Application of MAPK-related proteins in regulating plant architecture and growth and development

Abstract

This invention discloses the application of MAPK-related proteins that regulate plant architecture and growth. This invention belongs to the field of botany, specifically relating to the application of MAPK-related proteins that regulate plant architecture and growth. This invention provides a method for regulating plant architecture and growth, including regulating the activity and / or content of proteins in the target plant, and / or regulating the expression level of the protein-encoding genes, to regulate plant architecture and growth. The protein is a composition consisting of protein FvMAPK3 (SEQ ID No. 3) and protein FvMAPK6 (SEQ ID No. 6). Experiments have shown that reducing the expression of the two proteins FvMAPK3 and FvMAPK6 can regulate strawberry plant architecture and growth, which has important theoretical significance for strawberry breeding.

Description

Technical Field

[0001] This invention relates to the field of botany, specifically to the application of MAPK-related proteins that regulate plant architecture and growth and development. Background Technology

[0002] Strawberries have become the fastest-growing sector in my country's fruit industry. Their economic and social benefits have significantly increased, not only playing a positive role in boosting farmers' income but also promoting the integrated development of primary, secondary, and tertiary industries as a fruit suitable for agricultural tourism and fruit picking. The quality characteristics of strawberries, including the accumulation, morphology, and texture of nutrients and flavor compounds, affect their quality and nutritional value.

[0003] For many years, efforts have been made to improve the quality of horticultural crops. Recently developed gene-editing technologies and their application in horticultural crops offer an effective strategy for rapidly and efficiently improving crop quality. CRISPR / SpCas9, derived from Streptococcus pyogenes, is currently the most widely used genome editing system among CRISPR / Cas9 technologies, exhibiting high specificity and editing efficiency. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to regulate the plant shape and growth and development of strawberries.

[0005] To address the problems existing in the prior art, this invention provides a method for regulating plant shape and growth and development.

[0006] The method for regulating plant architecture and growth and development provided by the present invention includes regulating plant architecture and growth and development by simultaneously regulating the expression of the encoding genes of proteins FvMAPK3 and FvMAPK6 in the target plant, or regulating the activity and / or content of the proteins, or regulating the activity and / or content of the encoding genes of the proteins.

[0007] The protein is any of the following proteins:

[0008] G1) A composition consisting of a protein whose amino acid sequence is SEQ ID No. 3 and a protein whose amino acid sequence is SEQ ID No. 6;

[0009] G2) The amino acid sequence of the protein is SEQ ID No. 3 or the amino acid sequence of the protein is SEQ ID No. 6;

[0010] G3) The proteins of G1) and G2) are obtained by substituting and / or deleting and / or adding amino acid residues, resulting in proteins with more than 80% identity with the proteins shown in A1) and with functions related to regulating plant architecture and growth and development.

[0011] G4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1) or G2).

[0012] The protein whose amino acid sequence is SEQ ID No. 3 described above is named FvMAPK3.

[0013] The protein whose amino acid sequence is SEQ ID No. 6 described above is named FvMAPK6.

[0014] This invention also provides a method for cultivating plants with altered plant shape and growth and development.

[0015] The method for cultivating plants with altered plant type and growth and development provided by the present invention includes downregulating, inhibiting or reducing the expression level of the coding genes of proteins FvMAPK3 and FvMAPK6 in the target plant, and / or downregulating, inhibiting or reducing the activity and / or content of the coding genes of the proteins, to obtain plants with altered plant type and growth and development.

[0016] In the above cultivation method, the downregulation, inhibition, or reduction of the activity and / or content of the protein in the target plant, or / and the expression level of the protein encoding gene can be obtained by introducing a recombinant expression vector containing nucleic acid molecules that inhibit, reduce, or silence the expression of the encoding genes of the proteins FvMAPK3 and FvMAPK6 into the recipient plant, thereby obtaining the target plant with altered plant type and growth and development.

[0017] In the above method, the protein is any of the following proteins:

[0018] G1) A composition consisting of a protein whose amino acid sequence is SEQ ID No. 3 and a protein whose amino acid sequence is SEQ ID No. 6;

[0019] G2) The amino acid sequence of the protein is SEQ ID No. 3 or the amino acid sequence of the protein is SEQ ID No. 6;

[0020] G3) The proteins of G1) and G2) are obtained by substituting and / or deleting and / or adding amino acid residues, resulting in proteins with more than 80% identity with the proteins shown in A1) and with functions related to regulating plant architecture and growth and development.

