Method for increasing the parthenocarpy rate and yield of tomatoes and the biological material of the protein tsp4.2 used therefor

Abstract

The application discloses a method for improving the parthenocarpy rate and / or yield of tomatoes, comprising improving the parthenocarpy rate and / or yield of tomatoes by inhibiting or reducing the expression amount of a gene encoding a TSP4.2 protein in the genome of tomatoes. The TSP4.2 protein can be specifically the 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 obtained by substitution, deletion and / or addition of one or more amino acid residues in the protein of A1), the protein having more than 90% identity with the protein shown in A1) and having the same activity; and A3) a fusion protein obtained by connecting a protein tag to the N terminal and / or C terminal of the protein of A1) or A2). The TSP4.2 protein and related biological materials can be used for regulating the parthenocarpy rate of tomatoes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for improving the parthenocarpy rate and / or yield of tomatoes, and related biomaterials containing the protein TSP4.2 used therein. Background Technology

[0002] Self-pollinating tomatoes (Solanum lycopersicum L.) rely on wind and insects for pollination. Only after fertilization can the tomato fruit grow and develop normally. In greenhouse cultivation, tomatoes often suffer from poor pollination, leading to decreased fruit set and reduced yield. Plant growth regulators are frequently used to improve fruit set. However, the use of plant growth regulators not only imposes additional economic and labor costs on farmers, but also increases the rate of deformed fruit under high-temperature conditions.

[0003] Parthenocarpy is a desirable trait that increases yield under adverse conditions and is one of the important breeding goals for tomatoes. Parthenocarpy is a phenomenon in which fruits can still expand normally without fertilization, eliminating the need for flower dipping with plant growth regulators, and offering advantages such as stable yield and savings in labor and resources. In addition, parthenocarpy fruits also have many advantages such as superior quality, better taste, lower rate of deformed fruits, better marketability, and better storage resistance.

[0004] Studies have shown that regulating auxin synthesis, transport, and signaling can lead to parthenocarpy in tomatoes. The differentially expressed gene *Aucsia* in the flower buds of parthenocarpy tomatoes (DefH9-iaaM) is involved in regulating auxin synthesis. This gene inhibits auxin synthesis, but RNAi promotes it, leading to parthenocarpy (Molesini et al. 2009a). The auxin efflux transporter *SlPIN* can transport auxin from the ovary to the outside; therefore, silencing *SlPIN* increases auxin levels in the ovary, resulting in parthenocarpy (Mounet et al. 2012). *TIR1* and its homologs act as auxin receptors; overexpression of *TIR1* alters the transcriptional levels of some auxin response factors, leading to parthenocarpy (Ren et al. 2011).

[0005] The auxin-responsive factor SlARF5 may play an important role in regulating auxin and gibberellin signaling pathways during fruit development; RNAi lines can induce parthenocarpy in tomatoes (Liu, et al. 2018). The complex formed by SlARF8 and SlIAA9 inhibits the transcription of genes controlling fruit development; silencing this complex leads to parthenocarpy in tomatoes (Goetz, et al. 2007). SlIAA9 is a major regulator of leaf morphogenesis and fruit development; both RNAi and knockout lines can induce parthenocarpy in tomatoes (Wang, et al. 2005). Inhibition or downregulation of ARFs (auxin-responsive factors) and Aux / IAA (auxin transcription factors) can induce parthenocarpy in tomatoes. Changes in SlARF2 expression in fruits significantly affect abscisic acid, cytokinin, and salicylic acid; silencing this SlARF2 leads to parthenocarpy in tomatoes (Breitel, et al. 2016). SlARF7 regulates the auxin and GA signaling pathways, participating in the formation and development of tomato fruit. RNAi lines produce parthenocarpic tomatoes (de Jong, et al. 2011).

[0006] Although recent years have seen some progress in research on tomato parthenocarpy, with the identification of some QTL loci, the number of cloned genes is very limited, which restricts its application in breeding practices to some extent. Analyzing the genetic basis and regulatory network of tomato parthenocarpy at the molecular level is not only of great scientific significance for elucidating the biological mechanisms of tomato parthenocarpy, but also of great practical significance for improving tomato breeding standards in my country and accelerating the cultivation of parthenocarpy quality tomatoes. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to improve the parthenocarpy ability of tomatoes in order to increase the yield of tomatoes in greenhouse cultivation.

