Gene for improving tomato fruit firmness and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a gene for enhancing the hardness of tomato fruits and its application. Specifically, it discloses the application of the Solyc02g077940 gene in regulating the hardness of tomato fruits, and the amino acid sequence of the Solyc02g077940 gene is shown in SEQ ID NO: 1. The present invention discloses for the first time the application of Solyc02g077940 in regulating the hardness of tomato fruits. By screening target genes and combining with the CRISPR / Cas9 technology to knockout / knockdown Solyc02g077940, it proves the importance of Solyc02g077940 in enhancing the hardness of tomato fruits.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a gene for improving the firmness of tomato fruits and an application thereof. Background Art

[0002] Tomato (Solanum lycopersicum), native to South America, is an important vegetable crop in the Solanaceae family, ranking first in the world in annual vegetable production. Rich in essential nutrients and bioactive substances, tomato fruits play a crucial role in human health, diet, and cooking.

[0003] my country leads the world in both tomato cultivation area and production. However, collisions during long-distance transportation and a short shelf life can cause fruit spoilage, resulting in significant resource waste and economic losses. Therefore, reducing post-harvest tomato losses has significant economic benefits and production needs.

[0004] Fruit firmness is a key quality trait of tomatoes, closely related to transportation losses, storage stability, and shelf life. Improving fruit firmness can reduce losses caused by long-distance transportation and spoilage, while also extending shelf life. Improving tomato firmness is a key breakthrough in addressing excessive postharvest losses and is also a production requirement for adapting to mechanized harvesting. In actual production, natural mutations have been introduced to increase fruit firmness, but this results in a significant decrease in lycopene and soluble solids content, affecting fruit qualities such as color and taste. Therefore, a method for increasing tomato fruit firmness without affecting fruit qualities such as color and taste is urgently needed.

[0005] The CRISPR / Cas9 system, which combines clustered regularly interspaced short palindromic repeats (CRISPR) with the Cas9 (CRISPR-associated) protein, is a bacterial immune system that removes foreign viral genetic material from its own genome. Its ability to introduce mutations during double-stranded DNA cleavage and repair makes it a valuable tool for gene editing. Compared to other earlier gene editing technologies, the CRISPR / Cas9 system is easier to use and more scalable, enabling more precise and efficient mutations in genomic sequences. This makes the acquisition of mutants more efficient and convenient, greatly facilitating the study of gene function. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a use of Solyc02g077940 in regulating tomato fruit firmness.

[0007] The second aspect of the present invention aims to provide a use of the Solyc02g077940 inhibitor.

[0008] The third aspect of the present invention aims to provide an sgRNA.

[0009] The fourth aspect of the present invention aims to provide biological materials related to the sgRNA of the third aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide the use of the sgRNA of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, the CRISPR / Cas system containing the sgRNA of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, or the gRNA of the third aspect of the present invention, the biomaterial reagent of the fourth aspect of the present invention.

[0011] The sixth aspect of the present invention aims to provide a method.

[0012] The seventh aspect of the present invention is to provide a CRISPR / Cas system,

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The tomato Solyc02g077940 gene is a coding sequence located at interval 42753490 to 42753654 on chromosome 2. Its function in fruit firmness has not been previously reported.

[0015] In a first aspect of the present invention, a use of Solyc02g077940 in regulating tomato fruit firmness is provided. The amino acid sequence of Solyc02g077940 is shown in SEQ ID NO: 1.

[0016] The second aspect of the present invention provides the use of the Solyc02g077940 inhibitor in any one of a1) to a4);

[0017] a1) Improve the firmness of tomato fruits;

[0018] a2) screening and / or preparing a product for increasing the firmness of tomato fruit;

[0019] a3) Breeding of high-hardness tomato varieties;

[0020] a4) preparing products cultivated from tomato varieties with high hardness.

[0021] In some embodiments of the present invention, the amino acid sequence of Solyc02g077940 is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO: 2.

[0022] In some embodiments of the present invention, the Solyc02g077940 inhibitor is at least one of a substance that degrades the Solyc02g077940 gene or a substance that reduces the expression level of the Solyc02g077940 gene.

