Gene SlYABBY1 for regulating and controlling tomato stigma exposure as well as encoding protein and application thereof

Abstract

The invention relates to a gene SlYABBY1 for regulating and controlling stigma exsertion as well as an encoding protein and application thereof. A tomato material of a stigma exsertion type male sterile line can be obtained by utilizing related biological materials of the gene through gene editing. The gene and the encoded protein thereof have important theoretical and practical significance for research on a tomato stigma exsertion regulation and control molecular mechanism. Experiments prove that the gene editing method is effective, a wild tomato material can be quickly converted into a stigma-exposed tomato material, and an economical, quick and effective way is provided for creating a tomato stigma-exposed male sterile line material. The invention has important application and market prospects in the field of agriculture.

Description

Technical Field

[0001] The present invention relates to plant genes, their encoded proteins and applications, in particular to a gene SlYABBY1 that regulates tomato stigma exsertion, its encoded protein and applications. Biomaterials related to this gene or its encoded protein can be used to cultivate tomato stigma-exserted male sterile plants and for tomato breeding. Background Art

[0002] Tomato (Solanum lycopersicum) is a vegetable crop widely cultivated worldwide. Hybrid seed production is a key link in the development of the tomato seed industry. Compared with traditional hybrid seed production, the use of male sterile lines can eliminate manual emasculation, reduce the cost of seed production, and the pollen is aborted, avoiding self-pollination and ensuring the seed purity of hybrid seed production. Among the functional sterility types, the stigma-exserted type can avoid manual emasculation in hybrid seed production, and the pistil and pollen viability are normal. Its sterile trait can be maintained by artificial assisted self-pollination, which is beneficial to the popularization of tomato male sterile lines and has certain advantages and application prospects in the process of hybrid seed production.

[0003] The tomato stigma-exserted trait belongs to a quantitative trait. Genes controlling stigma exsertion vary among different materials, and this trait is also affected by the external environment. Currently, three genes related to controlling stigma exsertion have been cloned: SE3.1, style2.1, and SlLst. Studies have shown that Style2.1 and SE3.1 control stigma exsertion in a dominant inheritance manner, which is difficult to apply in production, while SlLst is the major gene controlling stigma exsertion in the material 'T431'.

[0004] Previous studies have shown that the YABBY gene family has functions in determining the boundary properties of meristems and determining the polar morphogenesis of lateral organs, especially flower organs (Golz and Hudson, 1999; Bowman, 2000). For example, the mutant fil of AtYABBY1 produces flowers with clusters of filamentous structures, and the number and shape of flower organs have changed. In particular, the morphology of anthers, filaments, styles, and stigmas has changed or even become shorter. This gene plays a key role in the morphogenesis of flower organs. There are nine members of the YABBY gene family in tomato, namely YAB1a, YAB3 / YAB1b, YAB2a, FAS / YAB2b, YAB5a, YAB5b, INO, CRCa, and CRCb, and their functions have not been fully clarified (Huang et al., 2013). Currently, only YAB2b and CRCa have been reported to be involved in regulating the size of flowers and fruits (Sun et al., 2013; Yang et al., 2022). However, there is currently no report on the function of YABBY1 (Solyc01g091010). Summary of the Invention

[0005] Aiming at the defects of the prior art, the object of the present invention is to provide a gene for regulating stigma exsertion in tomatoes, its encoded protein and application. Through a large amount of analysis, the inventors found that the SlYABBY1 gene or protein has the function of regulating stigma exsertion in tomatoes, and thus the present invention is proposed. A tomato material with stigma exsertion can be obtained by using biological materials related to the protein SlYABBY1. This material can be used as a male sterile line material for hybrid seed production. At the same time, the male sterile line material with stigma exsertion can maintain its sterile trait through artificial assisted self-pollination, which is beneficial to the breeding of tomato male sterile lines.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a protein SlYABBY1 for regulating stigma exsertion in tomatoes, which has the amino acid sequence described in any one of the following (1) to (3):

[0008] (1) The amino acid sequence shown in Sequence 2 in the sequence listing;

[0009] (2) The amino acid sequence shown in Sequence 2 in the sequence listing, which has been substituted, deleted or added with one or several amino acid residues and has the function of regulating stigma exsertion in flowering tomatoes;

[0010] (3) An amino acid sequence derived from tomatoes, which has at least 75% identity with the amino acid sequence in (1) or (2) and has the function of regulating stigma exsertion in tomatoes.

[0011] This protein is called SlYABBY1, and Sequence 2 in the sequence listing consists of 242 amino acid residues. The maintenance of the gene function can prevent the stigma of tomatoes from being exserted, while the deletion of the gene function will lead to the exsertion of the tomato stigma.

