Application of PBB2 protein and related biological materials thereof in regulation and control of plant fruit size

Abstract

The invention discloses application of PBB2 protein and related biological materials thereof in regulation and control of plant fruit size, and belongs to the technical field of biology. The amino acid sequence of the PBB2 protein is as shown in SEQ ID No. 1. Experiments prove that after SlPBB2 is silenced in tomato fruits, cell division and cell expansion in the development process of the fruits are promoted, and finally the fruits become larger, and the weight is increased.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of PBB2 protein and related biological materials in regulating the size of plant fruits. Background Art

[0002] Fruits are important reproductive organs of plants, rich in nutrients such as carbohydrates, inorganic salts, and vitamins, and are important food sources for humans and animals, being closely related to human life. Fruit size is one of the important factors determining fruit yield. Fruit size is affected by environmental conditions and water and fertilizer conditions, but is mainly controlled by related genes. Exploring genes that regulate or affect fruit size is of great significance for understanding the molecular mechanism of fruit size and biological breeding. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a new use of PBB2 protein in regulating fruit size. The technical problems to be solved are not limited to the described technical topics, and those skilled in the art can clearly understand other technical topics not mentioned herein through the following description.

[0004] To solve the above technical problems, the present invention provides the following technical solutions:

[0005] The present invention provides the application of a protein or a substance that regulates the expression of the coding gene of the protein or a substance that regulates the activity and / or content of the protein in any one of the following:

[0006] M1) Regulating the size of plant fruits;

[0007] M2) Preparing a product for regulating the size of plant fruits;

[0008] M3) Increasing the size of fruits;

[0009] M4) Cultivating a product for increasing the size of fruits;

[0010] M5) Cultivating a plant with an increased size of fruits;

[0011] M6) Preparing a product for cultivating a plant with an increased size of fruits;

[0012] M7) Plant breeding;

[0013] The protein is any one of the following:

[0014] A1) A protein with an amino acid sequence of SEQ ID No.1;

[0015] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1), having more than 80% identity with the shown protein and having the same function;

[0016] A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in either A1) or A2).

[0017] The tag proteins include, but are not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0018] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding protein PBB2 of the present invention by using known methods, such as directed evolution or site-directed mutagenesis. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of protein PBB2 isolated from the present invention, as long as they encode protein PBB2 and have the function of protein PBB2, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0019] The above 75% or more identity can be 80%, 85%, 90% or more than 95% identity.

[0020] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of the amino acid sequence, and then the identity value (%) can be obtained.

[0021] In this article, the above 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0022] The size of the fruit can be reflected by the transverse diameter. The transverse diameter is the longest diameter of the cross-sectional circle perpendicular to the fruit stalk in the fruit.

[0023] Increasing the size of the fruit includes increasing the single fruit weight, transverse diameter and / or pericarp thickness of the fruit. The transverse diameter (diameter) includes the transverse diameter during the entire development process of the plant fruit.

[0024] In this article, the substance that regulates the activity and / or content of the protein can be a substance that regulates gene expression, and the gene encodes the protein. The substance that regulates gene expression can be a substance that performs at least one of the following six regulations: 1) Regulation at the transcriptional level of the gene; 2) Regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0025] In the above application, the substance that regulates the expression of the coding gene of the above protein or the substance that regulates the content or activity of the above protein includes a substance that reduces the expression of the coding gene of the above protein or a substance that increases the expression of the coding gene of the above protein.

[0026] The substance that reduces the expression of the coding gene of the above protein in the cell is any one of the following:

[0027] B1), an RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein or an RNA molecule that inhibits or reduces or down-regulates the activity or content of the protein;

[0028] B2), a coding gene that expresses the RNA molecule described in B1);

[0029] B3), an expression cassette containing the gene described in B2);

[0030] B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0031] B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

[0032] B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4);

[0033] B7), a transgenic plant tissue containing the gene described in B2), or a transgenic plant tissue containing the expression cassette described in B3), or a transgenic plant tissue containing the recombinant vector described in B4);

[0034] B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0035] The substance B1) that reduces the expression of the coding gene of the above-mentioned protein in cells targets the mRNA transcribed from the gene of the above-mentioned protein.

