Application of TST3b protein and related biological materials thereof in regulating and controlling soluble sugar content of tomato

Abstract

The invention discloses a substance for reducing the activity or content of a protein TST3b, or application of a substance for inhibiting or reducing the expression of a coding gene of the protein TST3b. The TST3b protein can be specifically a protein of A1), A2) or A3) as follows: A1) a protein with an amino acid sequence shown as SEQ ID No.2 in a sequence table, A2) a protein with an amino acid sequence shown as SEQ ID No.2 in a sequence table, and A3) a protein with an amino acid sequence shown as SEQ ID No.2 in the sequence table, or A3) a protein with an amino acid sequence shown as SEQ ID No.2 in the sequence table; a2) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the protein of A1), has 90% or more of identity with the protein of A1) and has the same activity as the protein of A1); and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The TST3b protein and related biological materials thereof can be used for regulating and controlling the soluble sugar content and the single fruit weight of tomatoes.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of TST3b protein and related biological materials in regulating the soluble sugar content of tomatoes. Background Art

[0002] Soluble sugar is the main component that affects the sweetness and taste of tomatoes. It plays an important role in the formation of fruit flavor quality and is an important indicator for evaluating tomato fruit quality. However, current research on regulating the soluble sugar content of tomato fruit is still limited. Very few genes involved in sugar content regulation have been cloned, and there is a lack of effective molecular marker-assisted breeding selection. Therefore, discovering new genes that regulate the soluble sugar content of tomato fruit and utilizing them is the basis and key to solving this problem, which is of great significance to high-yield and high-quality tomato breeding.

[0003] Tomato fruit is a typical sink organ. 80% of the soluble sugar in the fruit is assimilated and transported from photosynthates produced by source organs such as leaves, and 20% comes from photosynthesis of the fruit itself. Sucrose is the main assimilated product of tomato photosynthesis and the main form of long-distance transport. During the development period of tomato fruit, photosynthates are transported long distances from source organs such as mature leaves to fruits through phloem in the form of sucrose and unloaded through the symplast and apoplast pathways. The MYB transcription factor SlGLK2 regulates the accumulation of chloroplasts and chlorophyll in the part of tomato fruit close to the tomato stalk (fruit shoulder), making the fruit shoulder dark green, while improving fruit photosynthesis, increasing the starch content in immature fruits, and thus increasing the soluble sugar content in mature fruits. SlLIN5 encodes a cell wall sucrose invertase, which participates in the apoplast pathway of sucrose unloading in tomato fruit. A SNP in this gene leads to a non-synonymous substitution of the encoded amino acid, which affects the activity of the enzyme, reduces the conversion efficiency of sucrose in tomato fruit, and thus reduces the soluble sugar content in the fruit.

[0004] The soluble sugar content of tomato fruit is a complex process, which is regulated by the external environment and internal genes. Although previous studies have been conducted on the soluble sugar content of tomato fruit, these genes will bring other unfavorable traits in the improvement of soluble sugar content in tomato fruit, and there is a bottleneck in their utilization. Therefore, mining and cloning genes that regulate the soluble sugar content of tomato fruit and utilizing them is an effective way to solve this problem. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to increase the soluble sugar content and single fruit weight of tomatoes, thereby improving the quality and yield of tomatoes.

[0006] In order to solve the above technical problems, the present invention first provides a substance that reduces the activity or content of protein TST3b, or an application of a substance that inhibits or reduces the expression of a gene encoding the protein TST3b, wherein the application is any of the following:

[0007] P1. Application in increasing soluble sugar content in tomatoes;

[0008] P2. Application in improving the sweetness of tomatoes;

[0009] P3, application in improving the taste of tomatoes;

[0010] P4. Application in improving tomato quality;

[0011] P5. Application in increasing single fruit weight of tomatoes;

[0012] P6. Application in increasing tomato yield;

[0013] P7, application in tomato breeding;

[0014] The protein TST3b may be the following protein A1), A2) or A3):

[0015] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0016] A2) a protein derived from A1) or having 80% or more identity with the protein shown in A1) and having the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1);

[0017] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1), A2) or A3).

[0018] In the above application, the protein TST3b can be derived from tomatoes.

[0019] In the above application, the tomato breeding is to select varieties with high soluble sugar content, and / or high sweetness, and / or excellent quality, and / or high single fruit weight, and / or high yield.

[0020] In the above application, SEQ ID No. 2 in the sequence listing consists of 725 amino acid residues.

