CN117106817A - Application of strawberry FvMAPK12 gene and its encoded protein and biological materials in regulating fruit yield and quality - Google Patents

Abstract

The invention discloses the application of strawberry FvMAPK12 gene and its encoded protein and biological materials in regulating fruit yield and quality. One technical solution to be protected by the present invention is the application of protein in regulating the number of plant fruiting branches, fruit yield and/or fruit sugar content. The protein is the protein shown in SEQ ID No. 1 in the sequence list. In the embodiments of the present invention, phenotypic observations were conducted on the T ₁ generation transgenic FvMAPK12 gene overexpressing strawberry strain, wild-type strawberry and gene-edited strawberry, and it was found that the number of fruits and total yield of the FvMAPK12 overexpressing strain were significantly higher than those of the wild type, and the gene The fruit number and yield of the edited plants are significantly lower than those of the wild type. At the same time, overexpression of FvMAPK12 gene will increase the fructose content and anthocyanin accumulation in strawberry fruits, and promote fruit ripening. The invention can be applied to strawberry breeding and variety improvement.

Description

Strawberry FvMAPK12 gene and its encoded proteins and biological materials play a role in regulating fruit yield and quality applications

Technical field

The invention belongs to the field of biotechnology, and specifically relates to the application of strawberry FvMAPK12 gene and its encoded protein and biological materials in regulating strawberry fruit yield and quality.

Background technique

Strawberry (Fragaria × ananassa Duch.) belongs to the perennial herbaceous plant of the genus Fragaria in the Rosaceae family. Its edible part is an enlarged receptacle, and the fruit is brightly colored, sweet and sour.

Sugar content is an important indicator of strawberry fruit quality. Strawberries are of the direct sugar accumulation type, that is, the photosynthetic products are soluble sugars, which are stored in cell vacuoles. Soluble sugars mainly include fructose, sucrose and glucose. Fructose has the highest sweetness, followed by sucrose, and glucose has the lowest sweetness. As the fruit matures, the content of soluble sugar increases rapidly and the sweetness of the fruit increases. Improving strawberry fruit yield and quality is of great significance to the strawberry industry.

Contents of the invention

The technical problem to be solved by the present invention is how to control the fruit yield of strawberry/or how to control the quality of strawberry fruit and/or how to control the sugar content of strawberry fruit.

In order to solve the above technical problems, the present invention first provides any of the following applications of protein:

P1. Application of the protein in regulating the number of plant fruiting branches;

P2. Application of the protein in increasing the number of plant fruiting branches;

P3. Application of the protein in regulating fruit yield of plant plants,

P4. Application of the protein in increasing plant fruit yield;

P5. Application of the protein in regulating plant fruit sugar content;

P6. Application of the protein in increasing plant fruit sugar content;

P7. Application of the protein in regulating the soluble sugar content and/or fructose content and/or sucrose content of plant fruits;

P8. Application of the protein in increasing the soluble sugar content and/or fructose content and/or sucrose content of plant fruits;

P9. Application of the protein in plant breeding;

P10. Application of the protein in improving plant quality.

The protein may be the following protein A1), A2) or A3):

A1) The amino acid sequence is the protein of SEQ ID No. 1 in the sequence listing;

A2) The amino acid sequence shown in SEQ ID No. 1 in the sequence listing is obtained by substituting and/or deleting and/or adding one or several amino acid residues and having the same function, derived from A1) or with A1) The proteins shown are proteins with more than 80% identity and the same function;

A3) A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2).

Among the above-mentioned proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, etc.

In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage. For example, in advanced BLAST2.1, you can use blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost, and Lambda ratio to respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate, and then the identity value (%) can be obtained.

In the above-mentioned protein, the identity of more than 80% can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, the protein can be derived from strawberry.

In the above application, the plant can be any of the following:

C1) Monocotyledonous plants;

C2)Dicotyledonous plants,

C3) Plants of the order Rosales,

C4) Rosaceae plants,

C5)Fragaria plants,

C6)Strawberry.

In the above application, the substance that regulates gene expression can 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 the gene is transcribed. (That is, the regulation of the splicing or processing of the primary transcript of the gene); 3) The regulation of the RNA transport of the gene (that is, the 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 mRNA degradation of the gene; 6) Post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene) control).

In order to solve the above technical problems, the present invention also provides any of the following applications of biological materials related to the above-mentioned proteins:

Q1. Application of the biological materials in regulating the number of plant fruiting branches;

Q2. The application of the biological materials in increasing the number of plant fruiting branches;

Q3. Application of the biological materials in regulating plant fruit yield;

Q4. Application of the biological materials in improving plant fruit yield;

Q5. Application of the biological materials in regulating the sugar content of plant fruits;

Q6. The application of the biological materials in increasing the sugar content of plant fruits;

Q7. Application of the biological materials in regulating the soluble sugar content and/or fructose content and/or sucrose content of plant fruits;

Q8. The application of the biological materials in increasing the soluble sugar content and/or fructose content and/or sucrose content of plant fruits;

Q9. Application of the biological materials in plant breeding;

Q10. Application of the biological materials in improving plant quality.

