Application of protein encoded by SlGA20ox7 gene in creating tomato male sterile line

Abstract

This invention belongs to the field of molecular biology technology, specifically involving SlGA20ox7 Application of gene-encoded proteins in the creation of male-sterile tomato lines. The technical problem this invention aims to solve is to provide a method for creating male-sterile tomato lines. The technical solution of this invention is a tomato... SlGA20ox7 The application of gene-encoded proteins in the creation of male-sterile tomato lines. This invention also provides a method for creating male-sterile tomato lines by reducing the concentration of certain proteins in tomatoes. SlGA20ox7 This is achieved through gene expression levels; SlGA20ox7 The nucleotide sequence of the gene is shown in SEQ ID No. 2 or SEQ ID No. 5. This invention utilizes... SlGA20ox7 SlGA20ox7 Gene editing can rapidly create new male-sterile tomato germplasm, which is of great significance for the utilization of heterosis in tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving SlGA20ox7 Application of gene-encoded proteins in the creation of male-sterile tomato lines. Background Technology

[0002] tomato( Solanum lycopersicum Tomato, also known as male-sterile tomato, is a sweet and sour fruit with high nutritional and edible value. It plays a vital role in my country's "vegetable basket" project and industrial restructuring, and has become one of the leading industries in modern vegetables. Commercial tomato varieties are mainly hybrids. Currently, global tomato hybridization breeding and seed production still rely entirely on manual pollination. The labor cost of this work accounts for more than 25% of the total tomato breeding cost, with manual emasculation alone accounting for 40% of the cost of tomato hybridization pollination. Furthermore, the purity of the resulting hybrid seeds cannot be guaranteed, severely restricting the development of the tomato seed industry. However, using male-sterile line technology for hybrid seed production can eliminate the manual emasculation step, significantly reducing breeding costs and effectively ensuring seed purity and preventing parental loss. Therefore, obtaining ideal sterile types through biotechnology will greatly improve economic efficiency and has a transformative and original innovative significance for tomato production.

[0003] Male sterility is characterized by abnormal stamen development, resulting in the inability to produce functional pollen, while the pistil develops normally. Therefore, the plant can achieve fertilization through fertilization with foreign normal pollen. Male sterility is widespread in the plant kingdom and is generally classified into two types: genic male sterility (GMS) and cytoplasmic male sterility (CMS). GMS is usually caused by nuclear gene mutations and is widely present in over 610 plant species. CMS, on the other hand, originates from abnormal nucleocytoplasmic interactions caused by mutations in cytoplasmic genetic material and has been identified and applied in various crops such as cabbage, rapeseed, radish, rice, corn, cotton, and wheat. For crops whose main products are seeds or fruits, the CMS mechanism is more complex. In hybrid seed production, it relies on restorer line nuclear genes to achieve fertility restoration (i.e., a three-line system of male-sterile line, maintainer line, and restorer line), and also presents challenges such as susceptibility to diseases. Therefore, GMS is gradually receiving more attention.

[0004] Male sterility in tomatoes was first reported in 1915 and has been a research hotspot for scholars both domestically and internationally since the 1930s. According to literature, nearly 60 male-sterile mutants of tomatoes have been discovered. Based on their phenotypic differences, these materials can be divided into three types of sterility: spore sterility, structural sterility, and functional sterility. Spore sterility is mainly characterized by abnormalities during pollen development, resulting in the inability to produce viable pollen, such as mutants.hydra ( hyd ), male sterile10 ( ms10 ), male sterile5 ( ms5 Structural sterility refers to malformed stamens, mostly manifested as the absence or degeneration of stamens, resulting in the inability to form pollen. Examples include mutants. tomatoagamous 1 ( tag1 ), stamenless-2 ( sl-2 Both types exhibit carpelization of stamens and abnormal anther development; functional sterility is not true pollen sterility, but rather the ability to produce normal pollen, but pollination and fertilization are hindered due to abnormal stamen tissue development. style2.1 , exerted ( ex The mutant stigma protrudes significantly from the anther tube, preventing mature pollen from settling on the stigma after release from the anther, thus causing pollination failure. However, currently, there is a severe shortage of production-ready male-sterile tomato resources, research on key genes regulating male reproductive development is weak, and ideal male-sterile lines are yet to be created. Therefore, identifying male-sterile genes applicable to tomato hybridization breeding has significant practical implications. In recent years, gene editing technology has made breakthrough progress, with methods such as CRISPR-Cas9, TALEN, and ZFN becoming widely used. Gene editing technology is a genetic manipulation technique that can modify DNA sequences at the genomic level. Gene editing technology will be widely applied in all agricultural production fields, and its application prospects in tomato breeding are broad. It can not only reduce seed production costs and improve seed purity by creating male-sterile lines, but also cultivate parthenocarpic seedless tomatoes by knocking out specific genes, providing new strategies and methods for tomato breeding. Therefore, gene editing technology has a more comprehensive and far-reaching impact on the creation of male-sterile tomato lines. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for creating male-sterile lines of tomatoes.