[0021] G4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1) or G2).

[0022] The protein whose amino acid sequence is SEQ ID No. 3 described above is named FvMAPK3.

[0023] The protein whose amino acid sequence is SEQ ID No. 6 described above is named FvMAPK6.

[0024] The FvMAPK3 and FvMAPK6 genes encode the FvMAPK3 and FvMAPK6 proteins.

[0025] In one embodiment of the present invention, the method for cultivating plants with altered plant type and growth and development includes the following steps:

[0026] 1) Construct a recombinant expression vector containing DNA molecules shown in SEQ ID No. 2 and SEQ ID No. 4 that inhibit, reduce, or silence them;

[0027] 2) Transform the recombinant expression vector constructed in step 1) into the recipient plant (such as crop or strawberry);

[0028] 3) Transgenic plants with altered plant type and growth and development were obtained through screening and identification.

[0029] The importation refers to the importation through recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, in-planta technology, and so on.

[0030] By using any vector capable of guiding the expression of exogenous genes in plants, the coding genes or gene fragments for knocking out proteins FvMAPK3 and FvMAPK6 provided in this invention can be introduced into plant cells or recipient plants, resulting in transgenic cell lines and transgenic plants with altered plant architecture and growth development. Expression vectors carrying the coding genes for knocked-out proteins FvMAPK3 and FvMAPK6 can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0031] The microorganisms described in this article can be yeast, bacteria, algae, or fungi. Among them, bacteria can originate from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*. Specifically, *Agrobacterium tumefaciens* EHA105 is an example.

[0032] This invention also provides the application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating plant architecture and growth and development.

[0033] The application provided by this invention is the use of proteins or substances that regulate gene expression, or substances that regulate the activity or content of said proteins, in any of the following:

[0034] 1) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating plant architecture and growth and development.

[0035] 2) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that regulate plant architecture and growth and development.

[0036] 3) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the cultivation of plants with altered plant type and growth and development.

[0037] 4) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that cultivate plants with altered plant type and growth and development.

[0038] 5) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in plant breeding;

[0039] The protein is any of the following proteins:

[0040] G1) A composition consisting of a protein whose amino acid sequence is SEQ ID No. 3 and a protein whose amino acid sequence is SEQ ID No. 6;

[0041] G2) The amino acid sequence of the protein is SEQ ID No. 3 or the amino acid sequence of the protein is SEQ ID No. 6;

[0042] G3) The proteins of G1) and G2) are obtained by substituting and / or deleting and / or adding amino acid residues, resulting in proteins with more than 80% identity with the proteins shown in A1) and with functions related to regulating plant architecture and growth and development.

[0043] G4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1) or G2).

[0044] The protein whose amino acid sequence is SEQ ID No. 3 described above is named FvMAPK3.

[0045] The protein whose amino acid sequence is SEQ ID No. 6 described above is named FvMAPK6.

[0046] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0047] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0048] The protein mentioned in the above application is derived from strawberry (Fragaria vesca).

[0049] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the proteins FvMAPK3 and FvMAPK6.

[0050] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0051] In this paper, the expression of the gene regulating the protein can be achieved by inhibiting, reducing, or downregulating the expression of the coding gene. Inhibition, reduction, or downregulation of the coding gene expression can be achieved through gene knockout or gene silencing.

[0052] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence, including but not limited to zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a site in the genome containing multiple short repeat sequences, where the Cas9 protein, mediated by RNA, can cleave target sequences recognized by crRNA–tracrRNA.

[0053] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0054] In the above applications, the substances that regulate gene expression and the substances that regulate the activity or content of the protein can be biological materials related to the protein, and the biological materials can be any of the following:

[0055] c1) The nucleic acid molecule that encodes the protein described above;

[0056] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0057] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0058] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0059] 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);

[0060] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0061] 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);

[0062] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0063] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0064] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0065] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0066] 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);

[0067] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0068] 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).

