Solyc02g161260 gene for improving tomato fruit firmness and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a Solyc02g161260 gene for improving the hardness of tomato fruits and an application thereof, and specifically discloses the application of the Solyc02g161260 gene in regulating the hardness of tomato fruits, wherein the amino acid sequence of the Solyc02g161260 gene is shown in SEQ ID NO: 1. The present invention discloses for the first time the application of Solyc02g161260 in regulating the hardness of tomato fruits, and knocks out / knocks down Solyc02g161260 by target gene screening and combining CRISPR / Cas9 technology, thereby proving the importance of Solyc02g161260 in improving the hardness of tomato fruits.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Solyc02g161260 gene for improving the firmness of tomato fruits and an application thereof. Background Art

[0002] Tomato (Solanum lycopersicum) is native to South America and is an important vegetable crop of the Solanaceae family. Its annual output ranks first among vegetables in the world. Tomato fruit is rich in many basic nutrients and bioactive substances required by the human body, and plays an important role in human health, diet and cooking.

[0003] my country ranks first in the world in terms of tomato cultivation area and production. However, collisions during long-distance transportation and short shelf life cause fruit spoilage, resulting in a large waste of resources and economic losses. Therefore, reducing post-harvest tomato losses has huge economic benefits and production needs.

[0004] Fruit hardness is an important quality trait of tomatoes, which is closely related to transportation loss, storage resistance and shelf life. Improving fruit hardness can reduce losses caused by long-distance transportation and deterioration, while extending the shelf life. Improving tomato hardness is an important breakthrough point to solve the problem of excessive post-harvest losses, and it is also a production requirement to adapt to mechanized harvesting. In actual production, natural mutations have been introduced to increase fruit hardness, but this will affect the normal ripening of the fruit and cause a significant decrease in lycopene content, affecting fruit quality such as fruit color. Therefore, there is an urgent need for a method to increase the hardness of tomato fruit without affecting normal ripening and fruit quality such as fruit color.

[0005] The CRISPR / Cas9 system, which combines clustered regularly interspaced shortpalindromic repeats (CRISPR) with Cas9 (CRISPR associated) protein, is an immune system for bacteria to remove foreign viral genetic material inserted into their own genome. Its ability to introduce mutations during double-stranded DNA cutting and repair makes it an important gene editing tool. Compared with other previous gene editing technologies, the CRISPR / Cas9 system is easier to operate, has stronger scalability, and is more accurate and efficient in mutating genome sequences, making the acquisition of mutants more efficient and convenient. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide the use of Solyc02g161260 in regulating the hardness of tomato fruit.

[0007] The second aspect of the present invention aims to provide the application of Solyc02g161260 inhibitor.

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

[0009] The purpose of the fourth aspect of the present invention is 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 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, and 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 aims 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 Solyc02g161260 gene has a total of 398 bases and contains two exons. The ITAG4.1 gene model of the tomato genome (https: / / solgenomics.net / ) shows that Solyc02g161260 is located in the range 40733534 to 40733931 on chromosome 2, and its protein contains 84 amino acids. This gene has not been reported before.

[0015] In a first aspect of the present invention, a use of Solyc02g161260 in regulating tomato fruit firmness is provided, wherein the amino acid sequence of Solyc02g161260 is shown in SEQ ID NO:1.

[0016] The second aspect of the present invention provides the use of the Solyc02g161260 inhibitor in any one of a1) to a4);

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

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

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

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

[0021] In some embodiments of the present invention, the amino acid sequence of Solyc02g161260 is as shown in SEQ ID NO:1, and the nucleotide sequence encoding the amino acid is as shown in SEQ ID NO:2.

[0022] In some embodiments of the present invention, the Solyc02g161260 inhibitor is at least one of a substance that degrades the Solyc02g161260 gene or a substance that reduces the expression level of the Solyc02g161260 gene.

