Use of a mapk-related protein to regulate flowering and fertility in plants

Abstract

The application discloses a MAPK related protein for regulating flowering and fertility of plants, and belongs to the field of botany, and particularly relates to the application of a MAPK related protein for regulating flowering and fertility of plants.A method for regulating flowering and fertility of plants is provided, which comprises regulating the activity and / or content of a protein in a target plant, or / and regulating the expression amount of a gene coding the protein, so as to regulate the flowering and fertility of the plant, wherein the protein is a composition composed of a protein FvMAPK3 with an amino acid sequence of SEQ ID No.3 and a protein FvMAPK6 with an amino acid sequence of SEQ ID No.6.Experiments prove that reducing the expression of the two proteins FvMAPK3 and FvMAPK6 can regulate the flowering and fertility of strawberries, and has important theoretical significance for strawberry breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of botany, in particular to the application of MAPK related protein in regulating the flowering and fertility of plants. BACKGROUND

[0002] MAPK pathway is considered as one of the key components of signal cascade, which regulates many cellular processes, such as cell division, developmental program, hormone response, biotic and abiotic stress response. MAPK, located downstream of MAPK cascade system, is a kind of serine / threonine protein kinase, which can be divided into two categories according to the 'TXY' motif in the protein sequence: TEY and TDY. Studies have shown that MAPK3 and MAPK6 of TEY group play a crucial role in jointly mediating the response of low temperature, salt stress, melatonin, pathogen, etc. In addition, in rice, MAPK6 can also regulate the size and weight of rice grains.

[0003] Strawberry is a perennial herb of Rosaceae genus Fragaria, which has strong adaptability and is one of the most widely cultivated fruits in the world. It has high nutritional, medicinal value and anti-aging effect, and is loved by people. However, strawberry production is easily affected by low temperature, weak light or short-term high temperature stress, which causes poor growth of strawberry plants, poor pollination and fertilization, and poor fruit quality, and seriously affects the economic benefits of strawberry.

[0004] Therefore, cloning or knocking out the key genes that regulate the morphological development, flower and fruit development and quality formation of strawberry plants through molecular biology methods can lay a foundation for improving the quality of strawberry in the future. SUMMARY

[0005] The technical problem to be solved by the present application is how to regulate the flowering and fertility of strawberry.

[0006] In order to solve the problems existing in the prior art, the present application provides the application of protein or expression material of regulatory gene or material for regulating the activity or content of the protein in regulating the flowering and fertility of plants.

[0007] The application provided by the present application is the application of protein or expression material of regulatory gene or material for regulating the activity or content of the protein in any one of the following:

[0008] 1) the application of protein or expression material of regulatory gene or material for regulating the activity or content of the protein in regulating the flowering and fertility of plants;

[0009] 2) the application of protein or expression material of regulatory gene or material for regulating the activity or content of the protein in preparing products for regulating the flowering and fertility of plants;

[0010] 3) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in a plant whose flowering period and fertility are changed;

[0011] 4) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in the manufacture of a product for a plant whose flowering period and fertility are changed;

[0012] 5) Use of a protein or a substance that regulates expression of a gene or a substance that regulates activity or content of the protein in plant breeding;

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

[0014] G1) a composition of a protein whose amino acid sequence is SEQ ID No. 3 and a protein whose amino acid sequence is SEQ ID No. 6;

[0015] G2) a protein whose amino acid sequence is SEQ ID No. 3 or a protein whose amino acid sequence is SEQ ID No. 6;

[0016] G3) a protein that has more than 80% identity with the protein of A1) and has a function of regulating flowering period and fertility of a plant, which is obtained by substitution and / or deletion and / or addition of amino acid residues of the proteins of G1) and G2);

[0017] G4) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein of G1) or G2).

[0018] The protein whose amino acid sequence is SEQ ID No. 3 is named FvMAPK3.

[0019] The protein whose amino acid sequence is SEQ ID No. 6 is named FvMAPK6.

[0020] The above protein can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0021] In the above protein, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using DNA in vitro recombination technology, so as to facilitate expression, detection, tracking and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0022] The protein in the above use is derived from strawberry (Fragaria vesca, cv Fragola di Bosco).

[0023] In the present context, the substance that modulates the activity and / or the content of the protein can be a substance that modulates the expression of a gene that encodes the protein FvMAPK3 and FvMAPK6.

[0024] In the above, the substance that modulates the expression of a gene can be a substance that performs at least one of the following six kinds of modulation: 1) modulation performed at the transcription level of the gene; 2) modulation performed after the transcription of the gene (that is, modulation performed on the splicing or processing of the primary transcript of the gene); 3) modulation performed on the RNA transport of the gene (that is, modulation performed on the transport of mRNA of the gene from the nucleus to the cytoplasm); 4) modulation performed on the translation of the gene; 5) modulation performed on the degradation of mRNA of the gene; and 6) modulation performed on the post-translation of the gene (that is, modulation performed on the activity of the protein translated from the gene).