[0069] In the above-mentioned biological materials, c1) the nucleic acid molecule is any of the following DNA molecules:

[0070] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 2;

[0071] d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0072] d3) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 5;

[0073] d4) The coding region sequence is the DNA molecule of SEQ ID No. 4 in the sequence listing.

[0074] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0075] In the above e3), the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be the pYLCRISPR / Cas9Pubi-H vector.

[0076] As a specific embodiment, the recombinant vector is the recombinant vector mapk3 / mapk6-cr. The recombinant vector mapk3 / mapk6-cr is obtained by inserting the expression cassette sequences (nucleotide sequences of SEQ ID No. 7 in the sequence listing) of sgRNA1 and sgRNA2 into the restriction endonuclease BsaI site, while keeping the other nucleotide sequences of the vector pYLCRISPR / Cas9Pubi-H unchanged. The recombinant plasmid is named the recombinant vector mapk3 / mapk6-cr.

[0077] The expression cassette sequences of sgRNA1 and sgRNA2 consist of 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), forming a complete sgRNA expression cassette.

[0078] The microorganism mentioned in e4) above can be Agrobacterium. The Agrobacterium is EHA105. The recombinant Agrobacterium can be EHA105 / mapk3 / mapk6-cr.

[0079] Those skilled in the art can readily mutate the nucleotide sequences of the genes encoding the proteins FvMAPK3 and FvMAPK6 of this invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the proteins FvMAPK3 and FvMAPK6 isolated in this invention, provided they encode and function as proteins FvMAPK3 and FvMAPK6, are derived from and equivalent to the nucleotide sequences of this invention.

[0080] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0081] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0082] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0083] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0084] Recombinant expression vectors containing the FvMAPK3 and FvMAPK6 genes can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmids (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0085] When constructing recombinant plant expression vectors using the FvMAPK3 and FvMAPK6 genes, any enhancing or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0086] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0087] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0088] The proteins and / or the biological materials described in the preceding application are also within the scope of protection claimed by this invention.

[0089] In this invention, the plant is any one of the following:

[0090] C1) Dicotyledons;

[0091] C2) Plants of the Rosales order;

[0092] C3) Rosaceae plants;

[0093] C4) Strawberry plants;

[0094] C5) Strawberry.

[0095] The strawberry mentioned above can be the strawberry variety Fragaria vesca, cv Fragola di Bosco. The MAPK3 and MAPK6 proteins of this invention have the function of regulating strawberry plant architecture and growth development. By downregulating, weakening, reducing, or knocking out the expression of MAPK3 and MAPK6 genes using CRISPR-Cas9, and verifying their gene function, it was found that knocking out only FvMAPK3 (i.e., line 1) and homozygous knockout of FvMAPK3 (i.e., lines 4 and 7) resulted in mottling of strawberry leaves. FvMAPK3 may affect the synthesis of pigments such as chlorophyll in strawberry leaves. Double knockout of FvMAPK3 / mapk6 (lines 6 and 7) severely affected the normal growth and development of strawberry plants. This invention creates transgenic strawberry materials with double knockout of mapk3 / mapk6 using CRISPR / SpCas9 gene editing technology, providing valuable genetic material for future improvement of strawberry quality. Attached Figure Description

[0096] Figure 1 This is a diagram of the pYLCRISPR / Cas9Pubi-H vector.

[0097] Figure 2 Mutation types of transgenic strawberry lines with different knockout types of MAPK3 and MAPK6 are shown. A represents WT plants. B represents the mutation site results for line 1; C represents the mutation site results for line 2; D represents the mutation site results for line 3; E represents the mutation site results for line 4; F represents the mutation site results for line 5; G represents the mutation site results for line 6; and H represents the mutation site results for line 7.

[0098] Figure 3Phenotypic results of transgenic strawberry lines with different knockout types of mapk3 / mapk6-cr. Detailed Implementation

[0099] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0101] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0102] The strawberry variety Fragaria vesca,cv Fragola di Bosco used in the following examples (sourced from THEHEIRLOOM SEED STORE, available at: https: / / www.theheirloomseedstore.com / product / strawberry-fragola-di-bosco).