[0021] The one or more amino acid residues mentioned above may specifically be within ten amino acid residues.

[0022] In the above application, the coding gene of the protein TST3b can be a DNA molecule as shown in a1) or a2) or a3) below:

[0023] a1) The coding sequence is the DNA molecule shown in SEQ ID No.1 in the sequence list;

[0024] a2) a DNA molecule having 90% or more identity with the nucleotide sequence defined in a1) and encoding the protein TST3b described above;

[0025] a3) A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in a1) or a2) and encodes the protein TST3b described above.

[0026] In the above application, the gene encoding the protein TST3b can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0027] In the above application, the substance that reduces the activity or content of protein TST3b can be a substance that knocks out the gene encoding the protein TST3b, and / or a substance that inhibits or reduces the expression of the gene encoding the protein TST3b.

[0028] In the above application, inhibiting or reducing the expression of the gene encoding the protein TST3b can be achieved by gene knockout or gene silencing.

[0029] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0030] The gene silencing refers to the phenomenon of making the gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0031] In the above application, the inhibition or reduction of the expression of the gene encoding the protein TST3b may be an agent that inhibits or reduces the expression of the gene. The agent that inhibits or reduces the expression of the gene may be an agent that knocks out the gene, such as an agent that knocks out the gene by homologous recombination, or an agent that knocks out the gene by CRISPR-Cas9. The agent that inhibits or reduces the expression of the gene may include a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0032] In the above application, the substance that reduces the activity or content of protein TST3b, or the substance that inhibits or reduces the expression of the gene encoding the protein TST3b, can be any of the following substances c1)-c4):

[0033] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TST3b;

[0034] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0035] c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0036] c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

[0037] The nucleic acid molecule described in c1) may be an sgRNA that targets the protein TST3b encoding gene and / or protein B encoding gene described above, or a DNA molecule that expresses the sgRNA.

[0038] The sgRNAs are sgRNA named sgRNA1 and sgRNA named sgRNA2, the target sequence of sgRNA1 is positions 59-77 of SEQ ID No.1, and the target sequence of sgRNA2 is positions 117-135 of SEQ ID No.1.

[0039] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The identity of 90% or more can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0040] In order to solve the above technical problems, the present invention also provides a method for increasing the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes, comprising increasing the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes by inhibiting or reducing the expression level of the gene encoding the protein TST3b in the tomato genome.

[0041] The above-mentioned inhibition or reduction of the expression level of the gene encoding the protein TST3b in the tomato genome can be achieved by any method in the prior art, so as to cause deletion mutation, insertion mutation or base change mutation of the gene, thereby achieving reduction or loss of gene function, specifically chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination, etc.

[0042] In the above-mentioned genome fixed-site editing method, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology or clustered regularly interspaced short palindromic repeats and their related systems (Clustered regularly interspaced short palindromic repeats / CRISPR associated, CRISPR / Cas9 system) technology, and other technologies that can achieve genome fixed-site editing can be used. Regardless of which method is adopted, the entire coding gene of the protein described above can be used as a target, and each element that regulates the expression of the protein coding gene described above can be used as a target, as long as the gene function loss or reduction can be achieved. For example, the exon or 5'UTR of the coding gene of the protein described above can be used as a target.

[0043] The method described above may include introducing into the tomato a substance that reduces or inhibits the activity of the protein TST3b described above, or introducing a substance that reduces or inhibits the expression of the gene encoding the protein TST3b. The substance that reduces or inhibits the activity of the protein TST3b described above, or the substance that reduces or inhibits the expression of the gene encoding the protein TST3b may be any of the following substances c1)-c4):

[0044] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TST3b described above;

[0045] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0046] c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0047] c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

[0048] The nucleic acid molecule described in c1) is a sgRNA targeting the gene encoding the protein TST3b described above or a DNA molecule expressing the sgRNA.

[0049] The sgRNAs are sgRNA named sgRNA1 and sgRNA named sgRNA2, the target sequence of sgRNA1 is positions 59-77 of SEQ ID No.1, and the target sequence of sgRNA2 is positions 117-135 of SEQ ID No.1.

