Application of SlPRX3 gene in regulation and control of tomato flowering time

Abstract

The invention discloses application of an SlPRX3 gene in regulation and control of tomato flowering time, and belongs to the field of gene engineering. The technical problem to be solved by the invention is how to regulate and control the flowering time of plants and / or the ripening time of stem tip meristem. Therefore, the invention provides a method for regulating and controlling the flowering time and / or the ripening time of the stem apex meristem of the plant, and the method comprises the step of regulating and controlling the expression quantity of a coding gene of SlPRX3 protein in a receptor plant and / or regulating and controlling the activity or content of the SlPRX3 protein in the receptor plant so as to regulate and control the flowering time and / or the ripening time of the stem apex meristem of the receptor plant, the SlPRX3 protein is a protein of which the amino acid sequence is as shown in SEQ ID NO. 3. The invention discloses the effect of the SlPRX3 gene and the protein coded by the SlPRX3 gene in regulating and controlling the flowering time of plants and / or the maturation time of stem tip meristem for the first time, and the SlPRX3 gene plays an important role in improving germplasm resources of crops such as tomatoes and the like.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and specifically relates to SlPRX3 the application of genes in regulating the flowering time of tomatoes. Background Art

[0002] Tomato ( Solanum lycopersicum ) originated in the warm and humid regions of the Andes Mountains in South America. It is a day-neutral plant and can be widely cultivated in different regions. Therefore, tomato is one of the most popular fruit and vegetable crops. The modern cultivated tomato variety AC is often used as a control group in scientific research.

[0003] The fate of stem cells in the shoot apical meristem of plants is determined by the balance of cell proliferation and differentiation to maintain the stem cell population and organ formation. Once the endogenous and environmental signals are sensed and integrated, the shoot apical meristem undergoes a process of gradual maturation, which causes the plant to transition from the vegetative growth stage to the reproductive growth stage, accompanied by continuous leaf production. This process is called shoot apical meristem maturation. Shoot apical meristem maturation determines the flowering time and inflorescence structure of plants, and thus affects the fruit harvest time and yield. Tomato is an important economic crop, and its fruit harvest time is crucial for its economic value. Starting from genes and using genetic engineering means to create tomato plants with an ideal flowering time is helpful for tomato agricultural production. Summary of the Invention

[0004] The technical problem to be solved by this application is: how to regulate the flowering time of plants and / or the maturation time of shoot apical meristems. Specifically, the technical problem to be solved by this application is: how to regulate the flowering time of tomatoes and / or the maturation time of shoot apical meristems.

[0005] To solve the above technical problem, this application provides a method for regulating the flowering time of plants and / or the maturation time of shoot apical meristems. The method includes regulating the expression level of the gene encoding the SlPRX3 protein in the receptor plant and / or regulating the activity or content of the SlPRX3 protein in the receptor plant to regulate the flowering time of the receptor plant and / or the maturation time of the shoot apical meristem. The SlPRX3 protein can be any of the following proteins: a1), a protein with the amino acid sequence shown in SEQ ID NO.3; 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 amino acid sequence shown in a1), and having the same function; a3), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

[0006] Further, in the method described above, the regulation may be to increase or promote or up-regulate.

[0007] Further, in the method described above, the regulation may also be to decrease or inhibit or down-regulate.

[0008] In the present application, the maturation time of the plant shoot apical meristem can be reflected in the number of leaf primordia produced before the plant undergoes the transition to flowering. The more leaf primordia are produced, the later the maturation time of the shoot apical meristem.

[0009] In the present application, the flowering time of the plant can be reflected in the number of leaves before the first inflorescence of the plant. The more leaves there are, the later the flowering time.

[0010] In the present application, the protein may be derived from tomato.

[0011] In the present application, SEQ ID NO.3 consists of 347 amino acid residues.

[0012] The above-mentioned protein can be artificially synthesized, or its coding gene can be synthesized first and then obtained through biological expression.

[0013] a3) The connection can be achieved through a peptide bond.

[0014] The protein tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and / or a SUMO protein tag, etc.

[0015] Further, the method includes reducing the expression level of the coding gene of the SlPRX3 protein in the recipient plant and / or reducing the activity or content of the above-mentioned SlPRX3 protein in the recipient plant, so as to delay the flowering time and / or the maturation time of the shoot apical meristem of the recipient plant, and the recipient plant contains the coding gene of the SlPRX3 protein.