[0012] The second aspect of the present invention provides a gene for regulating stigma exsertion in tomatoes, named Solyc01g091010 (hereinafter referred to as SlYABBY1), which is derived from the tomato variety Alisa Craig, and the nucleotide sequence of its cDNA is any one of the following (a1) or (a2):

[0013] (a1) The DNA sequence encoding the amino acid sequence described in the first aspect of the present invention; preferably the DNA sequence shown in Sequence 1 in the sequence listing;

[0014] (a2) A DNA sequence that hybridizes with the DNA sequence described in (a1) under stringent conditions and encodes a protein molecule with the function of regulating stigma exsertion in tomatoes.

[0015] The stringent conditions may be: hybridizing and washing the membrane at 65°C in a solution of 0.1X SSPE (or 0.1X SSC) and 0.1% SDS.

[0016] The DNA molecule of Sequence 1 in the sequence listing consists of 729 nucleotides, and its coding sequence is the nucleotides at positions 1-729 from the 5' end. This DNA molecule encodes a protein with the amino acid residue sequence of Sequence 2 in the sequence listing.

[0017] The third aspect of the present invention provides a biological material related to the protein described in the first aspect above or the gene described in the second aspect above. The biological material is selected from the following (a) or (b):

[0018] (a) A substance for silencing or inhibiting the expression of the coding gene of the protein SlYABBY1 described in the first aspect of the present invention or the gene described in the second aspect of the present invention, or a substance for knocking out the coding gene of the protein SlYABBY1 described in the first aspect of the present invention or the gene described in the second aspect of the present invention;

[0019] (b) A substance for reducing or inhibiting the activity and / or content of the protein SlYABBY1 described in the first aspect of the present invention in tomatoes.

[0020] In the biological material of the third aspect of the present invention, the biological material can be any material that realizes the functions described in (a) or (b). Considering efficiency and operation, as a preferred embodiment, the biological material is a tomato CRISPR / Cas9 genome editing system, including: a recombinant expression vector, the recombinant expression vector includes a Cas9 expression cassette and an sgRNA expression cassette. The Cas9 expression cassette expresses Cas9, and the sgRNA expression cassette expresses sgRNA. The target gene of the sgRNA is a partial or all DNA fragment of the coding gene of the protein SlYABBY1 in tomatoes. More preferably, in the present invention, the sgRNA includes sgRNA1 and sgRNA2, and both sgRNA1 and sgRNA2 specifically target the 3rd exon of the SlYABBY1 gene.

[0021] Furthermore, the target sequence of sgRNA1 is the DNA molecule shown in Sequence 3 in the sequence listing (i.e., AATCACTCATGCCAGTCCG), or the target sequence of sgRNA1 is the reverse complementary sequence of the sequence shown in Sequence 3 in the sequence listing;

[0022] The target sequence of sgRNA2 is the DNA molecule shown in Sequence 4 in the sequence listing (i.e., CCAGTCGCAAACAGACGTA), or the target sequence of sgRNA2 is the reverse complementary sequence of the sequence shown in Sequence 4 in the sequence listing.

[0023] In a specific embodiment, the recombinant expression vector can be obtained by inserting the coding DNA of the target sequence binding region (i.e., the sgRNA expression cassette) into the BsaI restriction site of the CRISPR / Cas9 vector.

[0024] The biological material can also be a transgenic cell line containing the above recombinant expression vector, a host bacterium containing the above recombinant expression vector, and other products that can be used for gene editing. In addition, primers used to amplify any fragment of the gene related to regulating tomato stigma exsertion for screening tomato materials with expected gene mutations from the tomato materials obtained after transformation with the gene editing system also fall within the protection scope of the present invention.

[0025] In a specific embodiment of the present invention, the host bacterium containing the above recombinant expression vector is Agrobacterium tumefaciens GV3101 containing the recombinant expression vector pTX041::CR-SlYABBY1.

[0026] The fourth aspect of the present invention provides the application of the biological material described in the third aspect of the present invention in creating tomato materials with stigma exsertion or tomato breeding.

[0027] Furthermore, the tomato material with stigma exsertion is obtained by disabling the function of the protein SlYABBY1 in the wild-type tomato material AC using the biological material described in the third aspect above. When the obtained tomato material with stigma exsertion is a homozygous mutant of the SlYABBY1 gene, the tomato material with stigma exsertion can be used as a male sterile line for cross-breeding. In addition, since the pistils and pollen viability of this male sterile line are normal, the breeding of the sterile line can be achieved through artificial assisted self-pollination, and the breeding method is simple and reliable.

[0028] The fifth aspect of the present invention provides a method for regulating tomato stigma exsertion, the method comprising editing the gene SlYABBY1 in the genome of a tomato material using the tomato CRISPR / Cas9 genome editing system described in the third aspect of the present invention, thereby disabling the function of SlYABBY1 to obtain a tomato material with stigma exsertion; in the above method, as an implementable embodiment, the method specifically comprises:

[0029] S1 Introduce the tomato CRISPR / Cas9 genome editing system described in the third aspect of the present invention into the tomato material, and obtain successfully transformed plants (i.e., T0 generation regenerated tomato plants) through screening;

[0030] S2 Obtain gene SlYABBY1 mutant lines from the successfully transformed plants through identification, that is, obtain plants with edited genes.

