or 2 to reduce the male sterility of the plant.

7. A method for breeding male sterile plants, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein in claim 1 or 2 in the target plant, and / or the activity and / or content of the protein, thereby obtaining a male sterile plant.

8. The method according to claim 7, characterized in that: The steps include: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described in claim 1 or 2; (2) The recombinant expression vector constructed in step (1) is transferred into a recipient plant to obtain a male sterile plant.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: G1) monocots or dicots; G2) Cucurbitales; G3) Cucurbitaceae; G4) Citrullus; G5) Watermelon.

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