Application of alfalfa VIK protein in cultivation of phytophthora root rot resistant plants

Abstract

The invention discloses application of alfalfa VIK protein in cultivation of phytophthora root rot resistant plants, and belongs to the technical field of biology. The technical problem to be solved by the invention is how to improve the disease resistance of plants. The VIK protein disclosed by the invention is a protein of which the amino acid sequence is SEQ ID No. 1 or SEQ ID No. 4. Experiments prove that after the phytophthora phytopathogen is inoculated, compared with a wild plant, the mutant obtained by editing the VIK coding gene has the advantages that the disease resistance is enhanced, the survival rate is improved, the fresh weight, the overground part height, the root length and the plant height of the plant are increased, and the relative conductivity of leaves is reduced. Therefore, the resistance of the plant to the phytophthora phytopathogen is improved through the mutation of the VIK protein and the coding gene thereof, the coding gene of the VIK can be targeted through a gene editing technology, a VIK gene knockout plant is obtained, and a plant resisting the phytophthora phytopathogen is cultivated.

Description

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of alfalfa VIK protein in cultivating plants infected with Phytophthora root rot. Background Art

[0002] Most Phytophthora oomycetes are parasitic and are important plant pathogens that induce many important plant diseases. Phytophthora root rot is a plant disease caused by Phytophthora, a devastating disease in agricultural production. It causes serious economic losses to crops such as soybeans, potatoes, and vegetables every year, and seriously threatens global ecology and food security. Phytophthora is a eukaryotic organism that belongs to the oomycete group. Phytophthora is similar in appearance to filamentous fungi and is often called pseudofungi. The rapid variation and high genetic diversity of Phytophthora in the field pose major challenges to prevention and control, and current synthetic fungicides have limitations in controlling diseases caused by Phytophthora. Excessive use of fungicides can lead to environmental problems, pose risks to human health, and create an economic burden.

[0003] Alfalfa (Medicago L.) plants are annual or perennial herbs, rarely shrubs. Alfalfa is rich in protein, vitamins and various minerals, and its nutritional value ranks first among all kinds of forage grasses. Processed alfalfa hay, grass meal and grass pellets are easy to store and transport, and are good raw materials for livestock and poultry feed. As a legume plant, alfalfa has a nitrogen-fixing function in its root system, and is an important crop for crop rotation. Alfalfa has a well-developed root system that can prevent wind and sand, and play a role in soil and water conservation. Alfalfa plants include many types of alfalfa, including purple alfalfa (Medicago sativa), Medicago truncatula, and early flowering alfalfa (Medicago praecox). Purple alfalfa (Medicago sativa) is a perennial herb in the legume family. It is the most widely planted forage grass variety in the world and is known as the "Queen of Forage Grasses". Alfalfa root rot: Leaves turn yellow in the seedling stage, and the plants wilt and fall over; the typical symptoms of adult plants are that the root cortex tissue to the woody tissue turns yellow to brown, and the edges of the lesions on the main root are unclear. Alfalfa root rot mainly occurs in old-year alfalfa in high-altitude cold and humid areas and Sichuan and plateau irrigation areas. Especially in May and August, the low temperature, rain and irrigation make alfalfa very susceptible to root rot, causing large areas of alfalfa fields to die and become bare.

[0004] Therefore, using genetic improvement and molecular breeding to cultivate new varieties to control the occurrence of Phytophthora root rot is the most economical and effective measure, and it is also the only way for the development of the forage industry in the future. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the disease resistance of plants, especially the disease resistance to plant pathogens of the genus Phytophthora.

[0006] In order to solve the above technical problems, the present invention first provides any of the following applications of a protein or a substance for regulating the content or activity of the protein:

[0007] D1) Regulate plant disease resistance;

[0008] D2) preparing products for regulating plant disease resistance;

[0009] D3) Breeding plants with altered disease resistance;

[0010] D4) preparing products for breeding plants with altered disease resistance;

[0011] The protein is derived from alfalfa plants and is called VIK. VIK is as follows A1), A2) or A3):

[0012] A1) a protein whose amino acid sequence is SEQ ID No. 1 or SEQ ID No. 4;

[0013] A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 4 in the sequence list, with one or more amino acid residues substituted and / or deleted and / or added;

[0014] A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0015] The VIK protein in A2) above is a protein having 75% or more identity with the amino acid sequence of the protein shown in SEQ ID No.1 or SEQ ID No.4 and having the same function. Identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and searching for the identity of a pair of amino acid sequences to calculate, the identity value (%) can be obtained. The 75% or more identity is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] The VIK protein in A2) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0017] The gene encoding the VIK protein in A2) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No.2 or SEQ ID No.5, and / or performing missense mutation of one or several base pairs, and / or attaching a coding sequence of a tag at its 5′ end and / or 3′ end. The DNA molecule shown in SEQ ID No.2 encodes the VIK protein shown in SEQ ID No.1, and the DNA molecule shown in SEQ ID No.5 encodes the VIK protein shown in SEQ ID No.4.

[0018] The tag in A3) can be a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag and / or SUMO tag, etc.

[0019] In the above application, the substance for regulating VIK content or activity may be any one of the following B1) to B9):

[0020] B1) a nucleic acid molecule encoding VIK;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] 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);

[0024] 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);

[0025] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0026] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0027] B8) Nucleic acid molecules that reduce VIK content or activity;

[0028] B9) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B8).