[0036] The substance that increases the expression of the coding gene of the above-mentioned protein in cells is any one of the following:

[0037] B1), a nucleic acid molecule encoding the above-mentioned protein;

[0038] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0039] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0040] 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);

[0041] 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);

[0042] B6), a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2), or a transgenic plant tissue containing the recombinant vector described in B3);

[0043] B7), a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0044] Among the substances that increase the expression of the coding gene of the above-mentioned protein in cells, the nucleic acid molecule described in B1) is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No. 2.

[0045] In a specific embodiment of the present invention, the substance that regulates the expression of the coding gene of the above-mentioned protein or the substance that regulates the content or activity of the above-mentioned protein is a substance that reduces the expression of the coding gene of the above-mentioned protein.

[0046] In the above application, the plant is any one of the following:

[0047] G1) Monocotyledonous plants or dicotyledonous plants;

[0048] G2) Solanales plants;

[0049] G3) Solanaceae plants;

[0050] G4) Solanum;

[0051] G5) Tomato.

[0052] Among the above substances, the expression cassette containing a nucleic acid molecule described in B3) refers to a DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for the nucleic acid molecule expressing any one of the above proteins. The regulatory sequences can direct the coding sequence to express any one of the above proteins in a suitable host cell under its compatible conditions. The regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences should include a promoter and transcription and translation termination signals. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein, regulatory sequences with linkers can be provided. The regulatory sequences can be suitable promoter sequences, i.e., nucleic acid sequences recognizable by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences mediating protein expression. The promoter can be any nucleic acid sequence having transcriptional activity in the selected host cell, including mutated, truncated, and chimeric promoters, and can be derived from genes encoding extracellular or intracellular proteins homologous or heterologous to the host cell. The regulatory sequences can also be suitable transcription termination sequences, i.e., a sequence recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3'-end of the nucleic acid sequence encoding the protein. Any terminator functional in the selected host cell can be used in the present invention. The regulatory sequences can also be suitable leader sequences, i.e., the untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5'-end of the nucleic acid sequence encoding the protein. Any leader sequence functional in the selected host cell can be used in the present invention. The regulatory sequences can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein and can direct the encoded protein into the cell secretion pathway. Any signal peptide coding region capable of directing the expressed protein into the secretion pathway of the used host cell can be used in the present invention. It may also be necessary to add regulatory sequences capable of regulating protein expression according to the growth of the host cell. Examples of regulatory sequences are those systems that can respond to chemical or physical stimulants (including in the presence of regulatory compounds) to turn on or off gene expression. Other examples of regulatory sequences are those regulatory sequences capable of gene amplification.

[0053] The present invention also provides a method for increasing fruit size, the method comprising introducing an expression cassette into a target plant to regulate the fruit size of the target plant by reducing the expression level of the coding gene of the aforementioned protein and / or reducing the content of the protein and / or reducing the activity of the protein in the target plant; the expression cassette contains a fruit-specific promoter and a silencing gene driven by the fruit-specific promoter, and the silencing gene can transcribe an RNA that silences the gene of the aforementioned protein.

[0054] In the above method, in the expression cassette, the fruit-specific promoter may be SEQ ID No.3. The sequence of the silencing gene may be SEQ ID No.2 or the 330th to 627th bases of SEQ ID No.2.

[0055] In the above method, the expression cassette is introduced into the target plant through a silencing vector, and the silencing vector contains the expression cassette.

[0056] In the above method, the plant is any one of the following:

[0057] G1) Monocotyledonous plants or dicotyledonous plants;

[0058] G2) Plants of Solanales;

[0059] G3) Plants of Solanaceae;

[0060] G4) Solanum;

[0061] G5) Tomato.

[0062] The substances and proteins described in the above B1 to B5) also fall within the scope of protection of the present invention.

[0063] The present invention proves through experiments that after silencing SlPBB2 in tomato fruits, it promotes cell division and cell expansion during fruit development, ultimately resulting in larger fruits and increased weight. Description of the Drawings

[0064] Figure 1 It is the map of the RNAi-PPC2-SlPBB2 vector (pCambia-PPC2-SlPBB2).

[0065] Figure 2 It is the relative expression level of SlPBB2 in the fruits of each RNAi-SlPBB2 line at the green-ripe stage.

[0066] Figure 3 It is the phenotype of the RNAi-SlPBB2 transgenic plants.