[0008] To address the aforementioned technical problems, the present invention first provides a method for improving the parthenocarpy rate and / or yield of tomatoes, including improving the parthenocarpy rate and / or yield of tomatoes by inhibiting or reducing the expression level of the gene encoding the protein TSP4.2 in the tomato genome;

[0009] The protein TSP4.2 is a protein that is either A1), A2), or A3 as follows:

[0010] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0011] A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1).

[0012] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0013] The above-mentioned inhibition or reduction of the expression level of the gene encoding the protein TSP4.2 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.

[0014] 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.

[0015] In the method described above, inhibiting or reducing the expression level of the gene encoding the protein TSP4.2 in the tomato genome involves introducing any one of the following substances (c1)-c4) into the tomato:

[0016] c1) Inhibit or reduce the expression of the nucleic acid molecules encoding the TSP4.2 protein gene mentioned above;

[0017] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0018] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0019] 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).

[0020] c1) The nucleic acid molecule mentioned above is an sgRNA that targets the gene encoding the protein TSP4.2 described above, or a DNA molecule that expresses the sgRNA.

[0021] The sgRNAs are sgRNA1 and sgRNA2, with the target sequence of sgRNA1 being positions 66-84 of SEQ ID No. 3 and the target sequence of sgRNA2 being positions 70-88 of SEQ ID No. 3.

[0022] In the method described above, the inhibition or reduction of the expression level of the gene encoding the protein TSP4.2 in the tomato genome is achieved by performing at least one of the following mutations on the gene encoding the protein TSP4.2 shown in SEQ ID No. 1 in the tomato genome:

[0023] 1) Nucleotides 77-81 of the coding sequence of the TSP4.2 gene in tomato genomic DNA are deleted;

[0024] 2) Nucleotides 74-197 of the coding sequence of the TSP4.2 gene in tomato genomic DNA are deleted.

[0025] To address the aforementioned technical problems, the present invention also provides a protein TSP4.2, which is a protein of the following type: A1), A2), or A3):

[0026] A1) The amino acid sequence of this protein is SEQ ID No. 2;

[0027] A2) A protein derived from A1) or having more than 80% identity with and the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1).

[0028] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0029] Of the proteins mentioned above, TSP4.2 can be derived from tomatoes.

[0030] Of the proteins mentioned above, SEQ ID No. 2 in the sequence listing consists of 654 amino acid residues.

[0031] The term "more than one amino acid residue" as mentioned above can specifically refer to up to ten amino acid residues.

[0032] In the above applications, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identity.

[0033] The present invention also protects biological materials related to the protein TSP4.2, wherein the biological material related to the protein TSP4.2 is any one of B1) to B5) below:

[0034] B1) Nucleic acid molecules encoding TSP4.2;

[0035] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0036] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0037] 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);

[0038] 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).

[0039] 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.

[0040] 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 the nucleotide of SEQ ID No. 1.

[0041] Of these, SEQ ID No. 1 in the sequence listing consists of 1965 nucleotides and encodes the protein shown in SEQ ID No. 2.

[0042] In the aforementioned biological materials, the expression cassette (TSP4.2 gene expression cassette) containing the nucleic acid molecule described in B2) refers to a nucleic acid molecule capable of expressing TSP4.2 in host cells. This nucleic acid molecule may include not only a promoter to initiate TSP4.2 gene transcription but also a terminator to terminate TSP4.2 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 inducible 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 can be 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.

[0043] In the aforementioned biological materials, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0044] The present invention also provides the use of substances that reduce the activity or content of the protein TSP4.2, or substances that inhibit or reduce the expression of the gene encoding the protein TSP4.2, wherein the use is any one of the following:

[0045] P1. Application in improving the parthenocarpy ability of tomatoes or in the preparation of products that improve the parthenocarpy ability of tomatoes;

[0046] P2. Application in increasing tomato yield or in the preparation of products that increase tomato yield;

[0047] P3. Application in tomato breeding.

[0048] In the above applications, the tomato breeding involves selecting varieties with high parthenocarpy rates.