[0029] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0030] A person skilled in the art can easily mutate the nucleotide sequence encoding the VIK protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the VIK protein of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the VIK protein and have the function of the VIK protein.

[0031] In the above application, the nucleic acid molecule described in B1) may be the following b11) or b12) or b13):

[0032] b11) the coding sequence is the DNA molecule of SEQ ID No. 2 in the sequence list;

[0033] b12) the coding sequence is the DNA molecule of SEQ ID No.5 in the sequence list;

[0034] b13) A DNA molecule having 75% or more identity with the nucleotide sequence defined in b11) or b12) and having the same function.

[0035] b11) The DNA molecule may be the DNA molecule shown in SEQ ID No. 2, or the DNA molecule shown in SEQ ID No. 3. b12) The DNA molecule may be the DNA molecule shown in SEQ ID No. 5, or the DNA molecule shown in SEQ ID No. 6.

[0036] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.4. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0037] The above 75% or more identity may be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0038] In the above application, the expression cassette containing the nucleic acid molecule encoding the VIK protein (VIK gene expression cassette) described in B2) refers to a DNA capable of expressing the VIK protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the VIK gene, but also a terminator for terminating transcription of the VIK gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0039] In the above application, the vector can be a plasmid, a cosmid, a phage or a viral vector. The plasmid can specifically be a p6401 vector.

[0040] B8) The nucleic acid molecule that reduces the VIK content may be an sgRNA targeting the gene encoding VIK.

[0041] B9) The recombinant vector can be a recombinant vector prepared by using the CRISPR / Cas9 system that can edit the VIK gene. The recombinant vector can express the sgRNA targeting the nucleic acid molecule in B1). The target sequence of the sgRNA can be the 171-189th and / or the 364th-382th positions of SEQ ID No.2 in the sequence list, or the 51st-69th and / or the 146th-164th positions of SEQ ID No.5.

[0042] In one embodiment of the present invention, the recombinant vector B9) is p6401-MtVIK.

[0043] In another embodiment of the present invention, the recombinant vector in B9) is p6401-MsVIK.

[0044] In the above application, the microorganism can be yeast, bacteria, algae or fungi. Among them, the bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0045] In the above applications, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0046] In the above application, the substance regulating VIK content or activity may be a substance reducing VIK content or activity, the regulating plant disease resistance may be improving the disease resistance of the plant, and the plant with altered disease resistance may be a plant with improved disease resistance.

[0047] The present invention also provides any of the following methods:

[0048] X1) A method for improving plant disease resistance, comprising: reducing the content or activity of VIK in the plant, or knocking out the coding gene of VIK in the plant, or reducing the expression level of the coding gene of VIK in the plant, so as to improve the disease resistance of the plant;

[0049] X2) A method for cultivating plants with improved disease resistance, comprising: reducing the content or activity of VIK in the plant, or knocking out the coding gene of VIK in the plant, or reducing the expression level of the coding gene of VIK in the plant, to obtain the target plant with improved disease resistance.

[0050] In the above methods, the methods X1) and X2) can be achieved by editing the coding gene and changing the function of the protein encoded by the coding gene.

[0051] The editing can be performed using the CRISPR / Cas9 method. Gene editing of the coding gene using the CRISPR / Cas9 method can be performed by introducing a recombinant vector (such as p6401-MtVIK, p6401-MsVIK) encoding Cas9 and capable of transcribing sgRNA targeting the coding gene into a plant to screen for the target plant in which the coding gene is edited.

[0052] In one embodiment of the present invention, the editing is to delete one G in GGG at positions 365-367 of SEQ ID No.2, to delete C at position 368 of SEQ ID No.2, or to delete GGGC at positions 365-368 of SEQ ID No.2.

[0053] In another embodiment of the present invention, the editing is deletion of ACTCATA at positions 60-66 and TT at positions 147-148 of SEQ ID No.5 and / or deletion of ATAC at positions 64-67 of SEQ ID No.5;

[0054] Or, ACTCATA at positions 60-66 and TT at positions 147-148 of SEQ ID No.5 are deleted and / or ATAC at positions 64-67 of SEQ ID No.5 are deleted and / or ACTCATAC at positions 60-67 of SEQ ID No.5 are deleted.

[0055] The target plant is understood to include not only the first generation plant in which the VIK protein or its encoding gene is changed, but also its progeny. For the target plant, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species, especially commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus, complete plants and cells.

[0056] VIK or the substance for regulating the content or activity of VIK also falls within the protection scope of the present invention.

[0057] In the present invention, the disease resistance may be resistance to plant pathogens of the genus Phytophthora (such as Phytophthora medicaginis). In one embodiment of the present invention, the Phytophthora medicaginis is Phytophthora medicaginis 44390.

[0058] In the present invention, the plant may be any one of M1)-M5):

[0059] M1) dicotyledonous plants;

[0060] M2) plants of the order Rosales;

[0061] M3) Leguminosae;

[0062] M4) Medicago;

[0063] M5) Medicago truncatula or Medicago sativa.

[0064] Experiments have shown that VIK and its encoding gene can regulate the resistance of plants to Phytophthora plant pathogens: after inoculation with Phytophthora plant pathogens, the mutants edited with VIK have enhanced disease resistance compared with wild-type plants, as shown by increased survival rate, increased plant fresh weight, aboveground height, root length, plant height, and decreased leaf relative conductivity (plant electrolyte exudation rate). This indicates that mutations in VIK and its encoding gene increase plant resistance to Phytophthora plant pathogens, and gene editing technology can be used to target the encoding gene of VIK, obtain VIK gene knockout plants, and cultivate plants resistant to Phytophthora plant pathogens.