[0031] Furthermore, the identification is performed by using the genome of the successfully transformed plant as a template for PCR amplification of the gene SlYABBY1 fragment, and then detecting by gel electrophoresis or sequencing to obtain the lines with mutated SlYABBY1 gene.

[0032] Furthermore, the tomato material is tomato variety AC.

[0033] Since tomato is a diploid plant, when Cas9 starts to cut the specific SlYABBY1 gene, both alleles on the two homologous chromosomes in the same cell may be edited. Therefore, the gene-edited plants include SlYABBY1 gene homozygous mutants and heterozygous mutants. A SlYABBY1 homozygous mutant refers to a plant in which the SlYABBY1 genes on the two homologous chromosomes have the same mutation. The specific method of the identification in step S2 is as follows: PCR amplification and / or sequencing are performed on the T0 generation of regenerated tomato plants using the primers shown in Sequence 5 and Sequence 6. Compared with the wild-type plants, the T0 generation of regenerated tomato plants with nucleotide deletions or insertions in the target site 1 (the target sequence recognized by sgRNA1), the target site 2 (the target sequence recognized by sgRNA2), or the DNA fragment between the two target sites are the plants with the SlYABBY1 gene edited. Among them, the T0 generation of regenerated tomato plants with a single PCR product electrophoresis band and a product size different from that of the wild-type plants (i.e., the tomato material without the transformed recombinant expression vector) are the SlYABBY1 gene homozygous mutant plants; while the T0 generation of regenerated tomato plants with two PCR product electrophoresis bands and a product size different from that of the wild-type plant products are the SlYABBY1 gene heterozygous mutant plants. Using Sequence 5 and Sequence 6 as primers for PCR amplification of wild-type tomato plants, the PCR product is 295 bp.

[0034] In the specific embodiment of the present invention, using the above method, the obtained SlYABBY1 gene homozygous mutant is CR-slyabby1-1, which is a SlYABBY1 gene homozygous targeted plant. It is a plant obtained by deleting the 413-446th positions of the SlYABBY1 genes on the two homologous chromosomes of wild-type tomato (Alisa Craig) while keeping other sequences of the genome of wild-type tomato (Alisa Craig) unchanged. The nucleotide sequence of the mutated SlYABBY1 gene is as shown in Sequence 7 in the sequence listing. The anthers and styles of this plant are shorter, and the stigma is exposed. The PCR product electrophoresis band of this homozygous mutant is single and smaller than that of the wild-type plant.

[0035] The sixth aspect of the present invention provides a method for obtaining a non-transgenic tomato material with exposed stigma, and the obtaining method includes the following steps: Self-cross the gene SlYABBY1 mutant line (i.e., the tomato material with exposed stigma) obtained by the method described in the fifth aspect of the present invention to obtain self-cross progeny, and identify the self-cross progeny with homozygous mutation of the SlYABBY1 gene (the SlYABBY1 genes on two homologous chromosomes have the same mutation) and without exogenous DNA fragments, which is the non-transgenic tomato material with exposed stigma.

[0036] Further, the identification method includes:

[0037] A1: Using the genomic DNA of the self-cross progeny as a template, perform PCR cloning and electrophoresis on the SlYABBY1 gene fragment with the primer pairs shown in Sequence 5 and Sequence 6, and select the homozygous mutant line of the SlYABBY1 gene. For example, the one with a single and smaller electrophoretic band than the wild-type plant is the homozygous mutant plant of the SlYABBY1 gene;

[0038] A2: Using the sequences 10 and 11 in the sequence listing as primers, perform PCR amplification and electrophoresis on the Cas9 gene of the homozygous mutant line of the SlYABBY1 gene obtained in step A1, and select the homozygous mutant line of the SlYABBY1 gene without exogenous Cas9 gene fragment, which is the non-transgenic tomato material with exposed stigma that is homozygous mutant of the SlYABBY1 gene and without exogenous Cas9 gene fragment.

[0039] Even further, in step A2, the selection refers to selecting materials with PCR product sizes of 1 kb and 700 bp.

[0040] In the specific embodiment of the present invention, the genomic DNA of the self-cross progeny can be obtained by the following method: Self-cross the SlYABBY1 gene-edited plant with large nucleotide fragment deletion, harvest the seeds, sow the obtained seeds, and extract the genomic DNA from the leaves after true leaves grow out.

[0041] The seventh aspect of the present invention provides the application of the method described in the fifth aspect or the sixth aspect of the present invention in cultivating a male sterile line material with exposed stigma or tomato hybrid seed production.

[0042] The eighth aspect of the present invention provides a product for identifying whether a test tomato is a tomato material with exposed stigma or its progeny, and the product includes a pair of primers, and the sequences of the pair of primers are as shown in sequences 5 and 6 in the sequence listing.