[0006] The technical solution of this invention is tomato SlGA20ox7 The application of a gene-encoded protein in the creation of male-sterile tomato lines, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] Furthermore, the sequence of the nucleotides encoding the protein is shown in SEQ ID No. 2 or SEQ ID No. 5.

[0008] Specifically, the tomato male-sterile line is a tomato seed male-sterile line.

[0009] This invention also provides a method for creating male-sterile lines in tomatoes, by knocking out male-sterile cells in tomatoes.SlGA20ox7 Achieved through genes; SlGA20ox7 The nucleotide sequence of the gene is shown in SEQ ID No. 2 or SEQ ID No. 5.

[0010] Specifically, the tomato male-sterile line is a tomato seed male-sterile line.

[0011] Specifically, the knockout SlGA20ox7 The gene-related methods include at least one of genome editing, homologous recombination, or random insertion mutation.

[0012] Specifically, the genome editing method includes at least one of the following: giant nuclease method, ZFN method, TALEN method, or CRISPR-Cas method.

[0013] The CRISPR-Cas method includes the following steps:

[0014] a. Design targeting SlGA20ox7 sgRNA at gene target sites;

[0015] b. Construct a Cas editing expression vector that expresses sgRNA;

[0016] c. Convert the Cas editing expression vector into a tomato.

[0017] Furthermore, the CRISPR-Cas method is CRISPR-Cas9, CRISPR-Cas12a, or CRISPR-Cas12b.

[0018] In step a, the sequence of the target site is shown in positions 71-90 and / or positions 1003-1022 of SEQ ID No. 2.

[0019] Specifically, in step a, the sequence of the sgRNA is shown in SEQ ID No. 3 and SEQ ID No. 4.

[0020] Specifically, in step c, the transformed tomatoes are transformed using Agrobacterium-mediated transformation. The beneficial effects of this invention: This invention utilizes CRISPR-Cas9 gene editing technology to transform tomatoes using Agrobacterium-mediated transformation. SlGA20ox7 Genes were edited at specific sites, and after screening, homozygous male-sterile plants were obtained. slga20ox7 The plant's vegetative growth was inhibited, pollen malformation and fertility were significantly reduced, and the anthers were partially indehiscent and relatively small, resulting in some stigmas being exposed or level with the anthers; however, its female reproduction was normal. This invention, through... SlGA20ox7Gene editing can rapidly create new male-sterile tomato germplasm, which is of great significance for utilizing heterosis in tomatoes. The constructed male-sterile tomato lines can be hybridized with superior tomato breeding materials for hybrid seed production, saving hybridization costs. It is simple and efficient, significantly improving breeding and production efficiency, and has good application prospects in utilizing heterosis in tomatoes. Attached Figure Description

[0021] Figure 1 A diagram of mutation types in the T0 generation of tomato gene-edited plants.

[0022] Figure 2 mutant slga20ox7-1 Sequencing peak diagram.

[0023] Figure 3 mutant slga20ox7-2 Sequencing peak diagram.

[0024] Figure 4 Wild-type tomato WT and mutant slga20ox7-1 A diagram showing the comparison of plants, flowers, and fruits.

[0025] Figure 5 Wild-type tomato WT and mutant slga20ox7-2 Comparison of stigma and anther, where (a) is WT, slga20ox7-2 Phenotypic diagrams of the stigma and anthers of the plant, (b) for WT, slga20ox7-2 Statistical analysis of stigma and anther length of the plant, (c) represents WT, slga20ox7-2 The ratio of stigma to anther in a plant, reflecting the extent of stigma exposure.

[0026] Figure 6 Wild-type tomatoes (WT) and SlGA20ox7 Gene knockout lines slga20ox7-1 and slga20ox7-2 Paraffin sections of anthers during the budding and flowering stages.