The biological material may be any one of the following B1) to B9):

B1) Nucleic acid molecules encoding the proteins described above;

B2) An expression cassette containing the nucleic acid molecule described in B1);

B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

B7) Transgenic plant organs containing the nucleic acid molecule described in B1), or transgenic plant organs containing the expression cassette described in B2);

B8) Nucleic acid molecules that enhance or increase the gene expression of the protein described above;

B9) Expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

In the above application, the nucleic acid molecule described in B1) may be the coding gene for the protein shown in b1) b2) or b3) below:

b1) The coding sequence of the coding chain is a cDNA molecule or DNA molecule of the nucleotide of SEQ ID No. 2 in the sequence listing;

b2) The nucleotide is the cDNA molecule or DNA molecule of SEQ ID No. 2 in the sequence listing,

b3) A cDNA molecule or DNA molecule that hybridizes to the cDNA or DNA molecule defined in b2) and encodes a protein with the same function.

In the above application, the plant is any of the following:

C1) Monocotyledonous plants;

C2)Dicotyledonous plants,

C3) Plants of the order Rosales,

C4) Rosaceae plants,

C5)Fragaria plants,

C6)Strawberry.

Among the above-mentioned biological materials, the expression cassette containing nucleic acid molecules described in B2) refers to DNA that can express the protein described in the above-mentioned application in a host cell. This DNA can not only include a promoter for initiating the transcription of protein-encoding genes, but also can Includes terminators that terminate the transcription of protein-coding genes. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Existing plant expression vectors can be used to construct a recombinant expression vector containing the protein-encoding gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc.

Among the above biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

In order to solve the above technical problems, the present invention also provides a method for improving plant fruit yield and/or plant fruit branch number and/or plant fruit sugar content and/or plant fruit soluble sugar content and/or plant fruit fructose content and/or Methods for determination of sucrose content in plant fruits. The method may include enhancing or increasing the activity of the above-mentioned protein or/and the expression level of the gene encoding the above-mentioned protein in the target plant, thereby increasing the plant fruit yield and/or the number of plant fruiting branches and/or the plant fruit. Sugar content and/or plant fruit soluble sugar content and/or plant fruit fructose content and/or plant fruit sucrose content.

In the above method, the enhancement or improvement of the activity of the protein described above in the target plant or/and the expression level of the gene encoding the protein described above is achieved by introducing the gene encoding the protein described above into the target plant. of.

In the above method, the target plant can be any of the following:

C1) Monocotyledonous plants;

C2)Dicotyledonous plants,

C3) Plants of the order Rosales,

C4) Rosaceae plants,

C5)Fragaria plants,

C6)Strawberry.

In the above method, the transgenic plants are understood to include not only the first to second generation transgenic plants, but also their progeny. In the case of transgenic plants, the gene can be propagated in the species or transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, intact plants and cells.

The above-mentioned proteins or the above-mentioned biological materials also belong to the protection scope of the present invention.

The present invention finds that FvMAPK12 regulates the number of fruiting branches of strawberry fruits, thereby regulating the yield of strawberry fruits and promoting the sugar content of strawberry fruits, thereby providing genetic resources for improving the yield and quality of strawberry fruits.

Description of drawings

Figure 1 shows the expression level identification results of FvMAPK12 gene overexpression-positive plants. A. Green fluorescent protein GFP in FvMAPK12-OE fruits. B. The spots numbered p20, p21, p22 and p23 are the identification results of the control-transplanted plants; the spots numbered 23, 11, 12, 13, 15, 16, 21, 39, 40, 43 and 46 The sample wells are the identification results of FvMAPK12 overexpression T ₀ plants. C.WT is wild type, and the spots numbered 1-11 are the identification results of FvMAPK12 overexpression T ₁ generation.

Figure 2 shows the identification results of FvMAPK12 gene T ₁ editing material. A. Schematic diagram of FvMAPK12 gene sgRNA target design; B. FvMAPK12 gene editing method and target sequencing map. FvMAPK12-CRISPR-hz (heterozygote) is a hybrid material, and FvMAPK12-CRISPR-hm (Homozygous) is a homozygous material. Ref is the strawberry reference genome sequence, and Allele1 and Allele2 represent the two alleles on two homologous chromosomes.

Figure 3 is a photographic record of the phenotypes of wild-type strawberries (WT), FvMAPK12 gene overexpression materials (FvMAPK12-OE#4 and FvMAPK12-OE#5), and FvMAPK12 gene-edited homozygous materials (FvMAPK12-cr-hm) planted in solar greenhouses. . A is the phenotypic record of plant development progress at 74 days, 124 days and 154 days of colonization. The scale bar is 20cm. B is the single fruiting branch morphology diagram of WT, FvMAPK12-OE#4, FvMAPK12-OE#5 and FvMAPK12-cr-hm at 154 days of colonization.