[0043] In a specific embodiment of the present invention, when using this primer to perform PCR amplification on the genomic DNA of the tomato to be tested, if the size of the PCR product is 295 bp, the stigma of the tomato material to be tested is not exserted; if the size of the PCR product is 261 bp, the stigma of the tomato material to be tested is exserted, that is, the tomato material to be tested is a tomato material with exserted stigma.

[0044] Furthermore, the product further includes conventional PCR reagents.

[0045] Specifically, the product can be a kit containing the above primer pair and conventional PCR reagents.

[0046] The above tomato material with exserted stigma is a homozygous mutant of the SlYABBY1 gene.

[0047] The ninth aspect of the present invention provides a method for identifying whether a tomato to be tested is a tomato material with exserted stigma or its offspring.

[0048] The method provided by the present invention for identifying whether a tomato to be tested is the above tomato material with exserted stigma or its offspring includes the following steps: using a pair of primers to perform PCR amplification on the genomic DNA of the tomato to be tested to obtain a PCR amplification product, and judging whether the tomato to be tested is the above tomato material with exserted stigma or its offspring according to the sequencing result of the PCR amplification product; the sequences of the pair of primers are as shown in Sequence 5 and Sequence 6 in the sequence listing;

[0049] If the sequencing result shows that the DNA fragment after nucleotide deletion is consistent with that shown in Sequence 7 or a DNA fragment with a size of 261 bp is amplified, the tomato to be tested is the tomato material with exserted stigma CR-slyabby1-1 obtained by the above method or its offspring;

[0050] If the sequencing result is inconsistent with that shown in Sequence 7, the tomato to be tested does not belong to the tomato material with exserted stigma obtained by the above method or its offspring;

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] First of all, the present invention provides a gene SlYABBY1 that regulates the exsertion of tomato stigma and its encoded protein, and this gene and its encoded protein have important theoretical and practical significance for the study of the molecular mechanism of tomato stigma exsertion regulation.

[0053] In addition, a stigma-exserted tomato material is obtained using biological materials related to the protein YABBY1. This material can be used as a male sterile line material for hybrid seed production. At the same time, the stigma-exserted male sterile line material can maintain its sterile trait through artificial assisted self-pollination, which is beneficial to the breeding of tomato male sterile lines. Specifically, two sgRNA target sequences for CRISPR / Cas9 gene editing of the SlYABBY1 gene are obtained, and a gene editing vector containing the above two targets is obtained. Transforming this vector into tomato material (Alisa Craig) can precisely edit its SlYABBY1 gene, and non-transgenic stigma-exserted tomato materials with homozygous mutations of the SlYABBY1 gene can be screened from its self-crossed progeny. Experiments have proven that the gene editing method of the present invention is effective and can quickly transform wild-type tomato material (Alisa Craig) into stigma-exserted tomato material, providing an economical, rapid, and effective way to create tomato stigma-exserted male sterile line materials. The present invention has important application and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is the structural information of the SlYABBY1 gene and the positions of CRISPR / Cas9 gene editing sgRNA target 1 and target 2. Among them, E1-E7 are exons 1-7.

[0055] Figure 2 It is the sequencing result of the SlYABBY1 gene of T0 generation plants. Among them, WT is the untransformed wild-type plant, and 1-7 are T0 generation tomato plants.

[0056] Figure 3 It is the phenotype of stigma exsertion of T0 generation targeted plants. Among them, CR-Slyabby1-1 is the No. 1 homozygous targeted plant, and the SlYABBY1 of all cells of the plant is targeted; WT is the untransformed wild-type plant.

[0057] Figure 4 It is the measurement result of the anther and stigma lengths of T0 generation homozygous targeted plants. Among them, the wild-type plant is the Alisa Craig material, and the No. 1 homozygous mutant plant is the regenerated plant of Alisa Craig with homozygous mutation of SlYABBY1. The selected materials are the No. 1 and the T1 generation homozygous mutant plants self-crossed from No. 2 and No. 3.

[0058] Figure 5 It is the PCR detection of the SlYABBY1 gene and Cas9 gene of the self-crossed progeny of T0 targeted plants. Among them, - is the negative blank control, WT is the self-crossed progeny of the untransformed wild-type plant, and 1-14 are the self-crossed progeny of the No. 1 targeted plant. DETAILED DESCRIPTION OF THE INVENTION

[0059] Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions, such as those described in the Molecular Cloning Experiment Guide (Second Edition, written by J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002), or according to the conditions recommended by the manufacturer.

[0060] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0061] In the following examples, all quantitative tests were set up with three repeated experiments, and the results were averaged.

[0062] The tomato variety AC used in the following examples, also known as tomato variety Ailsa Craig, also known as wild-type tomato, is from the Tomato Genetics Resource Center (TGRC, http: / / tgrc.ucdavis.edu / ) in the United States, with the number LA2838A.

[0063] CRISPR / Cas9 vector: namely pTX041 in the literature "Deng et al., Efficient generation of pink-fruited tomatoes using CRISPR / Cas9 system, Journal of Genetics and Genomics 45(2018)51-54". The public can obtain it from the Beijing Academy of Agriculture and Forestry Sciences.