[0067] Figure 4To silence SlPBB2 affects tomato fruit size. A, Comparison of fruit size between SlPBB2 gene-silenced fruits and wild-type (WT) fruits. (Br stage, bar = 1 cm). B, Statistical results of the single fruit weight of SlPBB2 gene-silenced fruits. C, Trend of fruit diameter change during fruit development. D, Fruit cross-section diagram (37 dpa, bar = 1 cm). E, Statistical results of the pericarp thickness of SlPBB2 gene-silenced fruits. F, Scanning image of fruit pericarp tissue section (20 dpa). G, Statistical results of the number of pericarp cell layers and the area of a single cell (20 dpa). t-test, "**" indicates P < 0.01, "***" indicates P < 0.001. Detailed implementation manners

[0068] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0069] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0070] pCambia-PPC2 plasmid: The CaMV35S promoter sequence in the vector pCAMBIA-1300-221 described in the non-patent literature "Gao et al, 2020, Gao Y, Wei W, Fan Z, Zhao X, Zhang Y, Jing Y, Zhu B, Zhu H, Shan W, Chen J, Grierson D, Luo Y, T, Jiang CZ, Fu DQ. Re-evaluation of the nor mutation and the role of the NAC-NOR transcription factor in tomato fruit ripening. J Exp Bot. 2020 Jun 22;71(12):3560-3574." was replaced with the PPC2 promoter sequence (SEQ ID, No. 3). The replaced sequence is as follows:

[0071] 5’-AGATTAGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAGA GAGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACG-3’. The pCambia-PPC2 plasmid can be obtained from the Food Biotechnology Laboratory of the College of Food Science and Nutritional Engineering, China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0072] Wild-type tomato (Solanum lycopersicum cv.) variety Ailsa Craig (AC): described in the non-patent literature "Gao et al, 2020, Gao Y, Wei W, Fan Z, Zhao X, Zhang Y, Jing Y, Zhu B, Zhu H, Shan W, Chen J, Grierson D, Luo Y, T, Jiang CZ, Fu DQ. Re-evaluation of the normutation and the role of the NAC-NOR transcription factor in tomato fruit ripening. J Exp Bot. 2020 Jun 22;71(12):3560-3574.)". The public can obtain it from the Food Biotechnology Laboratory of the College of Food Science and Nutritional Engineering, China Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0073] Example 1. Silencing SlPBB2 promotes fruit enlargement

[0074] I. RNAi vector construction method

[0075] Download the SlPBB2 (Solyc05g013820) gene from the Solanaceae Genomic Database (https: / / solgenomics.net / ).

[0076] The protein sequence of SlPBB2 (SEQ ID No. 1)

[0077] MATIDVPQKGGFSFDLCRRNEMLVSKGLRSPSFLKTGTTIVGLIFKDGVILGADTRATEGPIVADKNCEKIHYMAPNIYCCGAGTAADTEAVTDMVSSQLKLHRYHTGR ESRVVTSLTLLKSHLFSYQGHVSAALVLGGVDVTGPHLHTIYPHGSTDTLPYATMGSGSLAAMAMFESKYREGMSKDEGIKLVADAILSGVFNDLGSGSNVDICVITKGNTEYLRNHLSPNPRTYPQKGYSFSKKTEVLLTKITPLREIVQVIEGGDAMEE

[0078] The CDS sequence of SlPBB2 (SEQ ID No. 2)

[0079] 5'-ATGGCAACAATCGATGTTCCCCAAAAGGGTGGGTTTAGTTTTGATTTGTGTAGAAGAAATGAGATGTTGGTCAGCAAAGGACTTCGTTCTCCCTCTTTTCTCAAGACTGGTACTACCATCGTCGGCTTAATTTTTAAGGATGGTGTTATCCTTGGAGCAGACACACGGGCAACTGAAGGACCTATTGTCGCGGATAAAAACTGTGAGAAAATTCATTACATGGCTCCTAATATATATTGCTGTGGAGCTGGGACAGCTGCTGATACAGAGGCTGTGACTGACATGGTCAGTTCCCAGTTAAAGCTTCACAGGTATCACACTGGTCGTGAATCCAGGGTTGTGACATCTCTTACTCTTTTGAAGTCCCACCTTTTCAGCTACCAAGGCCACGTCTCTGCTGCTTTGGTCCTTGGTGGTGTTGATGTCACAGGGCCACATCTGCATACTATTTATCCACATGGATGGAATGAGCAAAGATGAAGGTATAAAGCTAGTAGCAGACGCCATTTTGTCTGGAGTGTTCAATGACCTTGGTAGTGGCAGCAATGTCGATATCTGTGTGATAACAAAGGGAAACACGGAGTACTTACGAAATCATTTATCGCCCAATCCCCGCACCTATCCACAAAAGGGTTACTCGTTCTCTAAAAAGACTGAGGTTCTCCTTACAAAGATTACACCACTGAGAGAAATTGTGCAAGTGATTGAAGGTGGAGACGCCATGGAAGAATGA-3'