[0025] In the above application, the substance that modulates the expression of a gene and the substance that modulates the activity or the content of the protein can be a biological material related to the protein, and the biological material can be any one of the following:

[0026] c1) a nucleic acid molecule that encodes the protein described above;

[0027] c2) an expression cassette that contains the nucleic acid molecule described in c1);

[0028] c3) a recombinant vector that contains the nucleic acid molecule described in c1), or a recombinant vector that contains the expression cassette described in c2);

[0029] c4) a recombinant microorganism that contains the nucleic acid molecule described in c1), or a recombinant microorganism that contains the expression cassette described in c2), or a recombinant microorganism that contains the recombinant vector described in c3);

[0030] c5) a transgenic plant cell line that contains the nucleic acid molecule described in c1), or a transgenic plant cell line that contains the expression cassette described in c2);

[0031] c6) a transgenic plant tissue that contains the nucleic acid molecule described in c1), or a transgenic plant tissue that contains the expression cassette described in c2);

[0032] c7) a transgenic plant organ that contains the nucleic acid molecule described in c1), or a transgenic plant organ that contains the expression cassette described in c2);

[0033] e1) a nucleic acid molecule that inhibits or reduces or silences the expression of a gene that encodes the protein described above;

[0034] e2) an expression cassette that contains the nucleic acid molecule described in e1);

[0035] e3) a recombinant vector containing the nucleic acid molecule of e1), or a recombinant vector containing the expression cassette of e2);

[0036] e4) a recombinant microorganism containing the nucleic acid molecule of e1), or a recombinant microorganism containing the expression cassette of e2), or a recombinant microorganism containing the recombinant vector of e3);

[0037] e5) a transgenic plant cell line containing the nucleic acid molecule of e1), or a transgenic plant cell line containing the expression cassette of e2);

[0038] e6) a transgenic plant tissue containing the nucleic acid molecule of e1), or a transgenic plant tissue containing the expression cassette of e2);

[0039] e7) a transgenic plant organ containing the nucleic acid molecule of e1), or a transgenic plant organ containing the expression cassette of e2).

[0040] In the above biological materials, the nucleic acid molecule of c1) is any one of the following DNA molecules:

[0041] d1) the nucleotide sequence is a DNA molecule as shown in SEQ ID No. 2;

[0042] d2) the coding region sequence is a DNA molecule as shown in SEQ ID No. 1 in the sequence listing;

[0043] d3) the nucleotide sequence is a DNA molecule as shown in SEQ ID No. 5;

[0044] d4) the coding region sequence is a DNA molecule as shown in SEQ ID No. 4 in the sequence listing.

[0045] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0046] In the above e3), the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be pYLCRISPR / Cas9Pubi-H vector.

[0047] As a specific example, the recombinant vector is a recombinant vector mapk3 / mapk6-cr. The recombinant vector mapk3 / mapk6-cr is a recombinant vector obtained by inserting the sgRNA1 and sgRNA2 expression cassette sequence (nucleotide sequence is SEQ ID No. 7 in the sequence listing) into the restriction endonuclease BsaI site of the vector pYLCRISPR / Cas9Pubi-H, while keeping other nucleotide sequences of the vector pYLCRISPR / Cas9Pubi-H unchanged. The recombinant plasmid is named as the recombinant vector mapk3 / mapk6-cr.

[0048] wherein the sgRNA1 and sgRNA2 expression cassette sequence is the promoter sequence (positions 163-482 of SEQ ID No. 7) of the pYLsgRNA-AtU6-29 vector (transcribable into the AtU6-29 snRNA promoter), the MAPK3 target site 5'-AATCTCACGGAGCGTGCGCT-3', the MAPK6 target site 5'-GACACGGTGATGTCAGAGGC-3', and the pYLsgRNA-AtU6-29 vector sequence (positions 20-102 of SEQ ID No. 7) constitute a complete sgRNA expression cassette.

[0049] The microorganism of e4) above can be Agrobacterium. The Agrobacterium is EHA105. The recombinant Agrobacterium can be EHA105 / mapk3 / mapk6-cr.

[0050] Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein FvMAPK3 and FvMAPK6 isolated from the present application, as long as they encode the protein FvMAPK3 and FvMAPK6 and have the function of the protein FvMAPK3 and FvMAPK6, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0051] The 75% or more identity above can be 80%, 85%, 90% or 95% or more identity.

[0052] In the present context, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using homology search sites on the internet, such as the BLAST page of the NCBI home page website. For example, the value of identity (%) can be obtained by performing a search in Advanced BLAST 2.1 using blastp as the program, setting Expect value to 10, setting all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and then calculating the identity of the amino acid sequence.