[0023] In some embodiments of the present invention, the Solyc02g161260 inhibitor includes at least one of b1) to b13):

[0024] b1) siRNA, dsRNA, miRNA, ribozyme, shRNA, CRISPR / Cas system targeting Solyc02g161260 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 Solyc02g161260 or biological materials associated with the sgRNA.

[0038] In some embodiments of the present invention, the nucleotide sequence of the sgRNA is shown in 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 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 material 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 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 bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0072] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional 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 is particularly selected from Cas9, including any recombinant variants thereof.

[0074] According to 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 Solyc02g161260 and can knock out / knock down Solyc02g161260 together with the Cas protein.

[0077] In some embodiments of the present invention, the amino acid sequence of Solyc02g161260 is as shown in SEQ ID NO:1, and the nucleotide sequence encoding the amino acid is as 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 of 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 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 bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0097] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional 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 present invention or the biological material according to the fourth aspect of the present invention;

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

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

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

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

[0106] a4) preparing products bred 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 is particularly 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, 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 Solyc02g161260 gene in a tomato plant;

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

[0126] h1) Method for improving 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 Solyc02g161260 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 Solyc02g161260 gene in tomato plants 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 Solyc02g161260 gene.

[0136] In some embodiments of the present invention, knocking out / knocking down the Solyc02g161260 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 Solyc02g161260 gene through gene editing, homozygous plants with significantly improved fruit firmness and no exogenous gene insertion can be quickly obtained in two generations, thereby obtaining parental materials for subsequent tomato fruit hardness 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 biological material 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 material 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 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 bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0159] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional 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 is particularly selected from Cas9, including any recombinant variants thereof.

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

[0162] The present invention discloses for the first time the application of Solyc02g161260 in regulating the firmness of tomato fruit. By target gene screening and knocking out / knockdown of Solyc02g161260 in combination with CRISPR / Cas9 technology, the importance of Solyc02g161260 in improving the firmness of tomato fruit is proved.

[0163] The present invention uses the CRISPR / Cas system to edit Solyc02g161260 in tomato plants, making it unable to produce normal active proteins. While significantly improving the firmness of tomato fruits, the fruits mature normally and the ripening time is not affected. The yield and lycopene content and other traits that affect the fruit quality are not affected. The present invention has the potential to be applied to the molecular design breeding of tomato fruit firmness, and provides a new strategy for improving the firmness of tomato fruits and extending the storage period. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 Solyc02g161260 gene knockout mutation was generated for gene editing; A is a gene knockout mutation, a single base deletion in the cr-1 strain and a 61bp base deletion in the cr-2 strain. The sgRNA used is shown in the red sequence, and the start codon is shown in the blue dotted box. The dotted line indicates the position and length of the mutant base deletion; B is the change in the mutant protein. Solyc02g161260 contains 2 exons, and the wild-type protein contains 84 amino acids. The single base deletion in the cr-1 strain causes premature translation termination and produces a truncated protein containing 8 amino acids, of which only 5 amino acids are the same as the wild type. The 61bp base deletion in the cr-2 strain causes the start codon to be missing and cannot produce functional protein. The red arrow indicates the position of the sgRNA.

[0165] Figure 2 It is the fruit ripening phenotype of the mutant strain 7 days after breaking color; A is the time (days) required for the fruits of the cr-1 and cr-2 homozygous strains isolated in the T1 generation to break color; B is the fruits of the cr-1 and cr-2 homozygous strains completely turning red 7 days (Br+7d) after breaking color. In the figure, WT represents the wild-type tomato plant, cr-1 and cr-2 are the homozygous strains of the Solyc02g161260 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 (Newton) at the Br+7d stage; B is the fruit hardness (Newton) at the Br+15d stage; in the figure, WT represents wild-type tomato plants, cr-1 and cr-2 are homozygous strains of Solyc02g161260 gene knockout mutations isolated in the T1 generation, * represents p < 0.05, *** represents p < 0.001.

[0167] Figure 4 The results of the fruit yield and other fruit quality tests of the mutant strains are shown in Table 1. A is the yield of the mutant strain (kg); B is the relative lycopene content in the fruits of the mutant strains. All fruits were picked on Br+7d. 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 used to limit the scope of the present invention.