[0065] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The structure of the MtVIK gene (a) and protein domain (b). The MtVIK gene contains 11 exons. The amino acids 2-13 of the MtVIK protein are the low-complexity region; the amino acids 51-80 are the ANK domain; and the amino acids 143-401 are the Pkinase Tyr domain.

[0067] Figure 2 This is a diagram of the vector structure of the Medicago truncatula editing vector p6401-MtVIK.

[0068] Figure 3 The results of gene editing detection of three Mtvik mutant plants.

[0069] Figure 4 Morphological photos were taken of the growth status of Medicago truncatula R108, Mtvik mutant, grown in soil for 3 weeks (control group (CK), inoculated group (Pm)).

[0070] Figure 5 The growth status of Medicago truncatula R108 and Mtvik mutant after 3 weeks of growth in soil (control group (CK), inoculated group (Pm)). a, morphological observation after taking out the soil and washing the soil; b, test results of survival rate (1), fresh weight (2), height of aboveground part (3), root length (4), plant height (5), and relative conductivity of leaves (6). Data marked with different letters have significant differences, and data marked with the same letter have no significant differences.

[0071] Figure 6 The MsVIK gene structure (a) and protein domain (b). The MsVIK gene contains 11 exons. The 2nd to 13th amino acids of the MsVIK protein are the low-complexity region; the 51st to 80th amino acids are the ANK domain; and the 143rd to 401st amino acids are the Pkinase_Tyr domain.

[0072] Figure 7 This is a diagram of the vector structure of the alfalfa expression vector p6401-MsVIK.

[0073] Figure 8 The results of gene editing detection of two Msvik mutant plants.

[0074] Fig. 9 The growth status of the Msvik mutant cuttings of alfalfa 'Zhongmu 1' after growing in soil for 2 weeks (control group (CK), inoculated group (Pm)). a, morphological observation; b, test results of fresh weight (1), aboveground height (2), root length (3), and plant height (4). Data marked with different letters have significant differences, and data marked with the same letter have no significant differences. DETAILED DESCRIPTION

[0075] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and SPSS and GraphPad Prism were used for data analysis. The experimental results are expressed as mean ± standard deviation, and one-way analysis of variance (ANOVA), Kruskal-Wallis, and nonparametric test Tukey's test were used for pairwise comparison.

[0076] The Medicago truncatula R108, Medicago sativa 'Zhongmu No. 1', p6401 vector and p5CBC vector in the following embodiments are all recorded in the article "Lihua Zheng, Jiangqi Wen, Jinling Liu, Xiangzhao Meng, Peng Liu, Na Cao, Jiangli Dong, Tao Wang. From model to alfalfa: Gene editing to obtain semidwarfand prostrate growth habits. The Crop Journal 10 (2022) 932-941." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0077] The alfalfa phytophthora in the following examples is the alfalfa phytophthora (Phytophthora medicaginis) 44390 strain, which is recorded in the following document "Zhang Zhengguang, Wang Yuanchao, Zheng Xiaobo. Analysis of rDNA ITS sequences of soybean phytophthora and alfalfa phytophthora. Fungal System, 2003, 22 (4): 542-548.", which is available to the public from the applicant and can only be used to repeat the experiments of the present invention.

[0078] SH3a liquid culture medium (1L): SH macromolecular solution (10×) 100mL, SH micromolecular solution (1000×) 1mL, SH organicmother solution (1000×) 1mL, EDFS iron salt mother solution (50×) 20mL, inositol (Myo-inositol) 0.1g, 2,4-D stock solution (10mg / mL) 0.4mL, 6-BAP stock solution (1mg / mL) 0.5mL, sucrose 30g, add H2O to make up to 1L, pH 5.85.

[0079] SH3a solid medium was obtained by adding 8 g / L of agar powder (BD) to SH3a liquid medium.

[0080] SH9 solid culture medium (1L): SH macromolecular solution (10×) 100mL, SH micromolecular solution (1000×) 1mL, SH organic solution (1000×) 1mL, EDFS iron salt solution (50×) 20mL, inositol (Myo-inositol) 0.1g, sucrose 20g, agar powder (BD) 8g, add H2O to make up to 1L, pH 5.85.

[0081] Among them, SH bulk mother liquor (10×) (1L): KNO3 28.30g, (NH4)2SO4 4.63g, KH2PO4 4g, MgSO4·7H2O1.85g, CaCl2·2H2O 1.66g, H2O is added to 1L.

[0082] SH trace mother solution (1000×) (1L): MnSO4·H2O 10g, H3BO3 5g, ZnSO4·7H2O 1g, KI 1g, CuSO4·5H2O0.2g, NaMoO4·2H2O 0.1g, CoCl2·6H2O 0.1g, H2O is added to 1L.

[0083] SH organic mother liquor (1000×) (1L): Nicotinic acid 5g, Thiamine HCl 5g, PyridoxineHCl 5g, H2O is added to make up to 1L.

[0084] EDFS iron salt mother solution (50×) (1L): 6.97g NaFe·EDTA, add H2O to make up to 1L.

