Method for improving unisexual maturing rate and yield of tomatoes and related biological material of protein TSP4.2 used by method

Abstract

The invention discloses a method for improving the unisexual maturing rate and / or yield of tomatoes. The method comprises the step of improving the unisexual maturing rate and / or yield of the tomatoes by inhibiting or reducing the expression quantity of a coding gene of protein TSP4.2 in a tomato genome. The TSP4.2 protein can be specifically the following protein A1), A2) or A3): A1) a protein with an amino acid sequence shown as SEQ ID No.2 in a 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 TSP4.2 protein and related biological materials thereof can be used for regulating and controlling the unisexual maturing rate of tomatoes.

Description

Technical Field

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

[0002] Self-pollinating tomatoes (Solanum lycopersicum L.) rely on wind and insects for pollination. After fertilization is completed, the tomato fruit can grow and develop normally. Facility-cultivated tomatoes often have a reduced fruit set rate due to poor pollination, resulting in reduced yields. Plant growth regulators are often used in production to increase the fruit set rate. The use of plant growth regulators not only brings additional economic and labor costs to farmers, but also increases the rate of deformed fruits when used under high temperature conditions.

[0003] Parthenocarpy is an excellent trait that can increase yield under adverse conditions and is one of the important breeding goals of tomatoes. Parthenocarpy is a phenomenon in which the fruit can still expand normally without fertilization. It does not require plant growth regulators to dip the flowers, and has the advantages of stable yield and saving manpower and material resources. In addition, parthenocarpy fruits also have many advantages such as excellent quality, good taste, low rate of deformed fruits, good commercial quality, and fruit storage resistance.

[0004] Studies have shown that tomato parthenocarpy can be formed by regulating auxin synthesis, transport and signaling. 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. After RNAi, it can promote auxin synthesis to form tomato parthenocarpy (Molesini, et al. 2009a). The auxin transporter SlPIN can transport auxin from the ovary to the outside of the ovary. Therefore, after silencing, the auxin content in the ovary can be increased to form tomato parthenocarpy (Mounet, et al. 2012). TIR1 and its homologs act as auxin receptors. After overexpression, they change the transcriptional levels of some auxin response factors to form tomato parthenocarpy (Ren, et al. 2011).

[0005] The auxin response factor SlARF5 may play an important role in regulating the auxin and gibberellin signaling pathways during fruit development and development. The RNAi line can form tomato parthenocarpy (Liu, et al. 2018). SlARF8 forms a complex with SlIAA9 to inhibit the transcription of genes that control fruit development; silencing can form tomato parthenocarpy (Goetz, et al. 2007). SlIAA9 is a major regulator of leaf morphogenesis and fruit development. Both RNAi and knockout lines can form tomato parthenocarpy (Wang, et al. 2005). Inhibition or downregulation of ARFs (auxin response factors) and Aux / IAA (auxin transcription factors) can induce tomato parthenocarpy. Changes in the expression of SlARF2 in the fruit have a significant effect on abscisic acid, cytokinin and salicylic acid, and silencing can form tomato parthenocarpy (Breitel, et al. 2016). SlARF7 regulates the auxin and GA signaling pathways to participate in the formation and development of tomato fruits, and the RNAi line causes tomato parthenocarpy (de Jong, et al. 2011).

[0006] Although some progress has been made in the study of tomato parthenocarpy in recent years and some QTLs have been located, the cloned genes are very limited, which to some extent restricts its application in breeding practice. Analyzing the genetic basis and regulatory network of tomato parthenocarpy at the molecular level is not only of great scientific significance for further elucidating the biological mechanism of tomato parthenocarpy, but also of great practical significance for improving the level of tomato breeding in my country and accelerating the cultivation of parthenocarpy quality tomatoes. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to improve the parthenocarpy ability of tomatoes so as to increase the yield of tomatoes during facility cultivation.

[0008] In order to solve the above technical problems, the present invention first provides a method for improving the parthenocarpy rate and / or yield of tomatoes, comprising increasing the parthenocarpy rate and / or yield of tomatoes by inhibiting or reducing the expression amount of the gene encoding the protein TSP4.2 in the tomato genome;

[0009] The protein TSP4.2 is the following protein A1), A2) or A3):

[0010] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0011] 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);

[0012] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and 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 that the gene produces a deletion mutation, an insertion mutation or a base change mutation, thereby achieving a reduction or loss of gene function, specifically chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination, etc.

[0014] 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.

[0015] In the above method, the inhibition or reduction of the expression of the gene encoding the protein TSP4.2 in the tomato genome is to introduce any one of the following substances c1)-c4) into the tomato:

[0016] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TSP4.2 described 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) 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).