[0170] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

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

[0172] Example 1

[0173] The plant material used in the embodiment is tomato (cv. Ailsa Craig), 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 Solyc02g161260 gene

[0175] The Solyc02g161260 gene has a total of 398 bases and contains two exons. The ITAG4.1 gene model of the tomato genome (https: / / solgenomics.net / ) shows that Solyc02g161260 is located in the 40733534-40733931 interval of chromosome 2, and its protein contains 84 amino acids. Through the online tool website CRISPOR (http: / / crispor.gi.ucsc.edu / ), small guide RNA (sgRNA) was designed based on the nucleotide sequence of Solyc02g161260 (SEQ ID NO: 2) and the nucleotide sequence encoded by it (SEQ ID NO: 2), and two were selected based on the work efficiency and off-target probability prediction. The sgRNA sequence is as follows:

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

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

[0178] Solyc02g161260 amino acid sequence:

[0179] MKLALRGKVKLGFVDGNCTKRSTVTADLMPSIVYASIAKKVWDEYKERFDRSN LTRIYYLCAEIANLKQGTDSVTSYYSKMKNL (SEQ ID NO: 1).

[0180] Solyc02g161260 nucleotide sequence:

[0181] CTTCTGATTC TCCAGACTTA GTAATAACTC CGATCAAACT CACAGGACCA GAGAACTACTCTCTATGGAG CAAATCGATG AAACTAGCAC TGCGAGGCAA AGTAAAACTT GGATTTGTGG ATGGAAACTGCACAAAGAGT AAGTTTAAAG GAGACCTAGA AGCGTAATGG GAAAAATTTA ATGCAATTGT GTATCATGGATAGGAAGTA CTGTTACAGC TGATTTGATG CCAAGCATAG TTTATGCTTC AATTGCGAAG AAGGTATGGGATGAATACAA GGAGCGTTTT GATAGATCGA ATTTGACGAG GATTTATTAT TTGTGCGCAG AAATTGCTAATTTGAAACAA GGAACAGACT CTGTGACAAG CTATTATTCC AAAATGAAGA ATCTATGA (SEQ ID NO: 2).

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

[0183] sgRNA1 and sgRNA2 were connected to the vector containing Arabidopsis U6 promoter, electroporated into Escherichia coli DH5a, and the plasmid was extracted after overnight culture in liquid medium containing carbenicillin. The primer sgRNA-R (5'-GACCCTGTGTTGCATGCCAT-3', SEQ ID NO: 5) was used for sequencing to ensure that the sgRNA sequence was correct. The vector containing sgRNA and vector was digested with 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 liquid medium containing kanamycin. The electroporated plasmid was transformed into Agrobacterium strain GV3101, coated on a plate medium containing rifampicin and kanamycin for screening, and a monoclonal plaque was picked and placed in a liquid medium containing rifampicin and kanamycin for culture. This method constructs two sgRNAs into one vector at the same time, and only requires one genetic transformation to obtain multiple different mutations located at different locations.

[0184] (3) Genetic transformation of tomato

[0185] Soak tomato seeds in 1% sodium hypochlorite solution for disinfection, wash with sterile water and inoculate in 1 / 2MS medium (MS + 1wt% sucrose + 0.8wt% agar), place in a culture room, 16h light / 8h, 25℃ for 5 days. Cut the cotyledons, place in medium (MS + 3wt% sucrose + 0.8wt% agar) and culture for 1 day in dark. Cultivate Agrobacterium overnight to OD 600 ≈0.8, transfer the cotyledons to the Agrobacterium solution obtained in (2) + 10 μM / mL acetosyringone for 10 min, then transfer the cotyledons to sterile 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) for 7-8 weeks, during which the culture medium is replaced every 2 weeks; when the regenerated plants grow to about 1 cm, cut them off and place them in rooting medium (MS + 15wt% sucrose + 0.8wt% agar + 0.25mg / L Indole-3-butyric acid) acid (IBA) + 320mg / L timentin) to take root; about 2 weeks later, the well-rooted regenerated seedlings were transplanted to asbestos for further cultivation.