[0023] In some embodiments of the present invention, the Solyc02g077940 inhibitor includes at least one of b1) to b13):

[0024] b1) siRNA, dsRNA, miRNA, ribozyme, shRNA, and CRISPR / Cas system targeting the Solyc02g077940 gene;

[0025] b2) a nucleic acid molecule encoding b1);

[0026] b3) an expression cassette containing the nucleic acid molecule described in b2);

[0027] b4) a recombinant vector containing the nucleic acid molecule described in b2);

[0028] b5) a recombinant vector containing the expression cassette described in b3);

[0029] b6) a recombinant cell containing the nucleic acid molecule described in b2);

[0030] b7) a recombinant cell containing the expression cassette described in b3);

[0031] b8) a recombinant cell containing the recombinant vector described in b4);

[0032] b9) a recombinant cell containing the recombinant vector described in b5);

[0033] b10) a recombinant microorganism containing the nucleic acid molecule described in b2);

[0034] b11) a recombinant microorganism containing the expression cassette described in b3);

[0035] b12) a recombinant microorganism containing the vector described in b4);

[0036] b13) A recombinant microorganism containing the vector described in b5).

[0037] In some embodiments of the present invention, the CRISPR / Cas system includes an sgRNA targeting Solyc02g077940 or biological materials associated with the sgRNA.

[0038] In some embodiments of the present invention, the nucleotide sequence of the sgRNA is shown as SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0039] In some embodiments of the present invention, the biological material associated with the sgRNA includes at least one of c1) to c12):

[0040] c1) a nucleic acid molecule encoding an sgRNA;

[0041] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0042] c3) a recombinant vector containing the nucleic acid molecule described in c1);

[0043] c4) a recombinant vector containing the expression cassette described in c2);

[0044] c5) a recombinant cell containing the nucleic acid molecule described in c1);

[0045] c6) a recombinant cell containing the expression cassette described in c2);

[0046] c7) a recombinant cell containing the recombinant vector described in c3);

[0047] c8) a recombinant cell containing the recombinant vector described in c4);

[0048] c9) a recombinant microorganism containing the nucleic acid molecule described in c1);

[0049] c10) a recombinant microorganism containing the expression cassette described in c2);

[0050] c11) a recombinant microorganism containing the recombinant vector described in c3);

[0051] c12) A recombinant microorganism containing the recombinant vector described in c4).

[0052] In some embodiments of the present invention, the CRISPR / Cas system further comprises a Cas protein and / or biological materials associated with the Cas protein;

[0053] In some embodiments of the present invention, the biological material associated with the Cas protein includes at least one of d1) to d12):

[0054] d1) a nucleic acid molecule encoding a Cas protein;

[0055] d2) an expression cassette containing the nucleic acid molecule described in d1);

[0056] d3) a recombinant vector containing the nucleic acid molecule described in d1);

[0057] d4) a recombinant vector containing the expression cassette described in d2);

[0058] d5) a recombinant cell containing the nucleic acid molecule described in d1);

[0059] d6) a recombinant cell containing the expression cassette described in d2);

[0060] d7) a recombinant cell containing the recombinant vector described in d3);

[0061] d8) a recombinant cell containing the recombinant vector described in d4);

[0062] d9) a recombinant microorganism containing the nucleic acid molecule described in d1);

[0063] d10) a recombinant microorganism containing the expression cassette described in d2);

[0064] d11) a recombinant microorganism containing the recombinant vector described in d3);

[0065] d12) A recombinant microorganism containing the recombinant vector described in d4).

[0066] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0067] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0068] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0069] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0070] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0071] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0072] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0073] In some embodiments of the invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14, including any recombinant variants thereof, and in particular selected from Cas9, including any recombinant variants thereof.

[0074] In a third aspect of the present invention, an sgRNA is provided, the nucleotide sequence of the sgRNA being as shown in SEQ ID NO: 3 and / or SEQ ID NO: 4, wherein A, C, G and T are nucleotide components including modified nucleotide components.