[0053] In the present context, the identity of more than 80% 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%.

[0054] The vectors described herein are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors.

[0055] The recombinant expression vectors comprising the FvMAPK3 and FvMAPK6 genes can be constructed using existing plant expression vectors. The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vectors can also comprise a 3' untranslated region of a foreign gene, i.e., a DNA segment comprising a polyadenylation signal and any other DNA segment involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylation of the 3' end of the mRNA precursor, such as the 3' untranslated region of Agrobacterium tumefaciens Ti plasmid genes (such as the nopaline synthase Nos gene), plant genes (such as the soybean storage protein gene), etc.

[0056] When the recombinant plant expression vector is constructed using the FvMAPK3 and FvMAPK6 genes, any one of the enhanced promoters or constitutive promoters can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of corn, which can be used alone or in combination with other plant promoters; in addition, when the plant expression vector is constructed using the genes of the present application, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be the ATG start codon or the adjacent region start codon, but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0057] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or chemical reagent-resistant marker genes (such as herbicide-resistant genes), etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0058] The present application also provides a method for regulating the flowering and fertility of plants.

[0059] The method for regulating the flowering and fertility of plants provided by the present application comprises regulating the expression of the coding genes of the proteins FvMAPK3 and FvMAPK6 or regulating the activity and / or content of the proteins or regulating the activity and / or content of the coding genes of the proteins to regulate the flowering and fertility of plants.

[0060] In the present application, the regulation is down-regulation or inhibition or reduction.

[0061] The present application also provides a method for cultivating plants with changed flowering and fertility.

[0062] The method for cultivating plants with changed flowering and fertility provided by the present application comprises down-regulating or inhibiting or reducing the expression amount of the coding genes of the proteins FvMAPK3 and FvMAPK6 in the target plants, or / and down-regulating or inhibiting or reducing the activity and / or content of the coding genes of the proteins, to obtain plants with changed flowering and fertility.

[0063] In the above breeding method, the down-regulation or inhibition or reduction of the activity and / or content of the protein in the plant of interest, or / and the expression amount of the coding gene of the protein, can be achieved by introducing into the recipient plant a recombinant expression vector comprising a nucleic acid molecule that inhibits or reduces or silences the expression of the coding gene of the protein FvMAPK3 and FvMAPK6, to obtain a plant of interest with altered flowering and fertility.

[0064] The FvMAPK3 and FvMAPK6 genes encode the FvMAPK3 and FvMAPK6 proteins.

[0065] In an embodiment of the present application, the method for breeding a plant with altered flowering and fertility comprises the following steps:

[0066] 1) constructing a recombinant expression vector comprising a DNA molecule as shown in SEQ ID No. 2 and SEQ ID No. 4;

[0067] 2) transforming the recombinant expression vector constructed in step 1) into a recipient plant (such as a crop or strawberry);

[0068] 3) obtaining a transgenic plant with altered flowering and fertility through screening and identification.

[0069] The introduction refers to introduction by recombination means, including but not limited to Agrobacterium-mediated transformation, biolistic method, electroporation, in planta technique, etc.

[0070] The coding gene or fragment of the protein FvMAPK3 and FvMAPK6 provided by the present application for knocking out can be introduced into a plant cell or a recipient plant using any vector that can guide the expression of a foreign gene in a plant, to obtain a transgenic cell line and transgenic plant with altered flowering and fertility. The expression vector carrying the coding gene of the protein FvMAPK3 and FvMAPK6 for knocking out can be transformed into a plant cell or tissue by using a Ti plasmid, a Ri plasmid, a plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. conventional biological methods, and the transformed plant tissue is cultivated into a plant.

[0071] The microorganism described herein can be a yeast, a bacterium, an alga or a fungus. Among them, the bacterium can be from Escherichia, Erwinia, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it can be Agrobacterium tumefaciens EHA105.

[0072] In the present invention, the regulation can be up-regulation or enhancement or increase. The regulation can also be down-regulation or weakening or decrease.

[0073] The proteins used in the above-mentioned applications and / or the biological materials also belong to the scope of the present invention.

[0074] In the present invention, the plant is any one of the following:

[0075] C1) a dicotyledonous plant;

[0076] C2) a Rosales plant;

[0077] C3) a Rosaceae plant;

[0078] C4) a Fragaria plant;

[0079] C5) a strawberry.

[0080] In the above, the strawberry can be Fragaria vesca, cv Fragola di Bosco.