[0050] The above-mentioned reduction or inhibition of the expression of the protein TST3b encoding gene is to replace the protein TST3b encoding gene shown in SEQ ID No.1 in the tomato genome with the TST3b-1 gene or the TST3b-2 gene, wherein the TST3b-1 gene is a DNA molecule obtained by deleting the nucleotide G at position 132 of SEQ ID No.1 in the sequence list, while keeping the other nucleotides of SEQ ID No.1 unchanged; the TST3b-2 gene is a DNA molecule obtained by deleting the nucleotide TG at positions 131-132 of SEQ ID No.1 in the sequence list, while keeping the other nucleotides of SEQ ID No.1 unchanged.

[0051] The present invention also protects the above protein TST3b.

[0052] The present invention also protects biological materials related to protein TST3b, wherein the biological materials related to protein TST3b are any one of the following B1) to B5):

[0053] B1) a nucleic acid molecule encoding TST3b;

[0054] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0055] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0056] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0057] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3).

[0058] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0059] In the above biological material, the nucleic acid molecule in B1) is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.1.

[0060] Among them, SEQ ID No.1 in the sequence list consists of 2178 nucleotides and encodes the protein shown in SEQ ID No.2.

[0061] In the above-mentioned biological material, the expression cassette (TST3b gene expression cassette) containing the nucleic acid molecule described in B2) refers to a nucleic acid molecule capable of expressing TST3b in a host cell, and the nucleic acid molecule may include not only a promoter for initiating transcription of the TST3b gene, but also a terminator for terminating transcription of TST3b. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 20071 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminators (see, for example, Odell et al. (1996) EMBO J. 4:3047-3053). 985)Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0062] In the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0063] The present invention discloses a method for knocking out the TST3b gene of tomatoes to improve the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes. Specifically, the TST3b gene in the starting tomatoes is edited using the CRISPR / Cas9 system, and the TST3b gene is mutated to cause the translation protein to terminate prematurely, thereby obtaining transgenic tomatoes and realizing the editing of the TST3b gene in the starting tomatoes. The present invention uses CRISPR / Cas9-mediated gene editing technology to perform site-directed knockout of specific targets on the tomato TST3b gene, and obtains tomato mutant materials with significantly improved soluble sugar content and single fruit weight, providing new materials for tomato variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is the TST3b gene expression pattern in Example 1 of the present invention.

[0065] Figure 2 This is the editing vector of the TST3b gene in Example 1 of the present invention.

[0066] Figure 3 This is the mutation type of the TST3b gene-edited plant in Example 1 of the present invention.

[0067] Figure 4 This is the soluble sugar content phenotype of the fruit of the tst3b-cr mutant in Example 1 of the present invention.

[0068] Figure 5 This is the fruit weight phenotype of the tst3b-cr mutant in Example 1 of the present invention. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0070] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are conventional biochemical reagents and can be obtained from commercial sources unless otherwise specified.

[0071] 1. Vectors and strains

[0072] In the following examples, Agrobacterium tumefaciens EHA105 is a product of Beijing Quanshijin Biotechnology Co., Ltd.

[0073] In the following examples, Agrobacterium AGL1 is a product of Beijing Quanshijin Biotechnology Co., Ltd.

[0074] 2 Plant strains

[0075] In the following examples, wild currant tomato (Solanum pimpinellifolium), referred to as PP, is available to the public at the Tomato Genetics Resource Center of the University of California, Davis (https: / / tgrc.ucdavis.edu / ), with the accession number PI365967.

[0076] The formula of the LB solid medium containing 50 mg / L kanamycin described in the following examples is: 50 mg / L kanamycin, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, and the pH is adjusted to 7.5 with NaOH.

[0077] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0078] The following examples use Excel tables to process data, and the experimental results are expressed as mean ± standard deviation, using Student's t test, P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a very significant difference. The quantitative tests in the following examples, unless otherwise specified, were repeated three times and the results were averaged.

[0079] Example 1: Spatiotemporal expression of TST3b gene in different tissues and organs of tomato

[0080] The nucleotide sequence of the TST3b gene (Solyc03g032040) cDNA from wild currant tomato PP is shown as SEQ ID No.1, and the amino acid sequence of the protein TST3b encoded by it is shown as SEQ ID No.2.

