Application of MAPK related protein for regulating and controlling flowering phase and fertility of plants

Abstract

The invention discloses an application of an MAPK related protein for regulating and controlling the flowering phase and fertility of plants. The invention belongs to the field of botany, and particularly relates to application of MAPK related protein for regulating and controlling the flowering phase and fertility of plants. The invention provides a method for regulating and controlling the flowering phase and fertility of a plant, which comprises the step of regulating and controlling the activity and / or content of protein in a target plant, or / and regulating and controlling the expression quantity of a coding gene of the protein, so as to regulate and control the flowering phase and fertility of the plant. The protein is the following protein: a composition consisting of a protein FvMAPK3 of which the amino acid sequence is SEQ ID No. 3 and a protein FvMAPK6 of which the amino acid sequence is SEQ ID No. 6. Experiments prove that the flowering phase and fertility of the strawberries can be regulated and controlled by reducing the expression of the two proteins FvMAPK3 and FvMAPK6, and important theoretical significance is achieved for strawberry breeding.

Description

Technical Field

[0001] The invention relates to the field of botany, and in particular to the application of MAPK-related proteins for regulating the flowering period and fertility of plants. Background Art

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

[0003] Strawberry is a perennial herbaceous plant of the genus Fragaria in the Rosaceae family. It has strong adaptability and is one of the most widely cultivated fruits in the world. It has extremely high nutritional and medicinal value and anti-aging effects, and is loved by people. However, during the production process of strawberries, they are 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 deformed fruits and poor fruit quality, which seriously affects the economic benefits of strawberries.

[0004] Therefore, cloning or knocking out key genes that regulate strawberry plant morphology, flower and fruit development, and quality formation through molecular biological methods can lay the foundation for improving strawberry quality in the future. Summary of the invention

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

[0006] In order to solve the problems existing in the prior art, the present invention provides the use of proteins or substances for regulating gene expression or substances for regulating the activity or content of the proteins in regulating the flowering period and fertility of plants.

[0007] The application provided by the present invention is the application of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in any of the following:

[0008] 1) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in regulating the flowering period and fertility of plants;

[0009] 2) Use of proteins or substances that regulate gene expression or substances that regulate the activity or content of the proteins in the preparation of products that regulate plant flowering period and fertility;

[0010] 3) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in cultivating plants with altered flowering period and fertility;

[0011] 4) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in the preparation of products for cultivating plants with altered flowering period and fertility;

[0012] 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding;

[0013] The protein is any of the following:

[0014] G1) a composition consisting of a protein having an amino acid sequence of SEQ ID No. 3 and a protein having an amino acid sequence of 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 obtained by replacing and / or deleting and / or adding amino acid residues of the proteins of G1) and G2) and having more than 80% identity with the protein shown in A1) and having functions related to regulating plant flowering period and fertility;

[0017] G4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus 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 proteins can be artificially synthesized, or their encoding genes 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 with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing 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 described in the above application is derived from strawberry (Fragaria vesca, cv Fragola di Bosco).

[0023] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the proteins FvMAPK3 and FvMAPK6.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0040] In the above biological material, the nucleic acid molecule described in c1) is a DNA molecule shown in any of the following:

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

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

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

[0044] d4) The coding region sequence is the DNA molecule of 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 may be a plant gene editing vector. The plant gene editing vector may be a pYLCRISPR / Cas9Pubi-H vector.

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

[0048] The sgRNA1 and sgRNA2 expression cassette sequences are the promoter sequence of the pYLsgRNA-AtU6-29 vector (positions 163-482 of SEQ ID No. 7) (which can be transcribed 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.

[0049] The microorganism described in e4) above may be Agrobacterium. The Agrobacterium is EHA105. The recombinant Agrobacterium may be EHA105 / mapk3 / mapk6-cr.

[0050] A person skilled in the art can easily mutate the nucleotide sequences encoding proteins FvMAPK3 and FvMAPK6 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequences of proteins FvMAPK3 and FvMAPK6 isolated from the present invention are all derived from the nucleotide sequences of the present invention and are equivalent to the sequences of the present invention as long as they encode proteins FvMAPK3 and FvMAPK6 and have the functions of proteins FvMAPK3 and FvMAPK6.