[0103] The pYLCRISPR / Cas9Pubi-H plasmid used in the following examples was kindly provided by Professor Liu Yaoguang's research group at South China Agricultural University and has been documented in: Zeng Dongchang, Ma Xingliang, Xie Xianrong, Zhu Qinlong, Liu Yaoguang. Operational methods for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vectors [J]. Science in China: Life Sciences, 2018, 48(07):783-794. This biological material is available to the public from the applicant and is intended solely for the replication of experiments in this invention; it may not be used for any other purpose.

[0104] The AtU6-29 plasmid used in the following examples was kindly provided by Professor Liu Yaoguang's research group at South China Agricultural University and is described in: Zeng Dongchang, Ma Xingliang, Xie Xianrong, Zhu Qinlong, Liu Yaoguang. Operational methods for construction and mutation analysis of plant CRISPR / Cas9 multi-gene editing vectors [J]. Science in China: Life Sciences, 2018, 48(07):783-794. This biological material is available to the public from the applicant and is intended solely for the replication of experiments in this invention; it may not be used for any other purpose.

[0105] Example 1: Construction of the CRISPR-Cas9 recombination vector mapk3 / mapk6-cr

[0106] The coding sequence of the FvMAPK3 gene in the strawberry variety Fragaria vesca, cv Fragola di Bosco is shown in SEQ ID No. 1 (nucleotide sequence), the genomic sequence of the FvMAPK3 gene is shown in SEQ ID No. 2 (genomic sequence), and the amino acid sequence of the encoded protein is shown in SEQ ID No. 3 (amino acid sequence).

[0107] The coding sequence of the FvMAPK6 gene in the strawberry variety Fragaria vesca, cv Fragola di Bosco is shown in SEQ ID No. 4, the genomic sequence of the FvMAPK6 gene is shown in SEQ ID No. 5, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 6.

[0108] 1. Construction of the recombinant vectors mapk3 / mapk6-cr

[0109] 1) Target Design. Targets for the FvMAPK3 and FvMAPK6 genes were designed using the website http: / / crispr.hzau.edu.cn / CRISPR / . High-scoring target sequences with no off-target effects were selected, and primers were synthesized for vector construction. Using pYLCRISPR / Cas9Pubi-H as the plant expression vector, the reverse complementary sequence located at positions 242-261 of sequence 1 in the CDS sequence of FvMAPK3 was selected as one target (target sequence: 5'-AATCTCACGGAGCGTGCGCT-3') for primer design and vector construction. Similarly, the target sequence located at positions 25-44 of sequence 4 in the CDS sequence of FvMAPK6 (target sequence: 5'-GACACGGTGATGTCAGAGGC-3') was selected for primer design and vector construction. The primer sequences are as follows:

[0110] AtU6-29MAPK3-:5'-AGCGCACGCTCCGTGAGATTCAATCTCTTAGTCGACT-3'

[0111] gRTMAPK3+:5'-AATCTCACGGAGCGTGCGCTGTTTTAGAGCTAGAAAT-3'

[0112] AtU6-29MAPK6-:5'-GCCTCTGACATCACCGTGTCCAATCTCTTAGTCGACT-3'

[0113] gRTMAPK6+:5'-GACACGGTGATGTCAGAGGCGTTTTAGAGCTAGAAAT-3'

[0114] UF:5'-CTCCGTTTTACCTGTGGAATCG-3'

[0115] gR-R:5'-CGGAGGAAAATTCCATCCAC-3'

[0116] 2) Construction of sgRNA expression cassette. Using sgRNA as a template, and the corresponding target site (U#T# / gRT#) and adapter (UF / gR-R) as primers, a PCR reaction system was prepared. The system is shown in Table 1 below.

