Application of alfalfa VIK protein in cultivating plants resistant to Phytophthora root rot

Abstract

The present invention discloses the application of alfalfa VIK protein in cultivating plants resistant to Phytophthora root rot, and belongs to the field of biotechnology. The technical problem solved by the present invention is how to improve the disease resistance of plants. The VIK protein disclosed in the present invention is a protein whose amino acid sequence is SEQ ID No.1 or SEQ ID No.4. Experiments have shown that after inoculation with Phytophthora plant pathogens, compared with wild-type plants, the mutants obtained by editing the VIK encoding gene have enhanced disease resistance and improved survival rate, increased plant fresh weight, above-ground part height, root length, and plant height, and decreased leaf relative conductivity. Therefore, mutations in the VIK protein and its encoding gene improve the plant's resistance to Phytophthora plant pathogens. The VIK encoding gene can be targeted by gene editing technology to obtain VIK gene knockout plants and cultivate plants resistant to Phytophthora plant pathogens.

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 disease in the field pose major challenges to prevention and control. 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 types 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, alfalfa has a nitrogen-fixing 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), clover (Medicago truncatula), early flowering alfalfa (Medicago praecox), etc. 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: Yellowing leaves and wilting and lodging of the plants occur in the seedling stage. Typical symptoms in mature plants are yellow to brown discoloration from the root cortex to the xylem, with unclear lesion margins on the taproot. Alfalfa root rot primarily occurs in older alfalfa in high-altitude, humid regions and irrigated areas of the highlands. Low temperatures, rainy weather, and flooding in May and August make alfalfa particularly susceptible to root rot, resulting in large areas of dead and bare alfalfa fields.

[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 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] 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 named VIK. VIK is as follows A1), A2) or A3):

[0012] A1) a protein having an amino acid sequence of SEQ ID No. 1 or SEQ ID No. 4;

[0013] A2) a protein having the same function as the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 4 in the sequence listing, wherein one or more amino acid residues are 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 that has an amino acid sequence identity of 75% or greater to the protein represented by SEQ ID No. 1 or SEQ ID No. 4 and has the same function. Identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained. The 75% or greater 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 more amino acid residue codons from the DNA sequence set forth in SEQ ID No. 2 or SEQ ID No. 5, and / or performing missense mutations of one or more base pairs, and / or attaching a tag to the coding sequence at its 5' and / or 3' end. The DNA molecule set forth in SEQ ID No. 2 encodes the VIK protein set forth in SEQ ID No. 1, and the DNA molecule set forth in SEQ ID No. 5 encodes the VIK protein set forth in SEQ ID No. 4.

[0018] The tag in A3) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology to facilitate expression, detection, tracing, and / or purification of the target protein. The tag can be a 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, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or 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] Those skilled in the art can readily mutate the VIK protein-encoding nucleotide sequence of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the VIK protein sequence of the present invention are derived from and are equivalent to the nucleotide sequences of the present invention, as long as they encode the VIK protein and possess VIK protein function.

[0031] In the above application, the nucleic acid molecule 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 listing;

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

[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 SEQ ID No. 3. b12) The DNA molecule may be the DNA molecule shown in SEQ ID No. 5 or SEQ ID No. 6.

[0036] As used herein, the term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identical to a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 4 of the present invention. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences.

[0037] The aforementioned 75% or greater 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 a nucleic acid molecule encoding a VIK protein (VIK gene expression cassette) described in B2) refers to DNA capable of expressing the VIK protein in a host cell. This 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, cosmid, phage or viral vector. Specifically, the plasmid can 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 using the CRISPR / Cas9 system and capable of editing the VIK gene. The recombinant vector can express an sgRNA targeting the nucleic acid molecule described in B1). The target sequence of the sgRNA can be positions 171-189 and / or positions 364-382 of SEQ ID No. 2 in the sequence listing, or positions 51-69 and / or positions 146-164 of SEQ ID No. 5.

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

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

[0044] In the above applications, the microorganism may be yeast, bacteria, algae or fungi. Among them, the bacteria may 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 reproductive 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 a plant, or knocking out a gene encoding VIK in a plant, or reducing the expression level of a gene encoding VIK in a plant, thereby improving plant disease resistance;

[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 gene encoding VIK in the plant, or reducing the expression level of the gene encoding 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 an sgRNA targeting the coding gene into a plant to screen for target plants in which the coding gene has been edited.

[0052] In one embodiment of the present invention, the editing is deleting one G in GGG at positions 365-367 of SEQ ID No. 2, deleting C at position 368 of SEQ ID No. 2, or deleting 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 of plants in which the VIK protein or its encoding gene has been altered, but also its progeny. The target plant can be propagated within the species in which the gene was altered, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus, whole plants, and cells.