[0186] (4) Screening of Solyc02g161260 gene knockout mutant strains that can be stably inherited

[0187] The leaves of the transformed plants were collected and DNA was extracted using the Phire hot start PCR kit (Thermo Fisher). Primers F1 (5'-GCTACAGATGGAAGTTCCAGC-3', SEQ ID NO: 6) and R1 (5'-AACTGTAACAATCGTTGTGACTTCC-3', SEQ ID NO: 7) were designed at about 200 bp upstream and downstream of the Solyc02g161260 gene, and Phire hot start II DNA polymerase was used to PCR amplify the DNA fragment containing the mutation region. After sequencing, the mutation was detected based on sequence comparison with the wild type Solyc02g161260. Plants containing mutations were retained and their seeds were collected for T1 generation Mendelian segregation.

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

[0189] PCR reaction conditions: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 5 seconds, annealing at 63.7°C for 5 seconds, extension at 72°C for 10 seconds, and 40 cycles.

[0190] Depend on Figure 1 It can be seen that Solyc02g161260 contains 2 exons, and the Solyc02g161260 protein of wild-type tomato plants contains 84 amino acids. The single base deletion in the cr-1 strain leads to premature termination of translation, producing a truncated protein containing 8 amino acids, of which only 5 amino acids are the same as the wild type. The 61bp base deletion in the cr-2 strain leads to the loss of the start codon, and no functional protein can be produced.

[0191] Leaves of T1 plants were collected, and DNA was extracted using the Phire hot start PCR kit (Thermo Fisher). Primers F1 and R1 were used to PCR amplify DNA fragments containing the mutation region, and sequencing was used to detect whether the mutation was homozygous. At the same time, primers CAS9-F (5'-CTTTGGCAATATCGTGGACG-3', SEQ ID NO: 8) and CAS9-R (5'-CGTTCTTCTTCTCCCCAGGG-3'SEQ ID NO: 9) were used to detect whether Cas9 was contained, and homozygous mutant lines without Cas9 were selected for continued planting. This method can ensure that all homozygous mutant plants do not contain exogenous gene insertions. Reaction conditions: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 5 seconds, 65℃ annealing for 5 seconds, 72℃ extension for 10 seconds, and 40 cycles.

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

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

[0194] The homozygous mutant strains obtained in Example 1 were further planted. The 2nd, 3rd, and 4th flower branches were selected for each strain. Five flowers were marked on each branch. The flowers were marked and vibrated to assist pollination on the day when the petals were fully unfolded. The time required for 15 flowers to develop into fruit color was recorded to represent the time required for maturity (days). Wild-type and mutant fruits were collected on the 7th and 15th days after the fruit color broke, and the texture was analyzed using a texture analyzer ( The fruit hardness was measured by squeezing the fruit at the equator of the fruit in Cape Town, South Africa. Nine fruits were collected from each strain, and the average was used to represent the fruit hardness (Newton, N) of this strain. The measurement parameters were: pre-measurement speed of 30 mm / s, measurement speed of 10 mm / s, measurement depth of 2 mm, and post-measurement speed of 40 mm / s.

[0195] The results are as follows Figure 2As shown, the fruits of the two mutant strains showed a fruit ripening phenotype. The fruits of both strains broke color normally (Breaker), and the time required for the fruit to break color (days) was not significantly different from that of the wild type. Both strains turned completely red 7 days after breaking color (Br+7d). Figure 2 The fruit firmness was measured by 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 strain, the 2nd, 3rd and 4th fruits were selected, 5 fruits were retained on each branch, and 15 fruits were weighed to represent the yield (kg) of the strain.

[0198] On the 7th day after the fruit broke color, 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 was taken to represent the relative lycopene content of the fruit of this strain.

