Application of tst3b protein and related biological materials in regulating soluble sugar content of tomato

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of TST3b protein and related biological materials in regulating the soluble sugar content of tomato. BACKGROUND

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

[0003] Tomato fruit is a typical sink organ, and 80% of the soluble sugar in the fruit is produced by photosynthesis and assimilation of source organs such as leaves, and 20% is from photosynthesis of the fruit itself. Sucrose is the main assimilation product of photosynthesis in tomato, and is also the main form of long-distance transport. During the development period of tomato fruit, photosynthetic products in the form of sucrose are transported from mature leaves and other source organs to fruits through phloem, and are unloaded through plasmodesmata and apoplast pathways. MYB transcription factor SlGLK2 regulates the accumulation of chloroplasts and chlorophyll in the part of tomato fruit near the fruit stalk (shoulder), making the shoulder dark green, and increases the photosynthesis of the fruit, increases the starch content in the immature fruit, and further increases the soluble sugar content in the mature fruit. SlLIN5 encodes a cell wall sucrose invertase involved in the apoplast pathway of the sucrose unloading pathway in tomato fruit. A SNP in this gene leads to a non-synonymous substitution of the encoded amino acid, affecting the activity of the enzyme and reducing the sucrose conversion efficiency of tomato fruit, thereby reducing the soluble sugar content of 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 made some progress in the soluble sugar content of tomato fruit, these genes may bring other adverse traits in the improvement of soluble sugar content of tomato fruit, and there is a bottleneck in utilization. Therefore, mining and cloning the genes regulating the soluble sugar content of tomato fruit and utilizing them are effective ways to solve this problem. SUMMARY

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

[0006] To solve the above technical problems, the present application first provides a substance for reducing the activity or content of a protein TST3b, or a substance for inhibiting or reducing the expression of a gene encoding the protein TST3b, and the substance is any one of the following:

[0007] P1, application in increasing the soluble sugar content of tomatoes;

[0008] P2, application in increasing the sweetness of tomatoes;

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

[0010] P4, application in increasing the quality of tomatoes;

[0011] P5, application in increasing the single fruit weight of tomatoes;

[0012] P6, application in increasing the yield of tomatoes;

[0013] P7, application in tomato breeding;

[0014] The protein TST3b can be a protein of A1), A2) or A3) as follows:

[0015] A1) a protein with an amino acid sequence of SEQ ID No. 2;

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

[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 breeding a variety with high soluble sugar content, and / or high sweetness, and / or high 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 described above can be specifically ten or less amino acid residues.

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

[0023] a1) the coding sequence is a DNA molecule as depicted in SEQ ID No. 1 of the sequence listing;

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

[0025] a3) a DNA molecule hybridizing under stringent conditions to the nucleotide sequence as defined in a1) or a2) and encoding the protein TST3b as described above.

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

[0027] In the above use, the substance decreasing the activity or the amount of the protein TST3b can be a substance knocking out the gene encoding the protein TST3b and / or a substance inhibiting or decreasing the expression of the gene encoding the protein TST3b.

[0028] In the above use, the inhibition or decrease of the expression of the gene encoding the protein TST3b can be achieved by gene knockout or by gene silencing.

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

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

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

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

[0033] c1) Nucleic acid molecules that inhibit or reduce the expression of the TST3b gene encoding the protein;

[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) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

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

[0038] The sgRNAs are sgRNA1 and sgRNA2, with the target sequence of sgRNA1 being positions 59-77 of SEQ ID No. 1 and the target sequence of sgRNA2 being 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 visually or using 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. Having 90% or more identity can mean at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0040] To address the aforementioned technical problems, the present invention also provides a method for improving the soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield of tomatoes, comprising improving 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 induce deletion mutations, insertion mutations or base change mutations in the gene, thereby reducing or losing gene function. Specifically, this can be achieved by chemical mutagenesis, physical mutagenesis, RNAi, site-directed genome editing or homologous recombination, etc.

