OsCYP51H4 Protein Related to Rice Disease Resistance, Its Encoding Gene and Application

Abstract

The present invention discloses an OsCYP51H4 protein related to rice disease resistance, its coding gene and applications. The amino acid sequence of the OsCYP51H4 protein is as shown in Sequence 2. In the present invention, OsCYP51H4 overexpression lines were obtained by overexpressing the OsCYP51H4 protein in the wild-type rice Zhonghua 11. Two OsCYP51H4 knockout mutant materials, Oscyp51h4-2 and Oscyp51h4-4, were obtained by knocking out the OsCYP51H4 gene in wild-type rice. Through the analysis of the disease resistance of the OsCYP51H4 overexpression lines and the OsCYP51H4 knockout mutant materials, it was found that after the OsCYP51H4 gene mutated, the plants were more susceptible to the infection of Fusarium oxysporum and developed diseases, while overexpressing this gene could improve the resistance of rice to Fusarium oxysporum. It shows that the OsCYP51H4 protein can regulate the disease resistance of rice.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an OsCYP51H4 protein related to rice disease resistance, its coding gene and applications. Background Art

[0002] Rice is an important food crop, and rice damping-off is one of the most serious diseases in the seedling stage of dry nursery seedlings of rice. In recent years, the incidence of the disease has been increasing year by year in the northern rice-growing areas, posing a great threat to the growth of rice seedlings in the seedling stage and the cultivation of strong dry nursery seedlings. The main incidence period of rice damping-off is the one-leaf-one-heart stage. The performance in the seedbed is that the rice seedlings are unevenly distributed; the roots of the diseased plants turn yellow-brown, and brown lesions appear at the base of the stem, which then gradually turn gray-white and rot, and it is not easy to pull out the seedlings by the roots; the leaves do not unfold, the central leaves of the leaves turn yellow and withered, and the leaf tips do not exude water. It is reported that Fusarium oxysporum has relatively strong pathogenicity, and it is determined to be one of the main pathogenic bacteria of rice damping-off in Heilongjiang Province.

[0003] At present, by means of chemical control and agricultural control, the occurrence of the disease cannot be effectively controlled, and the cost is high and the environmental pollution is large. Therefore, deeply exploring disease-resistant genes will be beneficial to promoting molecular-assisted breeding of rice against damping-off and accelerating the process of rice disease-resistant breeding. Improving plant disease resistance is of great significance for crop breeding and agricultural production. Summary of the Invention

[0004] An object of the present invention is to provide a protein. The protein provided by the present invention is derived from rice, and its name is OsCYP51H4. The OsCYP51H4 protein is any one of the following (a1)-(a4):

[0005] (a1) The protein shown in Sequence 2 in the sequence listing;

[0006] (a2) A fusion protein obtained by connecting a tag to the N-terminal and / or C-terminal of the protein described in (a1);

[0007] (a3) A protein related to plant disease resistance obtained by substituting and / or deleting and / or adding one or several amino acid residues to (a1);

[0008] (a4) A protein having more than 98% identity with (a1) and related to plant disease resistance.

[0009] In the protein described in the above (a2), the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag, etc.

[0010] In the protein described in (a3) above, the substitution and / or deletion and / or addition of the one or several amino acid residues is the substitution and / or deletion and / or addition of no more than 10 amino acid residues or the substitution and / or deletion and / or addition of no more than 9 amino acid residues or the substitution and / or deletion and / or addition of no more than 8 amino acid residues or the substitution and / or deletion and / or addition of no more than 7 amino acid residues or the substitution and / or deletion and / or addition of no more than 6 amino acid residues or the substitution and / or deletion and / or addition of no more than 5 amino acid residues or the substitution and / or deletion and / or addition of no more than 4 amino acid residues or the substitution and / or deletion and / or addition of no more than 3 amino acid residues or the substitution and / or deletion and / or addition of no more than 2 amino acid residues or the substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0011] In the protein described in (a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined by using a homology search site on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0012] The protein described in any one of (a1)-(a4) above can be artificially synthesized, or its coding gene can be synthesized first and then obtained by biological expression.

[0013] Another object of the present invention is to provide a nucleic acid molecule encoding the OsCYP51H4 protein.

[0014] The nucleic acid molecule described above is a DNA molecule described in any one of the following (b1) or (b2):

[0015] (b1) The DNA molecule shown in Sequence 1 or Sequence 3 in the Sequence Listing;

[0016] (b2) A DNA molecule having more than 75% identity with (b1) and encoding the protein.

[0017] Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the OsCYP51H4 protein of the present invention. Those artificially modified nucleotides having 75% or higher identity with the OsCYP51H4 nucleotide sequence isolated from the present invention, as long as they encode the OsCYP51H4 protein and have the same function, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0018] The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 2 of the present invention. 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.

[0019] Expression cassettes, recombinant vectors or recombinant microorganisms containing the above nucleic acid molecules also fall within the scope of protection of the present invention.

[0020] The expression cassette refers to DNA capable of expressing the OsCYP51H4 protein in a host cell, which DNA may not only include a promoter that initiates the transcription of OsCYP51H4, but also include a terminator that terminates the transcription of OsCYP51H4. Further, the expression cassette may also include enhancer sequences.

[0021] The vector may be a plasmid, cosmid, phage or viral vector. The recombinant vector may be a vector containing the DNA molecule for encoding the OsCYP51H4 protein shown in Sequence 1. In order to facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vector can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes and can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or anti-chemical reagent marker genes (such as herbicide-resistant genes).