[0027] Figure 7 Wild-type tomatoes (WT) and SlGA20ox7 Gene knockout lines slga20ox7-1 and slga20ox7-2 Observation diagram of TTC staining in pollen.

[0028] Figure 8 Wild-type tomatoes (WT) and SlGA20ox7 Gene knockout lines slga20ox7-1 and slga20ox7-2 Scanning electron microscopy observation of mature pollen. Figure 9Recombinant vector structure diagram; TRNA: transfer RNA; gRNA scaffold: gRNA backbone; CaMV 35S promoter: 35S promoter of cauliflower mosaic virus (CaMV); U6-26: U6 small nuclear RNA promoter; Cas9: Cas9 endonuclease; Hygr: hygromycin resistance selection tag; NeoR / KanR: neomycin / kanamycin resistance selection tag; KanR: kanamycin resistance selection tag. Detailed Implementation

[0029] This invention utilizes CRISPR-Cas9 technology to silence tomatoes. SlGA20ox7 Gene expression was used to obtain homozygous male-sterile plants. slga20ox7 Through observation of horticultural traits and fertility, it was found that... slga20ox7 Compared to the wild type, females reproduce normally, but the plants are abnormally stunted, with significantly reduced pollen fertility and incomplete anther dehiscence, resulting in some stigmas being exposed or level with the anthers. Utilizing this gene's ability to regulate male sterility in tomatoes, it can be applied to heterosis utilization, saving hybridization costs and significantly improving breeding and production efficiency.

[0030] Example 1 Targeting SlGA20ox7 Gene sgRNA design and vector construction

[0031] The tomato gene Solyc06g050110 encodes a gibberellin 20-oxidase. Based on its specificity in the tomato genome, the protein encoded by this gene was named SlGA20ox7 to distinguish it from other homologous proteins. SlGA20ox7 ( Solyc06g050110 Information can be found in the Sol Genomics Network database (https: / / solgenomics.net / ). The amino acid sequence of the GA20ox homolog is obtained from Phytozome v13 (https: / / phytozome-next.jgi.doe.gov / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ). SlGA20ox7 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 1, the mRNA sequence is shown in SEQ ID No. 2, and the genome sequence is shown in SEQ ID No. 5.

[0032] SEQ ID No. 1 SlGA20ox7 The amino acid sequence of the gene-encoded protein:

[0033] MDIDSIITNDKSSMVDEKNSLIFDASQMKHGKKDCAIPQELHAPLIDLGGFFSGDPTTMQEASRLVGEACKSHGFFLVVNHGVDENLISNAHRNMDMFFSLPLSEKLKAERKIGEHCGYASSFTGTFLSKLPWKETLSFQYSAKEHSSHIVEEYFQKTMGKSFNNLGKVYQEYCNSMSKLSL RIMELLGMSLGVQKENFKDFFEDNESIMRINYYPPCHNPELTLGTWPHYDPTSLTILHQDGVSGLQVFVDNEWHSINPNYNAFVVNIGDTFMALSNGIFKSCLHRAVVNNYIPRKSLVFFLCPDKDKVVRPPTELVDFNKPRIYPDFTWPTLLEFTQKHHPADTNTLQAFSNWLQHNNVQP.

[0034] Using the CRISPR-Cas9 system to construct vector knockout SlGA20ox7 Genes. Based on the mRNA sequence and corresponding genomic sequence information, specific target site sgRNAs were designed using computational software according to the target sequence. Two CRISPR target sites were designed here to improve targeting efficiency. Target PCR amplification primer sequences were designed, and after primer synthesis, the fragment containing the target was amplified by PCR. This PCR fragment was cloned into the final CRISPR vector. After constructing the vector, it was transformed into *E. coli* DH5α, single colonies were selected for culture, and then colony PCR was performed to screen for positive clones. The specific steps are as follows:

[0035] (1) Design of CRISPR target sites and target site adapter primers

[0036] in accordance with SlGA20ox7 The mRNA sequence (SEQ ID No. 2) and its genome sequence information were used to design specific target site sgRNAs based on the target sequence using computational software. Two CRISPR target sites were designed here to improve targeting efficiency.