[0016] Further, in the method described above, reducing the expression level of the coding gene of the above-mentioned SlPRX3 protein in the recipient plant and / or reducing the activity or content of the SlPRX3 protein in the recipient plant can be achieved by the following methods M1) or M2), M1), knocking out the coding gene of the SLPRX3 protein in the recipient plant through the CRISPR / Cas system; M2), mutating the genes in the recipient plant as follows: in the two homologous chromosomes of the recipient tomato genome SlPRX3Insert a thymine deoxynucleotide (T) between positions 3132 and 3133 of SEQ ID No.1 of the gene.

[0017] Furthermore, knocking out the coding gene of the SlPRX3 protein in the recipient plant by the CRISPR / Cas system can be achieved by introducing a nucleic acid molecule targeting the coding gene of the SlPRX3 protein and the coding gene of the Cas protein into the recipient plant.

[0018] Furthermore, the target sequence of the nucleic acid molecule targeting the coding gene of the SlPRX3 protein can be the reverse complementary sequence of positions 3116 - 3135 of SEQ ID NO.1 (TCTTGCCTAGGATCATTCGT) and / or positions 3185 - 3204 of SEQ ID NO.1 (GTGGAAGTGCAAACGGAGGA).

[0019] Furthermore, in the method, the nucleic acid molecule and the coding gene of the Cas protein are introduced into the recipient plant in the form of a vector.

[0020] In some embodiments of the present application, the vector containing the nucleic acid molecule and the coding gene of the Cas protein described in M1) can be the recombinant vector pDIRECT - 22C - SlPRX3. The pDIRECT - 22C - SlPRX3 vector can encode an sgRNA targeting the DNA molecule shown in SEQ IDNO.1 and an effector protein of the CRISPR / Cas9 system: Cas9 protein.

[0021] Furthermore, in the method, the plant is selected from dicotyledonous plants.

[0022] Furthermore, in the method, the dicotyledonous plant is selected from solanaceous plants.

[0023] Furthermore, in the method, the solanaceous plant is selected from solanum plants.

[0024] Furthermore, in the method, the solanum plant is selected from tomato ( Solanum lycopersicum )

[0025] The present application also provides a method for obtaining a target tomato with altered flowering time and / or maturation time of the shoot apical meristem. The method includes obtaining a target tomato with delayed flowering time and / or maturation time of the shoot apical meristem by reducing the expression level of the coding gene of the SlPRX3 protein in the recipient plant and / or reducing the activity or content of the SlPRX3 protein in the recipient tomato, and the recipient tomato contains the coding gene of the SlPRX3 protein.

[0026] Furthermore, reducing the expression level of the coding gene of the SlPRX3 protein in the recipient tomato and / or reducing the activity or content of the SlPRX3 protein in the recipient tomato can be achieved by the methods described in M1) or M2) above.

[0027] This application also provides the use of the SlPRX3 protein or a substance that regulates the expression of the coding gene of the SlPRX3 protein or a substance that regulates the activity or content of the SlPRX3 protein in any of the following: A1), in regulating the flowering time of plants; A2), in the application of preparing a product for regulating the flowering time of plants; A3), in regulating the maturation time of the shoot apical meristem of plants; A4), in the application of preparing a product for regulating the maturation time of the shoot apical meristem of plants; A5), in plant breeding or plant-assisted breeding; A6), in the application of preparing a product for plant breeding or plant-assisted breeding.

[0028] In the above applications, the indicators of the plant breeding may include the flowering time and / or the maturation time of the shoot apical meristem.

[0029] In the above applications, the purpose of the plant breeding may include cultivating plants with changed flowering time and / or maturation time of the shoot apical meristem.

[0030] In the above applications, the regulation may be to increase or promote or up-regulate.

[0031] In the above applications, the regulation may also be to decrease or inhibit or down-regulate.

[0032] Furthermore, in the above applications, the substance that regulates the expression of the coding gene of the SlPRX3 protein or the substance that regulates the activity or content of the SlPRX3 protein is a biological material, and the biological material may be any of the following: B1), a nucleic acid molecule that inhibits or reduces the expression of the above SlPRX3 protein coding gene; 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 B2); 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); 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); 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); B8), a nucleic acid molecule encoding the SlPRX3 protein; B9), an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue, and / or a transgenic plant organ containing the gene encoding the nucleic acid molecule described in B8).

[0033] Furthermore, the nucleic acid molecule described in B1) can be an RNA targeting the coding gene of the above-mentioned SlPRX3 protein or a DNA encoding the RNA; The nucleic acid molecule described in B8) can be a DNA molecule described in any one of the following g1)-g3): g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID NO.2; g2), a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID NO.1; g3), a DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the flowering time of plants.