[0049] In the above application, SEQ ID No. 2 in the sequence listing consists of 349 amino acid residues.

[0050] In the above applications, the gene encoding the protein TSP4.2 can be a DNA molecule as shown in a1), a2), or a3) below:

[0051] a1) The coding sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0052] a2) has 90% or more identity with the nucleotide sequence defined in a1) and encodes a DNA molecule that encodes the protein TSP4.2 described above;

[0053] a3) hybridizes under strict conditions to the nucleotide sequence defined by a1) or a2) and encodes a DNA molecule that encodes the protein TSP4.2 described above.

[0054] In the above applications, the gene encoding the protein TSP4.2 can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0055] In the above applications, the substance that reduces the activity or content of the protein TSP4.2 may be a substance that knocks out the coding gene of the protein TSP4.2, and / or a substance that inhibits or reduces the expression of the coding gene of the protein TSP4.2.

[0056] In the above applications, inhibiting or reducing the expression of the gene encoding the protein TSP4.2 can be achieved by gene knockout or gene silencing.

[0057] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0058] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0059] In the above applications, the inhibition or reduction of the expression of the gene encoding the protein TSP4.2 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.

[0060] In the above applications, the substance that reduces the activity or content of the protein TSP4.2, or the substance that inhibits or reduces the expression of the gene encoding the protein TSP4.2, can be any one of the following c1)-c4):

[0061] c1) Nucleic acid molecules that inhibit or reduce the expression of the TSP4.2 protein encoding gene;

[0062] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0063] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0064] 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).

[0065] c1) The nucleic acid molecule may be an sgRNA that expresses the gene encoding the protein TSP4.2 described above, or a DNA molecule that expresses the sgRNA.

[0066] The sgRNAs are sgRNA1 and sgRNA2, with the target sequence of sgRNA1 being positions 66-84 of SEQ ID No. 1 and the target sequence of sgRNA2 being positions 70-88 of SEQ ID No. 1.

[0067] The yield mentioned above refers to the yield under greenhouse cultivation.

[0068] This invention discloses a method for knocking out the TSP4.2 gene in tomatoes to improve the parthenocarpic rate. Specifically, the TSP4.2 gene in the starting tomato is edited using the CRISPR / Cas9 system, causing a mutation in the TSP4.2 gene that leads to premature termination of translation, resulting in transgenic tomatoes and achieving TSP4.2 gene editing in the starting tomato. This invention utilizes CRISPR / Cas9-mediated gene editing technology to perform site-specific knockout of the tomato TSP4.2 gene, obtaining tomato mutant materials with high parthenocarpic rate, providing new materials for tomato variety breeding. Attached Figure Description

[0069] Figure 1 This is the expression pattern of the TSP4.2 gene in Example 1 of the present invention.

[0070] Figure 2 This describes the subcellular localization of the TSP4.2 gene in Example 1 of this invention.

[0071] Figure 3This is the editing vector for the TSP4.2 gene in Example 1 of this invention.

[0072] Figure 4 This refers to the mutation type of the TSP4.2 gene-edited plant in Example 1 of this invention.

[0073] Figure 5 The image shows the fruit phenotype of the TSP4.2 gene mutant in Example 1 of this invention. ** indicates that the significance analysis result is P<0.01. Detailed Implementation

[0074] 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.

[0075] 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.

[0076] 1. Vector and strain

[0077] In the following examples, the vector pCAMBIA2300_YFP_HA is a product of Beijing Huayueyang Biotechnology Co., Ltd.

[0078] In the following examples, Agrobacterium tumefaciens EHA105 is a product of Beijing TransGen Biotech Co., Ltd.

[0079] In the following examples, Agrobacterium AGL1 is a product of Beijing TransGen Biotechnology Co., Ltd.

[0080] 2 plant strains

[0081] In the following examples, the tomato variety 'Money Maker' is a product of the Tomato Genetics Resource Center (https: / / tgrc.ucdavis.edu / ) at the University of California, Davis. Its Accession number is LA2706.

[0082] The public can obtain the materials from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to replicate the experiments described in this application.

[0083] The LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin in the following examples is an LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin. The formulation of the LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin is as follows: rifampicin 50 mg / L, 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.

[0084] 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.