[0037] The sgRNA1 and sgRNA2 sequences of the two CRISPR target sites are as follows:

[0038] sgRNA1 (SEQ ID No. 3): 5'-GATGCATCCCAAATGAAACA-3' (target sequence is positions 71-90 of SEQ ID No. 2);

[0039] sgRNA2 (SEQ ID No. 4): 5'-CCTTGAGTTTACACAGAAGC-3' (target sequence is positions 1003-1022 of SEQ ID No. 2).

[0040] SEQ ID No.2 shows SlGA20ox7 The mRNA sequence; the target sequence is positions 71-90 and 1003-1022; the PAM sequence is positions 91-93 and 1000-1002;

[0041]

[0042] (2) Preparation of target connector

[0043] An expression cassette sequence (SEQ ID No. 10) containing sgRNA1 and sgRNA2 was designed, with the sequence unit structure as follows: sgRNA1 - TRNA - gRNA backbone - sgRNA2. The fragment containing the sgRNA expression cassette sequence was amplified by PCR. This sequence was synthesized by Heilongjiang Jiansu Gene Technology Co., Ltd.

[0044] The expression cassette sequence of SEQ ID No. 10 sgRNA:

[0045] GATGCATCCCAAATGAAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACCTTGAGTTTACACAGAAGC.

[0046] The expression vector pv58-k was digested with a single enzyme. The reaction mixture was 50 μL: 1 μL KpnI; 5 μL 10× buffer; 2 μg plasmid DNA; and ddH2O to a final volume of 50 μL. The mixture was pipetted and incubated at 37°C for 30 minutes.

[0047] (3) Homologous recombination

[0048] The sgRNA expression cassette was recombined with the expression vector pv58-k. The expression vector pv58-k was linearized with the restriction endonuclease Kpn I, and then expression cassette sequences containing sgRNA1 and sgRNA2 were inserted to obtain the recombinant vector. Figure 9 The reaction system (20 μL) consisted of: 10 ng of sgRNA expression cassette, 200 ng of expression vector pv58-k, 2 μL of Exnase II, and 4 μL of 5×CEII Buffer. Sterile ddH2O was then added to bring the total volume to 20 μL. The components were gently and thoroughly mixed using a pipette. The mixture was incubated at 37°C in a metal bath for 30 minutes. Immediately after the reaction, the tube was transferred to ice to rapidly stop enzyme activity.

[0049] (4) Validation of positive clones

[0050] The recombinant vector obtained above was transformed into *E. coli* DH5α. The specific steps are as follows: The ligation product was incubated on ice for 30 minutes to allow the DNA to fully bind to competent cells. Then, it was heat-shocked in a 42°C water bath for 60 seconds and quickly transferred to ice for 2 minutes. Next, 600 µL of sterile LB liquid medium was added to the tube, and the culture was incubated at 37°C and 150 rpm for 1 hour for recovery. After recovery, an appropriate amount of bacterial culture was taken and evenly spread onto an LB agar plate containing kanamycin using a spreader. The plate was inverted and incubated overnight at 37°C. Finally, single colonies with good growth were picked from the plate and sent for sequencing verification. The recombinant vector was detected by PCR using primers SlGA20ox7-F2: AATCACAAAATGATAAATCGTCTATGGT (SEQ ID No. 6); SlGA20ox7-R2: TAAGGTTGCACATTATTGTGTTGAA (SEQ ID No. 7) to analyze the presence of the target sequence.

[0051] The PCR system consisted of 20 μL: 1 μL bacterial culture, 2 μL 10×Buffer, 0.4 μL dNTP Mixture (2 mM), 0.4 μL SlGA20ox7-F2 (10 μM), 0.4 μL SlGA20ox7-F2 (10 μM), 0.1 μL Taq (5 U / μL), and ddH2O to a final volume of 20 μL. The PCR program was as follows: 98℃ for 3 min; 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 34 cycles; 72℃ for 10 min; 25℃ for 2 min.

[0052] If both target sequences are present in the recombinant expression vector, it indicates that the recombinant vector expressing sgRNA1 and sgRNA2 has been successfully transferred into the expression vector pv58-K.

[0053] Successful sequencing enabled the transformation of Agrobacterium GV3101 for tomato genetic transformation. The plasmid with completely correct sequencing was transformed into Agrobacterium GV3101 and plated on LB solid medium containing rifampin and kanamycin to obtain an Agrobacterium strain that can be used to construct CRISPR-Cas9 gene editing materials.