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

[0052] Herein, 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 a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0053] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0054] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (ie, cosmid), Ti plasmid or viral vector.

[0055] The recombinant expression vector containing 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. The plant expression vectors may also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as carmine synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0056] When using FvMAPK3 and FvMAPK6 genes to construct recombinant plant expression vectors, any enhanced promoter or constitutive promoter can be added before the transcription start nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start 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 be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

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

[0059] The method for regulating the flowering period and fertility of plants provided by the present invention comprises regulating the flowering period and fertility of plants by simultaneously 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.

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

[0061] The present invention also provides a method for cultivating plants with altered flowering period and fertility.

[0062] The method for cultivating plants with altered flowering period and fertility provided by the present invention comprises down-regulating or inhibiting or reducing the expression level of the genes encoding the proteins FvMAPK3 and FvMAPK6 in the target plant, or / and down-regulating or inhibiting or reducing the activity and / or content of the genes encoding the proteins, to obtain plants with altered flowering period and fertility.

[0063] In the above-mentioned breeding method, the down-regulation or inhibition or reduction of the activity and / or content of the protein in the target plant, or / and the expression level of the gene encoding the protein can be obtained by introducing a recombinant expression vector containing a nucleic acid molecule that inhibits or reduces or silences the expression of the gene encoding the proteins FvMAPK3 and FvMAPK6 into the recipient plant, thereby obtaining the target plant with altered flowering period and fertility.

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

[0065] In one embodiment of the present invention, the method for cultivating plants with altered flowering period and fertility comprises the following steps:

[0066] 1) constructing a recombinant expression vector comprising the DNA molecules shown in SEQ ID No. 2 and SEQ ID No. 4 for inhibiting, reducing or silencing;

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

[0068] 3) Transgenic plants with altered flowering period and fertility are obtained through screening and identification.

[0069] The introduction refers to introduction by recombinant means, including but not limited to Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta technology, and the like.

[0070] By using any vector that can guide the expression of foreign genes in plants, the coding genes or gene fragments for knocking out proteins FvMAPK3 and FvMAPK6 provided by the present invention are introduced into plant cells or recipient plants, and transgenic cell lines and transgenic plants with altered flowering period and fertility of plants can be obtained. The expression vector carrying the coding genes for knocking out proteins FvMAPK3 and FvMAPK6 can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues are cultivated into plants.

[0071] The microorganism described herein may be yeast, bacteria, algae or fungi. Among them, the bacteria may be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it may be Agrobacterium EHA105.

[0072] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be down-regulation, attenuation or reduction.

[0073] The proteins and / or biological materials in the above-mentioned applications also fall within the scope of protection claimed by the present invention.

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

[0075] C1) dicotyledons;

[0076] C2) Plants of the order Rosales;

[0077] C3) Rosaceae;

[0078] C4) Fragaria plants;

[0079] C5) Strawberry.

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

[0081] The MAPK3 and MAPK6 proteins of the present invention have the function of regulating the flowering period and fertility of strawberries. The expression of MAPK3 and MAPK6 genes is downregulated or weakened or reduced or knocked out by the CRISPR-Cas9 method, and the gene function thereof is verified. It is found that when only FvMAPK3 is knocked out (i.e., strain 4), the strawberry shows a late flowering phenotype, and the double knockout of Fvmapk3 / mapk6 (strain 6 and strain 7) seriously affects the normal growth and development of strawberry plants and leads to a late flowering phenotype. The homozygous plant with the FvMAPK6 gene knocked out (i.e., strain 5) shows a completely sterile phenotype, and the strawberry plant cannot be pollinated and fertilized normally, and cannot set fruit. The present invention provides available genetic materials for regulating the flowering period and fertility of strawberry plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 This is a map of the pYLCRISPR / Cas9Pubi-H vector.

[0083] Figure 2 The mutation types of transgenic strawberry lines with different knockout types of MAPK3 and MAPK6. 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; and D is the result of the mutation site of line 7.