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

[0057] In the present invention, the disease resistance can 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) to M5):

[0059] M1) dicotyledons;

[0060] M2) plants of the order Rosales;

[0061] M3) Leguminosae;

[0062] M4) Medicago;

[0063] M5) Medicago truncatula or Medicago sativa.

[0064] Experiments have demonstrated that VIK and its encoding gene can regulate plant resistance to Phytophthora plant pathogens. After inoculation with Phytophthora plant pathogens, VIK-edited mutants exhibited enhanced resistance compared to wild-type plants, as evidenced by increased survival, fresh weight, aboveground height, root length, and plant height, and decreased leaf relative conductivity (the rate of electrolyte exudation). This suggests that mutations in VIK and its encoding gene enhance plant resistance to Phytophthora plant pathogens. Gene editing could be used to target the VIK encoding gene, creating VIK knockout plants and cultivating plants resistant to Phytophthora plant pathogens.

[0065] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided 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 further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0068] Figure 3 The results of gene editing tests on 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 Figure 2 shows the growth status of Medicago truncatula R108 and the Mtvik mutant after three weeks of soil growth (control group (CK) and inoculated group (Pm)). a, Morphological observation after washing the soil; b, Survival rate (1), fresh weight (2), aboveground height (3), root length (4), plant height (5), and leaf relative conductivity (6). Data marked with different letters are significantly different, while data marked with the same letter are not significantly different.

[0071] Figure 6 The MsVIK gene structure (a) and protein domain structure (b) are shown. The MsVIK gene contains 11 exons. Amino acids 2-13 of the MsVIK protein form a low-complexity region; amino acids 51-80 form the ANK domain; and amino acids 143-401 form 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] Figure 9 Figure 2 shows the growth of Medicago 'Zhongmu 1', a mutant of Msvik, after two weeks of soil growth (control group (CK) and 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 are significantly different; data marked with the same letter are not significantly different. DETAILED DESCRIPTION

[0075] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field 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 experiments in the following examples were all repeated at least three times, and data analysis was performed using SPSS and GraphPad Prism. The experimental results are expressed as mean ± standard deviation, and pairwise comparisons were performed using one-way analysis of variance (ANOVA), Kruskal-Wallis, and nonparametric test Tukey's test.

[0076] The Medicago truncatula R108, Medicago sativa 'Zhongmu No. 1', p6401 vector and p5CBC vector in the following examples 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 semidwarf and prostrate growth habits. The Crop Journal 10 (2022) 932-941." The public can obtain them 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 Systematics, 2003, 22(4): 542-548." The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

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

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

[0080] SH9 solid medium (1 L): SH macromolecular solution (10×) 100 mL, SH micromolecular solution (1000×) 1 mL, SH organic solution (1000×) 1 mL, EDFS iron salt solution (50×) 20 mL, myo-inositol (Myo-inositol) 0.1 g, sucrose 20 g, agar powder (BD) 8 g, add H2O to make up to 1 L, 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×) (1 L): MnSO4·H2O 10 g, H3BO3 5 g, ZnSO4·7H2O 1 g, KI 1 g, CuSO4·5H2O 0.2 g, NaMoO4·2H2O 0.1 g, CoCl2·6H2O 0.1 g, H2O is added to make up to 1 L.

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

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

[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 medium (1 L): MS macromolecular solution (20×) 25 mL, MS micromolecular solution (200×) 5 mL, MS organic solution (200×) 5 mL, MS iron salt solution (200×) 5 mL, sucrose 20 g, agar (BD) 8 g, add H2O to make up to 1 L, 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, H3BO3 310 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, glycocine 100 mg, and H2O is added to make up to 250 mL.

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

[0092] SM4 liquid 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 1 L, pH 5.85.

[0093] SM4 solid medium was 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 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 Medicago truncatula's resistance to Phytophthora root rot. In Medicago truncatula R108, the genomic sequence of MtVIK is SEQ ID No. 3 in the sequence listing, 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 171-189 are target 1, and the reverse complementary sequence of nucleotides 364-382 is target 2.

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

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

[0103] Primers MtVIK-BsF, MtVIK-F0, MtVIK-R0, and MtVIK-BsR were synthesized by BGI. The four primers were reacted in the same reaction system. PCR amplification was performed using Novozymes' high-fidelity Phanta enzyme and 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 the 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 resulting recombinant Agrobacterium EHA105 / p6401-MtVIK was used for the next infection step as follows:

[0113] (1) Explant Preparation: The leaf disc method was used, and the explants were 3-week-old leaves of Medicago truncatula R108. R108 seeds were treated and planted in cans containing 1 / 2 MS solid culture medium. The R108 seedlings were grown aseptically for 3 weeks.