[0085] Example 1: Spatiotemporal expression of the TSP4.2 gene in different tissues and organs of tomato

[0086] The nucleotide sequence of the tomato TSP4.2 gene (Solyc04g077490) cDNA is shown in SEQ ID No. 1, and the amino acid sequence of the protein TSP4.2 encoded by it is shown in SEQ ID No. 2.

[0087] SEQ ID No. 1:

[0088] ATGAAGATGAAGTCTATGAATGATGATAGCAGAAGTAGCAACAATAATATCAG

[0089] TAATAATAACAGTGCAGCAGCTAATACTAACTGGTTGGGTTTTCTCTCACACC

[0090] CCACATGAAAATGGAGGTTACTAATTCTTCTGATACTCAACATCAACATCAATT

[0091] TGCACAAAGTTTCTATCTTTCATCTTCTCCTCCTCCCCCAATGAATGTATCAACA

[0092] ACCTCTGCTCTCTGTTATGAAAATAACCCTTTTCATTCAACCTTATCTGTCATGC

[0093] CTTTGAAATCTGATGTGTTCTCTTTGCATTATGGAAGCTCTATCAAGATCTCATG

[0094] CTGATGCTATGGTGCAAAGTTCTTCACCTAAGCTTGAGGATTTTTTAGGGGGTG

[0095] CAAGTATGGGGAATAGTCAATATGGAAGTCATGATAGGGAGGCTATGGCTTTG

[0096] AGCTTAGATAGTTTGTATTATCATCAAAATGAAGAGGAGATTCAAGTTCATAG

[0097] TCATCATCCTTACTATTCTCCCATGCATTGTCATGGTATGTATCATCAAGAAAC

[0098] ACTATTGGAAGAAACTAAACCAACTCAAATTTCAAACTGTGAAGCTCAAATGA

[0099] CTGGGAATGAGCTGAAAAGCTGGGGTCAGTATGCTGAACAGCATGCTATTGAC

[0100] CAACACATAAATGCCACCTGTAGTATGGTTGCTGCTGCTGCAGCAGCAGCATC

[0101] TGGTGGTGGTGGTGGTACCTCTGGTTGTAATGAGTTGCAGTCTTTAAGTTTGTC

[0102] TATGAATCCTGGTTCTCAGTCCAGCTGTGTCACTCCAAGACAAATTTCTCCTAG

[0103] TGGACTGGAATGTGTAGCTGTAGAATCAAAAAAGAGGGCCTCTGGTAAAGTTG

[0104] CTCAAAAACAACCTGTCCACAGGAAATCCATTGACACATTTGGTCAGAGAACT

[0105] TCTCAGTATAGAGGTGTCACTAGACATAGGTGGACAGGTAGGTATGAAGCACA

[0106] TTTATGGGATAATAGTTGCAAGAAGGAAGGGCAAACCAGGAAAGGGAGACAA

[0107] GTTTATCTTGGTGGCTATGATATGGAAGAAAAAGCTGCAAGAGCTTATGATCT

[0108] AGCTGCACTAAAATATTGGGGTCCTTCAACTCACATCAATTTCCCATTAGAAAA

[0109] TTATCAGAAAGAACTTGATGACATGAAGAATATGACCAGGCAAGAATATGTTG

[0110] CACATTTAAGAAGGAAAAGTAGTGGATTCTCAAGAGGTGCTTCTATTTACAGA

[0111] GGAGTGACAAGGCACCATCAACATGGAAGATGGCAGGCCAGAATTGGAAGAG

[0112] TTGCTGGGAACAAAGATCTTTATCTTGGCACTTTTAGCACACAAGAAGAAGCA

[0113] GCAGAGGCTTATGATGTTGCTGCAATCAAATTCAGGGGTGTAAATGCTGTTAC

[0114] AAACTTTGACATATCGCGATACGACGTTGAAAAGATCATGGCTAGTAATACTC

[0115] TGCCTGCTGGTGAATTAGCTAAGAGAACTAAAGAAAGAGAGTCAATTGAATAC

[0116] AACAATAACACAGGAGGTGTTGGTGGTCAAAAGAATGAGGAATGTGTTGACA

[0117] ACAACAACAATGGGACTATCACAGATTGGAAAATGGTGTTATATCAAACATCC

[0118] AATCCTTCACTAGGATCGAGTTATCGTAACCCTACATCATTCTCCATGGCATTA

[0119] CAAGATTTGATTGGCATCGATTCGATGACTAATTCAAATAACCATCATCATGCC

[0120] ACAATTCTTGATCATGAACAGAACAAGATTGGTAACCCTTTTTCAAATGCTTCA

[0121] TCTTTGGTAACTAGTCTTGGTAGTTCAAGAGAAGCAAGTCCTGATAAAAGTGCT

[0122] ACTGCTGCCTCATTAGTCTTTGCTAAGCCTACGAAATTCGCCGTCCCAACGGCT