[0117] Table 1. PCR reaction system for constructing sgRNA expression cassettes

[0118]

[0119] The PCR reaction conditions were: 98℃ for 30 s; 98℃ for 10 s, 56℃ for 5 s, 72℃ for 10 s, 32 cycles; 72℃ for 1 min. The PCR products were detected by 1% agarose gel electrophoresis, and a band of approximately 500 kb was excised. This fragment was recovered using Tiangen's agarose gel extraction kit. Using this fragment as a template, a second round of PCR amplification was performed using primers Pps-GGL, Pgs-GG2, Pps-GG2, and Pgs-GGR, with the same system as above. Electrophoresis was performed, and the fragments were recovered. The primer sequences are as follows:

[0120] Pps-GGL:5'-TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGCAAAGG-3'

[0121] Pgs-GG2:5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3'

[0122] Pps-GG2:5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3'

[0123] Pgs-GGR:5'-AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAAGCTC-3'

[0124] 3) Assemble the sgRNA expression cassette (PCR gel recovery product) from step 2) into the pYLCRISPR / Cas9Pubi-H vector (abbreviated as pYLCRISPR / Cas9 vector). The sgRNA expression cassette was ligated into the expression vector using the Golden Gate method; the system is shown in Table 2 below.

[0125] Table 2. Connection Reaction System

[0126]

[0127]

[0128] The PCR reaction conditions were: 37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min, 20 cycles, 37℃ for 5 min;

[0129] 4) The PCR product was transformed into DH5α Escherichia coli using the heat shock method. The transformation method was as follows: the gel-recovered product was added to 50 μl of DH5α Escherichia coli competent cells, placed on ice for 30 min, heat-shocked at 42℃ for 45 s, immediately cooled on ice for 2 min, 500 μl of antibiotic-free LB was added, and the mixture was placed on a shaker at 37℃ for 180 rpm for 40 min, centrifuged at 5000 rpm for 5 min to collect the bacteria, 400 μl of supernatant was discarded, the bacterial resuspended, and spread on LB solid medium containing 50 mg / L kanamycin using a spreader. The mixture was incubated overnight at 37℃. Single clones were picked and placed in LB liquid medium containing 50 mg / L kanamycin and incubated in a shaking incubator at 37℃ for 6-8 h. The bacterial culture was identified by PCR using primers SP-L1: 5'-GCGGTGTCATCTATGTTACTAG-3' and SP-R: 5'-TGCAATAACTTCGTATAGGCT-3', and positive clones were selected for sequencing. If the sequencing result matches the nucleotides shown in SEQ ID No. 7 of the sequence listing, the corresponding transformed bacteria are positive. The positive bacteria are recombinant *E. coli* DH5α / mapk3 / mapk6-cr containing the recombinant vector mapk3 / mapk6-cr. The correctly sequenced positive clones are cultured in shake flasks, and plasmids are extracted using a plasmid miniprep kit to obtain the recombinant vector mapk3 / mapk6-cr.

[0130] The recombinant vector mapk3 / mapk6-cr is a cassette consisting of sgRNA1 and sgRNA2 inserted into the BsaI restriction endonuclease site of the vector pYLCRISPR / Cas9Pubi-H, while keeping the other nucleotide sequences of the vector pYLCRISPR / Cas9Pubi-H unchanged. The recombinant plasmid was named the recombinant vector mapk3 / mapk6-cr.

[0131] The expression cassette sequences of sgRNA1 and sgRNA2 consist of 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), forming a complete sgRNA expression cassette.

[0132] 2. Obtaining transgenic strawberry plants

[0133] 1) Obtaining recombinant Agrobacterium EHA105 / mapk3 / mapk6-cr

[0134] The recombinant vector mapk3 / mapk6-cr was transformed into Agrobacterium competent cells EHA105 (Shanghai Weidi Biotechnology Co., Ltd.). The transformation method was as follows: 5 μg of plasmid was added to 50 μl of GV3101 Agrobacterium competent medium, gently mixed, and placed on ice for 5 min; the cells were then flash-frozen in liquid nitrogen for 5 min, and incubated in a 37℃ water bath for 5 min; 700 μl of antibiotic-free LB liquid medium was added, and the cells were cultured at 28℃ with shaking at 180 rpm for 4 h; the cells were collected by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the remaining 100 μl of cells was resuspended and plated on plates containing 25 mg / L rifampin and 50 mg / L kanamycin, and cultured at 28℃ for 2 days. Single colonies were picked and shaken, and bacterial culture PCR was performed using SP-L and SP-R primers. The PCR product of the positive clone was 1000 bp in size (i.e., the length of two sgRNA expression cassettes), and recombinant Agrobacterium EHA105 / mapk3 / mapk6-cr, which was successfully transformed into the recombinant vector mapk3 / mapk6-cr with double knockout of the mapk3 / mapk6 gene, was obtained.