[0042] Among the aforementioned site-specific genome editing methods, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas9 system) technology, and other technologies capable of site-specific genome editing can be employed. Regardless of the method used, the entire coding gene of the aforementioned proteins can be targeted, or individual elements regulating the expression of the coding gene can be targeted, as long as gene function loss or reduction is achieved. For example, exons or 5' UTRs of the coding genes of the aforementioned proteins can be used as targets.

[0043] The method described above may include introducing a substance into the tomato 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 one of the following c1)-c4):

[0044] c1) Nucleic acid molecules that inhibit or reduce the expression of the TST3b protein encoding gene 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) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

[0048] c1) The nucleic acid molecule mentioned above is an sgRNA that targets the TST3b protein encoding gene or a DNA molecule that expresses the sgRNA.

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

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

[0051] This invention also protects the aforementioned protein TST3b.

[0052] This invention also protects biological materials related to the protein TST3b, said biological material being any one of B1) to B5) below:

[0053] B1) Nucleic acid molecules 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) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms 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 can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

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

[0060] Of these, SEQ ID No. 1 in the sequence listing consists of 2178 nucleotides and encodes the protein shown in SEQ ID No. 2.

[0061] In the aforementioned biological materials, 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 host cells. This nucleic acid molecule may include not only a promoter to initiate TST3b gene transcription but also a terminator to terminate TST3b transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this 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: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120:979-992); chemically induced promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 20071)). 0099169.7), seed storage protein-specific promoters (e.g., promoters of bean globular protein, 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 full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator (see, e.g., Odell et al. (I 985Nature 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 aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0063] This invention discloses a method for knocking out the TST3b gene in tomatoes to improve soluble sugar content, and / or sweetness, and / or quality, and / or single fruit weight, and / or yield. Specifically, the TST3b gene in starting tomatoes is edited using the CRISPR / Cas9 system, causing a mutation that leads to premature termination of translation, resulting in transgenic tomatoes and achieving TST3b gene editing in starting tomatoes. This invention utilizes CRISPR / Cas9-mediated gene editing technology to perform site-specific knockout of the tomato TST3b gene, obtaining tomato mutant materials with significantly increased soluble sugar content and single fruit weight, providing new materials for tomato variety breeding. Attached Figure Description

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

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

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

[0067] Figure 4 This is the phenotype of soluble sugar content in 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 Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are conventional biochemical reagents and are commercially available.

[0071] 1. Vector and strain

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

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

[0074] 2 plant strains

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

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

[0077] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing 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 spreadsheets to process the data. Experimental results are expressed as mean ± standard deviation. Student's t-test was used, with P < 0.05 (*) indicating a significant difference and P < 0.01 (**) indicating a highly significant difference. Unless otherwise specified, all quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0079] Example 1: Spatiotemporal expression of the 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 in SEQ ID No. 1, and the amino acid sequence of the protein TST3b encoded by it is shown in SEQ ID No. 2.

[0081] SEQ ID No. 1:

[0082]

[0083] SEQ ID No. 2:

[0084] MRGAVLIALAAAIGNMLQGWDNATIAGSVLYIKKEFNLQTQPTMEGLIVAMSLIGATVITTFSGPVSDMLGRRPMLIISSVLYFLSGLVMLWAPNVYVLLLARLLDGFGIGLAVTLVPVYISETAPPEIRGQLNTFPQFTGSLGMFLSYCMVFGMSLTQAPSWRLMLGVLSIPSLAYFFLA LFYLPESPRWLVSKGRMKEAKQVLQRLRGREDVSGEMALLMEGLGVGGEVSIEEYIIGPDNELADNHDEKDQIKLYGAEEGLSWIAKPVTGQSTLGLVSRHGSMANQSMPLMDPLVTLFGSVHEKMPEMGSMRSMLFSNVGSMFNITENQGKTDNWDEESQKDEENHMSDGSGAESDDNLR SPLLSRQGTNAEGNMGPPTSLSMRQGSNFMQANGVGEQASMGIGGGWQLAYRKDEKKEGALKRIYLHEEGGSGSRRGSIISLPGDAHADQAEFIHAAALVSQSVLRAESVLGQQSIEEAIETQSETVTKKSVWKALLEPGVKHALIVGVGLQILQQFSGINGVLYYTPQILEQAGVGVLLS NMGIGSDSASFLISAVTTLLMLPTIGVAMRLMDLAGRRWLLLATLPVLLSSLIVLVLGNVINMGEVMHAVISTASVVVYFCTFVMGFGPIPNILCSEIFPTSVRGICIAICALTFWIGDIIVTYSLPVMLNSIGLGGVFAIYAVVCAVAWVFVFLKVPETKGMPLEVITEFFAVGAKKAATE