Figure 4 is a comparison of the phenotypic measurement results of wild-type strawberry (WT), FvMAPK12 gene overexpression materials (FvMAPK12-OE#4 and FvMAPK12-OE#5), and FvMAPK12 gene-edited homozygous materials (FvMAPK12-cr-hm). A is a comparison of plant height phenotypic results; B is a comparison of crown width phenotypic measurement results; C is a comparison of the yield of a single plant of different genotypes in 35 days from February 3 to March 11, 2023; D is a comparison of the yield of different genotypes in 35 days Comparison chart of the number of fruits per plant; (with the wild type as the control group, t test was used to analyze the significant differences between samples, "*" represents P<0.05, "**" represents P<0.01, ns represents no statistical difference ).

Figure 5 shows the statistics of the number of fruiting branches, flowers and fruits of the wild type, FvMAPK12-OE, and FvMAPK12-cr-hm. A. Number of fruiting branches; B. Total number of flowers and fruits; C. Number of first-level flowers and fruits; D. Number of second-level flowers and fruits; E. Number of third-level flowers and fruits; F. Number of fourth-level flowers and fruits (the wild type is used as the control group , use t test to analyze the significant differences between samples, *P<0.05, **P<0.01, ns means no statistical difference).

Figure 6 shows the post-flowering fruit ripening progress, anthocyanin and fructose content determination of wild type, FvMAPK12-OE and FvMAPK12-cr-hm. A. Post-flowering fruit ripening process of wild type, FvMAPK12-OE#4 and FvMAPK12-OE#5, FvMAPK12-cr-hm. Scale bar is 1cm; B. Anthocyanin content of wild type, FvMAPK12-OE#4, and FvMAPK12-cr-hm on days 29 and 31 after flowering. C. Determination of fructose content in wild type, FvMAPK12-OE#4, and FvMAPK12-cr-hm at 25, 29, and 31 days after flowering. (Wild type was used as the control group, and t test was used to analyze the significant differences between samples, *P<0.05, **P<0.01, ns means no statistical difference).

Figure 7 shows the universal primer sequence information for CRISPR vector construction.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in literature in the field or in accordance with product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

In the embodiments of the present invention, the experiments were repeated three times, and the data were analyzed and graphed using SPSS software and GraphPad Prism 6 software.

Example 1. Obtaining strawberry plants overexpressing FvMAPK12 gene.

1. Construction of strawberry FvMAPK12 gene overexpression vector.

The fruits of the strawberry diploid variety Dibosco (Fragaria vesca, cv. 'Dibosco') (from THEHEIRLOOM SEED STORE, website: https://www.theheirloomseedstore.com/product/strawberry-fragola-di-bosco) were used as experimental materials. , use Omega RNA extraction kit (Plant RNAKitR6827) to extract total RNA, and use M-MLV reverse transcriptase to reverse transcriptase to obtain cDNA. Design primers P1 and P2 based on the FvMAPK12 gene coding region sequence. Using the cDNA obtained by reverse transcription as a template, PCR amplification was performed using primers P1 and P2. The primer sequences are as follows:

Upstream primer P1: 5’-AAAAAGCAGGCTCGATGGACGGCCTGATTCGCTGG-3’;

Downstream primer P2: 5’-AGAAAGCTGGGTTGACACCACATTAATCATCTCTTTCT-3’.

PCR system:

PCR reaction conditions were: 98°C for 3 min; 33 cycles of 98°C for 10 s, 55°C for 15 s, 72°C for 1 min/kb; and 72°C for 2 min.

Conduct agarose gel electrophoresis detection and gel recovery of the PCR product. Use the gel recovery fragment as a template and use AttB1 and AttB2 primers to perform the second round of PCR amplification. The system is the same as above. The second round of PCR product is obtained by electrophoresis detection and recovery. The primer sequence as follows:

Upstream primer: AttB1: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3’;

Downstream primer AttB2: 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’.

The PCR products that have undergone two rounds of amplification are recovered by electrophoresis and then subjected to BP reaction:

React at 25°C for 1 hour, add 1 μL Proteinase K, and incubate at 37°C for 10 minutes to terminate the reaction. The reaction product (pDONR221-target gene) was transformed into Escherichia coli competent DH5α (full gold CD201-01), spread on LB solid medium containing Kan resistance, cultured at 37°C for 10 hours, then picked and shaken, and the bacteria were Liquid PCR identification was performed, and positive clones were sent for sequencing.