[0064] The pCBC-DT1T2_tomatoU6 vector is from the above-mentioned literature Deng et al, and the public can obtain it from the Beijing Academy of Agriculture and Forestry Sciences.

[0065] The Premix Taq DNA polymerase, KOD polymerase, and DNA Ligation Kit Ver.2.1 used in the following examples are all products of Dalian TaKaRa Company; the restriction endonuclease is a product of NEB Company; the PCR product purification kit is a product of Omega Company; the fast plant genomic DNA extraction kit is a product of Beijing Bomed Gene Technology Co., Ltd.; the primers are synthesized by Thermo Fisher Scientific Company; the sequencing is completed by Beijing Ruibo Xingke Company; the rest of the reagents are all analytical pure reagents.

[0066] Through functional analysis, the present invention found that the protein SlYABBY1 shown in Sequence 2 can regulate whether the stigma of tomatoes is exposed and the lengths of the anthers and styles. When the expression of the gene SlYABBY1 is inhibited or the gene is knocked out, the stigma of tomatoes is exposed, and both the anthers and styles become shorter. For details, please refer to Figure 3 and Figure 4。The pistils of the SlYABBY1 / AC mutant lines developed normally, and normal fruit setting and seed setting could occur after artificial pollination with wild-type pollen or the pollen of the mutant lines themselves.

[0067] The following examples describe the process of obtaining the gene SlYABBY1 provided by the present invention and obtaining stigma-exserted tomato materials through gene editing.

[0068] Example 1: Obtaining the sequence of wild tomato SlYABBY1

[0069] 1. Total RNA was extracted from the flowers of Ailsa Craig and then reverse transcribed into cDNA.

[0070] 2. Using the cDNA obtained in step 1 as a template, the open reading frame of the SlYABBY1 gene was amplified using primers SlYABBY1-F and SlYABBY1-R. The PCR reaction system was: 5 μL of KOD Plus Buffer, 5 μL of dNTP, 2 μL of MgCl2, 1.5 μL of each forward and reverse primer, 1 μL of cDNA, 1 μL of KOD Plus enzyme, and double-distilled water was added to 50 μL; the PCR reaction conditions were: denaturation at 94°C for 20 seconds, annealing at 55°C for 20 seconds, extension at 68°C for 40 seconds, for a total of 35 cycles. The primer sequences are as follows:

[0071] SlYABBY1-F: 5’-ATGATCAATCAATCAATCAAATC-3’ (Sequence 8);

[0072] SlYABBY1-R: 5’-TCAGTAAGGAGATACACCAATG-3’ (Sequence 9).

[0073] 3. The PCR product was detected by 1% agarose gel electrophoresis, and the 729 bp DNA fragment was recovered and purified.

[0074] 4. The recovered fragment was ligated to pJET1.2 using the CloneJET PCR Cloning kit and sent to Beijing Ruibo Xingke Company for sequencing (sequence 729 bp), and the nucleotide sequence is as shown in Sequence 1 in the sequence listing.

[0075] Example 2: Construction of the CRIPSR / Cas9 gene editing vector pTX041::CR-SlYABBY1

[0076] I. Obtaining the CRIPSR / Cas9 gene editing target sequence of the SlYABBY1 gene

[0077] According to the SlYABBY1 gene sequence registered in the SGN database (http: / / solgenomics.net / ) (accession number: Solyc01g091010.2, and the nucleotide sequence is Sequence 1 in the sequence listing), the structure of the SlYABBY1 gene was analyzed. The analysis results are as Figure 1 shown. The SlYABBY1 gene consists of 7 exons (labeled E1 - E7 respectively) and 6 introns. The sequence of exon 3 was submitted to the CRISPRdirect online target analysis database (http: / / crispr.dbcls.jp / ), with the PAM sequence set to NGG and the species data set to Tomato (Solanum lycopersicum) str. Heinz 1706 genome SL2.50 for CRIPSR / Cas9 target design. The two finally selected sgRNA targets are as Figure 1 shown, and the specific sequences are as follows:

[0078] SlYABBY1 gene sgRNA target 1: 5’-AATCACTCATGCCAGTCCG-3’ (Sequence 3);

[0079] SlYABBY1 gene sgRNA target 2: 5’-CCAGTCGCAAACAGACGTA-3’ (Sequence 4).

[0080] II. Construction of the CRISPR / Cas9 gene editing vector for the SlYABBY1 gene

[0081] 1. Design of sgRNA amplification primers

[0082] According to the selected target sequences, sgRNA amplification primers were designed, specifically:

[0083] SlYABBY1 DT1-F0 (Sequence 12):

[0084] 5’-ATATAT GTTTG AATCACTCATGCCAGTCCG GTTTTAGAGCTAGAAATAGC-3’, the boxed part is the recognition site of Bsa I restriction enzyme, and the underlined part is the sequence of target 1.