[0034] Furthermore, the target sequence of the nucleic acid molecule described in B1) can be the 3116-3135th position of SEQ ID NO.1 (TCTTGCCTAGGATCATTCGT) and / or the reverse complementary sequence of the 3185-3204th position of SEQ ID NO.1 (GTGGAAGTGCAAACGGAGGA).

[0035] In some embodiments of the present application, the expression cassette described in B2) can be an expression cassette for transcribing RNA targeting the coding gene of the above-mentioned SlPRX3 protein.

[0036] The recombinant vector described in B9) can be the pDIRECT-22C-SlPRX3 vector. The pDIRECT-22C-SlPRX3 vector can encode an sgRNA targeting the DNA molecule shown in SEQ ID NO.1 and an effector protein of the CRISPR / Cas9 system: Cas9 protein.

[0037] Furthermore, in the above application, the expression cassette in B9) refers to the DNA capable of expressing the SlPRX3 protein in a host cell. This DNA may not only include the promoter that initiates the transcription of the SlPRX3 protein-encoding gene, but also include the terminator or / and enhancer sequence that terminates the transcription of the SlPRX3 protein-encoding gene.

[0038] Furthermore, in the above application, the recombinant microorganism can specifically be yeast, bacteria, algae, and fungi.

[0039] Furthermore, in the above application, the plant tissue can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0040] Furthermore, in the above application, the transgenic plant organs can be the roots, stems, leaves, flowers, fruits, and seeds of transgenic plants.

[0041] Furthermore, in the above application, the transgenic plant cell line, transgenic plant tissue, and transgenic plant organ may or may not include propagation materials.

[0042] Furthermore, in the above application, the plant can be selected from dicotyledonous plants.

[0043] Furthermore, in the above application, the dicotyledonous plant is selected from solanaceous plants.

[0044] Furthermore, in the above application, the solanaceous plant is selected from solanum plants.

[0045] Furthermore, in the above application, the solanum plant is selected from tomato ( Solanum lycopersicum )

[0046] The above-mentioned SlPRX3 protein and / or the above-mentioned biological material are also the protection scope of this application.

[0047] In this application, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences (or nucleotide 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 the 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 a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0048] The above-mentioned identity of 80% or more can be an identity of 80%, 85%, 90% or more than 95%.

[0049] The identity of more than 80% can be an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The identity of more than 85% can be an identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The identity of more than 90% can be an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The identity of more than 95% can be an identity of at least 95%, 96%, 97%, 98% or 99%.

[0050] The beneficial technical effects obtained in this application are as follows: This application discloses for the first time SlPRX3 the application of the gene and the SlPRX3 protein encoded thereby in regulating the flowering time of plants and / or the maturation time of the shoot apical meristem of plants. The verification results show that this gene can significantly regulate the flowering time of plants and / or the maturation time of the shoot apical meristem. The tomato gene SlPRX3 can be widely applied to plant fields such as tomato genetic breeding, germplasm resource improvement, transgenic and genome editing breeding, and plays an important role in improving and modifying the germplasm resources of crops such as tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the statistical result of plant phenotypes. a. Schematic diagram of the target sites of the CRISPR / Cas9-edited SlPRX3 gene and the identification results of mutant genotypes. SlPRX3 There was an insertion of one base, resulting in the loss of gene function. b. Inflorescence meristem diagrams of AC plants and slprx3 mutant plants. Scale bar = 100 μm. c. Statistics of the number of leaf primordia produced by AC plants and slprx3 mutant plants before the transition to flowering. The quantitative results are the mean ± standard deviation of 32 and 39 replicates respectively, and the statistical data was analyzed by Student's t test, and *** indicates Student's t test P <0.001. e. Photos of AC plants and slprx3 mutant plants. Plant scale bar = 2 cm, inflorescence scale bar = 1 cm. f. AC plants and slprx3Statistics of the flowering time of mutant plants. The quantitative results are the mean ± standard deviation of 15 and 6 replicates respectively, and the statistical data were analyzed by Student's t test, and *** indicates Student's t test P < 0.001.

[0052] Figure 2 is the map of the recombinant vector pDIRECT-22C-SlPRX3. Detailed implementation manners

[0053] The present application will be further described in detail below in conjunction with the specific implementation manners. The examples given are only for clarifying the present application, rather than limiting the scope of the present application. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present application in any way.

[0054] The experimental methods in the following examples 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 examples can be obtained from commercial sources unless otherwise specified.

[0055] 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 / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0056] The quantitative tests in the following examples are all set with three replicates unless otherwise specified, and the results are averaged.

[0057] The following examples use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and are analyzed by t -test, *** ( P < 0.001) indicates a highly significant difference.