[0085] 2,4-D stock solution (10 mg / mL): phytotechlab, lot:15k0309021A.

[0086] 6-BAP stock solution (1 mg / mL): phytotechlab, lot: 15F0130022A.

[0087] 1 / 2MS solid culture medium (1L): MS macromolecular solution (20×) 25mL, MS micromolecular solution (200×) 5mL, MS organic solution (200×) 5mL, MS iron salt solution (200×) 5mL, sucrose 20g, agar (BD) 8g, add H2O to make up to 1L, pH 5.85.

[0088] Among them, MS bulk mother liquor (20×) (1L): KNO3 38g, NH4NO3 33g, CaCl2·2H2O 8.8g, MgSO4·7H2O7.4g, KH2PO4 3.4g, H2O is added to 1L.

[0089] MS trace mother solution (200×) (250 mL): MnSO4·H2O 845 mg, ZnSO4·7H2O 430 mg, H3BO3310 mg, KI 41.5 mg, NaMoO4·2H2O 12.5 mg, CuSO4·5H2O 1.25 mg, CoCl2·6H2O 1.25 mg, H2O is added to 250 mL.

[0090] MS organic mother solution (200×) (250 mL): inositol 5 g, Nicotinic acid 25 mg, Thiamine HCl 25 mg, Pyridoxine HCl 25 mg, Glycine 100 mg, H2O is added to 250 mL.

[0091] MS iron salt mother solution (200×) (250 mL): Na2EDTA·2H2O 1.865 g, FeSO4·7H2O 1.39 g, add H2O to 250 mL and adjust the pH to 5.5.

[0092] SM4 liquid culture medium (1 L): MS powder 4.43 g, 2,4-D (10 mg / mL) 0.4 mL, 6-BAP (1 mg / mL) 0.2 mL, sucrose 30 g, add H2O to make up to 1 L, pH 5.85.

[0093] SM4 solid medium is obtained by adding 8 g / L of agar powder (BD) to SM4 liquid medium.

[0094] MSBK regeneration medium (1 L): MS powder 4.43 g, KT kinetin (1 mg / mL) 1 mL, 6-BAP (1 mg / mL) 0.5 mL, sucrose 30 g, agar (BD) 8 g, add H2O to make up to 1 L, pH 5.85.

[0095] Example 1. Obtaining and phenotypic detection of Medicago truncatula Mtvik mutants

[0096] This example found that the VIK protein from Medicago truncatula (denoted as MtVIK) can regulate the resistance of Medicago truncatula to Phytophthora root rot. In Medicago truncatula R108, the genome sequence of MtVIK is SEQ ID No.3 in the sequence list, and its CDS sequence is SEQ ID No.2, encoding the MtVIK protein shown in SEQ ID No.1, Figure 1 .

[0097] 1. Construction of vector p6401-MtVIK

[0098] 1.1. Gene editing target design

[0099] The target sequences were obtained through the online target prediction website (http: / / crispor.tefor.net / crispor.py). Target 1 was 5′-CGTCGCTTCTCTTCACGGT-3′, and target 2 was 5′-ACGGGGGCGGAACCGCCCT-3′. Figure 2 shown.

[0100] In SEQ ID No. 2, nucleotides 171 to 189 are target 1, and the reverse complementary sequence of nucleotides 364 to 382 is target 2.

[0101] In SEQ ID No. 3, nucleotides 346 to 364 are target 1, and the reverse complementary sequence of nucleotides 712 to 730 is target 2.

[0102] 1.2. Construction of sgRNA module p5CBC-MtVIK

[0103] Primers MtVIK-BsF, MtVIK-F0, MtVIK-R0 and MtVIK-BsR were synthesized by BGI, and the four primers were reacted in the same system. The high-fidelity Phanta enzyme of Novozymes was used to perform PCR amplification with the p5CBC vector as a template to amplify the sgRNA module p5CBC-MtVIK with the target sequence. The primer sequences are as follows:

[0104] MtVIK-BsF: 5′-ATATATGGTCTCGCTTG CGTCGCTTCTCTTCACGGT GTT-3′;

[0105] MtVIK-F0:5′-G CGTCGCTTCTCTTCACGG TGTTTTAGAGCTAGAAATAGC-3′;

[0106] MtVIK-R0:5′-AAC AGGGCGGTTCCGCCCCCGT CAATTTAATGGTTCGCTTGTA-3′;

[0107] MtVIK-BsR: 5′-ATTATTGGTCTCGAAAC AGGGCGGTTCCGCCCCCGT C-3′.

[0108] 1.3. Construction of gene editing binary vector p6401-MtVIK

[0109] Golden Gate digestion and ligation reaction: the p5CBC-MtVIK fragment with the target sequence obtained in step 1.2 and the p6401 vector were digested with BsaI enzyme and ligated with T4 DNA ligase. The recombinant vector with the correct sequence was p6401-MtVIK( Figure 2 ).

[0110] 2. Obtaining the Mtvik mutant of Medicago truncatula

[0111] 2.1. Infection of Medicago truncatula with Agrobacterium p6401-MtVIK

[0112] The vector p6401-MtVIK was introduced into Agrobacterium EHA105, and the obtained recombinant Agrobacterium EHA105 / p6401-MtVIK was used for the next infection, the steps are as follows:

[0113] (1) Preliminary preparation of explants: The leaf disc transformation method is used, and the explants are leaves of Medicago truncatula R108 that have grown for 3 weeks. R108 seeds can be processed and planted in canning bottles containing 1 / 2MS solid culture medium to ensure that the R108 seedlings grow aseptically for 3 weeks.