[0081] SEQ ID No.1:

[0082]

[0083] SEQ ID No.2:

[0084] MRGAVLIALAAAIGNMLQGWDNATIAGSVLYIKKEFNLQTQPTMEGLIVAMSLIGATVITTFSGPVSDMLGRRPMLIISSVLYFLSGLVMLWAPNVYVLLLARLLDGFGIGLAVTLVPVYISETAPPEIRGQLNTFPQFTGSLGMFLSYCMVFGMSLTQAPSWRLMLGVLSIPSLAYFFLA LFYLPESPRWLVSKGRMKEAKQVLQRLRGREDVSGEMALLMEGLGVGGEVSIEEYIIGPDNELADNHDEKDQIKLYGAEEGLSWIAKPVTGQSTLGLVSRHGSMANQSMPLMDPLVTLFGSVHEKMPEMGSMRSMLFSNVGSMFNITENQGKTDNWDEESQKDEENHMSDGSGAESDDNLR SPLLSRQGTNAEGNMGPPTSLSMRQGSNFMQANGVGEQASMGIGGGWQLAYRKDEKKEGALKRIYLHEEGGSGSRRGSIISLPGDAHADQAEFIHAAALVSQSVLRAESVLGQQSIEEAIETQSETVTKKSVWKALLEPGVKHALIVGVGLQILQQFSGINGVLYYTPQILEQAGVGVLLS NMGIGSDSASFLISAVTTLLMLPTIGVAMRLMDLAGRRWLLLATLPVLLSSLIVLVLGNVINMGEVMHAVISTASVVVYFCTFVMGFGPIPNILCSEIFPTSVRGICIAICALTFWIGDIIVTYSLPVMLNSIGLGGVFAIYAVVCAVAWVFVFLKVPETKGMPLEVITEFFAVGAKKAATE

[0085] In order to clarify the role of TST3b in the development of tomato fruit, the cDNA of the fruit of wild currant tomato (Solanumpimpinellifolium, referred to as PP) on the day of flowering, 2, 5, 10, 20 days after flowering, and in the green, color change, orange, and red stages was used as a template to detect the expression changes of TST3b using qRT-PCR technology. The primer sequences are as follows:

[0086] TST3b-F: ACAAGCAGGCGTTGGAGTTC (same as the sequence at positions 1602-1621 of SEQ ID No. 1);

[0087] TST3b-R: AGCGGTGGAGATCACAGCAT (reverse complement to the sequence at positions 1832-1851 of SEQ ID No. 1).

[0088] The tomato gene SlUBI3 was selected as the internal reference gene, and the primers used were as follows:

[0089] Actin-F:TCTTCCGACACCATCGACAA;

[0090] Actin-R:AGAACTGCAACACAGTGAGC.

[0091] The results are as follows Figure 1 As shown, the expression level of TST3b gene was higher during fruit development and lower after fruit ripening, indicating that TST3b gene may play a role in fruit development.

[0092] Example 2: CRISPR / Cas9 editing to verify the function of the TST3b gene

[0093] 1. Construction of CRISPR / Cas9 editing vector for TST3b gene

[0094] According to the coding sequence of the TST3b gene (as shown in SEQ ID No. 1), two CRISPR / Cas9 editing target sequences were designed by CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR / ). The specific target sequences were named target 1 and target 2, respectively, as follows:

[0095] Target 1: 5'-GGGACAATGCGACGATAGC-3' (corresponding to positions 59-77 of SEQ ID No. 1);

[0096] Target 2: 5'-AACACAGCCAACCATGGAA-3' (corresponding to positions 117-135 of SEQ ID No. 1).

[0097] The sgRNA targeting target 1 in the CRISPR / Cas9 method is recorded as sgRNA1, and the sgRNA targeting target 2 is recorded as sgRNA2.

[0098] TST3b-CR-F1: ATATATGGTCTCGTTTGGGGACAATGCGACGATAGCGTTTTAGAGCTAGAAATAGC (the sequence indicated in italics is the sequence that binds to target 1);

[0099] TST3b-CR-R1: ATTATTGGTCTCGAAACTTCCATGGTTGGCTGTGTTCTGCACCAGCCGGGAATCGAA (the sequence indicated in italics is the sequence bound by target 2).

[0100] Using the pCBC_DT1T2_SlU6p vector (the vector is recorded in the following literature: Li R, Sun S, Wang HJ, Wang KT, Yu H, Zhou Z, Xin PY, Chu JF, Zhao TM, Wang HZ, Li JY, Cui X. 2020. FIS1 encodes aGA2-oxidase that regulates fruit firmness in tomato. Nature Communications 11.) as a template, PCR amplification was performed with primers TST3b-CR-F1 and TST3b-CR-R1 to obtain the sgRNA1_gRNAscaffold_sgRNA2 fragment, which contains the coding sequences of both sgRNA1 and sgRNA2.