[0085] SlYABBY1 DT1-R0 (Sequence 13):

[0086] 5’-ATTATT GAAAC CCAGTCGCAAACAGACGTA CAAACTACACTGTTAGATTC-3’, the boxed part is the recognition site of Bsa I restriction enzyme, and the underlined part is the sequence of target 2.

[0087] Two target sites plus primer adapters, using pCBC-DT1T2_tomatoU6 as a template, perform one-step amplification (Taq at 55 °C, 40 s), and recover and purify to obtain a 500-bp fragment.

[0088] 2. Take the CRISPR / Cas9 vector (pTX041), digest it with the restriction enzyme BsaI, and recover the 19300-bp vector backbone.

[0089] 3. Ligate the PCR amplification product obtained in step 1 and the vector backbone obtained in step 2 to obtain the recombinant plasmid pTX041::CR-SlYABBY1. The recombinant plasmid has been verified by sequencing.

[0090] Example 3: Obtaining tomato materials with exposed stigmas

[0091] I. Transform the recombinant plasmid in step 2 into Agrobacterium

[0092] Take 1 μg of the gene editing vector pTX-041::CR-SlYABBY1 prepared in Example 1 and place it in 100 μL of GV3101 competent cells (Beijing Huayuanyang Biotechnology, NRR01270), quickly freeze it in liquid nitrogen for 3 minutes, water bath at 37 °C for 5 minutes, then add 1 mL of YEB medium (YEB medium consists of solute and solvent, the solvent is water, and the solute and its concentration in the medium are: yeast extract 5 g / L, peptone 5 g / L, beef extract 5 g / L, magnesium sulfate heptahydrate 0.5 g / L, sucrose 1 g / L), culture at 28 °C for 2 - 4 hours; centrifuge at 10000×g for 30 seconds, discard the supernatant, add 0.1 mL of YEB medium to resuspend the cells, coat them on a YEB plate containing 50 μg / mL kanamycin, 500 μg / mL streptomycin and 50 μg / mL rifampicin, and culture in the dark at 28 °C for 2 - 3 days; pick single colonies, inoculate them in a YEB liquid medium containing 50 μg / mL kanamycin, 500 μg / mL streptomycin and 50 μg / mL rifampicin, and culture with shaking at 28 °C overnight to obtain transformants; perform colony PCR identification on the transformants, using pTX02R (5’-GCAGGCATGCAAGCTTATTGG-3’, sequence 10) and M13R (5’-CAGGAAACAGCTATGACC-3’, sequence 11) as primers, and the positive recombinant bacteria are those with amplified fragments of 1 kb and 700 bp in size. Name this recombinant bacterium pTX-041::CR-SlYABBY1 / GV3101 and store it at -80 °C for later use.

[0093] II. Obtaining and identification of T0 generation SlYABBY1 gene-edited plants

[0094] 1. Obtaining of T0 generation regenerated plants

[0095] The recombinant bacterium pTX-041::CR-SlYABBY1 / GV3101 was transformed into the explants (cotyledons) of the wild-type tomato material AC. Then, it was co-cultured for 48 hours at 25 ± 1.5 °C and a light intensity of 100 - 200 lx in an MS solid medium (Beijing Huayueyang Biotechnology, M519) containing 1 mg / L indoleacetic acid, 1.75 mg / L zeatin riboside, and a pH of 5.8. Then, it was transferred to an MS solid medium containing 1.0 mg / L indoleacetic acid, 1.75 mg / L zeatin, 200 mg / L ticarcillin, and 75 mg / L kanamycin, and a pH of 5.8, and cultured at 25 ± 1.5 °C, a photoperiod of 16 h / d, and a light intensity of 800 - 1200 lx until regenerated buds grew. When the regenerated buds grew to 2 - 3 cm, the regenerated buds were cut off and transferred to an MS solid medium containing 200 mg / L ticarcillin and 50 mg / L kanamycin, and a pH of 5.8, and cultured at 25 ± 1.5 °C, a photoperiod of 16 h / d, and a light intensity of 800 - 1200 lx until roots grew, obtaining 7 plants, which were the T0 generation regenerated plants.

[0096] 2. Obtaining of T0 generation SlYABBY1 gene-edited plants

[0097] The DNA of the above 7 T0 generation regenerated plants was extracted using a fast plant genomic DNA extraction kit. Using the nucleotide sequences shown in Sequence 5 and Sequence 6 as primers, the SlYABBY1 gene in different plants was cloned and sequenced. The PCR reaction system was: 10 μL of Premix Taq DNA polymerase Mix, 0.8 μL of each of the forward and reverse primers, 1.5 μL of DNA, and double-distilled water was added to 20 μL; the PCR reaction conditions were: denaturation at 94 °C for 20 seconds, annealing at 56 °C for 20 seconds, extension at 72 °C for 25 seconds, for a total of 35 cycles. The primer sequences are as follows:

[0098] SlYABB1-CR F: 5’-CACCTCACAATCTTCTGGT-3’ (Sequence 5);

[0099] SlYABB1-CR R: 5’-AGGCTAGCTAGACCTGCAT-3’ (Sequence 6).