[0084] Figure 3 The mapk3 / mapk6-cr phenotype diagram shows that A is the flowering phenotype of strawberry plants, and B and C are the fertility phenotypes of strawberry plants. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0086] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0087] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

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

[0089] The pYLCRISPR / Cas9Pubi-H plasmid in the following examples was kindly donated by the research group of Professor Liu Yaoguang of South China Agricultural University, and has been recorded in: Zeng Dongchang, Ma Xingliang, Xie Xianrong, Zhu Qinlong, Liu Yaoguang. Operation method for construction of plant CRISPR / Cas9 multi-gene editing vector and mutation analysis [J]. Chinese Science: Life Sciences, 2018, 48(07): 783-794. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0090] The AtU6-29 plasmid in the following examples was kindly donated by the research group of Professor Liu Yaoguang of South China Agricultural University, and has been recorded in: Zeng Dongchang, Ma Xingliang, Xie Xianrong, Zhu Qinlong, Liu Yaoguang. 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 the biological material from the applicant, and the biological material is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0091] Example 1. Construction of CRISPR-Cas9 recombinant vector mapk3 / mapk6-cr and acquisition of transformed strawberry plants

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

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

[0094] 1. Construction of recombinant vector MAPK3 / MAPK6-CR

[0095] 1) Design of target sites. Design the target sites of FvMAPK3 and FvMAPK6 genes on the website http: / / crispr.hzau.edu.cn / CRISPR / , select target sequences with high scores and no off-target, synthesize primers for vector construction. Using pYLCRISPR / Cas9Pubi-H as a plant expression vector, select the reverse complementary sequence at positions 242-261 of sequence 1 in the CDS sequence of FvMAPK3 as a target (target sequence: 5'-AATCTCACGGAGCGTGCGCT-3') for primer design and vector construction, and select a target at positions 25-44 of sequence 4 in the CDS sequence of FvMAPK6 (target sequence: 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] UF:5'-CTCCGTTTTACCTGTGGAATCG-3'

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

[0103] 2) Construction of sgRNA expression cassette. Using sgRNA as template, the corresponding target (U#T# / gRT#) and linker (UF / gR-R) as primers, configure the PCR reaction system. The system is shown in Table 1 below.

[0104] Table 1. PCR reaction system constructed with sgRNA expression cassette

[0105]

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

[0107] Pps-GGL:5'-TTCAGAGGTCTCCTCGACTAGTATGGAATCGGCAGCAAAGG-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) in step 2) into the pYLCRISPR / Cas9Pubi-H vector (referred to as pYLCRISPR / Cas9 vector). The sgRNA expression cassette was connected to the expression vector using the Golden Gate method. The system is shown in Table 2 below.

[0112] Table 2. Ligation reaction system

[0113]

[0114]

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

[0116] 4) The PCR reaction product was transformed into DH5α Escherichia coli using the heat shock method. The transformation method was as follows: the gel recovery product was added to 50 μL DH5α Escherichia coli competent cells, placed on ice for 30 min, heat shocked at 42°C for 45 s, immediately cooled on ice for 2 min, added with 500 μL of antibiotic-free LB, placed in a 37°C shaker at 180 rpm for 40 min, centrifuged at 5000 rpm for 5 min to collect the bacteria, discarded 400 μL of supernatant, resuspended the cells, spread on LB solid culture medium containing 50 mg / L kanamycin with a spreader, cultured at 37°C overnight, picked a single clone and placed it in LB liquid culture medium containing 50 mg / L kanamycin, placed in a 37°C shaking incubator for 6-8 h, used primers SP-L1: 5'-GCGGTGTCATCTATGTTACTAG-3' and SP-R: 5'-TGCAATAACTTCGTATAGGCT-3' for bacterial liquid PCR identification, and selected positive clones for sequencing. If the sequencing result is the nucleotide shown in sequence 7 in the sequence table, the corresponding transformed bacteria are positive bacteria. The positive bacteria are recombinant Escherichia coli DH5α / mapk3 / mapk6-cr containing the recombinant vector mapk3 / mapk6-cr. The positive clones with correct sequencing are cultured in shake flasks, and the plasmids are extracted with a plasmid mini-extraction kit to obtain the recombinant vector mapk3 / mapk6-cr.

[0117] The recombinant vector mapk3 / mapk6-cr is a recombinant vector obtained by inserting the expression cassettes 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 the recombinant vector mapk3 / mapk6-cr.