[0054] Example 2: Genetic transformation of tomatoes and screening of positive plants

[0055] 1. Genetic transformation of tomatoes

[0056] Gene-edited plants were obtained using leaf disc transformation. Tomato variety AC (Ailsa Craig) was used as material. After sterilization, seeds were sown on 1 / 2 MS medium and cultured in an incubator until the cotyledons were fully expanded to obtain sterile seedlings. Cotyledons of the sterile seedlings were cut, both ends removed, and the middle section was cut into small pieces as explants, which were pre-cultured on medium for 2-3 days. These pre-cultured explants were then infected with Agrobacterium carrying the target vector, co-cultured, and then transferred to differentiation medium. After Agrobacterium colonies appeared around the explants, they were transferred to callus induction medium containing antibacterial agents and antibiotics for selection. Approximately 2-3 weeks later, resistant adventitious shoots differentiated from the explants. When the adventitious shoots elongated to more than 1 cm, they were cut off and transferred to rooting medium to induce rooting. Rooted seedlings were hardened off and then transplanted into a seedling substrate. Leaves of the regenerated plants were then cut, and genomic DNA was extracted using the CTAB method. Specific primers for the selection marker gene were then used. Hpt PCR identification was performed using primers to amplify HygR.

[0057] Hpt The primer sequences are as follows:

[0058] hpt557-F: ACACTACATGGCGTGATTTCAT (SEQ ID No. 8);

[0059] hpt557-R: TCCACTATCGGCGAGTACTTCT (SEQ ID No. 9).

[0060] The PCR system consisted of 30 μL: 1.5 μL DNA sample, 27 μL Chloride T3Mix, 0.5 μL hpt557-F (10 μmol / L), 0.5 μL hpt557-R (10 μmol / L), and ddH2O to a final volume of 30 μL. The PCR program was: 98℃ for 3 min; 98℃ for 10 sec, 56℃ for 10 sec, 72℃ for 10 sec, 36 cycles; 72℃ for 5 min; 25℃ for 2 min.

[0061] Thirty-four regenerated T0 generation plants were obtained through seed germination, pre-culture, Agrobacterium-mediated genetic transformation, selection culture and differentiation, and resistant bud rooting. Using... Hpt Primer pairs were used to detect the T0 generation of regenerated plants by PCR, and 18 of them were positive.

[0062] 2. Further testing of positive plants

[0063] DNA was extracted from leaves of wild-type tomato variety AC and 18 T0 generation positive transgenic plants obtained above, with the DNA of wild-type tomato variety AC serving as a negative control.

[0064] Primers were designed upstream and downstream of two specific target sequence sites to perform PCR amplification on 18 transgenic positive plants. The products were purified, recovered, and sequenced. SlGA20ox7 Gene editing site detection.

[0065] The target detection primers are as follows:

[0066] SlGA20ox7-F2:AATCACAAATGATAAATCGTCTATGGT;

[0067] SlGA20ox7-R2: TAAGGTTGCACATTATTGTGTTGAA.

[0068] Based on sequencing results and SlGA20ox7 By comparing the original genome sequence (SEQ ID No. 5), T0 generation positive plants with editing at the target site were screened. To obtain a stable mutant line, the T0 generation plants were self-pollinated, and T1 generation seeds were harvested. Subsequently, two T1 generation seeds carrying different editing types were selected and sown, and cultivated to the seedling stage. When the plants had 2-3 true leaves, leaf samples were collected, and genomic DNA was extracted. The SlGA20ox7 gene target region was amplified by PCR using primers SlGA20ox7-F2 and SlGA20ox7-R2 to determine the homozygous / heterozygous status and specific mutation type of the T1 generation plants.

[0069] Sequencing analysis of T1 generation plants revealed multiple... SlGA20ox7 Gene editing types. Two representative mutation types were selected from these ( slga20ox7-1, slga20ox7-2 ), for subsequent functional research ( Figure 1 ).

[0070] The peak plot file from sequencing of T1 generation plants shows... slga20ox7-1 It is a homozygous mutation. slga20ox7-2 Biallelic mutation ( Figure 2 and Figure 3 ).

[0071] homozygous mutant slga20ox7-1 The gene sequence has been edited based on SEQ ID No. 5 as follows ( Figure 1 ): slga20ox7-1 An A base is inserted at position 1005 of the gene; biallelic mutant slga20ox7-2 The gene sequence has been edited based on SEQ ID No. 5 as follows: slga20ox7-2 An A base is inserted at position 1005 of one strand of a gene, and the T base at position 1005 of the other strand is mutated to an A base.