Select the plasmid sample returned by the company that exactly matches the target sequence for LR reaction:

React at 25°C for 1 hour, add 1 μL Proteinase K, and incubate at 37°C for 10 minutes to terminate the reaction. The exchange product was transformed into Escherichia coli competent DH5α (full gold CD201-01), spread on LB solid medium containing Spe resistance, cultured at 37°C for 10 hours, picked and shaken, and PCR identification of bacterial liquid was performed (identification primers : Upstream primer 12F: 5'ATGGACGGCCTGATTCG3' Downstream primer 12R: 5'CTATGACACCACATTAATCATCTCTTTCT3'), select the positive clone (PCR amplification product 1743bp) and extract the plasmid by shaking, that is, the coding sequence containing the coding chain is SEQ ID No. in the sequence list .2 FvMAPK12 gene and a recombinant plasmid that can express FvMAPK12 protein, named pH7WG2D-FvMAPK12. The amino acid sequence of FvMAPK12 protein is SEQ ID No. 1 in the sequence listing. The intermediate vector pDONR221 vector and the plant expression vector pH7WG2D are preserved in our laboratory. Related literature: Mao W, Han Y, Chen Y, Sun M, Feng Q, Li L, Liu L, Zhang K, Wei L, Han Z, Li B .Low temperature inhibits anthocyanin accumulation in strawberry fruit by activating FvMAPK3-induced phosphorylation of FvMYB10 and degradation of Chalcone Synthase 1. Plant Cell. 2022Mar 29; 34(4):1226-1249).

2. Construction of FvMAPK12-CRISPR gene editing vector.

According to the CRISPR-P website (http://crispr.hzau.edu.cn/cgi-bin/CRISPR/CRISPR#), enter the gene number gene04076 of diploid strawberry FvMAPK12 to get the target recommended by the website. Select the target with high score. Three target sites without off-targets were used as targets of FvMAPK12 and primers were designed. References for CRISPR vector construction steps: Zeng Dongchang, Ma Xingliang, Xie Xianrong, Zhu Qinlong, Liu Yaoguang. Operation methods for plant CRISPR/Cas9 multi-gene editing vector construction and mutation analysis[J ]. Chinese Science: Life Sciences, 2018, 48(07): 783-794.

gRTMAPK12#1+: 5’-cggtggaccttgacatgaggttttagagctagaaat-3’;

AtU3bTMAPK12#1-:5’-ctcatgtcaaggtccaccgTgaccaatgttgctcc-3’;

gRTMAPK12#2+: 5’-tgattggcaaaggtagctagttttagagctagaaat-3’;

AtU6-1TMAPK12#2-:5’-tagctacctttgccaatcaCaatcactacttcgtct-3’;

gRTMAPK12#3+: 5’-ccaccgtcgcaaacgaccaggttttagagctagaaat-3’;

AtU3dTMAPK12#3-:5’-ctggtcgtttgcgacggtggTgaccaatggtgctttg-3’.

1) Use Overlapping PCR method to construct sgRNA expression cassette. The first round of PCR introduces the target sequence downstream of the U3/U6 promoter and upstream of the sgRNA sequence. The reaction system is as follows:

PCR reaction conditions were: 98°C for 3 min; 33 cycles of 98°C for 10 s, 55°C for 15 s, 72°C for 1 min/kb; and 72°C for 2 min. The PCR products were recovered using Tiangen glue recovery kit (DP209-02).

Second round of PCR. The purpose of this step is to construct the promoter, target and sgRNA into a complete expression cassette. Use corresponding universal primers (Pps-GGL/Pgs-GG2, Pps-GG2/Pgs-GG3, Pps-GG3/Pgs-GGR) to add linkers to the three targets. The PCR reaction system is as shown in the table below. The PCR reaction conditions are the same as above, and the recovery is the same as above.

2) Clone the sgRNA expression cassette into the pYLCRISPR/Cas9 vector. Using the "Golden Gate" cloning method based on BsaI digestion and ligation, the sgRNA expression cassette was assembled into the pYLCRISPR/Cas9 Pubi-H vector using the "cutting and ligation" method. The PCR system is 10×Cutsmart buffer 1.5μL, 10×T4 DNAligase buffer 1.5μL, pYLCRISPR/Cas9Pubi-H plasmid 60-80ng, sgRNA expression cassette (AtU3b, AtU6-1 and AtU3d) 10-15ng each, Bsa I 1μL, T4DNAligase 1 μL, add ddH ₂ O to make up 15 μL. PCR reaction conditions were: 37°C for 5 min; 10°C for 5 min, 20°C for 5 min, 10-15 cycles; 37°C for 5 min. Transform the PCR reaction product into DH5α E. coli and spread it on a plate containing Kan resistance. After obtaining the positive clone, send it to the company for sequencing. Shake the correctly sequenced bacterial liquid to extract the plasmid to obtain the gene editing plasmid of FvMAPK12 gene FvMAPK12-CRISPR. . The vectors pYLCRISPR/Cas9Pubi-H, AtU3b, AtU6-1 and AtU3d used in CRISPR gene editing were all donated by the research group of Professor Liu Yaoguang of South China Agricultural University.