[0075] In some embodiments of the present invention, the modification is, for example, sugar modification, methylation modification, etc.

[0076] In some embodiments of the present invention, the sgRNA targets Solyc02g077940 and can knock out / knock down Solyc02g077940 together with the Cas protein.

[0077] In some embodiments of the present invention, the amino acid sequence of Solyc02g077940 is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO: 2.

[0078] A fourth aspect of the present invention provides a biological material related to the sgRNA of the third aspect of the present invention, wherein the biological material comprises at least one of e1) to e12):

[0079] e1) a nucleic acid molecule encoding the sgRNA according to the third aspect of the present invention;

[0080] e2) an expression cassette containing the nucleic acid molecule described in e1);

[0081] e3) a recombinant vector containing the nucleic acid molecule described in e1);

[0082] e4) a recombinant vector containing the expression cassette described in e2);

[0083] e5) a recombinant cell containing the nucleic acid molecule described in e1);

[0084] e6) a recombinant cell containing the expression cassette described in e2);

[0085] e7) a recombinant cell containing the recombinant vector described in e3);

[0086] e8) a recombinant cell containing the recombinant vector described in e4);

[0087] e9) a recombinant microorganism containing the nucleic acid molecule described in e1);

[0088] e10) a recombinant microorganism containing the expression cassette described in e2);

[0089] e11) a recombinant microorganism containing the recombinant vector described in e3);

[0090] e12) A recombinant microorganism containing the recombinant vector described in e4).

[0091] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0092] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0093] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0094] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0095] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0096] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0097] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0098] A fifth aspect of the present invention provides the use of any one of f1) to f4) in any one of a1) to a4):

[0099] f1) sgRNA according to the third aspect of the present invention;

[0100] f2) the biomaterial according to the fourth aspect of the present invention;

[0101] f3) a CRISPR / Cas system containing the sgRNA according to the third aspect of the invention or the biological material according to the fourth aspect of the invention;

[0102] f4) a reagent containing the sgRNA according to the third aspect of the present invention, the biological material according to the fourth aspect of the present invention, or the CRISPR / Cas system according to f3);

[0103] a1) Improve the firmness of tomato fruits;

[0104] a2) screening and / or preparing a product for increasing the firmness of tomato fruit;

[0105] a3) Breeding of high-hardness tomato varieties;

[0106] a4) preparing products cultivated from tomato varieties with high hardness.

[0107] In some embodiments of the present invention, the CRISPR / Cas system in f3) further comprises a Cas protein and / or biological materials associated with the Cas protein;

[0108] The biological material associated with the Cas protein includes at least one of d1) to d12):

[0109] d1) a nucleic acid molecule encoding a Cas protein;

[0110] d2) an expression cassette containing the nucleic acid molecule described in d1);

[0111] d3) a recombinant vector containing the nucleic acid molecule described in d1);

[0112] d4) a recombinant vector containing the expression cassette described in d2);

[0113] d5) a recombinant cell containing the nucleic acid molecule described in d1);

[0114] d6) a recombinant cell containing the expression cassette described in d2);

[0115] d7) a recombinant cell containing the recombinant vector described in d3);

[0116] d8) a recombinant cell containing the recombinant vector described in d4);

[0117] d9) a recombinant microorganism containing the nucleic acid molecule described in d1);

[0118] d10) a recombinant microorganism containing the expression cassette described in d2);

[0119] d11) a recombinant microorganism containing the recombinant vector described in d3);

[0120] d12) A recombinant microorganism containing the recombinant vector described in d4).

[0121] In some embodiments of the invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14, including any recombinant variants thereof, and in particular selected from Cas9, including any recombinant variants thereof.

[0122] In some embodiments of the present invention, the products include but are not limited to reagents, kits and drugs.

[0123] In some embodiments of the present invention, the product further comprises a pharmaceutically acceptable carrier, including but not limited to: a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation agent, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a colorant, a flavoring agent or an adsorption carrier.