[0100] The sequencing results of the PCR products are as Figure 2As shown. Since tomatoes are diploid plants, when Cas9 starts to cut specific genes, both alleles on the two homologous chromosomes in the same cell may be edited, resulting in the same type or different types of mutations. A homozygous mutant refers to a plant in which the SlYABBY1 genes on the two homologous chromosomes have the same mutation. As can be seen from the figure: among the 7 T0 generation regenerated plants, except for the 5th / 6th and 7th regenerated plants, the other plants are all plants with the SlYABBY1 gene edited. The targeting efficiency (the number of plants with the SlYABBY1 gene edited / the total number of regenerated plants) is 57.1% (4 / 7).

[0101] Among the plants with the SlYABBY1 gene edited, the 1st regenerated plant is a homozygous targeting plant of the SlYABBY1 gene (SlYABBY1 homozygous mutant, also known as CR-Slyabby1-1). After measurement, its style and anther have both shortened to a certain extent (see Figure 4 ), and the stigma is exserted (see Figure 3 ).

[0102] The mutant sequence of the SlYABBY1 gene in the 1st regenerated plant is shown as sequence 7 in the sequence listing. The 1st SlYABBY1 gene homozygous targeting plant is a plant obtained by deleting the 413-446th positions of the SlYABBY1 genes on the two homologous chromosomes of wild-type tomato (Alisa Craig) while keeping other sequences of the genome of wild-type tomato (Alisa Craig) unchanged.

[0103] Example 4. Obtaining of non-transgenic tomato materials with exserted stigma

[0104] I. Detection of the mutated SlYABBY1 gene

[0105] The 1st SlYABBY1 gene homozygous targeting plant obtained in Example 3 was artificially self-crossed to obtain F1 generation seeds; 50 F1 generation seeds were taken for sowing; after true leaves grew, the offspring (F1 generation) of the 1st SlYABBY1 gene homozygous targeting plant were obtained.

[0106] Using the genomic DNA of the wild-type tomato plants (Alisa Craig) and the progeny (No. 1-14) of the homozygous targeted knockout plants of the SlYABBY1 gene as templates, and the nucleotide sequences shown in Sequence 5 and Sequence 6 as primers, the SlYABBY1 gene fragment was amplified by PCR and electrophoresed. The PCR reaction system was: 10 μL of Premix Taq DNA polymerase Mix, 0.8 μL of each forward and reverse primer, 1.5 μL of DNA, and double-distilled water was added to 20 μL; the PCR reaction conditions were: denaturation at 94 °C for 20 seconds, annealing at 56 °C for 20 seconds, extension at 72 °C for 25 seconds, for a total of 35 cycles. The PCR product of the SlYABBY1 gene in the wild-type tomato plants was 295 bp, and the PCR product of the mutant SlYABBY1 gene was 261 bp, and the two could be distinguished by 3% agarose electrophoresis.

[0107] The electrophoresis results of the PCR products were as Figure 5 shown in A. The PCR product of the SlYABBY1 gene in the wild-type plants was 295 bp (see lane 1), and the PCR products of the progeny of the No. 1 targeted knockout plants (No. 1-14) were all 261 bp, smaller than that of the wild-type plants (295 bp), indicating that the SlYABBY1 genes of the progeny of the homozygous targeted knockout plants of the No. 1 SlYABBY1 gene were all homozygous mutant plants ( Figure 5 A).

[0108] II. Detection of exogenous DNA fragments and obtaining of non-transgenic stigma-exserted tomato materials

[0109] Using the genomic DNA of the homozygous mutant plants (No. 1-14) among the progeny of the homozygous targeted knockout plants of the SlYABBY1 gene obtained above as a template, and the pTX02R and M13R described in Example 3 as primers, the Cas9 gene of the homozygous mutant plants was amplified by PCR and electrophoresed.

[0110] The results showed that: the detection results of the No. 2, 5, 6, 10, 11, and 13 progeny among the homozygous mutant plants (No. 1-14) were negative, and they were stigma-exserted tomato materials without exogenous DNA fragments ( Figure 5 B), that is, the target non-transgenic stigma-exserted tomato materials.

[0111] Of course, other targeted heterozygous plants obtained in Example 3, such as CR-Slyabby1-2, CR-Slyabby1-3, and CR-Slyabby1-4, can also be self-crossed for one or more generations, and then the above-mentioned mutant SlYABBY1 gene detection and exogenous DNA fragment detection are carried out on the progeny plants. When the mutation of SlYABBY1 is a homozygous mutation and the exogenous DNA fragment detection is negative, it indicates that non-transgenic stigma-exserted tomato materials have been obtained.

[0112] From the above results, it can be seen that the gene editing method of the present invention can rapidly generate tomato materials with stigma exsertion, having important application value and breeding prospects.