[0135] 2) Stable genetic transformation of diploid strawberries

[0136] Preparation and activation of the infecting bacterial suspension: The EHA105 / mapk3 / mapk6-cr bacterial suspension was centrifuged at 5000 rpm for 10 min under vigorous shaking. The supernatant was discarded in a clean bench. MS activation solution (solvent is water, solute and its content is: MS 4.4 g / L, sucrose 30 g / L, pH adjusted to 5.8 with 1M NaOH, autoclaved, and acetylsuccione added to a final concentration of 100-200 μM before use) was added to suspend the bacterial cells, so that the OD value of the bacterial suspension is 0.4-0.6, and it was activated in a shaker at 28℃ for 40 min.

[0137] Healthy and plump Dibosco strawberry seeds were sterilized with 75% anhydrous ethanol and 1% NaClO, then spotted onto 1 / 2 MS medium. After being kept in the dark for 4-7 days, they were placed in a constant temperature and humidity tissue culture room for cultivation. The photoperiod was 12 / 12h, the temperature was 24℃, and the humidity was 45%. Diploid strawberry tissue culture seedlings were obtained after 40-50 days of cultivation.

[0138] Explant infection (leaf disc method): During the bacterial culture activation period, plant materials were cut as explants. Tissue culture seedlings of the diploid strawberry variety di Bosco (hereinafter referred to as wild strawberry, WT) that have grown for 50-60 days were taken. Tender leaves and petioles were cut into appropriate sizes and placed in MS activation solution. After a certain number of explants were cut, activated bacterial culture solution was added. Then, the solution was placed in a 50ml syringe and vacuumed until the leaves were water-soaked.

[0139] Explant culture: After infection, explants were transferred to co-culture medium (solvent: water; solutes and their concentrations: 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; pH adjusted to 5.8 with 1M NaOH; autoclaved) and cultured in the dark for 2 days. After dark culture, explants were carefully transferred to selection medium (solvent: water; solutes and their concentrations: 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; pH adjusted to 5.8 with 1M NaOH; autoclaved; Hyg 2 mg / L, Tim (Termetin) 400 mg / L) were added). After approximately 40 days of culture, shoot clusters emerged.

[0140] Callus rooting: When the callus sprouts, transfer it to rooting medium (solvent is water, solute and its content is MS 4.4g / L, sucrose 30g / L, agar powder 7g / L, 1M NaOH to adjust pH to 5.8, after high temperature and high pressure sterilization, add Hyg 2mg / L, Tim 400mg / L).

[0141] Transplanting of transgenic seedlings: Seedlings with roots that have grown to 4-5 cm and have well-developed fibrous roots are transplanted into the soil. The culture medium is carefully removed from the roots, and the seedlings are covered with plastic wrap to keep them moist. After 15 days, the plastic wrap is removed to obtain transgenic plants containing the recombinant vector mapk3 / mapk6-cr to be identified.

[0142] 3. Identification of transgenic positive lines

[0143] To identify positive transgenic plants, leaves from the transgenic strawberry plants in step 2 were taken, DNA was extracted, and PCR detection was performed using genomic DNA as a template.

[0144] 1) Extract DNA from the transgenic plants containing the recombinant vector mapk3 / mapk6-cr and identify them by PCR using the following primers:

[0145] mapk3-cr-f: 5'-CCACAGGATCGCTTTCGCCT-3'

[0146] mapk3-cr-r:5'-CAACTTTCACAAAACATACAC-3'

[0147] mapk6-cr-f:5'-AAACTTAGGGCTGAGCTTCA-3'

[0148] mapk6-cr-r:5'-AAGAAACGACAGACCAGACG-3'

[0149] 2) Send the PCR stock solution for sequencing, and analyze the peaks using Snap Gene software. If the peaks show a double peak 3-4 nt before the PAM sequence, or if the target sequence cannot be found, the sequence may have been edited. The editing method can be determined using the MDS Decode website (http: / / skl.scau.edu.cn / dsdecode / ), which can simultaneously analyze multiple sequence files.