[0118] The sgRNA1 and sgRNA2 expression cassette sequences are the promoter sequence of the pYLsgRNA-AtU6-29 vector (positions 163-482 of SEQ ID No. 7) (which can be transcribed 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 transgenic strawberry plants

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

[0121] The recombinant vector mapk3 / mapk6-cr was transformed into Agrobacterium competent EHA105 (Shanghai Weidi Biotechnology Co., Ltd.). The transformation method is as follows: 5 μg of plasmid was added to 50 μL EHA105 Agrobacterium competent medium, gently mixed and placed on ice for 5 minutes; liquid nitrogen quick freezing for 5 minutes, 37°C water bath for 5 minutes; 700 μL of LB liquid medium without resistance was added, and shaking culture was carried out at 28°C and 180 rpm for 4 hours; centrifugation at 5000 rpm for 5 minutes to collect the bacteria, discard the supernatant, and the remaining 100 μL of resuspended bacteria was spread on a plate containing 25 mg / L rifampicin and 50 mg / L kanamycin, and cultured at 28°C for 2 days. Single colonies were picked and identified by PCR using SP-L and SP-R primers. The PCR product size of the positive clone was 1000 bp (i.e., the length of two sgRNA expression cassettes), and the recombinant Agrobacterium EHA105 / mapk3 / mapk6-cr that was successfully transformed with the recombinant vector mapk3 / mapk6-cr with double knockout of mapk3 / mapk6 genes was obtained.

[0122] 2) Stable genetic transformation of diploid strawberry

[0123] Preparation and activation of the infectious bacterial solution: Centrifuge the EHA105 / mapk3 / mapk6-cr bacterial solution at 5000rpm for 10 minutes, pour off the supernatant after centrifugation in a clean bench, add MS activation solution (the solvent is water, the solute and its content are: MS4.4g / L, sucrose 30g / L, 1M NaOH to adjust the pH to 5.8, sterilize at high temperature and high pressure, add acetosyringone with a final concentration of 100-200μM before use) to suspend the bacteria, so that the OD value of the bacterial solution is 0.4-0.6, and place it in a shaker at 28℃ for activation for 40 minutes.

[0124] Healthy and plump di Bosco strawberry seeds were sterilized with 75% anhydrous ethanol and 1% NaClO, and then spotted on 1 / 2MS culture medium. After being protected from light for 4-7 days, they were placed in a constant temperature and humidity tissue culture room for culture, with a photoperiod of 12 / 12h, a temperature of 24°C, and a humidity of 45%. After culturing for 40d-50d, diploid strawberry tissue culture seedlings were obtained.

[0125] Explant infection (leaf disc method): During the activation of bacterial solution, plant materials were cut as explants. Di Bosco (hereinafter referred to as wild-type strawberry, WT) tissue culture seedlings of 50-60d-old strawberry diploid variety were taken, and their young leaves and petioles were cut into appropriate sizes and placed in MS activation solution. After a certain number of explants were cut, the activated bacterial solution was added, and then the explants were placed in a 50ml syringe and vacuumed until the leaves were in a water-soaked state.

[0126] Explant culture: After infection, the explants were transferred to co-culture medium (solvent was water, solute and its content were 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 adjusted pH to 5.8, high temperature and high pressure sterilization), and cultured in the dark for 2 days. After dark culture, the explants were carefully transferred to the screening medium (solvent was water, solute and its content were 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 adjusted pH to 5.8, high temperature and high pressure sterilization, Hyg 2 mg / L, Tim (Timentin) 400 mg / L), and cultured for about 40 days to grow buds.

[0127] Callus rooting: When callus sprouts, transfer to rooting medium (solvent is water, solute and its content are MS4.4g / L, sucrose 30g / L, agar powder 7g / L, 1M NaOH to adjust pH to 5.8, add Hyg 2mg / L, Tim 400mg / L after high temperature and high pressure sterilization).

[0128] Transplantation of transgenic seedlings: Move seedlings with roots growing to 4-5 cm and well-developed fibrous root systems into the soil, carefully remove the root culture medium, and cover with plastic wrap to keep moisture. Remove the plastic wrap after 15 days to obtain transgenic plants to be identified containing the recombinant vector mapk3 / mapk6-cr.

[0129] 3. Identification of transgenic positive strains

[0130] In order to identify transgenic positive plants, take leaves of the transgenic strawberry plants in step 2, extract DNA, and perform PCR detection using genomic DNA as a template.