[0124] A sixth aspect of the present invention provides a method comprising the steps of reducing the expression level of the Solyc02g077940 gene in a tomato plant;

[0125] The method is h1) and / or h2):

[0126] h1) Method for increasing the firmness of tomato fruit;

[0127] h2) Methods for breeding high-hardness tomato varieties.

[0128] In some embodiments of the present invention, the step of reducing the expression level of the Solyc02g077940 gene in the tomato plant is to introduce at least one of i1) to i4) into the tomato plant;

[0129] i1) sgRNA according to the third aspect of the present invention;

[0130] i2) the biomaterial according to the fourth aspect of the present invention;

[0131] i3) the CRISPR / Cas system of the fifth aspect of the present invention;

[0132] i4) The reagent according to the fifth aspect of the present invention.

[0133] In some embodiments of the present invention, the method for breeding high hardness tomato varieties includes the step of knocking out / knocking down the Solyc02g077940 gene in the tomato plant using gene editing technology.

[0134] In some preferred embodiments of the present invention, the gene editing technology includes ZFNs, TALENs or CRISPR / Cas9 technology.

[0135] In some embodiments of the present invention, CRISPR / Cas9 technology is used to knock out / knock down the Solyc02g077940 gene.

[0136] In some embodiments of the present invention, knocking out / knockdown of the Solyc02g077940 gene using CRISPR / Cas9 technology includes the following steps: introducing sgRNA and Cas9 protein into tomato plants, and obtaining high hardness tomato varieties after screening.

[0137] By knocking out the Solyc02g077940 gene through gene editing, homozygous plants with significantly improved fruit firmness and no exogenous gene insertions can be quickly obtained in two generations, thereby obtaining parental materials for subsequent tomato fruit firmness breeding.

[0138] The seventh aspect of the present invention provides a CRISPR / Cas system, comprising the sgRNA of the third aspect of the present invention or the biomaterial of the fourth aspect of the present invention.

[0139] In some embodiments of the present invention, the CRISPR / Cas system further comprises a Cas protein and / or biological materials associated with the Cas protein;

[0140] The biological material associated with the Cas protein includes at least one of d1) to d12):

[0141] d1) a nucleic acid molecule encoding a Cas protein;

[0142] d2) an expression cassette containing the nucleic acid molecule described in d1);

[0143] d3) a recombinant vector containing the nucleic acid molecule described in d1);

[0144] d4) a recombinant vector containing the expression cassette described in d2);

[0145] d5) a recombinant cell containing the nucleic acid molecule described in d1);

[0146] d6) a recombinant cell containing the expression cassette described in d2);

[0147] d7) a recombinant cell containing the recombinant vector described in d3);

[0148] d8) a recombinant cell containing the recombinant vector described in d4);

[0149] d9) a recombinant microorganism containing the nucleic acid molecule described in d1);

[0150] d10) a recombinant microorganism containing the expression cassette described in d2);

[0151] d11) a recombinant microorganism containing the recombinant vector described in d3);

[0152] d12) A recombinant microorganism containing the recombinant vector described in d4).

[0153] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0154] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0155] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0156] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0157] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0158] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0159] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0160] In some embodiments of the invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14, including any recombinant variants thereof, and in particular selected from Cas9, including any recombinant variants thereof.

[0161] The beneficial effects of the present invention are:

[0162] This invention discloses for the first time the application of Solyc02g077940 in regulating tomato fruit firmness. By screening target genes and combining CRISPR / Cas9 technology to knock out / knock down Solyc02g077940, the importance of Solyc02g077940 in improving tomato fruit firmness is demonstrated.

[0163] The present invention uses the CRISPR / Cas system to edit Solyc02g077940 in tomato plants, rendering it unable to produce normal active proteins. While significantly improving the firmness of tomato fruits, quality traits that determine fruit color and taste, such as yield, lycopene content, and soluble solids content, remain unaffected. This approach has the potential to be applied to molecular design breeding for tomato fruit firmness, providing a new strategy for improving tomato fruit firmness and extending storage life. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 The Solyc02g077940 gene knockout mutation was generated by gene editing; among them, A is the gene knockout mutation, the sgRNA used is shown in the red sequence, the start codon is shown in the yellow dotted box, and the dotted line indicates the position and length of the mutant base deletion; B is the change in the mutant protein. The wild-type Solyc02g077940 protein contains 55 amino acids after translation. Both deletion mutations result in the loss of the start codon and cannot produce functional protein.