[0150] 3) Based on the results of the analysis using the website and software, transgenic strawberry lines with different knockout types of MAPK3 and MAPK6 were obtained.

[0151] The results are as follows Figure 2 As shown: Wild-type WT showed no changes in the FvMAPK3 and FvMAPK6 gene sequences. (See attached image for details.) Figure 2 A.

[0152] Compared with wild-type WT, strain 1 showed no mutation in the FvMAPK3 gene on one chromosome, but a mutation occurred on the other chromosome: "5'-AGCGCACGCTCCGTGAGATT-3'" was changed to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 1, a single nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2). This insertion caused a frameshift, leading to premature translation termination and partial loss of function of the FvMAPK3 protein, thus knocking down FvMAPK3 gene expression. The results for this mutation site are shown in [link to results]. Figure 2 B.

[0153] Compared to wild-type WT, strain 2 showed no mutation in the FvMAPK6 gene on one chromosome, but a mutation occurred on the other chromosome: "5'-GACACGGTGATGTCAGAGGC-3'" was changed to "5'-GACACGGTGATGTGC-3'". In strain 2, five nucleotides "CAGAG" were deleted from positions 38-42 of sequence 4 (corresponding to positions 298-302 of sequence 5) in the sequence listing. This nucleotide insertion caused a frameshift, leading to premature translation termination and partial loss of FvMAPK6 protein function, thus knocking down FvMAPK6 gene expression. The results for this mutation site are shown in [link to results]. Figure 2 C.

[0154] Compared to wild-type WT, strain 3 showed no mutation in the FvMAPK3 gene on one chromosome, but a mutation occurred on the other chromosome: "5'-AGCGCACGCTCCGTGAGATT-3'" was changed to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 3, a single nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2). This insertion caused a frameshift, leading to premature translation termination and partial loss of FvMAPK3 protein function, thus knocking down FvMAPK3 gene expression. The results for this mutation site are shown in [link to results]. Figure 2 In the FvMAPK6 gene, a mutation occurred on one chromosome: "5'-GACACGGTGATGTCAGAGGC-3'" was changed to "5'-GACACGGTGATGTGC-3'". In strain 3, five nucleotides "CAGAG" were deleted from positions 38-42 of sequence 4 (corresponding to positions 298-302 of sequence 5). This nucleotide insertion caused a frameshift, leading to premature translation termination and a partial loss of the functional portion of the FvMAPK6 protein, thus knocking down the expression level of the FvMAPK6 gene. The results for this mutation site are shown in [link to results]. Figure 2 D.

[0155] Compared to wild-type WT, strain 4 exhibited the following mutation in the FvMAPK3 gene on two homologous chromosomes: "5'-AGCGCACGCTCCGTGAGATT-3'" was changed to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 4, a single nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2). This insertion caused a frameshift, leading to premature translation termination and loss of function of the FvMAPK3 protein, thus knocking out the FvMAPK3 gene. The results for this mutation site are shown in [link to results]. Figure 2 E in Chinese.

[0156] Compared to wild-type WT, strain 5 exhibited the following mutation in the FvMAPK6 gene on two homologous chromosomes: “5'-GACACGGTGATGTCAGAGGC-3'” was changed to “5'-GACACGGTGATGTGC-3'”. In strain 5, five nucleotides “CAGAG” were deleted from positions 38-42 of sequence 4 (corresponding to positions 298-302 of sequence 5). This nucleotide insertion caused a frameshift, leading to premature translation termination and loss of function of the FvMAPK6 protein, thus knocking out the FvMAPK6 gene. The results for this mutation site are shown in [link to results]. Figure 2 Middle F.

[0157] Compared to wild-type WT, strain 6 showed no mutation in the FvMAPK3 gene on one chromosome, but a mutation occurred on the other chromosome: "5'-AGCGCACGCTCCGTGAGATT-3'" was changed to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 6, a single nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2). This insertion caused a frameshift, leading to premature translation termination and partial loss of function of the FvMAPK3 protein, thus knocking down the FvMAPK3 gene. The results for this mutation site are shown in [link to results]. Figure 2 In the FvMAPK6 gene, the following mutation occurred in two homologous chromosomes: "5'-GACACGGTGATGTCAGAGGC-3'" was changed to "5'-GACACGGTGATGTGC-3'". In strain 6, five nucleotides "CAGAG" were deleted from positions 38-42 of sequence 4 (corresponding to positions 298-302 of sequence 5). This nucleotide insertion caused a frameshift, leading to premature termination of translation and loss of function of the FvMAPK6 protein, thus knocking out the FvMAPK6 gene. The sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 G.