[0101] Then, the sgRNA1_gRNA scaffold_sgRNA2 fragment and pCAMBIA2300_35S_Cas9_SlU6p_sgRNA vector (described in the following literature: Song, J., Zhang, S., Wang, X., Sun, S., Liu, Z., Wang, K., Wan, H., Zhou, G., Li, R., Yu, H., and Cui, X. (2020). Variations in BothFTL1 and SP5G, Two Tomato FT Paralogs, Control Day-Neutral Flowering. Molecular plant.) were digested with BsaI, and finally connected using T4 DNA ligase to obtain a connection product. Cultured in LB solid medium containing kanamycin, colony PCR was used for positive clone screening, sequencing verified the correctness of the sequence, and the plasmid was extracted for standby use, which was the CRISPR / Cas9 vector of the TST3b gene, named TST3b_CRISPR. Figure 2 Shown is the TST3b_CRISPR structure.

[0102] 2. Obtaining CRISPR / Cas9 edited mutants

[0103] The CRISPR vector TST3b_CRISPR successfully constructed in step 1 was transferred into Agrobacterium AGL1, and wild currant tomato (PP) was used as the recipient for Agrobacterium-mediated genetic transformation.

[0104] The 8 regenerated plants were subjected to PCR detection of the Cas 9 gene, and the detection primers were Cas 9-F and Cas9-R:

[0105] Cas 9-F:CACTATCCTTCGCAAGACCC;

[0106] Cas9-R: GAGATTCCCGAACAAGCCG.

[0107] The PCR products were subjected to gel electrophoresis, and the Cas9 gene was detected in 6 plants.

[0108] The editing site of the TST3b gene was amplified by PCR, and the amplification primers were a primer pair consisting of CR-TST3b-F and CR-TST3b-R:

[0109] CR-TST3b-F: AACATGTTGCAAGGATGGG;

[0110] CR-TST3b-R: TGACAAAAACATTCCAAGGGA.

[0111] The PCR products were sequenced and two homozygous mutant plants were finally obtained.

[0112] The T0 homozygous mutant plants were kept as seeds to obtain T1 seeds for sowing, and T1 plants without Cas9 gene (detection primers were Cas 9-F and Cas9-R) were selected for self-pollination to obtain two independent CRISPR strains with different TST3b mutation types, named tst3b-cr1 and tst3b-cr2. The gene editing site sequences are as follows: Figure 3 As shown:

[0113] Compared with wild type (WT), tst3b-cr1( Figure 3In the genome of the human embryonic stem cell line tst3bcr1, the TST3b gene mutated: at target 2 (Target2), the nucleotide G was deleted at position 132 of SEQ ID No.1, with a total deletion of 1 bp, resulting in a frameshift mutation in the TST3b gene and obtaining the TST3b-1 gene, which was unable to encode the protein TST3b with the amino acid sequence of SEQ ID No.2, and ultimately caused the loss of TST3b function, thereby knocking out the TST3b gene.

[0114] Compared with wild type (WT), tst3b-cr2 ( Figure 3 In the genome of the human embryonic stem cell line tst3bcr2, the TST3b gene mutated: at target 2 (Target2), the nucleotide TG was deleted at positions 131-132 of SEQ ID No.1, with a total deletion of 2bp, resulting in a frameshift mutation in the TST3b gene and obtaining the TST3b-2 gene, which was unable to encode the protein TST3b with the amino acid sequence of SEQ ID No.2, and ultimately caused the loss of TST3b function, thereby knocking out the TST3b gene.

[0115] 3. Investigate the soluble sugar content phenotype of CRISPR mutants

[0116] In order to identify the phenotype of CRISPR materials, homozygous mutant CRISPR materials without Cas 9 (tst3b-cr1 and tst3b-cr2) and wild-type PP (WT) were planted in a solar greenhouse with three replicates, and five plants were planted in each replicate for each line.

[0117] The peels of red-ripe fruits from the 2nd to 4th ears were taken respectively, frozen with liquid nitrogen, ground and mixed, and then the soluble sugar content was detected by liquid chromatography-mass spectrometry.

[0118] The results are as follows Figure 4 As shown, compared with the wild-type control, the CRISPR mutant plants (tst3b-cr1 and tst3b-cr2) showed a significant increase in soluble sugar content. This result proves that TST3b is an important regulatory gene for the soluble sugar content of tomato fruit.