[0165] Figure 2The fruit ripening phenotype of the mutant strain 7 days after color breaking. In the figure, WT represents the wild-type tomato plant, cr-1 and cr-2 are the homozygous strains of the Solyc02g077940 gene knockout mutation isolated in the T1 generation, and the scale bar is 2 cm.

[0166] Figure 3 The changes in fruit hardness of mutant strains; A is the fruit hardness (Newtons) at the Br+7d stage, and B is the fruit hardness (Newtons) at the Br+15d stage; in the figure, WT represents wild-type tomato plants, cr-1 and cr-2 are homozygous strains of the Solyc02g077940 gene knockout mutation isolated in the T1 generation, * represents p < 0.05, and *** represents p < 0.001.

[0167] Figure 4 The following table shows the results of fruit yield and other fruit quality tests of the mutant strains. A is the yield of the mutant strain (kg); B is the relative lycopene content in the mutant strain's fruit; and C is the soluble solids content (Brix) in the mutant strain's fruit. All fruits were harvested on the Br+7th day. DETAILED DESCRIPTION

[0168] The present invention is further described in detail below through specific examples.

[0169] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0170] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0171] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0172] Example 1

[0173] The plant material used in the embodiment is tomato (cv. Moneyberg), which was planted in the ecological greenhouse of the Zhongluotan Base of the Institute of Facility Agriculture of Guangdong Academy of Agricultural Sciences.

[0174] (1) Design of sgRNA for the Solyc02g077940 gene

[0175] The tomato gene Solyc02g077940 is a coding sequence located between 42753490 and 42753654 on chromosome 2. Using the online tool CRISPOR (http: / / crispor.gi.ucsc.edu / ), small guide RNAs (sgRNAs) were designed based on the amino acid sequence (SEQ ID NO: 1) and coding sequence (SEQ ID NO: 2) of Solyc02g077940. Two sgRNAs were selected based on their efficiency and off-target probability. The sgRNA sequences are as follows:

[0176] sgRNA1: 5'-CTCGTTGTCTCTCTCGCAAA-3' (SEQ ID NO: 3);

[0177] sgRNA2: 5'-CGTCAGCCACCAGCCCCCAC-3' (SEQ ID NO: 4).

[0178] Solyc02g077940 amino acid sequence:

[0179] MVSNRSDARSSSNVSHQPPPAKKTQPTTIGKHSERRIWIRADLYRSVRGILLFI (SEQ ID NO: 1).

[0180] Solyc02g077940 nucleotide sequence:

[0181] ATGGTGAGCA ACCGCTCCGA CGCACGCAGC AGCAGCAACG TCAGCCACCA GCCCCCACCGGCGAAGAAGA CACAGCCAAC AACCATTGGC AAACACTCTG AGCGAAGAAT ATGGATAAGG GCAGATCTTTATAGATCTGT CCGAGGTATT CTTTTATTCA TATAG (SEQ ID NO: 2).

[0182] (2) Construction of CRISPR / Cas9 gene editing vector

[0183] sgRNA1 and sgRNA2 were respectively connected to a vector containing the Arabidopsis U6 promoter, electroporated into Escherichia coli DH5a, and the plasmid was extracted after overnight culture in a liquid medium containing carbenicillin. The primer sgRNA-R (5'-GACCCTGTGTTGCATGCCAT-3', SEQ ID NO: 5) was then sequenced to ensure that the sgRNA sequence was correct. The sgRNA and vector were digested using the endonuclease BbsI-HF (New England Biolabs, Inc) and linked to the CRISPR / Cas9 gene editing vector pAGM4723, electroporated into Escherichia coli DH5a, and the plasmid was extracted after overnight culture in a liquid medium containing kanamycin. The electroporated plasmid was transformed into the Agrobacterium tumefaciens strain GV3101, plated onto a plate culture medium containing rifampicin and kanamycin for screening, and monoclonal plaques were picked and cultured in a liquid culture medium containing rifampicin and kanamycin. This method constructs two sgRNAs into one vector at the same time, and only one genetic transformation is required to obtain multiple different mutations located at different positions.