[0114] (2) Preparation of Agrobacterium culture: 200 μL of EHA105 / p6401-MtVIK Agrobacterium culture was added to 200 mL of YEP liquid medium containing 75 mg / L rifampicin and 50 mg / L kanamycin. The culture was shaken at 28°C and 230 rpm for about 10 h until the OD 600nm It is 0.6-0.8.

[0115] (3) Preparation of infection fluid: Pour 200 mL of Agrobacterium culture fluid into a 250 mL sterile centrifuge bottle and centrifuge at 5000 rpm for 6 min at room temperature. Discard the supernatant in a clean bench and resuspend the cells in 200 mL of SH3a liquid culture medium (containing 0.1 mM acetosyringone) to prepare SH3a infection fluid.

[0116] (4) Infection: Cut 3-week-old sterile R108 leaves into 0.5 cm 2 Immerse the large and small fragments in a canning bottle containing SH3a infection solution, mix thoroughly, and evacuate to -0.1pka for 30 minutes. Then infect for 1.5 hours (80rpm, room temperature, away from light). In a clean bench, use sterile absorbent filter paper to remove the bacterial solution on the surface of the leaves, spread the leaves flat on a single layer of filter paper-covered solid culture medium containing 0.1mM acetosyringone SH3a, and culture in the dark at 22°C for 3 days.

[0117] (5) Callus induction: The leaves cultured in the dark for 3 days in step (4) were transferred to new SH3a solid medium (containing 10 mg / L hygromycin and 200 mg / L timentin) and cultured in the dark for 6 weeks (subcultured every 2 weeks).

[0118] (6) Inducing bud differentiation: After 6 weeks of dark culture in step (5), the induced callus was transferred to SH9 solid medium (containing 5 mg / L hygromycin and 200 mg / L timentin) and cultured under light conditions for 9 weeks (subcultured every 3 weeks).

[0119] (7) Rooting stage: The seedlings that have grown 2-3 small leaves are transferred to canned bottles containing 1 / 2MS culture medium. During this period, they are transferred to new canned bottles containing 1 / 2MS culture medium every 3 weeks. After the plants have taken root, they are transferred to the greenhouse for cultivation. After the positive seedlings are identified, the seeds are collected.

[0120] 2.2. Detection of Mtvik mutant plants

[0121] (1) Identification of positive seedlings: Detection of positive plants at the genomic DNA level. The genomic DNA of the plants was extracted by the CTAB method, and PCR amplification was performed using the genomic DNA as a template and MtVIK-F0 and MtVIK-R0 as primers. The p6401-MtVIK vector was used as a positive control, and the Medicago truncatula R108 genomic DNA and MilliQ H2O were used as negative controls. If a target band of 808 bp was obtained, the identification result was positive, which means that the plant was regenerated by the p6401-MtVIK vector.

[0122] (2) Identification of mutation patterns

[0123] The genomic DNA of the regenerated plant transferred with the p6401-MtVIK vector in step (1) was used as a template, and MtVIK-F and MtVIK-R were used as primers for PCR amplification. The amplified product was subjected to 1% nucleic acid gel electrophoresis, and the amplified band was cut for subsequent Sanger sequencing. The size of the control group R108 band was 856 bp. If a single peak was shown during sequencing, the sequence was directly compared with the control group R108; if a double peak was shown, the corresponding PCR amplification product was connected to the PLB vector, and multiple single clones were selected for sequencing again to determine the Mtvik mutant in which the MtVIK gene was edited.

[0124] MtVIK-F: 5′-GAAATCCAATCAATCAAGTAATGAGTTCAGGAAG-3′;

[0125] MtVIK-R: 5′-ACTTTAAACTATCCGTGAATGATCACTACACAAC-3′.

[0126] The three Mtvik mutant plants are Mtvik-6, Mtvik-16, and Mtvik-21, and their sequencing results are shown in ( Figure 3 ). The genotype mutations corresponding to the Mtvik mutant are as follows:

[0127] Mutant Mtvik-6: It is a biallelic homozygous mutant. Compared with the MtVIK gene in Medicago truncatula R108, this mutant lacks one G in GGG at positions 365-367 of SEQ ID No.2.

[0128] Mutant Mtvik-16: It is a biallelic homozygous mutant. Compared with the MtVIK gene in Medicago truncatula R108, this mutant lacks the C at position 368 of SEQ ID No.2.

[0129] Mutant Mtvik-21 is a biallelic homozygous mutant. Compared with the MtVIK gene in Medicago truncatula R108, this mutant lacks GGGC at positions 365-368 of SEQ ID No.2.

[0130] 3. Phenotypic detection of Mtvik mutants

[0131] Test seeds: T1 generation seeds of Mtvik-6, Mtvik-16, and Mtvik-21 strains, and Medicago truncatula R108 alfalfa seeds.

[0132] (1) The test seeds were placed in a 2 mL centrifuge tube, 1 mL of concentrated sulfuric acid was added and gently shaken for 8 min, rinsed 8 times with sterile water, treated with 1 mL of sodium hypochlorite solution (CLOROX) for 12 min, rinsed 10 times with sterile water in a clean bench, spread on a 0.8% water agar plate, and placed upside down in a dark refrigerator at 4°C for 3 days. The seeds germinated after 12 hours in the dark at room temperature.