[0080] The vector construction method is as follows:

[0081] 1. Linearize the pCambia-PPC2 plasmid. After double digestion of the original plasmid with BamHⅠ and SacⅠ, the vector backbone sequence was recovered by gel extraction.

[0082] The nucleotide sequence of the PPC2 promoter in the pCambia-PPC2 plasmid (SEQ ID No.3)

[0083]

[0084] 2. PCR amplification of the target gene fragment: Using specific primers with homologous arms and tomato cDNA as the template, a 217-bp DNA fragment with the nucleotide sequence at positions 331-547 of SEQ DI No.2 was PCR amplified and gel recovered to obtain the target fragment with homologous arms.

[0085] F: 5'-tttgattttctagtggatccTCCAGGGTTGTGACATCTCT-3';

[0086] R: 5'-gatcggggaaattcgagctcATTGCTGCCACTACCAAGGT-3';

[0087] 3. Using the Infusion method, the target fragment with homologous arms amplified in step 2 was reversely inserted between the two restriction enzyme sites BamHⅠ and SacⅠ of the target vector to obtain the ligation product.

[0088] 4. Transformation of the ligation product into Escherichia coli. Using Trans 5α chemically competent cells, the specific operation is as follows:

[0089] (1) Add the ligation product to 50 μL of Escherichia coli Trans5α competent cells melted on ice, gently stir and mix with a pipette tip, and incubate on ice for 30 min;

[0090] (2) Heat shock at 42 °C for 90 s, immediately incubate on ice for 2 min; add 800 μL of LB liquid medium without antibiotics, and shake culture at 37 °C at 200 r / min for 1 h;

[0091] (3) Centrifuge at 5000 rpm for 3 min to collect the bacteria, discard part of the supernatant, resuspend the bacteria and spread them on solid LB medium containing kanamycin antibiotic, and incubate inverted in a 37 °C constant temperature incubator overnight;

[0092] (4) Pick single colonies for PCR detection, and detect the colony PCR products by 1% agarose gel electrophoresis;

[0093] (5) Pick positive single colonies identified by colony PCR into liquid LB medium containing the corresponding antibiotic and culture overnight, and the bacterial solution is used for plasmid extraction.

[0094] 5. Send the extracted plasmid to the company for Sanger sequencing. The plasmid with the correct sequence verified by sequencing is named the recombinant expression vector pCambia-PPC2-SlPBB2. This recombinant expression vector is obtained by replacing the base between the BamHⅠ and SacⅠ enzyme digestion recognition sites of pCambia-PPC2 with the fragment of the 331-547th bases of SEQ DI No.2 while keeping the other nucleotides of the vector unchanged (the map is as Figure 1 shown).

[0095] 6. Agrobacterium transformation. Transform the recombinant expression vector pCambia-PPC2-SlPBB2 with correct sequencing into Agrobacterium tumefaciens GV3101 by the freeze-thaw method. The specific operations are as follows:

[0096] (1) Add the plasmid into 50 μL of Agrobacterium tumefaciens GV3101 competent cells melted on ice, gently stir and mix with a pipette tip, and incubate on ice for 5 min;

[0097] (2) Quick-freeze in liquid nitrogen for 5 min, immediately place in a 37 °C metal bath and keep for 5 min, and then incubate on ice for 5 min;

[0098] (3) Add 800 μL of LB liquid medium without antibiotics, place on a shaker at 28 °C and shake at 200 rpm for 2.5 h;

[0099] (4) Centrifuge at 5000 rpm for 3 min to collect the bacteria, leave about 100 μL of supernatant, gently pipette to resuspend the bacteria, take an appropriate amount of the bacterial solution and spread it on an LB plate containing the corresponding antibiotics, and place it in an incubator at 28 °C and incubate upside down for 48 h;

[0100] (5) Pick single colonies for PCR detection, and detect the colony PCR products by 1% agarose gel electrophoresis;

[0101] (6) Pick the positive single colonies identified by colony PCR and name them GV3101 / pCambia-PPC2-SlPBB2.