[0123] ACTAGTGTCAATGCTTGTATTCCCTCAGCCCAATTAAGGCCAATTCCAGTCTCC

[0124] ATGGCTCACCTACCAGTCTTTGCAGCTTTGAATGATGCATGA

[0125] SEQ ID No.2:

[0126] MKMKSMNDDSRSSNNNISNNNSAAANTNWLGFSLTPHMKMEVTNSSDTQHQHQ

[0127] FAQSFYLSSSPPPPMNVSTTSALCYENNPFHSTLSVMPLKSDGSLCIMEALSRSHAD

[0128] AMVQSSSPKLEDFLGGASMGNSQYGSHDREAMALSLDSLYYHQNEEEIQVHSHHP

[0129] YYSPMHCHGMYHQETLLEETKPTQISNCEAQMTGNELKSWGQYAEQHAIDQHIN

[0130] ATCSMVAAAAAAASGGGGGTSGCNELQSLSLSMNPGSQSSCVTPRQISPSGLECV

[0131] AVESKKRASGKVAQKQPVHRKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSC

[0132] KKEGQTRKGRQVYLGGYDMEEKAARAYDLAALKYWGPSTHINFPLENYQKELD

[0133] DMKNMTRQEYVAHLRRKSSGFSRGASIYRGVTRHHQHGRWQARIGRVAGNKDL

[0134] YLGTFSTQEEAAEAYDVAAIKFRGVNAVTNFDISRYDVEKIMASNTLPAGELAKRT

[0135] KERESIEYNNNTGGVGGQKNEECVDNNNNGTITDWKMVLYQTSNPSLGSSYRNPT

[0136] SFSMALQDLIGIDSMTNSNNHHHATILDHEQNKIGNPFSNASSLVTSLGSSREASPD

[0137] KSATAASLVFAKPTKFAVPTATSVNACIPSAQLRPIPVSMAHLPVFAALNDA

[0138] To clarify the role of TSP4.2 in tomato fruit development, cDNA from the roots, stems, leaves, flowers, and fruits at different stages of the tomato variety 'Money Maker' (MM) was used as a template, and qRT-PCR was employed to detect the expression level of TSP4.2. The primer sequences are as follows:

[0139] TSP4.2-F: TGGGGTCCTTCAACTCACATC (identical to the sequence of positions 1087-1107 of SEQ ID No. 1);

[0140] TSP4.2-R: GGTGCCTTGTCACTCCTCTG (inverse complementary to the sequence of SEQ ID No. 1, positions 1227-1246).

[0141] The internal reference gene is UBI, and the primer sequences are as follows:

[0142] UBI-F:TCTTCCGACACCATCGACAA;

[0143] UBI-R: AGAACTGCAACACAGTGAGC.

[0144] The results are as follows Figure 1As shown, the TSP4.2 gene is highly expressed in fruit but lowly expressed in roots, stems, leaves, and flowers. This suggests that the TSP4.2 gene may play a role in tomato parthenocarpy.

[0145] Example 2: Subcellular localization of the TSP4.2 gene

[0146] cDNA was extracted from leaves of the tomato variety 'Money Maker' (MM). Using this as a template, the full-length cDNA of the TSP4.2 gene was amplified by PCR (as shown in SEQ ID No. 1). The primer sequences used are as follows:

[0147] TSP4.2-F1: (The italicized positions 9-38 are identical to the sequence of positions 1-36 of SEQ ID No. 1, and the underlined position indicates the BamH1 enzyme recognition sequence);

[0148] TSP4.2-R1: (The italicized positions 9-38 are inversely complementary to the sequence of positions 1933-1962 of SEQ ID No. 1, and the wavy line indicates the Sal1 enzyme recognition sequence).