[0114] (2) Preparation of Agrobacterium culture: 200 μL EHA105 / p6401-MtVIK Agrobacterium culture was added to 200 mL YEP liquid medium containing 75 mg / L rifampicin and 50 mg / L kanamycin, and cultured at 28°C with shaking at 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 bacteria 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 large and small fragments in a canning jar containing SH3a infection solution, mix thoroughly, and vacuum at -0.1 pka for 30 minutes. Then, inoculate for 1.5 hours (80 rpm, room temperature, protected from light). In a laminar flow hood, wipe the surface of the leaf with sterile absorbent filter paper to remove the infection solution. Spread the leaf flat on a single layer of filter paper-covered solid medium containing 0.1 mM acetosyringone SH3a and incubate at 22°C in the dark 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: The induced callus tissue after 6 weeks of dark culture in step (5) 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).

[0119] (7) Rooting stage: The seedlings that have grown 2-3 small 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. 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. Genomic DNA from the plants was extracted using the CTAB method. 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 Medicago truncatula R108 genomic DNA and MilliQ H2O were used as negative controls. If an 808 bp target band was obtained, the identification result was positive, indicating that the regenerated plant had been transformed with the p6401-MtVIK vector.

[0122] (2) Identification of mutation patterns

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

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

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

[0126] Among them, three Mtvik mutant plants are Mtvik-6, Mtvik-16, and Mtvik-21, and their sequencing results are shown in ( Figure 3 The genotypes corresponding to the Mtvik mutants are as follows:

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

[0128] Mutant Mtvik-16: 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, and 1 mL of concentrated sulfuric acid was added and gently shaken for 8 min. The seeds were rinsed 8 times with sterile water, treated with 1 mL of sodium hypochlorite solution (CLOROX) for 12 min, and rinsed 10 times with sterile water in a laminar flow hood. The seeds were 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), 2.5 g of calcium carbonate, 15 g of agar, and the balance 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 medium to ensure that each flower pot (11 cm high, 9 cm wide) had one plate of V8 solid medium. In the inoculated group, the soil matrix (vermiculite:perlite=5:2) was mixed with the V8 solid medium containing bacteria to ensure that each flower pot (11 cm high, 9 cm wide) had one plate of V8 solid medium.

[0136] (5) Plant five Medicago truncatula plants in each pot. Three weeks later, morphological photographs of Medicago truncatula R108 and the Mtvik mutant 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 electrical conductivity of the plant leaves (such as Figure 5 ), culture conditions: 28°C, 16 h light / 8 h dark.

[0137] The results are as follows Figure 4 , Figure 5 As shown in the figure, there was no significant difference in the growth and development of the Mtvik mutant in the control group compared with Medicago truncatula R108. 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 (e.g. 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 Msvik mutants in alfalfa

[0139] This example discovered that the VIK protein (denoted as MsVIK) from alfalfa (Medicago sativa) can regulate alfalfa's resistance to Phytophthora root rot. In the alfalfa variety 'Zhongmu 1', the genomic sequence of MsVIK is SEQ ID No. 6 in the sequence listing, 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 the sgRNA module p5CBC-MsVIK

[0146] Primers MsVIK-BsF, MsVIK-F0, MsVIK-R0, and MsVIK-BsR were synthesized by BGI. PCR amplification of these four primers using the p5CBC vector as a template using high-fidelity Phanta enzyme from Novozymes was performed in the same system to amplify the sgRNA module p5CBC-MsVIK with the 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 Msvik mutants in alfalfa

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

[0155] The vector p6401-MsVIK was introduced into Agrobacterium EHA105, and the resulting recombinant Agrobacterium EHA105 / p6401-MsVIK was used for the next infection step 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 EHA105 / p6401-MsVIK Agrobacterium culture was added to 200 mL YEP liquid medium containing 75 mg / L rifampicin and 50 mg / L kanamycin, and cultured at 28°C in a shaker at 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 bacteria in 200 mL of SM4 liquid culture medium (containing 0.1 mM acetosyringone) to prepare SM4 infection fluid.

[0159] (4) Infection: Place leaves of alfalfa 'Zhongmu No. 1' in a 0.1% Tween 20 solution (1 mL Tween in 1 L deionized water) 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 in 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, ultrasonicate 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, wipe the bacterial solution on the leaf surface with sterile absorbent filter paper, spread the leaves flat on SM4 solid medium containing 0.1 mM acetosyringone without the selection antibiotic, and culture in the dark at 22°C for 3 days.