[0020] The nucleic acid molecule described in c1) is a sgRNA targeting the gene encoding the protein TSP4.2 described above or a DNA molecule expressing the sgRNA.

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

[0022] In the above method, the inhibition or reduction of the expression of the gene encoding the protein TSP4.2 in the tomato genome is to perform 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) The nucleotides 77-81 of the coding sequence SEQ ID No. 1 corresponding to the TSP4.2 gene in the tomato genomic DNA are missing;

[0024] 2) Nucleotides 74-197 of the coding sequence SEQ ID No. 1 corresponding to the TSP4.2 gene in the tomato genomic DNA are missing.

[0025] In order to solve the above technical problems, the present invention also provides a protein TSP4.2, which is the following protein A1), A2) or A3):

[0026] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0027] 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);

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

[0029] Among the above proteins, the protein TSP4.2 may be derived from tomatoes.

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

[0031] The one or more amino acid residues mentioned above may specifically be within ten amino acid residues.

[0032] In the above applications, the above 80% identity may 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 protein TSP4.2, wherein the biological materials related to protein TSP4.2 are any one of the following B1) to B5):

[0034] B1) a nucleic acid molecule 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) 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);

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

[0040] In the above biological material, the nucleic acid molecule in B1) is a cDNA molecule or a DNA molecule whose coding sequence is the nucleotide of SEQ ID No.1.

[0041] Among them, SEQ ID No.1 in the sequence list consists of 1965 nucleotides and encodes the protein shown in SEQ ID No.2.

[0042] In the above biological material, 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 a host cell, and the nucleic acid molecule may include not only a promoter for initiating transcription of the TSP4.2 gene, but also a terminator for terminating transcription of TSP4.2. 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).

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

[0044] The present invention also provides a substance that reduces the activity or content of the protein TSP4.2, or an application of a substance that inhibits or reduces the expression of a gene encoding the protein TSP4.2, wherein the application is any of the following:

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

[0046] P2. Use in increasing tomato yield or in preparing products for increasing tomato yield;

[0047] P3. Application in tomato breeding.

[0048] In the above application, the tomato breeding is to select varieties with high parthenocarpy rate.

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

[0050] In the above application, the coding gene of the protein TSP4.2 may be a DNA molecule as shown in a1) or a2) or a3) below:

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

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

[0053] a3) A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in a1) or a2) and encodes the protein TSP4.2 described above.

[0054] In the above application, the gene encoding the protein TSP4.2 that regulates the protein 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 the 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 mRNA degradation of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

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

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

[0057] 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.

[0058] 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.

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

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

[0061] c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TSP4.2;

[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) 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).

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

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

[0067] In the above text, the yield mentioned is the yield during facility cultivation.

[0068] The present invention discloses a method for knocking out the TSP4.2 gene of tomatoes to improve the parthenocarpy rate of tomatoes. Specifically, the TSP4.2 gene in the starting tomatoes is edited using the CRISPR / Cas9 system, and the TSP4.2 gene is mutated to cause the translation protein to terminate prematurely, thereby obtaining transgenic tomatoes and realizing the editing of the TSP4.2 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 TSP4.2 gene, obtain tomato mutant materials with a high parthenocarpy rate, and provide new materials for tomato variety breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0073] Figure 5 This is the fruit phenotype of the TSP4.2 gene mutant in Example 1 of the present invention. ** represents the significance analysis result of P<0.01. DETAILED DESCRIPTION

[0074] 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.

[0075] 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.

[0076] 1. Vectors and strains

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

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

[0079] In the following examples, Agrobacterium AGL1 is a product of Beijing Quanshijin 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 of the University of California, Davis (URL: https: / / tgrc.ucdavis.edu / ), with the accession number LA2706.

[0082] , the public can obtain it from the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences to repeat the experiments of this application.

[0083] The LB solid medium containing 50 mg / L of rifampicin and 50 mg / L of kanamycin in the following examples is an LB solid medium containing 50 mg / L of rifampicin and 50 mg / L of kanamycin. The formula of the LB solid medium containing 50 mg / L of rifampicin and 50 mg / L of kanamycin is: 50 mg / L of rifampicin, 50 mg / L of kanamycin, 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, 15 g / L of agar, and the pH is adjusted to 7.5 with NaOH.

[0084] 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.