[0081] The MAPK3 and MAPK6 proteins of the present invention have the function of regulating the flowering and fertility of strawberries. By down-regulating or weakening or decreasing or knocking out the expression of MAPK3 and MAPK6 genes through the CRISPR-Cas9 method, and verifying the gene function discovery, it is found that only knocking out FvMAPK3 (i.e. line 4), the strawberry appears the phenotype of late flowering, and Fvmapk3 / mapk6 double knockout (line 6 and line 7) will seriously affect the normal growth and development of strawberry plants and lead to the late flowering phenotype, and the homozygous plant of knocking out FvMAPK6 gene (i.e. line 5) will show the phenotype of complete abortion, which cannot pollinate and fertilize normally and cannot set fruit. The present invention provides useful genetic materials for the regulation of flowering and fertility of strawberry plants. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 It is a pYLCRISPR / Cas9Pubi-H vector map.

[0083] Figure 2 It is the mutation type of the transgenic strawberry lines of different knock-out types of MAPK3 and MAPK6. Among them, A is the result of the mutation site of line 4; B is the result of the mutation site of line 5; C is the result of the mutation site of line 6; D is the result of the mutation site of line 7.

[0084] Figure 3 It is a mapk3 / mapk6-cr phenotype diagram. Among them, A is the flowering phenotype of the strawberry plant; B and C are the fertility phenotype of the strawberry plant. DETAILED DESCRIPTION

[0085] The application will be further described in conjunction with the specific embodiments, the examples given are only for illustrating the application, but not for limiting the scope of the application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0086] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0087] The quantitative experiments in the following examples, unless otherwise specified, are all set up for three repeated experiments.

[0088] The strawberry variety Fragaria vesca, cv Fragola di Bosco (from THE HEIRLOOM SEED STORE, purchase website: https: / / www.theheirloomseedstore.com / product / strawberry-fragola-di-bosco) in the following examples.

[0089] The pYLCRISPR / Cas9Pubi-H plasmid in the following examples is a gift from Professor Liu Yaoguang's research group of South China Agricultural University, which has been recorded in: Zeng D C, Ma X L, Xie X R, Zhu Q L, Liu Y G. Operation method of plant CRISPR / Cas9 multi-gene editing vector construction and mutation analysis [J]. Chinese Science: Life Sciences, 2018, 48(07): 783-794. The public can obtain this biological material from the applicant, which is only used for repeating the experiments of the application and cannot be used for other purposes.

[0090] The AtU6-29 plasmid in the following examples is a gift from Professor Liu Yaoguang's research group of South China Agricultural University, which has been recorded in: Zeng D C, Ma X L, Xie X R, Zhu Q L, Liu Y G. Operation method of plant CRISPR / Cas9 multi-gene editing vector construction and mutation analysis [J]. Chinese Science: Life Sciences, 2018, 48(07): 783-794. The public can obtain this biological material from the applicant, which is only used for repeating the experiments of the application and cannot be used for other purposes.

[0091] Example 1, construction of CRISPR-Cas9 recombinant vector mapk3 / mapk6-cr and obtaining of strawberry transformed plants

[0092] The coding sequence of FvMAPK3 gene in Fragaria vesca, cv Fragola di Bosco is a nucleotide sequence as shown in SEQ ID No. 1, the genomic sequence of FvMAPK3 gene is as shown in SEQ ID No. 2, and the amino acid sequence of the encoded protein is as shown in SEQ ID No. 3.

[0093] The coding sequence of FvMAPK6 gene in Fragaria vesca, cv Fragola di Bosco is a nucleotide sequence as shown in SEQ ID No. 4, the genomic sequence of FvMAPK6 gene is as shown in SEQ ID No. 5, and the amino acid sequence of the encoded protein is as shown in SEQ ID No. 6.

[0094] 1. Construction of recombinant vector mapk3 / mapk6-cr

[0095] 1) Design target. The target of FvMAPK3 and FvMAPK6 gene is designed on the website http: / / crispr.hzau.edu.cn / CRISPR / , and the high-score and no-off-target target sequence is selected, the primer is synthesized, and is used for vector construction. The pYLCRISPR / Cas9Pubi-H is used as a plant expression vector, and the reverse complementary sequence of 242-261 of sequence 1 in the CDS sequence of FvMAPK3 is selected as a target (the target sequence is: 5'-AATCTCACGGAGCGTGCGCT-3') for primer design and vector construction, and a target of 25-44 of sequence 4 in the CDS sequence of FvMAPK6 is selected (the target sequence is: 5'-GACACGGTGATGTCAGAGGC-3') for primer design and vector construction.