[0158] Compared to wild-type WT, strain 7 exhibited the following mutation in the FvMAPK3 gene on two homologous chromosomes: "5'-AGCGCACGCTCCGTGAGATT-3'" was changed to "5'-AGCGGCACGCTCCGTGAGATT-3'". In strain 7, a single nucleotide "G" was inserted at positions 245-246 of sequence 1 (corresponding to positions 592-593 of sequence 2). This insertion caused a frameshift, leading to premature translation termination and loss of function of the FvMAPK3 protein, thus knocking out the FvMAPK3 gene. Sequencing results of this mutation site and surrounding nucleotides are shown in [link to sequencing data]. Figure 2 In the H section, regarding the FvMAPK6 gene, the FvMAPK3 gene on one chromosome remained unchanged, while the FvMAPK6 gene on the other homologous chromosome underwent the following mutation: "5'-GACACGGTGATGTCAGAGGC-3'" was changed to "5'-GACACGGTGATGTGC-3'". In strain 7, five nucleotides "CAGAG" were deleted from positions 38-42 of sequence 4 (corresponding to positions 298-302 of sequence 5) in the sequence listing. This nucleotide insertion caused a frameshift, leading to premature termination of translation and resulting in partial loss of function of the FvMAPK6 protein, thus knocking down the FvMAPK6 gene. The sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 H in the middle.

[0159] Example 2: Phenotypic identification of strawberry mutants with mapk3 / mapk6 gene knockout

[0160] The plants to be tested were diploid strawberry varieties di Bosco (abbreviated as wild type WT) and T1 generation plants of gene knockout lines 1-7.

[0161] The strawberry plants to be tested were planted in pots with a diameter of 230 mm and a depth of 230 mm in a substrate containing nutrient soil, vermiculite, and peat moss (2:1:1, v / v / v). The growing environment was set at a temperature of 25℃ / 18℃ (day / night), a relative humidity of 60%, a photoperiod of 16h / 8h (day / night), and a light density of 450 μmol / m³. -2 s -1 The phenotypes of each strain were observed.

[0162] The results are as follows Figure 3As shown, under the same planting conditions, the FvMAPK3 / MAPK6 gene affects the plant architecture of strawberry plants. Compared with the wild type, whether it is the heterozygous single knockout FvMAPK3 line (line 1), the heterozygous single knockout FvMAPK6 line (line 2), or the heterozygous double knockout FvMAPK3 and FvMAPK6 line (line 3), the size and growth status of the gene knockout plants are not significantly different from those of the wild type. Compared with the wild type, the homozygous FvMAPK3 knockout plants (i.e., lines 4 and 7) show mottling on the strawberry leaves. FvMAPK3 may affect the synthesis of pigments such as chlorophyll in strawberry leaves. The double knockout of FvMAPK3 / MAPK6 (lines 6 and 7) will seriously affect the normal growth and development of strawberry plants.

[0163] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a substance that knocks out a protein-coding gene: (The following is a list of possible applications.) Application of U1 in regulating plant architecture; Application of U2 in the preparation of products that regulate plant architecture; U3) Application in cultivating plants with altered plant structure; U4) Application in the preparation of products that cultivate plants with altered plant structure; The protein is either G1 or G2 as follows: G1) The amino acid sequence of the protein is SEQ ID No.

3. G2) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of G1); The controlled plant shape manifests as a mottled appearance on strawberry leaves; The plant's shape has changed, resulting in mottled leaves. The plant in question is a strawberry; The substance is a biomaterial related to the protein, and the biomaterial is any one of the following: e1) Knock out the nucleic acid molecule encoding the protein; 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) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms 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 molecules 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).

2. The application according to claim 1, characterized in that: e3) The expression cassette is a DNA molecule whose nucleotide sequence is shown in SEQ ID No. 7.

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