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

[0086] The nucleotide sequence of the cDNA of the TSP4.2 gene (Solyc04g077490) from tomato 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] In order to clarify the role of TSP4.2 in the development of tomato fruit, the expression of TSP4.2 was detected by qRT-PCR using cDNA from roots, stems, leaves, flowers, and fruits at different stages of the tomato variety 'Money Maker' (MM) as templates. The primer sequences are as follows:

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

[0140] TSP4.2-R: GGTGCCTTGTCACTCCTCTG (reverse complement to the sequence at positions 1227-1246 of SEQ ID No. 1).

[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 expression level of TSP4.2 gene is higher in fruit and lower in roots, stems, leaves and flowers, indicating that TSP4.2 gene may play a role in tomato parthenocarpy.

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

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

[0147] TSP4.2-F1: (Positions 9-38 indicated in italics are identical to positions 1-36 of SEQ ID No. 1, and the underline indicates the BamH1 enzyme recognition sequence);

[0148] TSP4.2-R1: (Positions 9-38 indicated in italics are reverse complementary to 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 basic plasmid pCAMBIA2300_YFP_HA linearized by digestion with BamH1 and Sal1 enzymes using T4 ligase. Specifically, the small fragment between the recognition sites of BamH1 and Sal1 enzymes on the plasmid pCAMBIA2300_YFP_HA was replaced with the sequence shown in positions 1-1962 of SEQ ID No. 1, and the other sequences of the plasmid pCAMBIA2300_YFP_HA were kept unchanged to obtain a recombinant expression vector expressing TSP4.2, which was named 35S::TSP4.2-YFP-HA.

[0150] The correctly constructed recombinant expression vector 35S::TSP4.2-YFP-HA was transferred into Agrobacterium tumefaciens EHA105, and after screening with LB solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin and colony PCR identification, it was injected into tobacco (Nicotiana benthamiana, from Li Jiayang's research group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) leaves. The 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. Construction of CRISPR / Cas9 editing vector for TSP4.2 gene

[0153] 1. Construction of CRISPR / Cas9 editing vector for TSP4.1 gene

[0154] According to the coding sequence of TSP4.1 gene (as shown in SEQ ID No.1), 4 sgRNAs of target sequences edited by CRISPR / Cas9 were designed by CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR / ). The target sequences of the sgRNAs are:

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

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

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

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

[0159] Design primers to amplify the DNA fragment containing the sgRNA:

[0160] For sgRNA1, the primer sequences are:

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

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

[0163] For sgRNA2, the primer sequences are:

[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:

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

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

[0169] Using the pCBC_DT1T2_SlU6p plasmid (the plasmid 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 using the above three pairs of primers to obtain sgRNA1 sequence fragment, sgRNA2 sequence fragment, and sgRNA3 and sgRNA4 sequence fragments, a total of three DNA fragments, which were then recovered and purified by gel recovery.

[0170] At the same time, the CRISPR-Cas9 vector (the name of the CRISPR-Cas9 vector is pTX041, which comes from Li Chuanyou's research group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and is recorded in the literature "Deng L, Wang H, Sun C, Li Q, Jiang H, DuM, 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 gel-recovered and purified.

[0171] Then, add the following to a 200uL RNase-free PCR tube: 10×T4 DNA ligase buffer, 1uL; 0.1% BSA (Takara), 1uL; purified linear vector pTX041, 100ng; 3 purified DNA fragments (sgRNA1 sequence fragment, sgRNA2 sequence fragment, sgRNA3 and sgRNA4 sequence fragment), 30ng each; BsaI enzyme (NEB), 0.5uL; T4 DNA ligase (Promega), 0.5uL; add ultrapure water to a total volume of 10uL. Place the above reaction system in a PCR instrument for connection and perform the following reaction: 37°C, 5min; 16°C, 10min; repeat the above reaction for 16 cycles; 50°C, 5min; 80°C, 5min; store at 10°C. The above ligation product was transformed into Escherichia coli DH5α competent cells (the method is the same as the transformation method in Example 2), kanamycin resistance, positive clone screening was performed, sequencing was performed to verify the correctness of the sequence (sequencing was completed by BGI), and the plasmid was 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, and the structure is shown in Figure 3 .