3. Obtaining recombinant Agrobacterium tumefaciens.

The overexpression recombinant plasmid pH7WG2D-FvMAPK12 obtained in step 1, the gene editing plasmid FvMAPK12-CRISPR obtained in step 2, and the overexpression empty vector pH7WG2D were transformed into Agrobacterium competent EHA105 (Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium EHA105. /pH7WG2D-FvMAPK12, EHA105/FvMAPK12-CRISPR and EHA105/pH7WG2D.

The transformation method is as follows: add 5 μg of plasmid to 50 μL of EHA105 Agrobacterium competent cells, mix gently and place on ice for 5 minutes; quick freeze in liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes; add 500 μL of LB liquid without resistance Culture medium, 28°C, 180rpm shaking culture for 4 hours; centrifuge at 6000rpm for 1 minute to enrich the bacteria; spread the enriched recombinant Agrobacterium liquid on an LB plate containing the corresponding antibiotics (wherein the recombinant Agrobacterium obtained by transforming plasmid pH7WG2D-FvMAPK12 was Take 100 μL of the bacillus liquid and spread it on an LB plate containing 25 mg/L rifampicin and 25 mg/L spectinomycin; take 100 μL of the recombinant Agrobacterium liquid obtained by transforming the plasmid FvMAPK12-CRISPR and spread it on an LB plate containing 25 mg/L rifampicin. , 50 mg/L kanamycin on LB plate), cultured at 28°C for 2 days. Pick a single colony and use P1 and P2 primers to identify the pH7WG2D-FvMAPK12 vector by PCR. Use step 2 universal primers PB-L/PB-R to identify the FvMAPK12-CRISPR vector by PCR, and select positive clones (PCR products). Size 1200bp) were shaken, saturated and maintained, and the Agrobacterium EHA105/pH7WG2D-FvMAPK12, which was successfully transformed into the FvMAPK12 gene overexpression vector, and the Agrobacterium EHA105/FvMAPK12-CRISPR and EHA105/pH7WG2D into the FvMAPK12 gene editing vector were successfully transformed.

2. Obtaining FvMAPK12 gene overexpression and gene editing strawberry plants.

2.1 Diploid strawberry tissue culture seedlings were obtained.

Take healthy and plump Dibosco strawberry seeds and sterilize them with 75% absolute ethanol and 1% NaClO, then place them on 1/2MS culture medium, avoid light for 4-7 days, and then place them in a constant temperature and humidity tissue culture room for culture, with a photoperiod of 12/12h. , temperature is 24°C, humidity is 45%, and diploid strawberry tissue culture seedlings are obtained after culturing for 40d-50d.

2.2 Procedures for stable genetic transformation.

Use scissors to cut the diploid strawberry tissue culture seedling leaves obtained in step 2.1 into MS liquid medium (containing 100uM acetosyringone), then transfer it to a 50mL sterile syringe, and add recombinant Agrobacterium EHA105/pH7WG2D and EHA105 respectively. /pH7WG2D-FvMAPK12 and EHA105/FvMAPK12-CRISPR, manually evacuate the recombinant Agrobacterium EHA105/pH7WG2D, EHA105/pH7WG2D-FvMAPK12 or EHA105/FvMAPK12-CRISPR Agrobacterium bacteria liquid to invade the leaf explants, and transfer the leaf explants to Co-cultivate the medium and cultivate in the dark for 2-3 days; transfer the explants to the induction medium to induce callus, wait for 2-4 weeks for the callus to grow, transfer to the normal light cycle, conduct screening and culture, and wait for the buds to be extracted. Finally, the bud clusters that survive on the selection medium containing 2.5 mg/L hygromycin may be positive bud clusters. Rooting and RNA or DNA extraction are continued for identification. The T ₀ generation transgenic plants identified as positive are then transplanted. and colonization.

Example 2. Obtaining and phenotypic identification of transgenic positive strawberry plants.

1. Identification of FvMAPK12 gene overexpression positive plants.

The T ₀ generation transgenic plants (numbered p20, p21, p22 and p23) that were to be identified and overexpressed the empty vector EHA105/pH7WG2D and the T ₀ generation transgenic plants transformed with the EHA105/pH7WG2D-FvMAPK12 vector (numbered 23, 11, 12, 13, 15, 16, 21, 39, 40, 43 and 46) RNA was reversed into cDNA and then used semi-quantitative PCR to identify the expression level of the FvMAPK12 gene. The internal reference gene Actin and FvMAPK12 full-length PCR primers were as follows:

ActinF: 5’-TCAAACGAGCTTTTACCCTT-3’

ActinR: 5’-GTCTACTATCCAGCGAAACCAC-3’

FvMAPK12F: 5’-ATGGACGGCCTGATTCGCT-3’;

FvMAPK12R: 5’-CTATGACACCACATTAATCATCTTCTTTCTTTTGC-3’.