[0133] (2) Prepare 1 L of V8 solid medium: 100 mL of V8 juice (Zinpro V8 fruit and vegetable juice from the United States), 2.5 g of calcium carbonate, 15 g of agar, and the remainder is water.

[0134] (3) Growth of Phytophthora alfalfa: 15 mL of V8 solid medium was added to a 90 mm culture dish (NEST). After solidification, Phytophthora alfalfa was inoculated on the V8 solid medium and cultured in an inverted oven at 28°C for 6 days.

[0135] (4) In the control group, the soil matrix (vermiculite: perlite = 5:2) was mixed with the blank V8 solid culture medium to ensure that each flower pot (11 cm high, 9 cm wide) had a plate of V8 solid culture medium. In the inoculated group, the soil matrix (vermiculite: perlite = 5:2) was mixed with the V8 solid culture medium with bacteria to ensure that each flower pot (11 cm high, 9 cm wide) had a plate of V8 solid culture medium.

[0136] (5) Five Medicago truncatula plants were planted in each pot. After 3 weeks, morphological photographs of Medicago truncatula R108 and Mtvik mutants growing in soil were taken (e.g. Figure 4 The plants were taken out of the soil and cleaned of soil for morphological photography. The survival rate, fresh weight of the whole plant, height of the aboveground part, root length, plant height (the length of the whole plant from the top of the plant to the bottom of the root), relative conductivity of plant leaves (such as Figure 5 ), culture conditions: 28°C, 16h light / 8h dark.

[0137] The results are as follows Figure 4 , Figure 5 As shown in the figure, there was no significant difference in growth and development between the Mtvik mutant and Medicago truncatula R108 in the control group. In the inoculated group, the survival rate of the Mtvik mutant was about 75%, and the survival rate of Medicago truncatula R108 was about 50%. The survival rate of the Mtvik mutant was significantly higher than that of R108 (as shown in the figure). Figure 5 b(1)); the fresh weight, aboveground height, root length and plant height of the Mtvik mutant in the inoculated group were significantly higher than those of R108 ( Figure 5 b(2)~(5)), the relative conductivity of leaves of Mtvik mutant was significantly lower than that of R108 (e.g. Figure 5 b(6)). This indicates that the mutation of the VIK gene in Medicago truncatula significantly improves the plant's resistance to Phytophthora alfalfa.

[0138] Example 2: Acquisition and phenotypic detection of alfalfa Msvik mutants

[0139] This example found that the VIK protein (denoted as MsVIK) derived from alfalfa (Medicago sativa) can regulate the resistance of alfalfa to Phytophthora root rot. In alfalfa 'Zhongmu 1', the genome sequence of MsVIK is SEQ ID No.6 in the sequence list, and its CDS sequence is SEQ ID No.5, encoding the MsVIK protein shown in SEQ ID No.4, Figure 6 .

[0140] 1. Construction of vector p6401-MsVIK

[0141] 1.1. Gene editing target design

[0142] The target list was generated by the online target prediction website (http: / / crispor.tefor.net / crispor.py), target 1 was 5′-TCGAACCTCACTCATACTA-3′, target 2 was 5′-GGAGTACGGTTATCGTAAT-3′, such as Figure 7 shown.

[0143] In SEQ ID No. 5, nucleotides 51 to 69 are target 1, and the reverse complementary sequence of nucleotides 146 to 164 is target 2.

[0144] In SEQ ID No.6, nucleotides 212-230 are target 1, and the reverse complementary sequence of nucleotides 307-325 is target 2.

[0145] 1.2. Construction of sgRNA module p5CBC-MsVIK

[0146] Primers MsVIK-BsF, MsVIK-F0, MsVIK-R0 and MsVIK-BsR were synthesized by BGI. The four primers were used in the same system to perform PCR amplification reaction using high-fidelity Phanta enzyme from Novozymes with p5CBC vector as template to amplify the sgRNA module p5CBC-MsVIK with target sequence. The primer sequences are as follows:

[0147] MsVIK-BsF: 5′-ATATATGGTCTCGCTTG TCGAACCTCACTCATACTA GTT-3′;

[0148] MsVIK-F0:5′-G TCGAACCTCACTCATACTA GTTTTAGAGCTAGAAATAGC-3′;

[0149] MsVIK-R0:5′-AAC ATTACGATAACCGTACTCC CAATTTAATGGTTCGCTTGTA-3′;

[0150] MsVIK-BsR: 5′-ATTATTGGTCTCGAAAC ATTACGATAACCGTACTCC C-3′.

[0151] 1.3. Construction of the gene editing binary vector p6401-MsVIK

[0152] Golden Gate digestion and ligation reaction: the p5CBC-MsVIK fragment with the target sequence obtained in step 1.2 and the p6401 vector were digested with BsaI enzyme and ligated with T4 DNA ligase. The recombinant vector with the correct sequence was p6401-MsVIK( Figure 7 ).

[0153] 2. Obtaining the Msvik mutant of alfalfa

[0154] 2.1. Infection of alfalfa with Agrobacterium p6401-MsVIK

[0155] The vector p6401-MsVIK was introduced into Agrobacterium EHA105, and the obtained recombinant Agrobacterium EHA105 / p6401-MsVIK was used for the next infection, the steps are as follows:

[0156] (1) Preliminary preparation of explants: The leaf disc transformation method was used, and the explants were healthy leaves of alfalfa 'Zhongmu No. 1'.