[0131] 1) Extract DNA from the transgenic plant to be identified containing the recombinant vector mapk3 / mapk6-cr and identify it by PCR. The primers are as follows:

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

[0133] mapk3-cr-r:5'-CAACTTTCACAAAACATACAC-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 graph of the sequencing results. If the peak graph shows double peaks 3-4 nt before the 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. The website can detect multiple sequence files at the same time.

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

[0138] Compared with the wild type WT, strain 4 had the following mutations in the FvMAPK3 gene in the two homologous chromosomes: the genes encoding the FvMAPK3 protein all underwent the following mutations: "5'-AGCGCACGCTCCGTGAGATT-3'" mutated to "5'-AGCGGCACGCTCCGTGAGATT-3'", and strain 4 inserted a nucleotide "G" at positions 245-246 of sequence 1 in the sequence list (corresponding to positions 592-593 of sequence 2). The insertion of the nucleotide caused a frameshift, resulting in premature termination of translation, resulting in loss of FvMAPK3 protein function, and thus knocking out the FvMAPK3 gene. The results of this mutation site are shown in Figure 2 Middle A.

[0139] Compared with the wild type WT, strain 5 had the following mutations in the FvMAPK6 gene in the two homologous chromosomes: the genes encoding the FvMAPK6 protein all underwent the following mutations: "5'-GACACGGTGATGTCAGAGGC-3'" mutated to "5'-GACACGGTGATGTGC-3'", and strain 5 deleted 5 nucleotides "CAGAG" from positions 38-42 of sequence 4 in the sequence list (corresponding to positions 298-302 of sequence 5). The insertion of nucleotides caused a frameshift, resulting in premature termination of translation, resulting in loss of FvMAPK6 protein function, and thus knocking out the FvMAPK6 gene. The results of this mutation site are shown in Figure 2 Middle B.

[0140] Compared with the wild-type WT, strain 6 had no mutation in the FvMAPK3 gene in one chromosome, but had the following mutation in the FvMAPK3 gene in the other chromosome: "5'-AGCGCACGCTCCGTGAGATT-3'" mutated to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 6, one nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2) in the sequence list. The insertion of the nucleotide caused a frameshift, resulting in premature termination of translation and partial loss of FvMAPK3 protein function, thereby knocking down the FvMAPK3 gene. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2 C; For the FvMAPK6 gene, the following mutations occurred in the FvMAPK6 gene in the two homologous chromosomes: the genes encoding the FvMAPK6 protein all underwent the following mutations: "5'-GACACGGTGATGTCAGAGGC-3'" mutated to "5'-GACACGGTGATGTGC-3'", and strain 6 deleted 5 nucleotides "CAGAG" from positions 38-42 of sequence 4 in the sequence list (corresponding to positions 298-302 of sequence 5). The insertion of nucleotides caused a frameshift, resulting in premature termination of translation, resulting in loss of FvMAPK6 protein function, and thus knocking out the FvMAPK6 gene. The results of this mutation site are shown in Figure 2 Middle C.

[0141] Compared with the wild type WT, strain 7 had the following mutations in the FvMAPK3 gene in both homologous chromosomes: the genes encoding the FvMAPK3 protein all had the following mutations: "5'-AGCGCACGCTCCGTGAGATT-3'" mutated to "5'-AGCGGCACGCTCCGTGAGATT-3'", and strain 7 had a nucleotide "G" inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2) in the sequence list. The insertion of the nucleotide caused a frameshift, resulting in premature termination of translation, causing loss of FvMAPK3 protein function, and thus knocking out the FvMAPK3 gene. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2 D; for the FvMAPK6 gene, the FvMAPK6 gene in one chromosome did not mutate, and the FvMAPK6 gene in the other homologous chromosome mutated as follows: "5'-GACACGGTGATGTCAGAGGC-3'" mutated to "5'-GACACGGTGATGTGC-3'", strain 7 deleted 5 nucleotides "CAGAG" from positions 38-42 of sequence 4 in the sequence list (corresponding to positions 298-302 of sequence 5), and the insertion of nucleotides caused a frameshift, resulting in premature termination of translation, causing partial loss of FvMAPK6 protein function, thereby knocking down the FvMAPK6 gene. The results of this mutation site are shown in Figure 2 Middle D.