[0149] The obtained PCR product was digested with BamH1 and Sal1 enzymes and then ligated to the linearized base plasmid pCAMBIA2300_YFP_HA, which was digested with BamH1 and Sal1 enzymes, using T4 ligase. Specifically, the small fragment between the BamH1 and Sal1 enzyme recognition sites on plasmid pCAMBIA2300_YFP_HA was replaced with the sequence shown in positions 1-1962 of SEQ ID No. 1, while keeping the other sequences of plasmid pCAMBIA2300_YFP_HA unchanged, to obtain the recombinant expression vector expressing TSP4.2, named 35S::TSP4.2-YFP-HA.

[0150] The correctly constructed recombinant expression vector 35S::TSP4.2-YFP-HA was transformed into Agrobacterium tumefaciens EHA105. After screening and colony PCR identification using LB solid medium containing 50 mg / L rifampin and 50 mg / L kanamycin, the bacteria were injected into tobacco leaves (Nicotiana benthamiana, from the Li Jiayang research group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). Fluorescence was observed after 36 hours. The results are as follows: Figure 2 As shown, the TSP4.2 gene is expressed in the cell nucleus.

[0151] Example 3: CRISPR / Cas9 editing to verify the function of the TSP4.2 gene

[0152] 1. Constructing a CRISPR / Cas9 editing vector for the TSP4.2 gene.

[0153] 1. Constructing a CRISPR / Cas9 editing vector for the TSP4.1 gene.

[0154] Based on the coding sequence of the TSP4.1 gene (as shown in SEQ ID No. 1), four sgRNAs targeting CRISPR / Cas9 editing sequences were designed using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR / ). The target sequences of the sgRNAs are as follows:

[0155] The target sequence of sgRNA1 is: 5'-TGCAGCAGCTAATACTAAC-3' (corresponding to positions 66-84 of SEQ ID No. 1).

[0156] The target sequence of sgRNA2 is: 5'-GCAGCTAATACTAACTGGT-3' (corresponding to positions 70-88 of SEQ ID No. 1).

[0157] The target sequence of sgRNA3 is: 5'-CTACACATTCCAGTCCACT-3' (corresponding to positions 805-823 of SEQ ID No. 1).

[0158] The target sequence of sgRNA4 is: 5'-CCCCCAATGAATGTATCAAC-3' (corresponding to positions 196-215 of SEQ ID No. 1).

[0159] Design primers to amplify DNA fragments containing sgRNA:

[0160] For sgRNA1, the primer sequence is:

[0161] TSP4.2-CR-F1: 5'-ATATATGGTCTCGTTTGTGCAGCAGCTAATACTAACGTTTTAGAGCTAGAAATAGC-3';

[0162] TSP4.2-CR-R1: 5'-ATTATTGGTCTCGCTGCCAAACTACACTGTTAGATT C-3'.

[0163] For sgRNA2, the primer sequence is:

[0164] TSP4.2-CR-F2: 5'-ATATATGGTCTCGGCAGCTAATACTAACTGGTGTTTTAGAGCTAGAAATAGC-3';

[0165] TSP4.2-CR-R2: 5'-ATTATTGGTCTCGGTAGCAAACTACACTGTTAGATT C-3'.

[0166] For sgRNA3 and sgRNA4, the primer sequences are as follows:

[0167] TSP4.2-CR-F3: 5'-ATATATGGTCTCGCTACACATTCCAGTCCACTGTTTTAGAGCTAGAAATAGC-3';

[0168] TSP4.2-CR-R3: 5'-ATTATTGGTCTCGAAACCCCCCAATGAATGTATCAACAAACTACACTGTTAGATTC-3'.

[0169] Using the pCBC_DT1T2_SlU6p plasmid (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 the above three pairs of primers to obtain three DNA fragments: sgRNA1, sgRNA2, sgRNA3, and sgRNA4. These fragments were then purified by gel extraction.