[0119] 4. Investigating the fruit weight phenotype of CRISPR mutants

[0120] In order to identify the phenotype of CRISPR materials, homozygous mutant CRISPR materials without Cas 9 (tst3b-cr1 and tst3b-cr2) and wild-type PP (WT) were planted in a solar greenhouse with three replicates, and five plants were planted in each replicate for each line.

[0121] After the fruits are ripe, the total weight and number of fruits in each ear of the 2nd to 4th ears are counted, and the average fruit weight is calculated.

[0122] The results are as follows Figure 5 As shown, compared with the wild-type control, the CRISPR mutant plants (tst3b-cr1 and tst3b-cr2) showed a significant increase in fruit weight. This result proves that after TST3b knockout, the soluble sugar content of tomato fruit was increased while the fruit weight was increased at the same time.

[0123] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A substance for reducing the activity or content of protein TST3b, or a substance for inhibiting or reducing the expression of a gene encoding the protein TST3b, characterized in that: The application is any of the following: P1. Application in increasing soluble sugar content in tomatoes; P2. Application in improving the sweetness of tomatoes; P3, application in improving the taste of tomatoes; P4. Application in improving tomato quality; P5. Application in increasing single fruit weight of tomatoes; P6. Application in increasing tomato yield; P7, application in tomato breeding; The protein TST3b may be the following protein A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 2; A2) a protein derived from A1) or having 80% or more identity with the protein shown in A1) and having the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1); A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1), A2) or A3).

2. The use according to claim 1, characterized in that: The protein TST3b is derived from tomato.

3. The use according to claim 1 or 2, characterized in that: The coding gene of the protein TST3b is a DNA molecule shown in a1) or a2) or a3) below: a1) The coding sequence is the DNA molecule shown in SEQ ID No.1 in the sequence list; a2) a DNA molecule having 90% or more identity with the nucleotide sequence defined in a1) and encoding the protein TST3b described above; a3) A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in a1) or a2) and encodes the protein TST3b described above.

4. The use according to any one of claims 1 to 3, characterized in that: The substance that reduces the activity or content of protein TST3b, or the substance that inhibits or reduces the expression of the gene encoding the protein TST3b, is any one of the following substances c1)-c4): c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TST3b; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

5. The use according to any one of claims 1 to 4, characterized in that: The nucleic acid molecule described in c1) is a sgRNA targeting the gene encoding the protein TST3b according to any one of claims 1 to 3 or a DNA molecule expressing the sgRNA.

6. The application according to claim 5, characterized in that: The sgRNAs are sgRNA named sgRNA1 and sgRNA named sgRNA2, the target sequence of sgRNA1 is positions 59-77 of SEQ ID No.1, and the target sequence of sgRNA2 is positions 117-135 of SEQ ID No.

1.

7. A method for increasing the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes, characterized in that: The method comprises increasing the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes by inhibiting or reducing the expression level of the gene encoding the protein TST3b described in any one of claims 1-3 in the tomato genome.

8. The method according to claim 7, characterized in that: The method comprises introducing into the tomato a substance that reduces the activity or content of the protein TST3b according to any one of claims 1 to 3, or introducing a substance that reduces the expression of the gene encoding the protein TST3b according to any one of claims 1 to 3; the substance that reduces the activity or content of the protein TST3b according to any one of claims 1 to 3, or introducing a substance that reduces the expression of the gene encoding the protein TST3b according to any one of claims 1 to 3 is any one of the following substances c1) to c4): c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TST3b; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3).

9. The method according to claim 8, characterized in that: The nucleic acid molecule described in c1) is a sgRNA targeting the gene encoding the protein TST3b according to any one of claims 1 to 3 or a DNA molecule expressing the sgRNA.

10. The method according to claim 7, characterized in that: The reduction or inhibition of the expression of the protein TST3b encoding gene is to replace the protein TST3b encoding gene shown in SEQ ID No.1 in the tomato genome with the TST3b-1 gene or the TST3b-2 gene, wherein the TST3b-1 gene is a DNA molecule obtained by deleting the nucleotide G at position 132 of SEQ ID No.1 in the sequence list, while keeping the other nucleotides of SEQ ID No.1 unchanged; the TST3b-2 gene is a DNA molecule obtained by deleting the nucleotide TG at positions 131-132 of SEQ ID No.1 in the sequence list, while keeping the other nucleotides of SEQ ID No.1 unchanged.

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