[0184] (3) Genetic transformation of tomato

[0185] Tomato seeds were sterilized by soaking in 1% sodium hypochlorite solution, then washed with sterile water and inoculated into 1 / 2 MS medium (MS + 1wt% sucrose + 0.8wt% agar), placed in a culture room, 16h light / 8h, 25°C for 5 days. Cotyledons were cut off and placed in a medium (MS + 3wt% sucrose + 0.8wt% agar) in the dark for 1 day. Agrobacterium was cultured overnight to an OD of 600 ≈0.8, transfer the cotyledons to the Agrobacterium solution obtained in (2) + 10μM / mL acetosyringone and incubate for 10 minutes, then transfer the cotyledons to sterilized filter paper, dry the solution, and then transfer the cotyledons to co-culture medium (MS + 3wt% sucrose + 0.05v / v% MES (2-N-morpholino ethanesulfonic acid) + 0.8wt% agar) for co-culture for 2 days, then transfer the cotyledons to meristem medium (MS + 1wt% glucose + 0.8wt% agar + 2mg / L zeatin + 500mg / L carbenicillin + 100mg / L kanamycin) and culture for 7-8 weeks, during which the culture medium was changed every 2 weeks; when the regenerated plants grew to about 1cm, they were cut and placed in rooting medium (MS + 15wt% sucrose + 0.8wt% agar + 0.25mg / L Indole-3-butyric acid) After about 2 weeks, the well-rooted regenerated seedlings were transplanted to asbestos for further cultivation.

[0186] (4) Screening of Solyc02g077940 gene knockout mutant strains that can be stably inherited

[0187] Leaves from transformed plants were collected and DNA was extracted using the Phire Hot Start PCR Kit (Thermo Fisher). Primers F1 (5'-TCTTCTTCTCCCTCTTCCGT-3', SEQ ID NO: 6) and R1 (5'-CAATGGGCATCAATCGACTG-3', SEQ ID NO: 7) were designed upstream and downstream of the Solyc02g077940 gene. DNA fragments containing the mutation region were amplified using Phire Hot Start II DNA Polymerase. Sequencing and comparison with wild-type Solyc02g077940 were used to detect the mutation. Plants harboring the mutation were retained and their seeds were collected for Mendelian segregation in the T1 generation.

[0188] PCR reaction system: 4 μL of 5× Phire buffer, 1 μL of F1 (10 μM / L), 1 μL of R1 (10 μM / L), 0.5 μL of DNA, 0.4 μL of Phire DNA polymerase, and 14.1 μL of sterile water.

[0189] PCR reaction conditions: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 5 seconds, annealing at 62.3°C for 5 seconds, and extension at 72°C for 10 seconds, for 40 cycles.

[0190] Depend on Figure 1 It can be seen that the wild-type Solyc02g077940 protein contains 55 amino acids after translation, and the two deletion mutations both result in the loss of the start codon and cannot produce functional proteins.

[0191] Leaves from T1 plants were collected and DNA was extracted using the Phire Hot Start PCR Kit (Thermo Fisher). Primers F1 and R1 were used to amplify the DNA fragment containing the mutation region, and sequencing was performed to confirm homozygous mutations. Simultaneously, primers CAS9-F (5'-CTTTGGCAATATCGTGGACG-3', SEQ ID NO: 8) and CAS9-R (5'-CGTTCTTCTTCTCCCCAGGG-3', SEQ ID NO: 9) were used to detect the presence of Cas9. Homozygous mutant lines lacking Cas9 were selected for further cultivation. This method ensures that all homozygous mutant plants do not contain exogenous gene insertions. Reaction conditions included pre-denaturation at 98°C for 30 seconds, followed by 40 cycles of denaturation at 98°C for 5 seconds, annealing at 65°C for 5 seconds, and extension at 72°C for 10 seconds.