[0170] Meanwhile, the CRISPR-Cas9 vector (named pTX041, from the Li Chuanyou research group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, described in the literature "Deng L, Wang H, Sun C, Li Q, Jiang H, Du M, Li CB, Li C. Efficient generation of pink-fruited tomatoes using CRISPR / Cas9 system. J Genet Genomics. 2018:45(1):51–54. https: / / doi.org / 10.1016 / j.jgg.2017.10.002") was digested with BsaI (NEB product), and the linearized CRISPR-Cas9 vector pTX041 was purified by gel extraction.

[0171] Then, in a 200 μL RNase-free PCR tube, the following were added sequentially: 10×T4 DNA ligase buffer, 1 μL; 0.1% BSA (Takara), 1 μL; purified linear vector pTX041, 100 ng; three purified DNA fragments (sgRNA1, sgRNA2, sgRNA3, and sgRNA4), 30 ng each; BsaI enzyme (NEB), 0.5 μL; T4 DNA ligase (Promega), 0.5 μL; and ultrapure water to a total volume of 10 μL. The above reaction system was placed in a PCR instrument for ligation, and the following reaction was performed: 37 °C, 5 min; 16 °C, 10 min; the above reaction was repeated 16 times; 50 °C, 5 min; 80 °C, 5 min; and stored at 10 °C. The ligation product was transformed into *E. coli* DH5α competent cells (using the same transformation method as in Example 2), kanamycin-resistant, and positive clones were screened. Sequencing was performed to verify the sequence accuracy (sequencing was performed by BGI Genomics). Plasmids were extracted to obtain a CRISPR / Cas9 vector targeting the TSP4.2 gene, named TSP4.2_CRISPR. The TSP4.2_CRISPR vector expresses sgRNA1, sgRNA2, sgRNA3, and sgRNA4. The structure is shown in [see attached diagram]. Figure 3 .

[0172] 2. Obtaining CRISPR / Cas9 editing mutants

[0173] The CRISPR vector TSP4.2_CRISPR successfully constructed in step 1 was transferred into Agrobacterium AGL1, and Agrobacterium-mediated genetic transformation was carried out using the cultivated tomato variety 'Money Maker' (MM) as the recipient.

[0174] The 13 regenerated plants were used as templates for genomic DNA analysis, and the Cas9 gene was detected by PCR using Cas9-F and Cas9-R primers.

[0175] Cas 9-F:CACTATCCTTCGCAAGACCC;

[0176] Cas9-R: GAGATTCCCGAACAAGCCG.

[0177] Using genomic DNA as a template, the PCR products were detected by gel electrophoresis, and the Cas9 gene was detected in 6 plants.

[0178] PCR amplification was performed on the editing site of the TSP4.2 gene using primers CR-TSP4.2-F and CR-TSP. The primer pair consisted of two R lines:

[0179] CR-TSP4.2-F: AAGTTCTTGATGTGAATCTTG;

[0180] CR-TSP4.2-R: CAGCATGAGATCTTGATAGAG.

[0181] The PCR products were sequenced, and two homozygous mutant plants were finally obtained.

[0182] 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 TSP4.2 mutation types, named tsp4.2cr-1 and tsp4.2cr-2, respectively. Their gene editing site sequences are as follows: Figure 4 As shown:

[0183] With wild-type MM ( Figure 4 Compared to genomic DNA labeled as Reference, tsp4.2cr-1 ( Figure 4 In the two homologous chromosomes labeled tsp4.2cr-1, the gene encoding the protein TSP4.2 underwent the following mutation: at target 1 (corresponding to sgRNA1), nucleotides (ATACT) were deleted at positions 77-81 of SEQ ID No.1, a total deletion of 5 bp, resulting in a frameshift mutation of the TSP4.2 gene, thereby knocking out the TSP4.2 gene.

[0184] With wild-type MM ( Figure 4 Compared to genomic DNA labeled as Reference, tsp4.2cr-2 ( Figure 4 In the two homologous chromosomes labeled tsp4.2cr-2, the gene encoding the protein TSP4.2 underwent the following mutation: between target 1 (corresponding to sgRNA1) and target 4 (corresponding to sgRNA4), a nucleotide deletion of 124 bp was found at positions 74-197 of SEQ ID No. 1, resulting in a frameshift mutation in the TSP4.2 gene, thereby knocking out the TSP4.2 gene.