[0058] Example 1. SlPRX3 can regulate the flowering time of tomatoes This example found that the SlPRX3 gene ( Solyc01g108320 ) of tomatoes can regulate their flowering time. In tomato AC, SlPRX3 the genomic sequence is as shown in SEQ ID No.1 in the sequence listing, its CDS sequence is as shown in SEQ ID No.2, and it encodes the SlPRX3 peroxidase shown in SEQ ID No.3.

[0059] I. Preparation of the gene editing target vector for tomato SlPRX3 ​Genetic editing of SlPRX3 genes was performed using the CRISPR-Cas9 system. SlPRX3 There are two target sequences for the

[0060] gene, namely target 1 (TCTTGCCTAGGATCATTCGT, positions 3116-3135 of SEQ ID NO.1) and target 2 (the reverse complementary sequence of positions 3185-3204 of SEQ ID NO.1, GTGGAAGTGCAAACGGAGGA). Using the pDIRECT-22C vector digested with BanI as a template, PCR amplification was performed using 22C-SlPRX3 F1 and 22C-SlPRX3R1 to obtain a PCR fragment, which is the vector promoter fragment and SlPRX3 the first 12 nucleotide sequences of the first target of the

[0061] gene. SlPRX3 Using the pDIRECT-22C vector as a template, PCR amplification was performed using 22C-SlPRX3 F2 and 22C-SlPRX3 R2 to obtain a PCR fragment, which is

[0062] the last 12 nucleotide sequences of the first target of the SlPRX3 gene and the first 12 nucleotide sequences of the second target.

[0063] Using the pDIRECT-22C vector as a template, PCR amplification was performed using 22C-SlPRX3 F3 and 22C-SlPRX3 R3 to obtain a PCR fragment, which is

[0064] the last 12 nucleotide sequences of the second target of the

[0063] gene.

[0064] Dilute the vector pDIRECT-22C to 50 ng, mix the obtained PCR fragments equally and then dilute to 5-7 ng. Add SapI, BanI, T4 ligase and 10×T4 ligase buffer, and make up the volume to 20 μL for the Golden Gate ligation reaction while cutting. The program is 37°C for 5 min, 16°C for 10 min, for 20 cycles.

[0064] Transform the ligated fragment into DH5α Escherichia coli competent cells, and screen for recombinant resistant bacteria on kanamycin medium. Use colony identification primers F / R to screen for correct positive clone plaques, and determine the correctness of the vector fragment by plasmid extraction and sequencing. Then transform the correct plasmid (i.e., recombinant vector pDIRECT-22C-SlPRX3) into AGL1 Agrobacterium competent cells.

[0065] The nucleotide sequence of the recombinant vector pDIRECT-22C-SlPRX3 is shown below, and its map is Figure 2 . The 4214th to 4559th positions of the nucleotide sequence of the recombinant vector pDIRECT-22C-SlPRX3 are the CaMV 35S promoter, the 5208th to 9311th positions are the coding sequence of Cas9, the 10132nd to 10151st positions are the sequence of SlPRX3-target1 (the first target of the SlPRX3 gene), the 10152nd to 10227th positions are the gRNA scaffold, the 10248th to 10267th positions are the sequence of SlPRX3-target2 (the second target of the SlPRX3 gene), and the 10268th to 10343rd positions are the gRNA scaffold.

[0066] The primers used are as follows: 22C-SlPRX3 F1: TGCTCTTCGCGCTGGCAGACATACTGTCCCAC; 22C-SlPRX3 R1: TGGTCTCC TCCTAGGCAAGA CTGCCTATACGGCAGTGAAC ; 22C-SlPRX3 F2: TGGTCTCA AGGATCATTCGT GTTTTAGAGCTAGAAATAGC ; 22C-SlPRX3 R2: TGGTCTCC TTGCACTTCCAC CTGCCTATACGGCAGTGAAC ; 22C-SlPRX3 F3: TGGTCTCA GCAAACGGAGGA GTTTTAGAGCTAGAAATAGC ; 22C-SlPRX3 R3: TGCTCTTCTGACCTGCCTATACGGCAGTGAAC.