[0157] (2) Preparation of Agrobacterium culture: 200 μL of EHA105 / p6401-MsVIK Agrobacterium culture was added to 200 mL of YEP liquid medium containing 75 mg / L rifampicin and 50 mg / L kanamycin. The culture was shaken at 28°C and 230 rpm for about 10 h until the OD 600nm It is 0.2-0.4.

[0158] (3) Preparation of infection fluid: Pour 200 mL of Agrobacterium culture fluid into a 250 mL sterile centrifuge bottle and centrifuge at 5000 rpm for 6 min at room temperature. Discard the supernatant in a clean bench and resuspend the cells in 200 mL of SM4 liquid culture medium (containing 0.1 mM acetosyringone) to prepare SM4 infection fluid.

[0159] (4) Infection: Place the leaves of alfalfa 'Zhongmu No. 1' in 0.1% Tween 20 solution (1 mL Tween is added to 1 L deionized water), treat for 5 minutes, rinse with deionized water 5 times, transfer to 75% ethanol and rinse for 15 seconds, rinse with deionized water 3 times, transfer the leaves to 30% bleach (300 mL Libai bleach is added to 700 mL deionized water), treat for 10 minutes, and rinse with deionized water 5 times. Soak the aseptically treated leaves of alfalfa 'Zhongmu No. 1' in a canning bottle containing SM4 infection solution, mix thoroughly, vacuumize to -0.1 pka for 5 minutes, ultrasonically treat for 3 minutes, and vacuumize for 5 minutes. Then infect for 0.5 hours (50 rpm, room temperature, away from light). In a clean bench, use sterile absorbent filter paper to remove the bacterial solution on the surface of the leaves, spread the leaves flat on SM4 solid culture medium containing 0.1 mM acetosyringone without selection antibiotics, and culture in the dark at 22°C for 3 days.

[0160] (5) Inducing callus: The leaves cultured in the dark for 3 days in step (4) were transferred to new SM4 solid culture medium (containing 10 mg / L hygromycin, 200 mg / L timentin, and 200 mg / L cephalexin), and cultured under light for 6 weeks (subcultured every 2 weeks).

[0161] (6) Callus regeneration: The callus in step (5) was transferred to MSBK regeneration medium (containing 10 mg / L hygromycin, 200 mg / L timentin, and 200 mg / L cephalexin) and grown for 3 weeks.

[0162] (7) Inducing bud differentiation: The regenerated callus after culturing for 3 weeks in step (6) was transferred to SH9 solid culture medium (containing 5 mg / L hygromycin and 200 mg / L timentin) and cultured under light conditions for 9 weeks (subcultured every 3 weeks).

[0163] (8) Rooting stage: The seedlings that have grown 2-3 leaves are transferred to cans containing 1 / 2MS culture medium. During this period, they are transferred to new cans containing 1 / 2MS culture medium every 3 weeks. After the plants have taken root, they are transferred to the greenhouse for cultivation and positive seedlings are identified.

[0164] 2.2. Detection of Msvik mutant plants

[0165] (1) Identification of positive seedlings: positive plants were detected at the genomic DNA level. The genomic DNA of the plants was extracted by the CTAB method, and PCR amplification was performed using the genomic DNA as a template and MsVIK-F0 and MsVIK-R0 as primers. The p6401-MsVIK vector was used as a positive control, and the genomic DNA of alfalfa 'Zhongmu No. 1' and MilliQ H2O were used as negative controls. If a target band of 808 bp was obtained, the identification result was positive, which means that the plant was regenerated by the p6401-MsVIK vector.

[0166] (2) Identification of mutation patterns

[0167] The genomic DNA of the regenerated plant transferred with the p6401-MsVIK vector in step (1) was used as a template, and MsVIK-F and MsVIK-R were used as primers for PCR amplification. The amplified product was subjected to 1% nucleic acid gel electrophoresis, and the amplified band was cut for subsequent Sanger sequencing. The band size of the control group 'Zhongmu No. 1' was 786 bp. If a single peak was shown in the sequencing, the sequence was directly compared with the control group 'Zhongmu No. 1'; if a double peak was shown, the corresponding PCR amplification product was connected to the PLB vector, and multiple single clones were selected for sequencing again to determine the Msvik mutant with the MsVIK gene edited.

[0168] MsVIK-F: 5′-ATGGCATGAACTGAACGCGGATACCCCACCCCACA-3′;

[0169] MsVIK-R: 5′-CAAGCTCAGAAGGATCAATTTCCCAATCACACTTATTAG-3′.

[0170] Two Msvik mutant plants were identified, namely Msvik-5 and Msvik-6. Their sequencing results are shown in ( Figure 8 ). The genotype mutations corresponding to the Msvik mutant are as follows:

[0171] Mutant Msvik-5 is a tetra-allelic heterozygous mutant. Compared with the MsVIK gene in alfalfa 'Zhongmu 1', the first and second chromosomes of the mutant lack ACTCATA at positions 60-66 and TT at positions 147-148 of SEQ ID No.5, and the third and fourth chromosomes lack ATAC at positions 64-67 of SEQ ID No.5.