[0192] Example 2 Fruit quality detection of mutant tomato strains

[0193] (1) Fruit firmness of mutant tomato strains

[0194] The homozygous mutant strain obtained in Example 1 was further planted, and wild-type and mutant fruits were collected on the 7th and 15th days after the fruit broke. The texture was analyzed using a texture analyzer ( Fruit firmness was measured by squeezing the fruit at the equator (Cape Town, South Africa). Nine fruits were collected from each strain, and the average value was used to represent the fruit firmness (Newtons) of that strain. Measurement parameters were: pre-test speed 30 mm / s, test speed 10 mm / s, measurement depth 2 mm, and post-test speed 40 mm / s.

[0195] The results are as follows Figures 2 and 3 As shown, the fruits of the two mutants showed a ripe fruit phenotype and turned completely red 7 days after breaking color (Br+7d), with no significant difference from the wild type ( Figure 2 The fruit firmness was measured using a texture analyzer. The results showed that the fruit firmness of the two mutant strains was significantly higher than that of the wild type on the 7th and 15th days after the fruit broke. Figure 3 ).

[0196] (2) Detection of tomato yield and fruit quality of mutant strains

[0197] For each line, the second, third, and fourth fruits were selected, and 5 fruits were retained on each branch. 15 fruits were weighed to represent the yield (kg) of the line.

[0198] On the 7th day after the fruit broke, 9 wild-type and mutant fruits were collected and measured at the fruit equator using a handheld diode array spectrophotometer (Pigment Analyzer PA1101, Germany). The average value was used to represent the relative lycopene content of the fruit of this strain.

[0199] On the 7th day after the fruit broke, 9 wild-type and mutant fruits were collected, the juice was squeezed out, and the Brix was measured using a low-concentration digital refractometer (PR-32a, ATAGO). The average value was taken to represent the soluble solids content of the fruit of this strain.

[0200] The results are as follows Figure 4 As shown, the tomato fruit yield, lycopene and soluble solids content in the mutant strain were not significantly different from those in the wild type, indicating that knocking out the Solyc02g077940 gene would not affect the tomato fruit yield and other fruit qualities.

[0201] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of Solyc02g077940 in regulating tomato fruit firmness, wherein the amino acid sequence of Solyc02g077940 is as shown in SEQ ID NO:

1.

2. Application of Solyc02g077940 inhibitor in any one of a1) to a4); a1) Improving the firmness of tomato fruits; a2) Screening and / or preparing products for improving the firmness of tomato fruits; a3) Cultivating tomato varieties with high firmness; a4) Preparing products for cultivating tomato varieties with high firmness; The amino acid sequence of the Solyc02g077940 is as shown in SEQ ID NO:1; The Solyc02g077940 inhibitor includes at least one of b1) to b13): b1) CRISPR / Cas system targeting Solyc02g077940; b2) Nucleic acid molecule encoding b1); b3) Expression cassette containing the nucleic acid molecule described in b2); b4) Recombinant vector containing the nucleic acid molecule described in b2); b5) Recombinant vector containing the expression cassette described in b3); b6) Recombinant cell containing the nucleic acid molecule described in b2); b7) Recombinant cell containing the expression cassette described in b3); b8) Recombinant cell containing the recombinant vector described in b4); b9) Recombinant cell containing the recombinant vector described in b5); b10) Recombinant microorganism containing the nucleic acid molecule described in b2); b11) Recombinant microorganism containing the expression cassette described in b3); b12) Recombinant microorganism containing the vector described in b4); b13) Recombinant microorganism containing the vector described in b5); The CRISPR / Cas system includes sgRNA targeting Solyc02g077940 or biomaterials related to sgRNA; The nucleotide sequence of the sgRNA is as shown in SEQ ID NO:3 and / or SEQ ID NO:

4.