[0067] II. Genetic transformation of tomatoes After introducing the recombinant vector pDIRECT-22C-SlPRX3 obtained in Step 1 into AGL1 Agrobacterium, use the obtained recombinant Agrobacterium to perform tomato genetic transformation by the leaf disc method. The specific steps are as follows: (1) Seed preparation: Disinfect tomato AC seeds (i.e., tomato Ailsa Craig seeds, hereinafter referred to as AC) with 75% ethanol for 1 min, rinse once with sterile distilled water, then treat with 10% sodium hypochlorite solution for 10 - 15 min, and then wash with sterile distilled water 3 - 5 times. Finally, sow the seeds into 1 / 2 MS medium (2.2 g / L MS powder, 30 g / L sucrose, 5 g / L phytagel), and place them in a dark environment at 26℃ for 48 h, then transfer them to an incubator at 26℃ with a photoperiod of 16 h light / 8 h dark for 5 - 7 days; (2) Explant preparation: When the cotyledons have extended and no true leaves have emerged, cut off the tips of the cotyledons and cut them into square explants. Immerse them in MSO liquid medium (4.4 g / L MS powder, 15 g / L sucrose) for 1 h, discard the liquid, and blot the remaining medium on the cotyledons with sterile filter paper. Then place the explants flat on D1 medium (4.4 g / L MS powder, 30 g / L sucrose, 5 g / L phytagel, 1 mg / L zeatin, 0.2 mg / L 2,4 - D, 0.1 mg / L kinetin) covered with sterile filter paper, with the adaxial side facing up, and culture in the dark for 2 days; (3) Bacterial liquid preparation: Transfer the plasmid into Agrobacterium tumefaciens strain AGL1. Pick a positive monoclonal colony and inoculate it into 3 mL LB liquid medium with the corresponding resistance, and then place it on a shaker at 28℃ and 220 rpm for overnight culture. The next day, inoculate it into 30 mL LB liquid medium containing the corresponding antibiotic at a ratio of 1:100, place it on a shaker at 28℃ and 220 rpm and continue to culture until the OD600 is 0.6 - 0.8. Then centrifuge at 6000 rpm for 10 min at room temperature to collect the bacteria, and resuspend the bacteria with MSO solution for standby; (4) Explant infection: Shake - culture the explants and the bacterial liquid for 15 min. After blotting the excess bacterial liquid with sterile filter paper, place them flat on D1 medium covered with sterile filter paper, and culture in the dark for 2 days; (5) Inducing callus and bud formation: Transfer the explants cultured in the dark from D1 medium to 2Z IAA medium (4.4 g / L MS powder, 20 g / L sucrose, 5 g / L phytagel, 2 mg / L zeatin, 200 mg / L ticarcillin, 150 mg / L kanamycin, 0.1 mg / L IAA, 500 μM ascorbic acid), and place them in an incubator at 26℃ with a photoperiod of 16 h light / 8 h dark for culture. Replace the medium every three weeks until callus and buds are formed on the explants; (6) Inducing rooting: Cut the buds that have grown to 1 - 2 cm and place them in the MSSV medium (4.4 g / L MS powder, 20 g / L sucrose, 5 g / L phytagel, 200 mg / L Timentin, 150 mg / L kanamycin, 0.1 mg / L IAA, 500 μM ascorbic acid) until roots grow. (7) When the rooted seedlings grow to 5 cm, extract the leaf DNA for gene editing identification, and transplant the identified positive seedlings. The primers used are as follows: pDIRECT - 22C vector Cas9 PCR identification primer F: GGAGAACCAGCTGTTGTTCCACAT; pDIRECT - 22C vector Cas9 PCR identification primer R: TCTGGTAGCCTCAGCAGTTTCACCA.

[0068] One slprx3 mutant was identified among the offspring of the positive seedlings, and this mutant is SlPRX3 a homozygous mutation of the SlPRX3 gene. The sequencing results showed that: compared with the wild type, a thymine deoxynucleotide (T) was inserted between the 3132nd and 3133rd positions of SEQ ID No.1 in one pair of homologous chromosomes of the SlPRX3 gene in this mutant, thereby knocking out the Figure 1 gene (a in

[0069] slprx3 The mutant self - crossed to obtain the homozygous T2 generation slprx3 mutant for phenotype observation.

[0070] III. Plant phenotype statistics and detection Test plants: Tomato AC, the T2 generation slprx3 mutant obtained in step II.

[0071] Pot the test plants, flower pot specifications: diameter 20.6 cm, height 17.5 cm, bottom 15 cm. Plant one plant in each pot.

[0072] (1) Statistics of the stem - tip meristem maturation time rate: Under a stereomicroscope, peel off the leaf primordia of the tomato plants, take pictures of the inflorescence meristem and count the number of leaf primordia produced before the inflorescence meristem.

[0073] The results showed that slprx3 compared with the AC plants, the number of leaf primordia produced by the Figure 1 mutant plants before the floral transition was significantly more than that of the AC plants (b, c in slprx3 ). This result indicates that: compared with the AC plants, the maturation time of the stem - tip meristem of the

[0074] (2)Flowering time statistics: When the first panicle inflorescence grows, count the number of true leaves below it.