[0085] To clarify the role of TST3b in tomato fruit development, cDNA samples from wild currant tomato (Solanumpimpinellifolium, abbreviated as PP) were collected on the day of flowering, and at 2, 5, 10, and 20 days after flowering, as well as at the green-ripe, veraison, orange-ripe, and red-ripe stages. The expression levels of TST3b were detected using qRT-PCR. Primer sequences are as follows:

[0086] TST3b-F: ACAGAGCAGGCGTTGGAGTTC (identical to the sequence of positions 1602-1621 of SEQ ID No. 1);

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

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

[0089] Actin-F:TCTTCCGACACCATCGACAA;

[0090] Actin-R:AGAACTGCAACACAGTGAGC.

[0091] The results are as follows Figure 1 As shown, the expression level of the TST3b gene is high during fruit development and low after fruit ripening, indicating that the 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. Constructing a CRISPR / Cas9 editing vector for the TST3b gene

[0094] Based on the coding sequence of the TST3b gene (as shown in SEQ ID No. 1), two CRISPR / Cas9 editing target sequences were designed using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR / ). These target sequences are specifically named Target 1 and Target 2, and are as follows:

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

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

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

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

[0099] TST3b-CR-R1: ATTATTGGTCTCGAAACTTCCATGGTTGGCTGTGTTCTGCACCAGCCGGGAATCGAA (The sequence indicated by italics is the sequence that binds to target 2).

[0100] Using the pCBC_DT1T2_SlU6p vector (described 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 using primers TST3b-CR-F1 and TST3b-CR-R1 to obtain the sgRNA1_gRNAscaffold_sgRNA2 fragment, which contains the coding sequences for both sgRNA1 and sgRNA2.

[0101] Subsequently, the sgRNA1_gRNA scaffold_sgRNA2 fragment and the 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 Both FTL1 and SP5G, Two Tomato FT Paralogs, Control Day-Neutral Flowering. Molecular plant.) were digested with BsaI. Finally, the ligation was performed using T4 DNA ligase to obtain the ligation product. The ligation was carried out in LB solid medium containing kanamycin, and positive clones were screened by colony PCR. Sequencing was used to verify the sequence correctness, and the plasmid was extracted for later use. This resulted in the CRISPR / Cas9 vector of the TST3b gene, named TST3b_CRISPR. Figure 2 The TST3b_CRISPR structure is shown.

[0102] 2. Obtaining CRISPR / Cas9 editing mutants

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

[0104] The Cas9 gene in the eight regenerated plants was detected by PCR using Cas9-F and Cas9-R primers.

[0105] Cas 9-F:CACTATCCTTCGCAAGACCC;

[0106] Cas9-R: GAGATTCCCGAACAAGCCG.

[0107] Gel electrophoresis of the PCR products revealed the Cas9 gene in 6 plants.

[0108] PCR amplification was performed on the editing site of the TST3b gene using 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 generation homozygous mutant plants were used as seed stock to obtain T1 generation seeds. T1 generation plants lacking the Cas9 gene (detection primers were Cas9-F and Cas9-R) were selected and self-crossed to obtain two independent CRISPR lines with different TST3b mutation types, named tst3b-cr1 and tst3b-cr2, respectively. Their gene editing site sequences are as follows: Figure 3 As shown:

[0113] Compared to wild type (WT), tst3b-cr1 ( Figure 3In the genome labeled tst3bcr1, the TST3b gene underwent a mutation: at target 2, nucleotide G was deleted at position 132 of SEQ ID No. 1, a total deletion of 1 bp, resulting in a frameshift mutation of the TST3b gene, resulting in the TST3b-1 gene, which cannot encode the protein TST3b with the amino acid sequence of SEQ ID No. 2, ultimately leading to the loss of TST3b function, thus knocking out the TST3b gene.