[0102] II. Preparation of RNAi plants

[0103] Transform the gene silencing vector pCambia-PPC2-SlPBB2 into wild-type tomato Solanum lycopersicum cv. Ailsa Craig (AC) by Agrobacterium-mediated leaf disc transformation method. All operations are carried out in a laminar flow hood.

[0104] The specific steps are as follows:

[0105] (1) Sowing: Take an appropriate amount of seeds of the wild-type tomato variety Solanum lycopersicum cv. Ailsa Craig (AC) in a sterile petri dish, soak them in 75% ethanol (shake to make uniform contact) for 5 min for disinfection, pour out the ethanol solution, then soak them in 4% sodium hypochlorite solution (shake to make uniform contact) for 10 min, and finally wash them with sterile water 7 - 8 times, soaking for 1 min each time. Then sow the seeds on the T0 seed germination medium.

[0106] Preparation of T0 medium: Weigh 1.77 g of MS salts and 12 g of sucrose, dissolve them in distilled water, adjust the pH to 5.8 with 1M NaOH, then add 5.6 g of agar, make up the volume to 800 mL, sterilize at 121 °C for 20 min, cool to about 55 - 65 °C, and pour it into tissue culture bottles for condensation and standby.

[0107] (2) Seed germination: Place the sown seeds in a dark room for culture for about 3 - 4 d, and then place them in the light for culture for 2 - 4 d after germination. Wait until the cotyledons are fully unfolded for the next operation.

[0108] (3) Pre-culture: Place the roots of tomato seedlings in sterile water, cut off the tips of the cotyledons, cut the remaining part of each cotyledon into two 5×5 mm squares, place them with the back facing up on the T1 pre-culture medium covered with a layer of filter paper, and obtain tomato explants after pre-culturing for 2 d.

[0109] (4) Preparation of infection solution: Pick a single colony of Agrobacterium tumefaciens GV3101 / pCambia - PPC2 - SlPBB2 into 3 mL of LB medium (containing 50 μg / mL kanamycin (kan), 50 μg / mL gentamicin (gen), and 50 μg / mL rifampicin (rif)), and culture it overnight with shaking at 28 °C for 16 h. The next day, take 300 μl of the bacterial solution into 20 mL of LB (containing 50 μg / mL kanamycin (kan), 50 μg / mL gentamicin (gen), and 50 μg / mL rifampicin (rif)) medium, and culture it with shaking at 28 °C for 6 - 7 h until the OD 600 reaches 0.5 - 0.6. Centrifuge at 5000 rpm for 10 min to collect the bacterial cells, and dilute the bacterial solution with sterile water to OD 600 = 0.1 - 0.2 to obtain the infection solution.

[0110] (5) Co - culture: Immerse the tomato explants pre - cultured for 2 d in the infection solution, gently shake for 5 min, then pour out the infection solution, blot the excess infection solution, and then place the explants back on the T1 pre - culture medium, and place them in a dark room for co - culture for 2 d;

[0111] Preparation of T1 pre-culture medium: Weigh 3.54 g of MS salts and 24 g of sucrose, dissolve them in distilled water, adjust the pH to 5.8 with 1 M NaOH, then add 5.6 g of agar, make up the volume to 800 mL, sterilize at 121 °C for 20 min, cool to about 55 - 65 °C, add 80 μL of IAA (indole-3-acetic acid, 0.1 mg / mL) and 800 μL of 6-BA (6-benzylaminopurine, 1 mg / ml), shake well, pour into petri dishes and let it condense for standby.

[0112] (6) Bud induction culture: Take out the co-cultured tomato explants after 2 days from the dark room, place them with the back facing down on the bud induction medium T21, culture them under the conditions of 25 °C, 16 h light / 8 h dark for 7 days, then transfer them to fresh T21 medium for subculture, change the fresh T21 medium every 2 weeks until the explants grow callus and small leaves.