[0142] Example 2: Identification of strawberry phenotypes of mapk3 / mapk6 gene knockout mutants

[0143] Plants to be tested: T1 plants of diploid strawberry varieties 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 the experiment was set up with 3 replicates.

[0144] The strawberry plants to be tested were planted in a pot with a diameter of 230 mm and a depth of 230 mm containing a matrix of nutrient soil, vermiculite, and peat (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 optical density was 450 μmol m -2 s -1 .

[0145] The results of observation of the phenotypes of each strain showed that the FvMAPK3 / MAPK6 gene affects the flowering period and fertility of strawberry plants. Compared with the wild type cultivated at the same time under the same planting conditions, the strain with homozygous knockout of FvMAPK3 (i.e. strain 4) showed a late flowering phenotype of strawberry, and the double knockout of FvMAPK3 / MAPK6 (i.e. strain 6 and strain 7) seriously affected the normal growth and development of strawberry plants and caused late flowering ( Figure 3 (A).

[0146] When the wild-type strains cultured under the same planting conditions entered the flowering and fruiting stage, the homozygous knockout strain of FvMAPK3 (i.e., strain 4) could bear fruit normally, while the homozygous knockout strain of FvMAPK6 (strain 5) could not bear fruit due to sterility ( Figure 3 However, in seasons with higher temperatures, the sterile line 5 occasionally produced a few deformed fruits ( Figure 3 Middle C).

[0147] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in any of the following: U1) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in regulating the flowering period and fertility of plants; U2) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in the preparation of products regulating plant flowering period and fertility; U3) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in cultivating plants with altered flowering period and fertility; U4) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for cultivating plants with altered flowering period and fertility; U5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding; The protein is any of the following: G1) a composition consisting of a protein having an amino acid sequence of SEQ ID No. 3 and a protein having an amino acid sequence of SEQ ID No. 6; G2) a protein whose amino acid sequence is SEQ ID No. 3 or a protein whose amino acid sequence is SEQ ID No. 6; G3) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the proteins of G1) and G2) and having more than 80% identity with the protein shown in A1) and having functions related to regulating plant flowering period and fertility; G4) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of G1) or G2).

2. The use according to claim 1, characterized in that: The protein is derived from strawberries.

3. The use according to claim 1 or 2, characterized in that: The substance regulating gene expression or the substance regulating the activity or content of the protein is a biological material related to the protein, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein according to claim 1; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein according to claim 1 or 2; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1), or a transgenic plant organ containing the expression cassette described in e2).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown in any of the following: d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No.2; d2) the coding region sequence is the DNA molecule shown in SEQ ID No. 1 in the sequence listing; d3) the nucleotide sequence is the DNA molecule shown in SEQ ID No.5; d4) The coding region sequence is the DNA molecule of SEQ ID No. 4 in the sequence listing.

5. The use according to claim 3, characterized in that: e3) The expression cassette is a DNA molecule whose nucleotide sequence is shown as SEQ ID No.

7.

6. A method for regulating the flowering period and fertility of plants, characterized in that: The method comprises regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and regulating the expression level of the gene encoding the protein described in claim 1 or 2 to regulate the flowering period and fertility of the plant.

7. A breeding method for cultivating plants with altered flowering period and fertility, comprising regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and regulating the expression level of the gene encoding the protein described in claim 1 or 2, to obtain plants with altered flowering period and fertility.

8. The method according to claim 7, characterized in that: The method of regulating the activity and / or content of the protein described in claim 1 or 2, or / and the expression level of the gene encoding the protein described in claim 1 or 2 in the target plant comprises introducing a recombinant expression vector containing a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein described in claim 1 or 2 into the recipient plant, thereby obtaining the target plant with changed plant flowering period and fertility; the encoding gene encodes the protein described in claim 1 or 2.

9. The protein for use in claim 1 or 2 and / or the biological material for use in claim 3 or 4.

10. The use according to any one of claims 1 to 4, the method according to any one of claims 6 to 8, characterized in that: The plant is any one of the following: C1) dicotyledons; C2) Plants of the order Rosales; C3) Rosaceae; C4) Fragaria plants; C5) Strawberry.

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