[0199] The results are as follows Figure 4 As shown, there was no significant difference in tomato fruit yield and lycopene content in tomato fruit between the mutant strain and the wild type, indicating that knocking out the Solyc02g161260 gene would not affect tomato fruit yield and other fruit qualities.

[0200] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of Solyc02g161260 in regulating tomato fruit hardness, the amino acid sequence of Solyc02g161260 is shown in SEQ ID NO:

1.

2. Use of Solyc02g161260 inhibitor in any one of a1) to a4); a1) Improve the firmness of tomato fruit; a2) screening and / or preparing products for improving the firmness of tomato fruits; a3) Breeding of high hardness tomato varieties; a4) Preparation of products bred from tomato varieties with high hardness; The Solyc02g161260 inhibitor includes at least one of b1) to b13): b1) CRISPR / Cas system targeting Solyc02g161260; b2) a nucleic acid molecule encoding b1); b3) an expression cassette containing the nucleic acid molecule described in b2); b4) a recombinant vector containing the nucleic acid molecule described in b2); b5) a recombinant vector containing the expression cassette described in b3); b6) a recombinant cell containing the nucleic acid molecule described in b2); b7) a recombinant cell containing the expression cassette described in b3); b8) a recombinant cell containing the recombinant vector described in b4); b9) a recombinant cell containing the recombinant vector described in b5); b10) a recombinant microorganism containing the nucleic acid molecule described in b2); b11) a recombinant microorganism containing the expression cassette described in b3); b12) a recombinant microorganism containing the vector described in b4); b13) a recombinant microorganism containing the vector described in b5); The CRISPR / Cas system includes an sgRNA targeting Solyc02g161260 or biological materials associated with the sgRNA; The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 3 and / or SEQ ID NO:

4.

3. The use according to claim 2, characterized in that: The biological material related to sgRNA includes at least one of c1) to c12): c1) Nucleic acid molecule encoding sgRNA; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1); c4) a recombinant vector containing the expression cassette described in c2); c5) a recombinant cell containing the nucleic acid molecule described in c1); c6) a recombinant cell containing the expression cassette described in c2); c7) a recombinant cell containing the recombinant vector described in c3); c8) a recombinant cell containing the recombinant vector described in c4); c9) a recombinant microorganism containing the nucleic acid molecule described in c1); c10) a recombinant microorganism containing the expression cassette described in c2); c11) a recombinant microorganism containing the recombinant vector described in c3); c12) A recombinant microorganism containing the recombinant vector described in c4).

4. The use according to claim 3, characterized in that: The CRISPR / Cas system also includes Cas protein and / or biological material associated with the Cas protein.

5. The use according to claim 4, characterized in that: The biological material related to the Cas protein includes at least one of d1) to d12): d1) Nucleic acid molecules encoding Cas proteins; 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. An sgRNA, the nucleotide sequence of which is shown in SEQ ID NO: 3 and / or SEQ ID NO:

4.

7. The 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 according to 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. Application of any of f1) to f4) in any of a1) to a4): f1) the sgRNA according to claim 6; f2) The biological material according to claim 7; f3) A CRISPR / Cas system containing the sgRNA of claim 6 or the biological material of claim 7; f4) a reagent containing the sgRNA of claim 6, the biological material of claim 7, or the CRISPR / Cas system of f3); a1) Improve the firmness of tomato fruit; a2) screening and / or preparing products for improving the firmness of tomato fruits; a3) Breeding of high hardness tomato varieties; a4) Preparation of products bred from tomato varieties with high hardness.

9. A method comprising the step of knocking out the expression level of the Solyc02g161260 gene in a tomato plant; The methods are h1) and / or h2): h1) Methods for improving the firmness of tomato fruits; h2) Methods for breeding high hardness tomato varieties.

10. The method according to claim 9, characterized in that The step of knocking out the expression level of the Solyc02g161260 gene in the tomato plant is to introduce at least one of i1) to i4) into the tomato plant; i1) the sgRNA according to claim 6; i2) The biological material according to claim 7; i3) The CRISPR / Cas system as described in claim 8; i4) The reagent as claimed in claim 8.

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