[0096] The primer sequences are as follows:

[0097] AtU6-29MAPK3-: 5'-AGCGCACGCTCCGTGAGATTCAATCTCTTAGTCGACT-3'

[0098] gRTMAPK3+: 5'-AATCTCACGGAGCGTGCGCTGTTTTAGAGCTAGAAAT-3'

[0099] AtU6-29MAPK6-: 5'-GCCTCTGACATCACCGTGTCCAATCTCTTAGTCGACT-3'

[0100] gRTMAPK6+: 5'-GACACGGTGATGTCAGAGGCGTTTTAGAGCTAGAAAT-3'

[0101] U-F: 5'-CTCCGTTTTACCTGTGGAATCG-3'

[0102] gR-R: 5'-CGGAGGAAAATTCCATCCAC-3'

[0103] 2) sgRNA expression cassette construction. The PCR reaction system was configured with sgRNA as the template and the corresponding target (U#T# / gRT#) and linker (U-F / gR-R) as primers. The system is shown in Table 1 below.

[0104] Table 1, PCR reaction system for sgRNA expression cassette construction

[0105]

[0106] The PCR reaction conditions were: 98°C for 30s; 98°C for 10s, 56°C for 5s, 72°C for 10s, 32 cycles; 72°C for 1 min. The PCR product was detected by 1% agarose gel electrophoresis, and a band of about 500 kb was cut off. The Tengen agarose gel recovery kit was used to recover the fragment. The fragment was used as a template, and Pps-GGL, Pgs-GG2, Pps-GG2, and Pgs-GGR primers were used for the second round of PCR amplification, and the system was the same as above. The primers were detected and recovered by electrophoresis, and the primer sequences were as follows:

[0107] Pps-GGL: 5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3'

[0108] Pgs-GG2: 5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3'

[0109] Pps-GG2: 5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3'

[0110] Pgs-GGR: 5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3'

[0111] 3) Assemble the sgRNA expression cassette (PCR gel recovery product) of step 2) into the pYLCRISPR / Cas9Pubi-H vector (pYLCRISPR / Cas9 vector for short). Use the Golden Gate method to connect the sgRNA expression cassette to the expression vector. The system is shown in Table 2 below.

[0112] Table 2, connection reaction system

[0113]

[0114]

[0115] The PCR reaction conditions are: 37°C for 5 min, 10°C for 5 min, 20°C for 5 min, 20 cycles, 37°C for 5 min;

[0116] 4) Transform the PCR reaction product into DH5a E. coli using the heat shock method. The transformation method is as follows: add the gel recovery product to 50 μL of DH5a E. coli competent cells, place on ice for 30 min, heat shock at 42°C for 45 s, immediately cool on ice for 2 min, add 500 μL of antibiotic-free LB, place in a 37°C shaker at 180 rpm for 40 min, centrifuge at 5000 rpm for 5 min to collect the bacteria, discard 400 μL of supernatant, resuspend the bacteria, use a spreader to spread on LB solid medium containing 50 mg / L kanamycin, incubate at 37°C overnight, pick single colonies in LB liquid medium containing 50 mg / L kanamycin, incubate in a 37°C shaking incubator for 6-8 h, use primers SP-L1: 5'-GCGGTGTCATCTATGTTACTAG-3' and SP-R: 5'-TGCAATAACTTCGTATAGGCT-3' for bacterial liquid PCR identification, select positive clones for sequencing. The sequencing result is the nucleotide sequence shown in sequence 7 in the sequence table, then the corresponding transformed bacteria are positive bacteria. The positive bacteria are recombinant E. coli DH5a / mapk3 / mapk6-cr containing the recombinant vector mapk3 / mapk6-cr. Shake flask culture the positive clones with correct sequencing, extract the plasmid using a plasmid extraction kit, and obtain the recombinant vector mapk3 / mapk6-cr.

[0117] The recombinant vector mapk3 / mapk6-cr is a recombinant vector obtained by inserting the expression cassette expressing sgRNA1 and sgRNA2 into the restriction endonuclease BsaI site of the vector pYLCRISPR / Cas9Pubi-H, while keeping the other nucleotide sequences of the vector pYLCRISPR / Cas9Pubi-H unchanged. The recombinant plasmid is named as the recombinant vector mapk3 / mapk6-cr.

[0118] The sgRNA1 and sgRNA2 expression cassette sequence is the promoter sequence of the pYLsgRNA-AtU6-29 vector (positions 163-482 of SEQ ID No. 7) (transcribable into AtU6-29 snRNA promoter), the MAPK3 target site 5'-AATCTCACGGAGCGTGCGCT-3', the MAPK6 target site 5'-GACACGGTGATGTCAGAGGC-3', and the pYLsgRNA-AtU6-29 vector sequence (positions 20-102 of SEQ ID No. 7) to form a complete sgRNA expression cassette.