[0172] Mutant Msvik-6 is a four-allelic heterozygous mutant. Compared with the MsVIK gene in alfalfa 'Zhongmu 1', the first chromosome of the four chromosomes of this mutant lacks ACTCATA at positions 60-66 and TT at positions 147-148 of SEQ ID No.5, the second and fourth chromosomes lack ATAC at positions 64-67 of SEQ ID No.5, and the third chromosome lacks ACTCATAC at positions 60-67 of SEQ ID No.5.

[0173] 2.3. Phenotypic detection of Msvik mutants

[0174] Test materials: Msvik-5, Msvik-6 T0 generation cuttings and alfalfa 'Zhongmu 1' cuttings.

[0175] (1) The T0 seedlings of Msvik-5 and Msvik-6 and alfalfa 'Zhongmu No. 1' were cut into soil matrix (vermiculite:perlite=5:2) and regenerated for 2 weeks until the plants were healthy and consistent.

[0176] (2) Prepare 1 L of V8 solid medium: 100 mL of V8 juice (Zinpro V8 fruit and vegetable juice from the United States), 2.5 g of calcium carbonate, 15 g of agar, and the remainder is water.

[0177] (3) Growth of Phytophthora alfalfa: 15 mL of V8 solid medium was added to a 90 mm culture dish (NEST). After solidification, Phytophthora alfalfa was inoculated on the V8 solid medium and cultured in an inverted oven at 28°C for 6 days.

[0178] (4) In the control group, the soil matrix (vermiculite: perlite = 5:2) was mixed with the blank V8 solid culture medium to ensure that each flower pot (11 cm high, 9 cm wide) had a plate of V8 solid culture medium. In the inoculated group, the soil matrix (vermiculite: perlite = 5:2) was mixed with the V8 solid culture medium with bacteria to ensure that each flower pot (11 cm high, 9 cm wide) had a plate of V8 solid culture medium.

[0179] (5) Transfer the 2-week-old seedlings from cuttings to pots, with 5 seedlings per pot (e.g. Fig. 9(a) After 2 weeks, the fresh weight, aboveground height, root length, and plant height (the length of the whole plant from the top of the plant to the bottom of the root) of alfalfa 'Zhongmu 1' and Msvik mutant plants were counted. The culture conditions were: 28°C, 16h light / 8h dark.

[0180] The results are as follows Fig. 9 As shown, there was no significant difference in growth and development between the Msvik mutant and 'Zhongmu No. 1' in the control group. In the inoculated group, the fresh weight, aboveground height, root length, and plant height of the Msvik mutant were significantly higher than those of 'Zhongmu No. 1'. This indicates that the mutation of the VIK gene in alfalfa significantly improves the plant's resistance to Phytophthora alfalfa.

[0181] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Any of the following uses of a protein or a substance for regulating the content or activity of the protein: D1) Regulate plant disease resistance; D2) preparing products for regulating plant disease resistance; D3) Breeding plants with altered disease resistance; D4) preparing products for breeding plants with altered disease resistance; The protein is the following A1), A2) or A3): A1) a protein whose amino acid sequence is SEQ ID No. 1 or SEQ ID No. 4; A2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 4 in the sequence list, with one or more amino acid residues substituted and / or deleted and / or added; A3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The use according to claim 1, characterized in that: The substance that regulates the protein content or activity is any one of the following B1) to B9): B1) a nucleic acid molecule encoding the protein; 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) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2); B8) a nucleic acid molecule that reduces the protein content or activity; B9) An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the nucleic acid molecule described in B8).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is the following b11) or b12) or b13): b11) the coding sequence is the DNA molecule of SEQ ID No. 2 in the sequence list; b12) the coding sequence is the DNA molecule of SEQ ID No.5 in the sequence list; b13) A DNA molecule having 75% or more identity with the nucleotide sequence defined in b11) or b12) and having the same function.

4. The use according to any one of claims 1 to 3, characterized in that: The substance that regulates the protein content or activity is a substance that reduces the protein content or activity, the substance that regulates plant disease resistance is to increase the disease resistance of the plant, and the plant with altered disease resistance is a plant with improved disease resistance.

5. The use according to any one of claims 1 to 4, characterized in that: The disease resistance is resistance to plant pathogens of the genus Phytophthora.

6. Any of the following methods: X1) A method for improving plant disease resistance, comprising: Reducing the content or activity of the protein described in claim 1 in a plant, or knocking out the gene encoding the protein described in claim 1 in a plant, or reducing the expression level of the gene encoding the protein described in claim 1 in a plant, thereby improving the disease resistance of the plant; X2) A method for cultivating plants with improved disease resistance, comprising: reducing the content or activity of the protein described in claim 1 in the plant, or knocking out the gene encoding the protein described in claim 1 in the plant, or reducing the expression level of the gene encoding the protein described in claim 1 in the plant, to obtain the target plant with improved disease resistance.

7. The method according to claim 6, characterized in that: The methods X1) and X2) are achieved by editing the coding gene and changing the function of the protein encoded by the coding gene.

8. The method according to claim 6 or 7, characterized in that: The disease resistance is resistance to plant pathogens of the genus Phytophthora.

9. The use according to any one of claims 1 to 5, or the method according to any one of claims 6 to 8, characterized in that: The plant is any one of M1)-M5): M1) dicotyledonous plants; M2) plants of the order Rosales; M3) Leguminosae; M4) Medicago; M5) Medicago truncatula or Medicago sativa.

10. The protein according to claim 1 or the substance for regulating the content or activity of the protein according to any one of claims 1 to 3.

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