Claims

1. The protein SlYABBY1 that regulates the stigma exsertion of tomatoes has an amino acid sequence as described in any one of the following (1) to (3): (1) The amino acid sequence shown in Sequence 2 of the sequence listing; (2) The amino acid sequence shown in Sequence 2 of the sequence listing with substitution, deletion or addition of one or several amino acid residues and having the function of regulating the stigma exsertion of tomato flowers; (3) An amino acid sequence derived from tomatoes, having at least 75% identity with the amino acid sequence of (1) or (2) and having the function of regulating the stigma exsertion of tomatoes.

2. The gene SlYABBY1 that regulates the stigma exsertion of tomatoes, and the nucleotide sequence of its cDNA is any one of the following (a1) or (a2): (a1) The DNA sequence encoding the amino acid sequence described in the first aspect of the present invention; preferably the DNA sequence shown in Sequence 1 of the sequence listing; (a2) A DNA sequence that hybridizes with the DNA sequence described in (a1) under stringent conditions and encodes a protein molecule with the function of regulating the stigma exsertion of tomatoes.

3. A biological material related to the protein described in claim 1 or the gene described in claim 2, and the biological material is selected from the following (a) or (b): (a) A substance for silencing or inhibiting the expression of the encoding gene of the protein SlYABBY1 described in claim 1 or the gene described in claim 2, or a substance for knocking out the encoding gene of the protein SlYABBY1 described in claim 1 or the gene described in claim 2; (b) A substance for reducing or inhibiting the activity and / or content of the protein SlYABBY1 described in claim 1 in tomatoes.

4. The biomaterial according to claim 3, characterized in that, The biological material is a tomato CRISPR / Cas9 genome editing system, including: a recombinant expression vector, the recombinant expression vector includes a Cas9 expression cassette and an sgRNA expression cassette, the Cas9 expression cassette expresses Cas9, the sgRNA expression cassette expresses sgRNA, and the target gene of the sgRNA is a partial or all DNA fragment of the encoding gene of the protein SlYABBY1 in tomatoes; Preferably, the sgRNA includes sgRNA1 and sgRNA2, and both sgRNA1 and sgRNA2 specifically target the 3rd exon of the SlYABBY1 gene; More preferably, the target sequence of sgRNA1 is the DNA molecule shown in Sequence 3 of the sequence listing, or the target sequence of sgRNA1 is the reverse complementary sequence of the sequence shown in Sequence 3 of the sequence listing; The target sequence of sgRNA2 is the DNA molecule shown in Sequence 4 of the sequence listing, or the target sequence of sgRNA2 is the reverse complementary sequence of the sequence shown in Sequence 4 of the sequence listing.

5. Use of the biological material described in claim 3 or 4 in creating stigma-exserted tomato materials or tomato breeding.

6. A method for regulating stigma exsertion in tomatoes, the method comprising: Using the tomato CRISPR / Cas9 genome editing system described in claim 4 to edit the gene SlYABBY1 in the genome of tomato materials, thereby causing the loss of function of SlYABBY1 and obtaining stigma-exserted tomato materials.

7. The method according to claim 6, wherein The method specifically includes: S1: Introduce the tomato CRISPR / Cas9 genome editing system described in claim 4 into the said tomato material, and obtain successfully transformed plants through screening; S2: Obtain gene SlYABBY1 mutant lines from the successfully transformed plants through identification; The said identification is to perform PCR amplification of the gene SlYABBY1 fragment using the genome of the successfully transformed plant as a template, and then detect by means of gel electrophoresis or sequencing to obtain the mutant lines with mutated gene SlYABBY1.

8. The method according to claim 7, characterized in that The primer pair used for the said PCR amplification is as shown in sequences 5 and 6 in the sequence listing.

9. A method for obtaining a non-transgenic tomato material with exposed stigma, the said obtaining method comprising the following steps: Self-cross the gene SlYABBY1 mutant lines obtained by the method according to any one of claims 6 - 8 to obtain self-cross progeny, and identify the self-cross progeny with homozygous mutation of the SlYABBY1 gene and without exogenous DNA fragments, which is the non-transgenic tomato material with exposed stigma.

10. The obtaining method according to claim 9, characterized in that, The said identification method comprises: A1: Using the genomic DNA of the self-cross progeny as a template, perform PCR cloning and electrophoresis of its SlYABBY1 gene fragment with the primer pair shown in sequences 5 and 6 in the sequence listing, and select the homozygous mutant lines of the SlYABBY1 gene, whose electrophoresis bands are single and smaller than those of wild-type plants; A2: Using sequences 10 and 11 in the sequence listing as primers, perform PCR amplification and electrophoresis of the Cas9 gene of the homozygous mutant lines of the SlYABBY1 gene obtained in step A1, and select the homozygous mutant lines of the SlYABBY1 gene without exogenous Cas9 gene fragments, which are the non-transgenic tomato materials with exposed stigma; In step A2, the said selection refers to selecting materials with PCR product sizes of 1 kb and 700 bp.

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