[0160] (5) Callus induction: The leaves cultured in the dark for 3 days in step (4) were transferred to new SM4 solid 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 tissue 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: Detect positive plants at the genomic DNA level. Genomic DNA from the plants was extracted using the CTAB method. PCR amplification was performed using the genomic DNA as a template and primers MsVIK-F0 and MsVIK-R0. The p6401-MsVIK vector was used as a positive control, and genomic DNA from alfalfa 'Zhongmu No. 1' and MilliQ H2O were used as negative controls. If an 808 bp target band was obtained, the identification result was positive, indicating that the regenerated plant had been transformed with the p6401-MsVIK vector.

[0166] (2) Identification of mutation patterns

[0167] Using the genomic DNA of the regenerated plant transformed with the p6401-MsVIK vector in step (1) as a template and primers MsVIK-F and MsVIK-R, PCR amplification was performed. The amplified product was subjected to 1% nucleic acid gel electrophoresis, and the amplified band was cut out for subsequent Sanger sequencing. The band size of the control group 'Zhongmu No. 1' was 786 bp. If a single peak was shown during sequencing, the sequence was directly aligned with the control group 'Zhongmu No. 1'; if a double peak was shown, the corresponding PCR amplified product was ligated to the PLB vector, and multiple single clones were selected and sequenced again to identify the Msvik mutant in which the MsVIK gene was 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, and their sequencing results are shown in ( Figure 8 The genotype mutations corresponding to the Msvik mutant are as follows:

[0171] Mutant Msvik-5: A quadri-allelic heterozygous mutant. Compared with the MsVIK gene in alfalfa 'Zhongmu 1', the first and second chromosomes of this 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: A quadri-allelic heterozygous mutant. Compared with the MsVIK gene in alfalfa 'Zhongmu 1', the first chromosome of the 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 No. 1' cuttings.

[0175] (1) Msvik-5, Msvik-6 T0 seedlings 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), 2.5 g of calcium carbonate, 15 g of agar, and the balance 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 medium to ensure that each flower pot (11 cm high, 9 cm wide) had one plate of V8 solid medium. In the inoculated group, the soil matrix (vermiculite:perlite=5:2) was mixed with the V8 solid medium containing bacteria to ensure that each flower pot (11 cm high, 9 cm wide) had one plate of V8 solid medium.

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

[0180] The results are as follows Figure 9 As shown, in the control group, the Msvik mutant showed no significant differences in growth and development compared to 'Zhongmu 1'. In the inoculated group, the Msvik mutant showed significantly higher fresh weight, aboveground height, root length, and plant height than 'Zhongmu 1'. This suggests that the mutation in the VIK gene in alfalfa significantly enhances 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 practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Any of the following uses of substances that reduce protein content or activity: D1) Improve disease resistance of alfalfa plants; D2) Preparation of products for improving disease resistance of alfalfa plants; D3) Breeding alfalfa plants with improved disease resistance; D4) preparing products for breeding alfalfa plants with increased disease resistance; The protein is as follows A1) or A2): A1) a protein having an amino acid sequence of SEQ ID No. 1 or SEQ ID No. 4; A2) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of A1); The disease resistance is to Phytophthora alfalfa ( Phytophthora medicaginis ) resistance; The alfalfa plant is Medicago truncatula or Medicago sativa.

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

3. The use according to claim 1, characterized in that: The nucleic acid molecule encoding the protein of claim 1 is as follows: b11) or b12) or b13): b11) The coding sequence is the DNA molecule of SEQ ID No. 2 in the sequence listing; b12) The coding sequence is a DNA molecule of SEQ ID No. 5 in the sequence listing; b13) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in b11) or b12) and encodes a protein with an amino acid sequence of SEQ ID No. 1 or SEQ ID No.

4.

4. Any of the following methods: X1) Methods for improving disease resistance in alfalfa plants, including: Reducing the content or activity of the protein described in claim 1 in alfalfa plants, or knocking out the gene encoding the protein described in claim 1 in alfalfa plants, or reducing the expression level of the gene encoding the protein described in claim 1 in alfalfa plants, thereby improving the disease resistance of alfalfa plants; X2) A method for breeding alfalfa plants with improved disease resistance, comprising: reducing the content or activity of the protein of claim 1 in the alfalfa plants, or knocking out the gene encoding the protein of claim 1 in the alfalfa plants, or reducing the expression level of the gene encoding the protein of claim 1 in the alfalfa plants, to obtain target alfalfa plants with improved disease resistance; The disease resistance is resistance to plant pathogens of the genus Phytophthora; The disease resistance is to Phytophthora alfalfa ( Phytophthora medicaginis ) resistance; The alfalfa plant is Medicago truncatula or Medicago sativa.

5. The method according to claim 4, 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.

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