[0113] Formulation of induction medium T21: Weigh 3.54 g of MS salts and 24 g of sucrose, dissolve them in distilled water, adjust the pH to 5.8 with 1 M NaOH, then add 5.6 g of agar, make up the volume to 800 mL, sterilize at 121 °C for 20 min, cool to about 55 - 65 °C, add 80 μL of IAA (indole-3-acetic acid, stock solution is 0.1 mg / mL), 800 μL of Ticarcillin (Timentin, 2 mg / mL), 800 μL of ZT (zeatin, 1 mg / mL), 160 μL of hygromycin (final concentration is 10 mg / L), shake well, pour into petri dishes and let it condense for standby.

[0114] (7) Bud elongation culture: After the explants grow small leaves, transfer them to the bud elongation medium T22, change the fresh T22 medium every two weeks until the stem elongates to 4 - 5 cm.

[0115] Formulation of bud elongation medium T22: Weigh 3.54 g of MS salts and 24 g of sucrose, dissolve them in distilled water, adjust the pH to 5.8 with 1 M NaOH, then add 5.6 g of agar, make up the volume to 800 mL, sterilize at 121 °C for 20 min, cool to about 55 - 65 °C, add 800 μL of Ticarcillin (Timentin, 2 mg / mL), 800 μL of ZT (zeatin, 1 mg / mL), 400 μL of GA (gibberellin, 1 mg / mL), 160 μL of hygromycin (final concentration is 10 mg / L), shake well, pour into petri dishes and let it condense for standby.

[0116] (8) Rooting culture: After the stem elongates, cut off the callus, transfer it to the rooting medium T3, culture for 3 - 4 weeks until the roots are well-developed.

[0117] Rooting medium T3 formulation: Weigh 3.54 g of MS salts and 24 g of sucrose, dissolve them in distilled water, adjust the pH to 5.8 with 1 M NaOH, add 5.6 g of agar, make up the volume to 800 mL, sterilize at 121 °C for 20 min, cool to about 55 - 65 °C, add 600 μL of Ticarcillin (Timentin, 2 mg / mL), 800 μL of IBA (Indole-3-butyric acid, 2 mg / mL), and 80 μL of hygromycin (final concentration 5 mg / L), then shake well and pour into tissue culture flasks for condensation and standby.

[0118] (9) Soil cultivation: Carefully remove the seedlings from the T3 medium, wash the residual medium on the roots with clear water, transfer the seedlings into a moist soil pot, support the surrounding with steel pins and wrap with plastic wrap for moisture preservation.

[0119] III. Identification of transgenic plants

[0120] 1. Identification by qRT-PCR

[0121] Extract the total RNA of the T2 generation of the aforementioned transgenic plants according to the aforementioned method and reverse transcribe to obtain cDNA.

[0122] Quantitative real-time PCR (qRT-PCR) is carried out with reference to the instruction manual of ChamQ SYBR qPCR Master Mix (Without ROX) (product number: Q321) of Nanjing Novoprotein Scientific Co., Ltd. The reaction system is as follows: 2×ChamQ SYBR qPCR Master Mix (Without ROX) 10 μL, fluorescence quantification F 0.4 μL, fluorescence quantification R 0.4 μL, template cDNA 1 μL, ddH2O 8.2 μL.

[0123] Actin-F: 5'-CAGCAGATGTGGATCTCAAA-3';

[0124] Actin-R: 5'-CTGTGGACAATGGAAGGAC-3';

[0125] SlPBB2-F2: 5'-GGGAATGAGCAAAGATGAAGG-3';

[0126] SlPBB2-R2: 5'-CCCTTTTGTGGATAGGTGCG-3';

[0127] For each gene, there are three biological replicates. Using the tomato Actin gene (Solyc03g078400) as an internal reference gene, the 2 -ΔΔCt method is used to analyze the qRT-PCR results and calculate the relative gene expression level.

[0128] The experimental data were processed using GraphPad Prism 9, and significant analysis was performed using IBM SPSS Statistics 22 statistical software. Student's t-test was used for comparison between two groups of data; one-way ANOVA with Duncan's multiple comparison was used for comparison among three or more groups of data. p < 0.05 indicates significant difference, p < 0.01 indicates very significant difference, and p < 0.001 indicates extremely significant difference.

[0129] As a result, three lines with relatively high silencing efficiency were screened out, namely RNAi-11, RNAi-13, and RNAi-16. The relative expression levels of the target genes in each line are as Figure 2 shown.