After identification, strains with higher expression levels of FvMAPK12 gene than plants (numbered p20, p21, p22 and p23 in Figure 1) transfected with the empty vector pH7WG2D (numbered 23, 12, 13, 16 and 39 in Figure 1) were obtained. , as the T ₀ generation FvMAPK12 gene overexpression positive plants, the T ₀ generation overexpression plants were selfed for one generation respectively, and the T ₁ generation transgenic overexpression lines were identified according to the above primers and methods.

2. Identification of FvMAPK12 gene editing materials.

Extract the DNA of the T ₀ generation transgenic plants to be identified and transferred to the EHA105/FvMAPK12-CRISPR vector, and use PCR to identify whether it contains the Cas9 gene fragment. The primers are as follows:

Cas9F: 5’-CTGACGCTAACCTCGACAAG-3’;

Cas9R: 5’-CCGATCTAGTAACATAGATGACACC-3’, using wild-type Dibosco strawberry as a negative control.

Continue to amplify the FvMAPK12 gene target sequence on the plant (A in Figure 2) containing the Cas9 gene fragment (PCR product 300 bp). The primers are as follows:

12 Target F: 5’-GGACTTCACAGAGTCAAACTGTTC-3’;

12 Target R: 5’-GCGGACCTTATCTTATCACAATACCAG-3’.

The PCR product (PCR product 500 bp) amplifying the FvMAPK12 target was sent for sequencing, and the editing type was analyzed based on the sequencing results. After identification, the T ₀ generation gene editing material plants have two effective edits (heterozygous mutation in which one chromosome is deleted by a 92bp fragment and homozygous mutation in which both chromosomes are deleted at the same time 50bp fragment). The T ₀ generation gene edited plants were selfed for 1 generation respectively. , the T ₁ generation gene editing strain was identified according to the above primers and methods. Finally, the homozygous strain (FvMAPK12-cr-hm) lacking 50 bp in gene editing was selected as the material for subsequent experiments.

3. Phenotypic identification.

The T ₁ generation FvMAPK12 gene overexpression lines (FvMAPK12-OE#4 and FvMAPK12-OE#5) and FvMAPK12 gene editing homozygous material lines (FvMAPK12-cr-hm) identified in steps 1 and 2 were planted in sunlight. In the greenhouse, 3-5 plants of each strain are maintained under the same temperature, humidity, soil, fertilizer and water management until the fruit matures.

3.1 Observation of strawberry fruit yield phenotypes.

Observing and recording the growth and development process, we found that wild type (WT in Figure 3), overexpression lines (FvMAPK12-OE#4 and FvMAPK12-OE#5 in Figure 3) and gene-edited homozygous material lines (FvMAPK12- in Figure 3 The plant growth and development process of cr-hm) is basically the same (A in Figure 3). After 154 days of colonization, the average plant height of the wild type was 19.33cm (WT in A in Figure 4), the average plant height of the overexpression line was 21.33cm (FvMAPK12-OE#5 in A in Figure 4), and the gene editing material strain The average plant height of the line (FvMAPK12-cr-hm of A in Figure 4) was 22.33cm, with no significant difference (A in Figure 4). The average crown widths of wild type, overexpressed OE#4OE#5 and gene-edited homozygotes were 41.17cm, 43.17cm, 43.33cm and 43.50cm respectively (B in Figure 4), with no significant difference. Further statistics on fruit yield from February to March 2023 showed that the fruit number and total yield of FvMAPK12-OE were significantly higher than the wild type, while the fruit number and yield of FvMAPK12-cr-hm were significantly lower than the wild type (Figure C and D in 4).

Strawberry is a typical dichotomous cyme. Multiple inflorescences can grow on one plant, and the inflorescences form fruiting branches. The same inflorescence has flowers of different levels. The lower the level, the earlier it blooms, and the larger the fruit. The higher the level, the later it blooms. , the smaller the fruit. Strawberry fruit yield is determined by the number of fruiting branches and the amount of fruit per branch. For this reason, this study systematically observed the number and shape of fruiting branches. It was found that the number of fruiting branches and the total amount of flowers and fruits of FvMAPK12-OE were significantly higher than those of the wild variety. type, the number of fruiting branches, the length of fruiting branches and the total number of flowers and fruits of FvMAPK12-cr-hm were significantly lower than those of the wild type (WT, A and B in Figure 5), and the secondary fruits, tertiary fruits and The number of fourth-grade fruits was significantly higher than that of the wild type (WT, D-F in Figure 5). The above results show that FvMAPK12 is an important gene that regulates strawberry fruit branching and can regulate strawberry fruit yield by affecting the number of fruiting branches and the number of single fruiting branches.

3.2 Determination of anthocyanin and fructose content in strawberry fruits.

Fruits of the wild type, overexpression lines and gene-edited materials were sampled on days 25, 29 and 31 after fruit setting, and their anthocyanin and fructose contents were detected. The corresponding kits of Suzhou Keming Biotechnology Co., Ltd. were used to determine the anthocyanin content (HSG-1-Y) and fructose (GT-1-Y) by micro-method.