[0119] 2. Obtaining of transgenic strawberry plants

[0120] 1) Obtaining of recombinant Agrobacterium EHA105 / mapk3 / mapk6-cr

[0121] The recombinant vector mapk3 / mapk6-cr was transformed into Agrobacterium competent EHA105 (Shanghai UDGene Biotechnology Co., Ltd.). The transformation method is as follows: 5 μg plasmid was added to 50 μL EHA105 Agrobacterium competent cells, mixed gently, and then placed on ice for 5 min; frozen in liquid nitrogen for 5 min, and then placed in a 37°C water bath for 5 min; 700 μL of LB liquid medium without resistance was added, and the mixture was cultured at 28°C and 180 rpm for 4 h; the bacterial cells were collected by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the remaining 100 μL of bacterial cells were resuspended and plated on a plate containing 25 mg / L rifampicin and 50 mg / L kanamycin, and cultured at 28°C for 2 d. Single colonies were picked and small-shaken, and SP-L and SP-R primers were used for bacterial liquid PCR identification. The positive clone had a PCR product of 1000 bp (i.e., the length of the two sgRNA expression cassettes), and the recombinant Agrobacterium EHA105 / mapk3 / mapk6-cr of the recombinant vector mapk3 / mapk6-cr double-knockout of the mapk3 / mapk6 gene was obtained.

[0122] 2) Stable genetic transformation of diploid strawberry

[0123] Preparation and activation of the infection bacterial liquid: the large-shaken EHA105 / mapk3 / mapk6-cr bacterial liquid was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded in a clean bench. MS activation liquid (solvent: water, solute and its content: MS 4.4 g / L, sucrose 30 g / L, 1 M NaOH to adjust pH to 5.8, high-temperature high-pressure sterilization, and 100-200 μM of acetyl-syringone was added before use) was added to suspend the bacterial cells, so that the OD value of the bacterial liquid was 0.4-0.6, and the bacterial liquid was activated at 28°C for 40 min.

[0124] Healthy and full di Bosco strawberry seeds were sterilized with 75% anhydrous ethanol and 1% NaClO, and then spotted on 1 / 2MS medium. After 4-7 days of dark culture, the seeds were placed in a constant temperature and humidity tissue culture room with a light cycle of 12 / 12 hours, a temperature of 24°C, and a humidity of 45%. After 40-50 days of culture, diploid strawberry tissue culture seedlings were obtained.

[0125] Explant infection (leaf disc method): During the activation of the bacterial solution, plant material was cut as explants. Strawberry di Bosco (hereinafter referred to as wild type strawberry, WT) tissue culture seedlings grown for 50-60 days were cut into young leaves and petioles, and then placed in MS activation solution. After a certain number of explants were cut, the activated bacterial solution was added, and then placed in a 50 ml syringe to vacuum. Finally, the leaves were waterlogged.

[0126] Explant culture: After infection, the explants were moved to co-culture medium (solvent: water, solute and its content: MS 4.4 g / L, sucrose 30 g / L, agar powder 7 g / L, 6-BA 0.1 mg / L, 2,4-D 0.01 mg / L, TDZ 2 mg / L, 1M NaOH to adjust pH to 5.8, high temperature and high pressure sterilization) and dark culture for 2 days. The explants after dark culture were carefully moved to the screening medium (solvent: water, solute and its content: MS 4.4 g / L, sucrose 30 g / L, agar powder 7 g / L, 6-BA 0.1 mg / L, 2,4-D 0.01 mg / L, TDZ 2 mg / L, 1M NaOH to adjust pH to 5.8, high temperature and high pressure sterilization, then add Hyg 2 mg / L, Tim (trimethoprim) 400 mg / L), and cultured for about 40 days to grow out the bud mass.

[0127] Callus rooting: When the callus grows out the bud mass, it is moved to the rooting medium (solvent: water, solute and its content: MS 4.4 g / L, sucrose 30 g / L, agar powder 7 g / L, 1M NaOH to adjust pH to 5.8, high temperature and high pressure sterilization, then add Hyg 2 mg / L, Tim 400 mg / L).

[0128] Transgenic seedling transplantation: The seedlings with root systems of 4-5 cm and well-developed fibrous root systems were transplanted into soil, the root medium was carefully removed, and the soil was covered with plastic wrap to keep it moist. After 15 days, the plastic wrap was removed, and the transgenic plants containing the recombinant vector mapk3 / mapk6-cr were obtained.

[0129] 3. Identification of transgenic positive lines

[0130] In order to determine the transgenic positive plants, the leaves of the transgenic strawberry plants in step 2 were taken, and the DNA was extracted. The genomic DNA was used as a template for PCR detection.

[0131] 1) Extract DNA of the transgenic plants to be identified containing recombinant vector mapk3 / mapk6-cr, and identify by PCR, primers as follows:

[0132] mapk3-cr-f: 5'-CCACAGGATCGCTTTCGCCT-3'

[0133] mapk3-cr-r: 5'-CAACTTTCACAAACATACAC-3'

[0134] mapk6-cr-f: 5'-AAACTTAGGGCTGAGCTTCA-3'

[0135] mapk6-cr-r: 5'-AAGAAACGACAGACCAGACG-3'

[0136] 2) Send the PCR stock solution for sequencing, and use Snap Gene software to analyze the peak chart. If the peak chart shows double peaks after 3-4 nt of PAM sequence, or the target sequence cannot be found, the sequence may have been edited. The MDS Decode website http: / / skl.scau.edu.cn / dsdecode / ) can be used to determine the editing method, and the website can detect multiple sequence files at the same time.