[0130] 2. Phenotypic observation

[0131] RNAi-11, RNAi-13, RNAi-16, and wild-type plants were planted under the same conditions (23 °C, 16 h of light and 8 h of darkness). After 30 days of planting, the inventor observed the growth of the screened transgenic lines and found that under the condition of using the PPC2 fruit-specific promoter, the vegetative growth of the transgenic plants was not affected and was consistent with the WT phenotype ( Figure 3 ).

[0132] IV. Silencing SlPBB2 affects tomato fruit size

[0133] Experimental method: RNAi-11, RNAi-13, RNAi-16, and wild-type plants were planted under the same conditions (23 °C, 16 h of light and 8 h of darkness). After flowering, relevant fruit data were started to be counted. Fifteen fruits were selected from 10 - 15 plants of each line starting from the 5 dpa stage and marked. The transverse diameter of the fruits was measured every 5 days starting from 5 dpa until 55 dpa.

[0134] (1) Effect of silencing SlPBB2 on tomato fruit size

[0135] The fruits of RNAi plants RNAi-11, RNAi-13, and RNAi-16 at the Br stage (i.e., the breaker stage (Br), also 85 days after germination) were compared with those of the WT. The results are as Figure 4 shown in A below. The fruits of RNAi plants RNAi-11, RNAi-13, and RNAi-16 with silenced SlPBB2 were significantly larger (P < 0.001).

[0136] (2) Effect of silencing SlPBB2 on the single fruit weight of tomatoes

[0137] The single fruit weight of the fruits was statistically analyzed. The results showed that the average single fruit weights of the three SlPBB2 gene-silenced lines (RNAi-11, RNAi-13, and RNAi-16) were 64.90 ± 5.60, 66.02 ± 8.37, and 65.28 ± 9.4 g, respectively, while the single fruit weight of the WT fruits was 49.06 ± 2.83 g( Figure 4 B), and the single fruit weight of the SlPBB2 gene-silenced lines was extremely significantly higher than that of the WT fruits (P < 0.001). Compared with the WT, the fruit weight increased by approximately 32.65% - 34.57% after silencing SlPBB2.

[0138] (3) Effect of silencing SlPBB2 on the transverse diameter during tomato fruit development

[0139] To further observe the change in fruit size, the transverse diameter of the fruits was measured every 5 days starting from 5 dpa (5 days after anthesis). The results are as Figure 4 shown in C. Starting from 10 dpa (10 days after anthesis), the transverse diameters of the fruits of the three SlPBB2 gene-silenced lines were larger than those of the WT and maintained this trend until the fruits matured( Figure 4 C).

[0140] (4) Effect of silencing SlPBB2 on the pericarp thickness of tomato fruits

[0141] By observing the fruit cross-section( Figure 4 D), it can be seen that the pericarp thickness of the SlPBB2 gene-silenced fruits increased. The statistical results are as Figure 4 shown in E. The pericarp thicknesses of the gene-silenced fruits (RNAi-11, RNAi-13, and RNAi-16) were 6.56 ± 0.14, 6.96 ± 0.14, and 7.51 ± 0.24 mm, respectively, all significantly greater than that of the WT (4.54 ± 0.28 mm).

[0142] (5) Effect of silencing SlPBB2 on the pericarp tissue of tomato fruits

[0143] To further observe the effect of silencing SlPBB2 on fruit size at the cellular level, pericarp tissues of fruits at the late stage of cell division and the early stage of cell expansion (20 dpa, 20 days after anthesis) were taken to make paraffin sections and observe cell morphology. The method is as follows:

[0144] ① Fixation: Cut the pericarp at the equatorial part of the fruit into small pieces about 1 cm 2 in size and immediately place them in 50% FAA fixative for more than 24 h;

[0145] ② Dehydration: Dehydration was carried out using a gradient of alcohol, 75% alcohol for 4 h → 85% alcohol for 2 h → 90% alcohol for 1.5 h → 95% alcohol for 1.5 h → 100% alcohol for 1 h → 100% alcohol for 1 h → alcohol-benzene mixture for 20 min → xylene for 20 min → xylene for 20 min → paraffin for 1 - 2 h → paraffin for 1 - 2 h → paraffin for 1 - 2 h.

[0146] ③ Embedding: Transfer the sample into an embedding cassette, immerse it in paraffin wax in an oven at 60 °C for 6 h, then take it out and let it solidify at room temperature.