[0072] SEQ ID No. 5SlGA20ox7 The genome sequence:

[0073]

[0074] Example 3 SlGA20ox7 The effect of gene deletion expression

[0075] (1) Morphological observation of gene knockout plants:

[0076] In order to study SlGA20ox7 Its function in tomato plant morphogenesis will result in the acquisition of tomatoes SlGA20ox7 The growth phenotypes of gene knockout lines were observed and compared with those of the wild-type tomato variety AC (WT). slga20ox7- 1 and slga20ox7-2 They are phenotypically similar, therefore in Figure 4 Only wild-type tomato WT and mutants were presented. slga20ox7-1 A comparison of the plant, flowers, and fruits revealed that the plant was severely stunted, the floral organs were smaller with exposed stigmas, and the fruits produced by assisted pollination were longer. Figure 5 Only wild-type tomato WT and mutants were shown. slga20ox7- 2 A comparison of the stigma and anthers reveals that the stigma length is not significantly different from that of the WT, but its anthers are relatively shorter, resulting in some stigmas being exposed or level with the anthers.

[0077] Paraffin sections of mature anthers were examined. Figure 6 Gene knockout strains were discovered. slga20ox7-1 and slga20ox7-2 Some anthers failed to dehisce normally, preventing pollen from being released. However, no obvious abnormalities were found in the appearance of the petals and pistils. This was observed in gene knockout strains. slga20ox7-1 and slga20ox7-2 The plants underwent assisted pollination, and the results showed that they could produce fruit and harvest seeds normally. However, compared to WT, the gene knockout lines... slga20ox7-1 and slga20ox7-2 The fruit is slightly long and irregular in shape, and the number of seeds is relatively small.

[0078] (2) Observation of pollen viability of gene knockout plants

[0079] Wild-type tomato variety AC (WT) and gene knockout lines slga20ox7-1 and slga20ox7-2 Mature pollen was stained with TTC (Total Transcriptional Chromium) Figure 7 It was found that WT pollen could be stained red, had a full pollen shape, and a pollen viability of 70%, while the gene knockout line... slga20ox7-1 and slga20ox7-2 The pollen count was significantly reduced, with very few pollen cells stained red; only 3.7% of the pollen cells were viable, a highly significant difference compared to the control. Further scanning electron microscopy was performed on the mature pollen cells.Figure 8 It was found that the pollen grains of WT were plump, while those of the gene knockout line were... slga20ox7-1 and slga20ox7-2 The pollen grains are in a collapsed state. The above embodiments are merely preferred embodiments of the present invention and not intended to limit the invention in any way. Although the above preferred embodiments have been disclosed, the scope of protection of the present invention is not limited thereto. Any equivalent substitutions, minor modifications, or adaptive adjustments made by those skilled in the art within the scope of the technical solutions disclosed in the present invention, as long as they do not depart from the essence of the present invention, shall be considered to be included within the scope of protection of the claims.

Claims

1. Tomato SlGA20ox7 The application of gene-encoded proteins in the creation of male-sterile tomato lines is characterized by: The amino acid sequence of the protein is shown in SEQ ID No. 1; the application involves knocking out the protein in tomatoes. SlGA20ox7 It is achieved through genes.

2. The application according to claim 1, characterized in that: The nucleotide sequence encoding the protein is shown in SEQ ID No. 2 or SEQ ID No.

5.

3. A method for creating male-sterile tomato lines, characterized in that: By knocking out the tomatoes SlGA20ox7 Achieved through genes; SlGA20ox7 The nucleotide sequence of the gene is shown in SEQ ID No. 2 or SEQ ID No.

5.

4. The method according to claim 3, characterized in that: The knockout SlGA20ox7 The gene-based method used was CRISPR-Cas9.

5. The method according to claim 4, characterized in that: The CRISPR-Cas9 method includes the following steps: a. Design targeting SlGA20ox7 sgRNA at gene target sites; b. Construct a Cas editing expression vector that expresses sgRNA; c. Convert the Cas editing expression vector into a tomato.

6. The method according to claim 5, characterized in that: In step a, the sequence of the sgRNA is shown in SEQ ID No. 3 and SEQ ID No. 4.

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