[0137] 3) According to the results of website combined with software analysis, MAPK3 and MAPK6 different knockout type transgenic strawberry lines were obtained: line 4, line 5, line 6 and line 7.

[0138] Line 4, compared with wild type WT, for FvMAPK3 gene, 2 homologous chromosomes FvMAPK3 gene mutation as follows: the gene encoding FvMAPK3 protein all have the following mutations: "5'-AGCGCACGCTCCGTGAGATT-3'" is mutated to "5'-AGCGGCACGCTCCGTGAGATT-3'", line 4 is to insert 1 nucleotide "G" at the 245-246th position of sequence 1 in the sequence table (corresponding to the 592-593th position of sequence 2), the insertion of nucleotide causes frame shift, leading to premature termination of translation, resulting in loss of function of FvMAPK3 protein, thereby knocking out FvMAPK3 gene, the mutation site result is shown in Figure 2 A.

[0139] Strain 5, compared with wild type WT, for FvMAPK6 gene, FvMAPK6 gene in two homologous chromosomes has the following mutations: the gene encoding FvMAPK6 protein has the following mutations: "5'-GACACGGTGATGTCAGAGGC-3'" is mutated into "5'-GACACGGTGATGTGC-3'", strain 5 is that 38-42 of sequence 4 in the sequence listing (corresponding to 298-302 of sequence 5) are deleted by 5 nucleotides "CAGAG", the insertion of nucleotides causes a frame shift, which leads to premature termination of translation, resulting in loss of function of FvMAPK6 protein, so as to knock out FvMAPK6 gene, the mutation site result is shown in Figure 2 Table B.

[0140] Strain 6, compared with wild type WT, for FvMAPK3 gene, FvMAPK3 gene in one chromosome has no mutation, and FvMAPK3 gene in the other chromosome has the following mutation: "5'-AGCGCACGCTCCGTGAGATT-3'" is mutated into "5'-AGCGGCACGCTCCGTGAGATT-3'", strain 6 is that 245-246 of sequence 1 in the sequence listing (corresponding to 592-593 of sequence 2) are inserted by 1 nucleotide "G", the insertion of nucleotides causes a frame shift, which leads to premature termination of translation, resulting in partial loss of function of FvMAPK3 protein, so as to knock down FvMAPK3 gene, the sequencing result of the mutation site and the surrounding nucleotides is shown in Figure 2 Table C; for FvMAPK6 gene, FvMAPK6 gene in two homologous chromosomes has the following mutations: the gene encoding FvMAPK6 protein has the following mutations: "5'-GACACGGTGATGTCAGAGGC-3'" is mutated into "5'-GACACGGTGATGTGC-3'", strain 6 is that 38-42 of sequence 4 in the sequence listing (corresponding to 298-302 of sequence 5) are deleted by 5 nucleotides "CAGAG", the insertion of nucleotides causes a frame shift, which leads to premature termination of translation, resulting in loss of function of FvMAPK6 protein, so as to knock out FvMAPK6 gene, the mutation site result is shown in Figure 2 Table C.

[0141] Strain 7 has the following mutation in the FvMAPK3 gene on both homologous chromosomes: the gene encoding the FvMAPK3 protein has the following mutation: "5'- AGCGCACGCTCCGTGAGATT-3'" is mutated into "5'-AGCGGCACGCTCCGTGAGATT-3'", strain 7 is an insertion of 1 nucleotide "G" at position 245-246 of SEQ ID NO: 1 (corresponding to positions 592-593 of SEQ ID NO: 2), the insertion of nucleotides causes a frame shift, resulting in premature termination of translation, causing the FvMAPK3 protein to lose function, thereby knocking out the FvMAPK3 gene, the sequencing results of the mutation site and the surrounding nucleotides are shown in Table 1. Figure 2 Strain 7 has the following mutation in the FvMAPK6 gene on one homologous chromosome: the gene encoding the FvMAPK6 protein has the following mutation: "5'- GACACGGTGATGTCAGAGGC-3'" is mutated into "5'-GACACGGTGATGTGC-3'", strain 7 is a deletion of 5 nucleotides "CAGAG" at positions 38-42 of SEQ ID NO: 4 (corresponding to positions 298-302 of SEQ ID NO: 5), the insertion of nucleotides causes a frame shift, resulting in premature termination of translation, causing the FvMAPK6 protein to partially lose function, thereby knocking down the FvMAPK6 gene, the results of the mutation site are shown in Table 2. Figure 2 Strain 7 has the following mutation in the FvMAPK6 gene on one homologous chromosome: the gene encoding the FvMAPK6 protein has the following mutation: "5'- GACACGGTGATGTCAGAGGC-3'" is mutated into "5'-GACACGGTGATGTGC-3'", strain 7 is a deletion of 5 nucleotides "CAGAG" at positions 38-42 of SEQ ID NO: 4 (corresponding to positions 298-302 of SEQ ID NO: 5), the insertion of nucleotides causes a frame shift, resulting in premature termination of translation, causing the FvMAPK6 protein to partially lose function, thereby knocking down the FvMAPK6 gene, the results of the mutation site are shown in Table 2.