[0075] The results showed that slprx3 Compared with AC plants, the number of true leaves produced by mutant plants before the first panicle inflorescence was significantly more than that of AC plants ( Figure 1 in d, e). This result indicates that slprx3 The flowering time of mutant plants was later than that of AC plants.

[0076] The above results indicate that SlPRX3 The gene and its encoded protein can regulate the flowering time and / or the maturation time of the shoot apical meristem of tomatoes, SlPRX3 The flowering time of the homozygous mutant obtained by gene editing was later than that of the wild type. It shows that SlPRX3 After gene knockout, the flowering time and / or the maturation time of the shoot apical meristem of tomatoes were delayed.

[0077] SEQ ID No.1 ( SlPRX3 ( Solyc01g108320 ), genomic sequence (including 3 kb upstream and 1 kb downstream)) is as follows: ATGGCGAGTCGTAGTTTGCTCCCCTGCCTTAC TGTTTTGTTCTGTATGATTAGTATTATGGCTCCTTTAACTTTTGGTCAGCTTGATTACAGTTACTATGATAGAGCA TGCCCCGTCTTGCCTAGGATCATTCGTTGGAACGTTTGGTCGGCTCTTCGTAATGATTCCAGAATAGCTGCATCCC TCCTCCGTTTGCACTTCCACGACTGTTTTGTAAAT GTAATTTCTCTAATAGTCGTTTTATTGTGTACTACTCATCCCAGTTTAATTGCATACTCTTAGGGTAATACTTTATTTATTCGTCAGTTTATGCTTATGTTTGACAG GGTTGTGATG GATCTGTGCTTCTTGATGACACAAATGATTTCAAGGGTGAGAAGAATGCTGCACCAAATCGCAATTCAGTTCGAGG ATTTGAGACAATCGATAACATAAAAGCAGACCTTGAAAGAGCTTGTCCATTTACTGTATCTTGTGTGGATATATTA ACTCTTGCTGCTAGAGAAGTTGTCGTCATG GTAAGTATCAACATATTTGACGTGCAGACAAGTATTGTAATAATGCCTGAATATGAACAGAAGACTATATGTTAATTTCCTTTCTTCCTTTACAG TCAGGAGGACCATTTTGGCCAGTTTTA CTTGGTCGACGAGATGGTCTAACTGCAAGTGAAAAAGCAGCAAATGAACAATTACCTTCACCCTTTGAGCCCCTGG ATAAAATCGCGGCCAAGTTTACTGATAAGGGCCTTAATCTAAGGGATGTAGTAGTTCTTTCAG GTACCTAGCTTCTATCCCCCAGTATCATATTTTTGTATACAAAGAATCCCTTTCAAAGTTTCCAGTGTAACAACATTTTAAGTATGGTTCTAATGAAATTTCTGACTCTACGACTAATATAG GAGCGCACACAATTGGTTTTGCTCAATGTTTCACATTCAAGAG GAGACTTTTCAACTATCAAGACTCTGGAAAGCCAGATCCACTTCTTGATTATTCAATGTTGTTAAATTTACAAAGC ACTTGTCCTGAAGAGGGACCAAACTCCAAAATTACTCCTCTAGATAACCAATCCGTTACGCGATTCGACAATGCAT ATTACAGAAACCTTATGAACAACACAGGACTACTTGAATCTGATCAAGCTCTAATGTCAAATTCTGAGACAGCTGA TATGGTTAAAGCCTACAGTTTGTACCCTTATCTTTTCTACCAAGACTTTGCTGCATCAATGGTAAAATTAGGAAAT ATTGGGGTCCTTACTGGAGAAAGTGGACAAATTAGAAAAGTATGTGGCTCTGTAAATTACTACTATTAAAGTGTTTATGTTAAGTATGTGTAGTTTAATTATTAGACAAAGTTTATCAAGTGTGTTTGCTTCAATATGTATTTTTGTACTAATAATATCAAATGATATCGTTCAAGTAGATATTTTCGTAGTTCGGTATAATTAATTTATTTATTTTGGATCATAAAAAAGTTTCGATTTTTTTTAAAAAAAATATTGTAACGAAGGAAGAAAATGGACAACGGGATCCCACTTTAGTAGGAAAACTTTGGTCTATTAGAATATATAATTTAAAGAAGGAAAAAAGGGTCCATCCATATGCTTGACTTTCGATGTGCTAAAAGAAAATATTGTACTCTTTTGTTCTTTATGCTCAACGCCCCTTCAATTTTATGTTGTCACTGATTCTATGTTCTCTAAATTCCCTTTTAATTATTCTATAGTTCGAATTTTATGTAGTATATTGTTTTTTTTTCTTTTTCTTTATATTTTTCATATGCCGTCTAATTTCTATATTAAAATCCTGATTAAATTCGTATCATGCATTATAGGACTTATTCAAAAGAGACTCTCTCAACATAAATATTCTTCATATACATATTTTAAACCCAAAACCTTTGATTTTACAAAGTTATGGAAAGATAAGGAAAGAAAGGGACAAGAAAATGATAGATTCGGAAAAATAAATTAAATTAAATTATTTTCTCTCTTATTTTACCGAATATAGTCACTTGATATGTATTTTTGTTAACGAGAAACGATGTGTTATCTATGAAATCAGTGGAAGCAAACATAAGCTAATCTAATCATAATCACATATTTAATATAATTTCATAAGTAAAACTCGGACTAAATAAAGTATTACATTTAACTCACGAAGAGACTTTTAACTACAAAGATAAAAAGATTGTTTCTAAAAAAAAAAAATTAGCAGGGTGGGGTGAGGCTGACTAAACCTATGTGTTATATATCAACATCAACCTATCAAGCTTTTAATTTATCCTTTGTCATCTCCTGCAAAAAAAGCATC; SEQ ID No.2 ( SlPRX3 ( Solyc01g108320 ),CDS sequence) is as follows: ATGGCGAGTCGTAGTTTGCTCCCCTGCCTTACTGTTTTGTTCTGTATGATTAGTATTATGGCTCCTTTAACTTTTGGTCAGCTTGATTACAGTTACTATGATAGAGCATGCCCCGTCTTGCCTAGGATCATTCGTTGGAACGTTTGGTCGGCTCTTCGTAATGATTCCAGAATAGCTGCATCCCTCCTCCGTTTGCACTTCCACGACTGTTTTGTAAATGGTTGTGATGGATCTGTGCTTCTTGATGACACAAATGATTTCAAGGGTGAGAAGAATGCTGCACCAAATCGCAATTCAGTTCGAGGATTTGAGACAATCGATAACATAAAAGCAGACCTTGAAAGAGCTTGTCCATTTACTGTATCTTGTGTGGATATATTAACTCTTGCTGCTAGAGAAGTTGTCGTCATGTCAGGAGGACCATTTTGGCCAGTTTTACTTGGTCGACGAGATGGTCTAACTGCAAGTGAAAAAGCAGCAAATGAACAATTACCTTCACCCTTTGAGCCCCTGGATAAAATCGCGGCCAAGTTTACTGATAAGGGCCTTAATCTAAGGGATGTAGTAGTTCTTTCAGGAGCGCACACAATTGGTTTTGCTCAATGTTTCACATTCAAGAGGAGACTTTTCAACTATCAAGACTCTGGAAAGCCAGATCCACTTCTTGATTATTCAATGTTGTTAAATTTACAAAGCACTTGTCCTGAAGAGGGACCAAACTCCAAAATTACTCCTCTAGATAACCAATCCGTTACGCGATTCGACAATGCATATTACAGAAACCTTATGAACAACACAGGACTACTTGAATCTGATCAAGCTCTAATGTCAAATTCTGAGACAGCTGATATGGTTAAAGCCTACAGTTTGTACCCTTATCTTTTCTACCAAGACTTTGCTGCATCAATGGTAAAATTAGGAAATATTGGGGTCCTTACTGGAGAAAGTGGACAAATTAGAAAAGTATGTGGCTCTGTAAATTACTACTATTAA; SEQ ID No.3 ( SlPRX3 ( Solyc01g108320 ), protein sequence) is as follows: MASRSLLPCLTVLFCMISIMAPLTFGQLDYSYYDRACPVLPRIIRWNVWSALRNDSRIAASLLRLHFHDCFVNGCDGSVLLDDTNDFKGEKNAAPNRNSVRGFETIDNIKADLERACPFTVSCVDILTLAAREVVVMSGGPFWPVLLGRRDGLTASEKAANEQLPSPFEPLDKIAAKFTDKGLNLRDVVVLSGAHTIGFAQCFTFKRRLFNYQDSGKPDPLLDYSMLLNLQSTCPEEGPNSKITPLDNQSVTRFDNAYYRNLMNNTGLLESDQALMSNSETADMVKAYSLYPYLFYQDFAASMVKLGNIGVLTGESGQIRKVCGSVNYYY*; The nucleotide sequence of the recombinant vector pDIRECT-22C-SlPRX3 is as follows:

[0078] The above has described the present application in detail. For those skilled in the art, without departing from the spirit and scope of the present application and without the need for unnecessary experiments, the present application can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present application are given, it should be understood that the present application can be further improved. In general, according to the principle of the present application, the present application is intended to include any modifications, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed in the present application.