[0147] ④ Sectioning: Use a microtome to section, and the thickness of the sections is 8 - 12 μm.

[0148] ⑤ Dewaxing: Put the sections into xylene Ⅰ for 20 min, xylene Ⅱ for 20 min, absolute ethanol Ⅰ for 5 min - absolute ethanol Ⅱ for 5 min, 75% alcohol for 5 min, and then wash with water.

[0149] ⑥ Staining: Immerse the tissue sections in toluidine blue staining solution for 3 - 6 min, then wash with water. After passing the microscopic examination, dry the sections in an oven.

[0150] ⑦ Clearing and mounting: Put the sections into xylene for 5 min for clearing. Take the sections out of xylene and let them dry slightly, then mount them with neutral balsam.

[0151] ⑧ Microscopic examination: Examine under a microscope and collect and analyze the images.

[0152] The results are as Figure 4 shown in F and G below. The pericarp thickness of the SlPBB2 gene-silenced fruits (RNAi-11) was significantly thicker than that of the WT. The number of cell layers in the WT pericarp was about 22 layers, while that of the gene-silenced fruits was about 29 layers. Representative cells were selected to statistically analyze the area of individual cells, and it was found that the area of the pericarp cells of the SlPBB2 gene-silenced fruits increased by about 40.97%. The above results indicate that silencing SlPBB2 in tomato fruits promotes cell division and cell expansion during fruit development.

[0153] The above has detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Application, characterized in that, Use of a protein, or a substance that regulates the expression of the coding gene of said protein, or a substance that regulates the activity and / or content of the protein in any of the following: M1) Regulating the size of plant fruits; M2) Preparing a product for regulating the size of plant fruits; M3) Increasing the size of fruits; M4) Cultivating a product for increasing the size of fruits; M5) Cultivating a plant with an increased size of fruits; M6) Preparing a product for cultivating a plant with an increased size of fruits; M7) Plant breeding; The protein is any of the following: A1) A protein with an amino acid sequence of SEQ ID No.1; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1), having more than 80% identity with the protein shown therein and having the same function; A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in either A1) or A2).

2. The application according to claim 1, wherein The substance that regulates the expression of the coding gene of the protein described in claim 1 or regulates the content or activity of the protein described in claim 1 is a substance that reduces the expression of the coding gene of the protein described in claim 1.

3. The application according to claim 2, wherein The substance is any of the following: B1) An RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein or inhibits or reduces or down-regulates the activity or content of the protein; B2) A coding gene that expresses the RNA molecule described in B1); B3) An expression cassette containing the gene described in B2); B4) A recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) A recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) A transgenic plant tissue containing the gene described in B2), or a transgenic plant tissue containing the expression cassette described in B3), or a transgenic plant tissue containing the recombinant vector described in B4); B8) A transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

4. The application according to claim 3, characterized in that, The RNA molecule described in B1) targets the mRNA transcribed from the gene of the protein described in claim 1.

5. The application according to any one of claims 1-4, characterized in that, The plant is any of the following: G1) Monocotyledonous plants or dicotyledonous plants; G2) Plants of the Solanales order; G3) Plants of the Solanaceae family; G4) The Solanum genus; G5) Tomatoes.

6. A method for increasing the size of fruits, characterized in that, The method includes introducing an expression cassette into a target plant to reduce the expression level of the coding gene of the protein described in claim 1 in the target plant and / or reduce the content of the protein and / or reduce the activity of the protein to regulate the fruit size of the target plant; The expression cassette contains a fruit-specific promoter and a silencing gene driven by the fruit-specific promoter, and the silencing gene can transcribe an RNA that silences the protein gene recited in claim 1.

7. The method according to claim 6, wherein The expression cassette is introduced into the target plant through a silencing vector, and the silencing vector contains the expression cassette.

8. The method according to claim 6 or 7, characterized in that, The plant is any one of the following: G1) Monocotyledonous plants or dicotyledonous plants; G2) Solanales plants; G3) Solanaceae plants; G4) Solanum; G5) Tomato.

9. A biological material related to the protein recited in claim 1, and the biological material is any one of the following: B1) An RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein, or an RNA molecule that inhibits or reduces or down-regulates the activity or content of the protein; B2) A coding gene that expresses the RNA molecule described in B1); B3) An expression cassette containing the gene described in B2); B4) A recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) A recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

10. The protein recited in claim 1.

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