3.2.1 Determination of anthocyanin content in strawberry fruits

Extraction of anthocyanins: According to the ratio of sample mass (g): extraction solution volume (mL) is 1:5 to 10 (it is recommended to weigh about 0.1g of sample and add 1mL of extraction solution), homogenize thoroughly and transfer to EP tube , adjust the volume of the extraction solution to 1 mL, cover tightly, conduct ultrasonic extraction for 2 hours, centrifuge at 8000g for 10 minutes at room temperature, and take the supernatant for testing.

Determination steps:

1. Preheat the microplate reader for more than 30 minutes; preheat reagent one and reagent two at 25°C (room temperature) for more than 10 minutes;

2. Take 20 μL of supernatant and 180 μL of reagent one (equivalent to dilution 10 times), bathe in 40°C water for 20 minutes, and measure the absorbance values at 530 nm and 700 nm, respectively, recorded as A1 and A2.

3. Take 20 μL of the supernatant and 180 μL of reagent two (equivalent to a 10-fold dilution), bathe in a 40°C water bath for 20 minutes, and measure the absorbance values at 530 nm and 700 nm, respectively, recorded as A3 and A4.

4. Calculate △A=(A1-A2)-(A3-A4)

Note: If A1 is greater than 1, the dilution factor can be appropriately increased to ensure that the total volume remains unchanged at 200 μL, such as 10 μL supernatant and 190 μL reagent 1 (equivalent to a 20-fold dilution); if A1 is less than 0.1, the dilution factor can be appropriately reduced to ensure that The total volume remains unchanged, such as 100 μL supernatant and 100 μL reagent 1 (equivalent to a 2-fold dilution), keeping A1 in the range of 0.1 to 1 can improve detection sensitivity; similarly adjust the volume ratio of supernatant and reagent 2; when calculating Substitute the actual dilution factor into the following formula.

Calculation of anthocyanin content:

Anthocyanin content (μg/g fresh weight) = [ΔA×V÷(ε×d)×M×F×10 ⁶ ]÷W

=33.4×ΔA×F÷W

V: Volume of extraction solution, 1×10 ^-3 L; ε: Molar extinction coefficient of anthocyanins, 2.69×10 ⁴ L/mol/cm; d: Optical path of 96-well plate, 0.5cm; M: Relative molecular mass of anthocyanins: 449.2g/mol; F: dilution factor; 10 ⁶ : 1g = 10 ⁶ μg; W: sample fresh weight in g.

ΔA linear range is 0.005-0.5.

3.2.2 Determination of fructose content in strawberry fruits

The method for determining the fructose content of strawberry fruits by micro-method is as follows:

(1) Grind the strawberry fruit with liquid nitrogen until it becomes powdery, weigh 0.1g into a cryovial, and quickly add 0.5mL of extraction solution;

(2) 80°C water bath for 10 minutes, shake 3 to 5 times, cool and centrifuge at 4000g for 10 minutes at 25°C, and take the supernatant;

(3) Add about 2 mg of reagent IV, decolorize at 80°C for 30 minutes; then add 0.5 mL of extraction solution, 4000g, centrifuge at 25°C for 10 minutes, and take the supernatant for measurement.

(4)Sampling system:

Mix well and react in a 95°C water bath for 30 minutes. After cooling, take 200 μL and measure OD _480nm in a 96-well plate. The blank tube, standard tube and measurement tube are recorded as A1, A2 and A3 respectively.

Fructose content calculation:

Fructose content (mg/g fresh weight) = (C standard tube × V1) × (A3-A1) ÷ (A2-A1) ÷ (W × V1 ÷ V2) = (A3-A1) ÷ (A2-A1) ÷ W.

Among them, C standard tube is the standard tube concentration of 1mg/mL; V1 is the added sample volume of 0.03mL; V2 is the added extraction liquid volume of 1mL; W is the fresh weight of the sample (unit g).

3.2.3 Analysis of anthocyanin content and fructose content of strawberry fruits.

The ripening progress of WT, FvMAPK12-OE#4, FvMAPK12-OE#5 and FvMAPK12-cr-hm fruits was analyzed. The results showed that compared with the wild type, the anthocyanin accumulation in FvMAPK12-OE#4 and FvMAPK12-OE#5 fruits was faster and the fruit maturity period was earlier, while the anthocyanin accumulation in FvMAPK12-cr-hm fruits was delayed and the fruit maturity period was delayed. Slow (Figure 6). The determination of anthocyanin content was consistent with the phenotype. This indicates that FvMAPK12 is an important gene that regulates the anthocyanin accumulation rate in strawberry fruits and controls the maturity period of strawberry fruits.