Claims

1. A method for regulating the flowering time and / or the maturation time of shoot apical meristems of plants, characterized in that, The method includes regulating the expression level of the coding gene of SlPRX3 protein in the receptor plant and / or regulating the activity or content of the SlPRX3 protein in the receptor plant to regulate the flowering time and / or the maturation time of the shoot apical meristem of the receptor plant, and the SlPRX3 protein is any one of the following proteins: a1), a protein with the amino acid sequence shown in SEQ ID NO.3; a2), a protein which is obtained by substituting, deleting and / or adding amino acid residues to the amino acid sequence shown in a1) and has more than 80% identity with the amino acid sequence shown in a1) and has the same function; a3), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of a1) or a2).

2. The method according to claim 1, wherein The method includes reducing the expression level of the coding gene of the SlPRX3 protein in the receptor plant and / or reducing the activity or content of the above-mentioned SlPRX3 protein in the receptor plant to delay the flowering time and / or the maturation time of the shoot apical meristem of the receptor plant, and the receptor plant contains the coding gene of the SlPRX3 protein.

3. The method according to claim 2, wherein The reduction of the expression level of the coding gene of the above-mentioned SlPRX3 protein in the receptor plant and / or the reduction of the activity or content of the SlPRX3 protein in the receptor plant are achieved by the following method M1) or M2), M1), knocking out the coding gene of the SlPRX3 protein in the receptor plant through the CRISPR / Cas system; M2), mutate the gene in the recipient plant as follows: Insert a thymidine monophosphate between the 3132nd and 3133rd positions of SEQ ID No.1 of the gene on two homologous chromosomes of the recipient tomato genome. SlPRX3 ​ 4. A method for obtaining a target tomato with a delayed flowering time and / or a delayed shoot apical meristem maturation time, characterized in that, The method includes obtaining a target tomato with a delayed flowering time and / or a delayed maturation time of the shoot apical meristem by reducing the expression level of the coding gene of the SlPRX3 protein in the receptor tomato and / or reducing the activity or content of the SlPRX3 protein in the receptor tomato, and the receptor tomato contains the coding gene of the SlPRX3 protein.

5. The method according to claim 4, characterized in that, The reduction of the expression level of the coding gene of the SlPRX3 protein in the receptor tomato and / or the reduction of the activity or content of the SlPRX3 protein in the receptor tomato are achieved by the above-mentioned method M1) or M2).

6. Use of the SlPRX3 protein described in claim 1 or a substance for regulating the expression of the coding gene of the SlPRX3 protein or a substance for regulating the activity or content of the SlPRX3 protein in any one of the following, A1), use in regulating the maturation time of the shoot apical meristem of plants; A2), use in preparing a product for regulating the maturation time of the shoot apical meristem of plants; A3), use in regulating the flowering time of plants; A4), use in preparing a product for regulating the flowering time of plants; A3), use in plant breeding or plant assisted breeding; A4), use in preparing a product for plant breeding or plant assisted breeding.

7. The application according to claim 6, characterized in that The substance for regulating the expression of the coding gene of the SlPRX3 protein or the substance for regulating the activity or content of the SlPRX3 protein is a biological material, and the biological material is any one of the following: B1), a nucleic acid molecule that inhibits or reduces the expression of the coding gene of the SlPRX3 protein described in 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 B2); 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); 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); 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); B8), a nucleic acid molecule encoding the SlPRX3 protein described in claim 1; B9), an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue and / or a transgenic plant organ containing the gene encoding the nucleic acid molecule described in B8).

8. The application according to claim 6, wherein The nucleic acid molecule described in B1) is an RNA targeting the coding gene of the above-mentioned SlPRX3 protein or a DNA encoding the RNA; The nucleic acid molecule described in B8) is a DNA molecule as described in any one of the following g1)-g3): g1), a DNA molecule whose coding sequence of the coding strand is SEQ ID NO.2; g2), a DNA molecule whose nucleotide sequence of the coding strand is SEQ ID NO.1; g3), a DNA molecule having more than 80% identity with the DNA molecule described in g1) or g2) and regulating the flowering time of plants.

9. The application according to any one of claims 1-3, characterized in that, The plant is selected from dicotyledonous plants.

10. The SlPRX3 protein described in claim 1 and the biological materials described in claims 7-9.

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