[0114] Compared to wild type (WT), tst3b-cr2 ( Figure 3 In the genome labeled tst3bcr2, the TST3b gene underwent a mutation: at target 2, nucleotide TG was deleted at positions 131-132 of SEQ ID No. 1, a total deletion of 2 bp, resulting in a frameshift mutation of the TST3b gene, resulting in the TST3b-2 gene, which cannot encode the protein TST3b with the amino acid sequence of SEQ ID No. 2, ultimately leading to the loss of TST3b function, thus knocking out the TST3b gene.

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

[0116] To identify the phenotype of CRISPR materials, homozygous mutant CRISPR materials without Cas9 (tst3b-cr1 and tst3b-cr2) and wild-type PP (WT) were planted in a greenhouse with three replicates and five plants per replicate for each line.

[0117] The pericarps of the 2nd to 4th ripe red fruits were taken separately, frozen and ground in liquid nitrogen, mixed, and 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) both exhibited a significant increase in soluble sugar content. This result demonstrates that TST3b is an important regulatory gene for soluble sugar content in tomato fruit.

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

[0120] To identify the phenotype of CRISPR materials, homozygous mutant CRISPR materials without Cas9 (tst3b-cr1 and tst3b-cr2) and wild-type PP (WT) were planted in a greenhouse with three replicates and five plants per replicate for each line.

[0121] After the fruit ripens to red, the total weight and number of fruits in each of the 2nd to 4th clusters 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) exhibited a significant increase in fruit weight. This result demonstrates that TST3b knockout increases both the soluble sugar content and fruit weight in tomato fruits.

[0123] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of a substance that knocks out the gene encoding the protein TST3b, characterized in that: The application is any one of the following: P1. Application in increasing the soluble sugar content of tomatoes; P2. Application in increasing the weight of a single tomato fruit; The protein TST3b is a protein with the amino acid sequence SEQ ID No.

2.

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

3. The application according to claim 1, characterized in that: The gene encoding the protein TST3b is a DNA molecule whose coding sequence is shown in SEQ ID No. 1 of the sequence listing.

4. The application according to any one of claims 1-3, characterized in that: The substance that knocks out the gene encoding the protein TST3b is any one of the following substances c1)-c4): c1) Knock out the nucleic acid molecule encoding the TST3b protein gene; 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) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

5. The application according to claim 4, characterized in that: c1) The nucleic acid molecule is an sgRNA that targets the TST3b encoding gene of any one of the proteins described in claims 1-3 or a DNA molecule that expresses the sgRNA.

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

1.

7. A method for increasing the soluble sugar content and / or single fruit weight of tomatoes, characterized in that, The soluble sugar content and / or single fruit weight of tomatoes can be increased by knocking out 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 includes introducing a substance into the tomato that knocks out the gene encoding the protein TST3b of any one of claims 1-3; the substance that knocks out the gene encoding the protein TST3b of any one of claims 1-3 is any one of the following substances c1)-c4): c1) Knock out the nucleic acid molecule encoding the TST3b protein gene; 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) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

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

10. The method according to claim 7, characterized in that: The method of knocking out the coding gene of the protein TST3b according to any one of claims 1-3 in the tomato genome is to replace the coding gene of the protein TST3b shown in SEQ ID No. 1 in the tomato genome with TST3b-1 Gene or TST3b-2 Genes, the ones mentioned TST3b-1 The gene is a DNA molecule obtained by deleting nucleotide G at position 132 of SEQ ID No. 1, while keeping the other nucleotides of SEQ ID No. 1 unchanged; TST3b-2 A gene is a DNA molecule obtained by deleting nucleotide TG from positions 131-132 of SEQ ID No. 1 while keeping the other nucleotides of SEQ ID No. 1 unchanged.

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