[0185] 3. Investigate the parthenocarpy phenotype of CRISPR mutants

[0186] To identify the phenotype of CRISPR materials, homozygous mutant CRISPR materials without Cas9 (tsp4.2c-r1 and tsp4.2cr-2) and wild-type (MM) were planted in a greenhouse, with 5 plants per replicate for each line.

[0187] Without artificial pollination or the application of growth hormones, the plants were transplanted in early April and the parthenocarpy rate of 2-4 fruit clusters was counted in mid-June.

[0188] The results are as follows Figure 5 As shown, compared with the wild-type control, the CRISPR mutant plants (tsp4.2cr-1 and tsp4.2cr-2) all exhibited varying degrees of parthenocarpy. This result further demonstrates that TSP4.2 is an important regulatory gene for parthenocarpy in tomatoes.

[0189] 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. A method of increasing the parthenocarpic rate of tomatoes, characterized in that, The application discloses a method for improving parthenocarpy rate of tomato by knocking out a gene encoding a protein TSP4.2 in a genome of the tomato. The protein TSP4.2 is a protein with an amino acid sequence of SEQ ID No.

2.

2. The method of claim 1, wherein: The knocking out of the gene encoding the protein TSP4.2 in the genome of the tomato is achieved by introducing any one of the following c1) to c4) into the tomato: c1) a nucleic acid molecule for knocking out the gene encoding the protein TSP4.2 according to claim 1; c2) an expression cassette containing the nucleic acid molecule according to c1); c3) a recombinant vector containing the nucleic acid molecule according to c1), or a recombinant vector containing the expression cassette according to c2); c4) a recombinant microorganism containing the nucleic acid molecule according to c1), or a recombinant microorganism containing the expression cassette according to c2), or a recombinant microorganism containing the recombinant vector according to c3).

3. The method of claim 2, wherein: The nucleic acid molecule according to c1) is an sgRNA targeting the gene encoding the protein TSP4.2 according to claim 1, or a DNA molecule expressing the sgRNA.

4. The method of claim 1, wherein: The knocking out of the gene encoding the protein TSP4.2 in the genome of the tomato is achieved by introducing any one of the following mutations into the gene encoding the protein TSP4.2 in the genome of the tomato: 1) a deletion of nucleotides 77-81 of the coding sequence of the gene corresponding to TSP4.2 nucleotides 77-81 of the coding sequence of SEQ ID No.

1. 2) the nucleotides 74-197 of the coding sequence of the gene SEQ ID No. 1 are deleted in the genomic DNA of the tomato. TSP4.2 2) the nucleotides 74-197 of the coding sequence of the gene SEQ ID No. 1 are deleted in the genomic DNA of the tomato.

5. Use of a substance which knocks out the gene coding for the protein TSP4.2 as claimed in claim 1, characterized in that: The application is any one of the following: P1, application in improving parthenocarpy rate of tomato, or application in preparing a product for improving parthenocarpy rate of tomato; P2, application in breeding of tomato, wherein the breeding is breeding of a variety with high parthenocarpy rate.

6. Use according to claim 5, characterized in that: The substance for knocking out the gene encoding the protein TSP4.2 according to claim 1 is any one of the following c1) to c4): c1) a nucleic acid molecule for knocking out the gene encoding the protein TSP4.2 according to claim 1; c2) an expression cassette containing the nucleic acid molecule according to c1); c3) a recombinant vector containing the nucleic acid molecule according to c1), or a recombinant vector containing the expression cassette according to c2); c4) a recombinant microorganism containing the nucleic acid molecule according to c1), or a recombinant microorganism containing the expression cassette according to c2), or a recombinant microorganism containing the recombinant vector according to c3).

7. Use according to claim 6, characterized in that: The nucleic acid molecule according to c1) is an sgRNA targeting the gene encoding the protein TSP4.2 according to claim 1, or a DNA molecule expressing the sgRNA; the sgRNA is an sgRNA with a name of sgRNA1 and an sgRNA with a name of sgRNA4, the target sequence of the sgRNA1 is 66-84 of SEQ ID No. 1, and the target sequence of the sgRNA4 is 196-215 of SEQ ID No. 1.

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