Sugar content is an important commercial trait that determines the quality of strawberry fruits. Previous studies have shown that although many genes can regulate the maturity stage of strawberry fruits, they may not affect the sugar content in strawberry fruits. To this end, this study analyzed the content changes of fructose, the sweetest sugar component, in WT, FvMAPK12-OE#4, and FvMAPK12-cr-hm fruits. The results showed that the fructose content in FvMAPK12-OE fruits was significantly higher than that in wild-type fruits at 25, 29 and 31 days after flowering, while the fructose content in FvMAPK12-cr-hm fruits was at 25, 29 and 31 days after flowering. The daily average was significantly lower than that of wild-type fruits (Figure 6). The above results indicate that FvMAPK12 is an important gene that regulates fructose content in strawberry fruits.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art.

Claims (10)

1. Any of the following applications of protein: P1. Application of the protein in regulating the number of plant fruiting branches; P2. Application of the protein in increasing the number of plant fruiting branches; P3. Application of the protein in regulating fruit yield of plant plants, P4. Application of the protein in increasing plant fruit yield; P5. Application of the protein in regulating plant fruit sugar content; P6. Application of the protein in increasing plant fruit sugar content; P7. Application of the protein in regulating the soluble sugar content and/or fructose content and/or sucrose content of plant fruits; P8. Application of the protein in increasing the soluble sugar content and/or fructose content and/or sucrose content of plant fruits; P9. Application of the protein in plant breeding; P10. Application of the protein in improving plant quality; The protein is the following protein A1), A2) or A3): A1) The amino acid sequence is the protein of SEQ ID No. 1 in the sequence listing; A2) The amino acid sequence shown in SEQ ID No. 1 in the sequence listing is obtained by substituting and/or deleting and/or adding one or several amino acid residues and having the same function, derived from A1) or with A1) The proteins shown are proteins with more than 80% identity and the same function; A3) A fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of A1) or A2). 2. The application according to claim 1, characterized in that: the protein is derived from strawberry. 3. Application according to claim 1 or 2, characterized in that: the plant is any of the following: C1) Monocotyledonous plants, C2)Dicotyledonous plants, C3) Plants of the order Rosales, C4) Rosaceae plants, C5)Fragaria plants, C6)Strawberry. 4. Any of the following applications of biological materials related to the protein of claim 1 or 2: Q1. Application of the biological materials in regulating the number of plant fruiting branches; Q2. The application of the biological materials in increasing the number of plant fruiting branches; Q3. Application of the biological materials in regulating plant fruit yield; Q4. Application of the biological materials in improving plant fruit yield; Q5. Application of the biological materials in regulating the sugar content of plant fruits; Q6. The application of the biological materials in increasing the sugar content of plant fruits; Q7. Application of the biological materials in regulating the soluble sugar content and/or fructose content and/or sucrose content of plant fruits; Q8. The application of the biological materials in increasing the soluble sugar content and/or fructose content and/or sucrose content of plant fruits; Q9. Application of the biological materials in plant breeding; Q10. Application of the biological materials in improving plant quality; The biological material is any one of the following B1) to B9): B1) Nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecule described in B1), or transgenic plant organs containing the expression cassette described in B2); B8) Nucleic acid molecules that enhance or increase the gene expression of the protein described in claim 1; B9) Expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8). 5. The application according to claim 4, characterized in that: B1) the nucleic acid molecule is the encoding gene of the protein shown in b1) b2) or b3) below: b1) The coding sequence of the coding chain is a cDNA molecule or DNA molecule of the nucleotide of SEQ ID No. 2 in the sequence listing; b2) The nucleotide is the cDNA molecule or DNA molecule of SEQ ID No. 2 in the sequence listing, b3) A cDNA molecule or DNA molecule that hybridizes to the cDNA or DNA molecule defined in b2) and encodes a protein with the same function. 6. Application according to claim 4 or 5, characterized in that: the plant is any of the following: C1) Monocotyledonous plants, C2)Dicotyledonous plants, C3) Plants of the order Rosales, C4) Rosaceae plants, C5)Fragaria plants, C6)Strawberry. 7. A method for increasing plant fruit yield and/or plant fruiting branch number and/or plant fruit sugar content and/or plant fruit soluble sugar content and/or plant fruit fructose content and/or plant fruit sucrose content, including enhancing Or increase the activity of the protein described in claim 1 or/and the expression level of the gene encoding the protein described in claim 1 in the target plant, thereby increasing the plant fruit yield and/or the number of plant fruiting branches and/or the sugar content of the plant fruit. and/or plant fruit soluble sugar content and/or plant fruit fructose content and/or plant fruit sucrose content. 8. The method according to claim 7, characterized in that: enhancing or increasing the activity of the protein of claim 1 or/and the expression level of the gene encoding the protein of claim 1 in the target plant is performed by This is achieved by introducing the gene encoding the protein described in claim 1 into the target plant. 9. The method according to claim 7 or 8, characterized in that: the target plant is any of the following: C1) Monocotyledonous plants, C2)Dicotyledonous plants, C3) Plants of the order Rosales, C4) Rosaceae plants, C5)Fragaria plants, C6)Strawberry. 10. The protein of claim 1 or the biological material of claim 4.