3. The application according to claim 2, characterized in that The biomaterials related to sgRNA include at least one of c1) to c12): c1) Nucleic acid molecule encoding sgRNA; c2) Expression cassette containing the nucleic acid molecule described in c1); c3) Recombinant vector containing the nucleic acid molecule described in c1); c4) Recombinant vector containing the expression cassette described in c2); c5) Recombinant cell containing the nucleic acid molecule described in c1); c6) Recombinant cell containing the expression cassette described in c2); c7) Recombinant cell containing the recombinant vector described in c3); c8) Recombinant cell containing the recombinant vector described in c4); c9) Recombinant microorganism containing the nucleic acid molecule described in c1); c10) Recombinant microorganism containing the expression cassette described in c2); c11) Recombinant microorganism containing the recombinant vector described in c3); c12) Recombinant microorganism containing the recombinant vector described in c4).

4. The application according to claim 2, characterized in that, The CRISPR / Cas system further includes Cas protein and / or biomaterials related to Cas protein.

5. The application according to claim 4, characterized in that The biomaterials related to Cas protein include at least one of d1) to d12): d1) Nucleic acid molecule encoding Cas protein; d2) An expression cassette containing the nucleic acid molecule described in d1); d3) A recombinant vector containing the nucleic acid molecule described in d1); d4) A recombinant vector containing the expression cassette described in d2); d5) A recombinant cell containing the nucleic acid molecule described in d1); d6) A recombinant cell containing the expression cassette described in d2); d7) A recombinant cell containing the recombinant vector described in d3); d8) A recombinant cell containing the recombinant vector described in d4); d9) A recombinant microorganism containing the nucleic acid molecule described in d1); d10) A recombinant microorganism containing the expression cassette described in d2); d11) A recombinant microorganism containing the recombinant vector described in d3); d12) A recombinant microorganism containing the recombinant vector described in d4).

6. A sgRNA, wherein the nucleotide sequence of the sgRNA is as shown in SEQ ID NO:3 and / or SEQ ID NO:

4.

7. A biological material related to the sgRNA according to claim 6, characterized in that, The biological material comprises at least one of e1) to e12): e1) A nucleic acid molecule encoding the sgRNA described in claim 6; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1); e4) A recombinant vector containing the expression cassette described in e2); e5) A recombinant cell containing the nucleic acid molecule described in e1); e6) A recombinant cell containing the expression cassette described in e2); e7) A recombinant cell containing the recombinant vector described in e3); e8) A recombinant cell containing the recombinant vector described in e4); e9) A recombinant microorganism containing the nucleic acid molecule described in e1); e10) A recombinant microorganism containing the expression cassette described in e2); e11) A recombinant microorganism containing the recombinant vector described in e3); e12) A recombinant microorganism containing the recombinant vector described in e4).

8. The application of any one of f1) to f4) in any one of a1) to a4): f1) The sgRNA described in claim 6; f2) The biological material described in claim 7; f3) A CRISPR / Cas system containing the sgRNA described in claim 6 or the biological material described in claim 7; f4) A reagent containing the sgRNA described in claim 6, the biological material described in claim 7 or the CRISPR / Cas system described in f3); a1) To increase the hardness of tomato fruits; a2) To screen and / or prepare products for increasing the hardness of tomato fruits; a3) Cultivation of high-hardness tomato varieties; a4) To prepare products for cultivation of high-hardness tomato varieties.

9. A method, comprising the step of knocking out the Solyc02g077940 gene in a tomato plant; The method is h1) and / or h2): h1) A method for increasing the hardness of tomato fruits; h2) A method for cultivating high-hardness tomato varieties; The amino acid sequence of the Solyc02g077940 is as shown in SEQ ID NO:

1.

10. The method according to claim 9, wherein The step of knocking out the Solyc02g077940 gene in the tomato plant is to introduce at least one of i1) to i4) into the tomato plant; i1) The sgRNA described in claim 6; i2) The biological material described in claim 7; i3) The CRISPR / Cas system described in claim 8; The reagent as described in claim 8.

Images