[0172] 2. Obtaining CRISPR / Cas9 edited mutants

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

[0174] The 13 regenerated plants were used as templates to perform PCR detection on the Cas 9 gene. The detection primers were Cas 9-F and Cas9-R:

[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] The editing site of TSP4.2 gene was amplified by PCR, and the primer pair composed of CR-TSP4.2-F and CR-TSP.2-R was used:

[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 homozygous mutant plants were kept as seeds to obtain T1 seeds for sowing. T1 plants without Cas9 gene (detection primers were Cas 9-F and Cas9-R) were selected and self-pollinated to obtain two independent CRISPR strains with different TSP4.2 mutation types, named tsp4.2cr-1 and tsp4.2cr-2. The gene editing site sequences are as follows: Figure 4 As shown:

[0183] Compared with wild-type MM ( Figure 4 Compared with the genomic DNA of tsp4.2cr-1 ( Figure 4 In the two homologous chromosomes (labeled as tsp4.2cr-1), the genes encoding the protein TSP4.2 have undergone the following mutations: at target site 1 (Target1, corresponding to sgRNA1), nucleotides (ATACT) are deleted at positions 77-81 of SEQ ID No.1, a total of 5 bp are deleted, resulting in a frameshift mutation in the TSP4.2 gene, thereby knocking out the TSP4.2 gene.

[0184] Compared with wild-type MM ( Figure 4 Compared with the genomic DNA of tsp4.2cr-2 ( Figure 4 In the two homologous chromosomes (labeled as tsp4.2cr-2), the genes encoding protein TSP4.2 have undergone the following mutations: between target 1 (Target1, corresponding to sgRNA1) and target 4 (Target4, corresponding to sgRNA4), the nucleotides CTAATACTAACTGGTTGGGTTTTTCTCTCACACCCCACATGAAAATGGAGGTT ACTAATTCTTCTGATACTCAACATCAACATCAATTTGCACAAAGTTTCTATCTTT CATCTTCTCCTCCTCC) are deleted at positions 74-197 of SEQ ID No.1, with a total deletion of 124 bp, resulting in a frameshift mutation in the TSP4.2 gene, thereby knocking out the TSP4.2 gene.

[0185] 3. Investigating the parthenocarpic 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 solar greenhouse, with 5 plants per replicate for each line.

[0187] Without artificial pollination or application of auxin, the seeds were planted in early April and the parthenocarpy rate of 2-4 ears of fruit 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 showed different degrees of parthenocarpy. This result once again proves that TSP4.2 is an important regulatory gene for tomato parthenocarpy.

[0189] 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 method for increasing the parthenocarpic fruit setting rate and / or yield of tomatoes, characterized in that: The method comprises increasing the parthenocarpy rate and / or yield of tomatoes by inhibiting or reducing the expression of a gene encoding protein TSP4.2 in the tomato genome; The protein TSP4.2 is 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 method according to claim 1, characterized in that: The method of inhibiting or reducing the expression of the gene encoding the protein TSP4.2 of claim 1 in the tomato genome is to introduce any one of the following substances c1) to c4) into the tomato: c1) a nucleic acid molecule that inhibits or reduces the expression of the gene encoding the protein TSP4.2 according to claim 1; 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).

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

4. The method according to claim 1, characterized in that: The method of inhibiting or reducing the expression of the gene encoding the protein TSP4.2 in the tomato genome is to perform 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: 1) The nucleotides 77-81 of the coding sequence SEQ ID No. 1 corresponding to the TSP4.2 gene in the tomato genomic DNA are missing; 2) Nucleotides 74-197 of the coding sequence SEQ ID No. 1 corresponding to the TSP4.2 gene in the tomato genomic DNA are missing.

5. The protein TSP4.2 as claimed in claims 1-4.

6. The biomaterial related to the protein TSP4.2 according to claim 5, characterized in that: The biological material is any one of the following B1) to B5): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1); 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); 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).

7. The biomaterial according to claim 6, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is the nucleotide sequence of SEQ ID No.

1.

8. Use of a substance for reducing the activity or content of the protein TSP4.2 according to claim 5, or a substance for inhibiting or reducing the expression of the gene encoding the protein TSP4.2 according to claim 5, characterized in that: The application is any of the following: P1. Application in improving the parthenocarpy of tomatoes or in preparing products that improve the parthenocarpy of tomatoes; P2. Use in increasing tomato yield or in preparing products for increasing tomato yield; P3. Application in tomato breeding.

9. The use according to claim 8, characterized in that: The substance that reduces the activity or content of the protein TSP4.2 according to claim 5, or the substance that inhibits or reduces the expression of the gene encoding the protein TSP4.2 according to claim 5, 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 TSP4.2 according to claim 5; 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).

10. The use according to any one of claims 8 or 9, characterized in that: c1) The nucleic acid molecule is a sgRNA targeting the gene encoding the protein TSP4.2 according to claim 5 or a DNA molecule expressing the sgRNA; the sgRNA is a sgRNA named sgRNA1 and a sgRNA named sgRNA2, the target sequence of the sgRNA1 is positions 66-84 of SEQ ID No.3, and the target sequence of the sgRNA2 is positions 70-88 of SEQ ID No.3.

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