[0142] Example 2, Phenotype identification of mapk3 / mapk6 gene knockout mutant strawberry

[0143] Plants to be tested: T1 generation plants of diploid strawberry variety di Bosco (Fragaria vesca, cv. di Bosco) (referred to as wild type WT), gene knockout strain 4, strain 5, strain 6 and strain 7. Several plants of each strain were planted, and 3 replicates were set up.

[0144] The strawberry plants to be tested were planted in planting pots with a diameter of 230 mm and a depth of 230 mm, containing nutrient soil, vermiculite and grass carbon (2:1:1, v / v / v). The planting environment temperature was 25°C / 18°C (day / night), the relative humidity was 60%, the photoperiod was 16h / 8h (day / night), and the light density was 450 μmol m -2 s -1 .

[0145] The observation results of the phenotypes of each line show that the FvMAPK3 / MAPK6 gene affects the flowering time and fertility of the strawberry plant. Compared with the wild type cultured under the same planting condition at the same time, the line of homozygous knockout of FvMAPK3 (i.e. line 4) has the phenotype of late flowering, and the double knockout of FvMAPK3 / MAPK6 (i.e. line 6 and line 7) seriously affects the normal growth and development of the strawberry plant and leads to late flowering. Figure 3 In the middle A).

[0146] When the wild type cultured under the same planting condition at the same time enters the flowering and fruiting period, the line of homozygous knockout of FvMAPK3 (i.e. line 4) can normally fruit, and the line of homozygous knockout of FvMAPK6 (line 5) cannot fruit due to abortion. Figure 3 In the middle B). But in the season with higher temperature, the line 5 with abortion occasionally can set a few abnormal fruits. Figure 3 In the middle C).

[0147] The above has described the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the purpose and scope of the present application and without unnecessary experiments. Although the present application gives a special example, 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 intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.

Claims

1. Use of a substance that knocks out a gene encoding a protein in any one of the following: U1) for delaying flowering and reducing fertility in a plant; U2) for the manufacture of a product for delaying flowering and reducing fertility in a plant; U3) for breeding a plant with delayed flowering and reduced fertility; U4) for the manufacture of a product for breeding a plant with delayed flowering and reduced fertility; said protein is any one of the following: G1) a composition of a protein with an amino acid sequence of SEQ ID No. 3 and a protein with an amino acid sequence of SEQ ID No. 6; G2) a fusion protein obtained by linking a protein tag to the N-terminus or / and C-terminus of G1); said plant is a strawberry; said substance that knocks out a gene encoding a protein is a biological material related to said protein, and said biological material is any one of the following: e1) a nucleic acid molecule that knocks out the expression of said gene encoding a protein; e2) an expression cassette containing the nucleic acid molecule of e1); e3) a recombinant vector containing the nucleic acid molecule of e1), or a recombinant vector containing the expression cassette of e2); e4) a recombinant microorganism containing the nucleic acid molecule of e1), or a recombinant microorganism containing the expression cassette of e2), or a recombinant microorganism containing the recombinant vector of e3); e5) a transgenic plant cell line containing the nucleic acid molecule of e1), or a transgenic plant cell line containing the expression cassette of e2).

2. Use according to claim 1, characterized in that: e3) the expression cassette is a DNA molecule with a nucleotide sequence of SEQ ID No.

7.

3. A method for regulating the flowering period and fertility of plants, characterized in that: A method for delaying flowering and reducing fertility in a plant, comprising knocking out a gene encoding a protein as claimed in claim 1; said plant is a strawberry.

4. A breeding method for breeding plants with altered flowering time and fertility, characterized in that: A method for breeding a plant with delayed flowering and reduced fertility, comprising knocking out a gene encoding a protein as claimed in claim 1; said plant is a strawberry.

5. The method of claim 4, wherein: A method for breeding a plant with delayed flowering and reduced fertility, comprising introducing into a recipient plant a recombinant expression vector containing a nucleic acid molecule that knocks out the expression of a gene encoding a protein as claimed in claim 1; said gene encoding a protein is as claimed in claim 1.

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