[0022] The recombinant microorganism may be yeast, bacteria, algae and fungi containing the above nucleic acid molecule, or the above expression cassette, or the above recombinant vector. The bacteria may specifically be Agrobacterium.

[0023] Another object of the present invention is to provide new uses of the above OsCYP51H4 protein, or the above nucleic acid molecule, or the above expression cassette, recombinant vector or recombinant microorganism.

[0024] The present invention provides the use of the above-mentioned OsCYP51H4 protein, or the above-mentioned nucleic acid molecule, or the above-mentioned expression cassette, recombinant vector or recombinant microorganism in any one of the following (c1)-(c3):

[0025] (c1) Regulating plant disease resistance;

[0026] (c2) Cultivating transgenic plants with improved disease resistance;

[0027] (c3) Plant breeding.

[0028] The regulation of plant disease resistance is specifically manifested as follows: when the activity of the OsCYP51H4 protein in the plant is inhibited or the OsCYP51H4 protein is not expressed, the disease resistance of the plant is reduced; when the content of the OsCYP51H4 protein in the plant is increased or the OsCYP51H4 protein is overexpressed, the disease resistance of the plant is increased.

[0029] Another object of the present invention is to provide a new use of a substance that inhibits the above-mentioned OsCYP51H4 protein.

[0030] The present invention provides the use of a substance that inhibits the above-mentioned OsCYP51H4 protein in any one of the following (d1)-(d3):

[0031] (d1) Reducing plant disease resistance;

[0032] (d2) Cultivating transgenic plants with reduced disease resistance;

[0033] (d3) Plant breeding.

[0034] Furthermore, the substance that inhibits the above-mentioned OsCYP51H4 protein can be a substance that inhibits the activity of the above-mentioned OsCYP51H4 protein, or a substance that inhibits the expression of the gene encoding the above-mentioned OsCYP51H4 protein, or a substance that knocks out the gene encoding the above-mentioned OsCYP51H4 protein.

[0035] The substance that inhibits the activity of the above-mentioned OsCYP51H4 protein can be any substance that can cause the loss of activity of the above-mentioned OsCYP51H4 protein in the plant, such as a substance that inhibits the synthesis of the above-mentioned OsCYP51H4 protein, or promotes the degradation of the above-mentioned OsCYP51H4 protein, or a protein, polypeptide or small molecule compound (such as a protein activity inhibitor) that inhibits the function of the above-mentioned OsCYP51H4 protein.

[0036] The substance that inhibits the expression of the gene encoding the above-mentioned OsCYP51H4 protein can be any substance that can prevent the gene encoding the above-mentioned OsCYP51H4 protein in the plant from being expressed, such as a substance that silences the gene encoding the above-mentioned OsCYP51H4 protein in the plant (such as miRNA, siRNA, dsRNA, shRNA, etc.).

[0037] The term "knockout" means that the host cell carrying the knockout substance does not produce the functional protein product of the gene. The knockout substance can be any substance that enables the host cell not to produce the functional protein product of the gene, such as removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or altering regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, or preventing translation by binding to mRNA, etc. Generally, the knockout is carried out at the genomic DNA level so that the progeny of the cell also permanently carry the knockout. Further, the substance for knocking out the gene encoding the above-mentioned OsCYP51H4 protein can be any substance that can mutate the gene encoding the above-mentioned OsCYP51H4 protein in plants (the mutation form can be deletion mutation and / or insertion mutation and / or base substitution) so as to lose its activity, such as zinc finger protein ZFN gene editing system or TALENs gene editing system or CRISPR / Cas9 gene editing system, etc.

[0038] Furthermore, the substance for knocking out the gene encoding the above-mentioned OsCYP51H4 protein is the CRISPR / Cas9 gene editing system.

[0039] In a specific embodiment of the present invention, the target sequence of the CRISPR / Cas9 gene editing system is as shown in Sequence 4.

[0040] Another object of the present invention is to provide a method for cultivating a transgenic plant with improved disease resistance.

[0041] The method for cultivating a transgenic plant with improved disease resistance provided by the present invention includes the step of increasing the expression level and / or activity of the OsCYP51H4 protein in the recipient plant to obtain a transgenic plant; the disease resistance of the transgenic plant is higher than that of the recipient plant.

[0042] Further, the method for increasing the expression level and / or activity of the OsCYP51H4 protein in the recipient plant is to overexpress the OsCYP51H4 protein in the recipient plant.

[0043] Furthermore, the method for overexpression is to introduce a nucleic acid molecule encoding the above-mentioned OsCYP51H4 protein into the recipient plant.

[0044] The last object of the present invention is to provide a method for cultivating a transgenic plant with reduced disease resistance.

[0045] The method for cultivating a transgenic plant with reduced disease resistance provided by the present invention is as follows 1) or 2):

[0046] 1) A step of obtaining a transgenic plant by inhibiting the above-mentioned OsCYP51H4 protein in a receptor plant; the disease resistance of the transgenic plant is lower than that of the receptor plant;

[0047] 2) Replacing the DNA molecule shown at positions 168 - 187 of Sequence 1 in the OsCYP51H4 gene of a plant with the DNA molecule shown in Sequence 5 or Sequence 6 to obtain a transgenic plant with reduced disease resistance.

[0048] In the above 1), the method for inhibiting the above-mentioned OsCYP51H4 protein in the receptor plant is to introduce the substance that inhibits the OsCYP51H4 protein into the receptor plant.

[0049] In the above 2), the replacement is a homozygous replacement, that is, the same replacement occurs in homologous chromosomes.

[0050] In any of the above applications or methods, the disease resistance is the resistance to diseases caused by Fusarium oxysporum. The Fusarium oxysporum can specifically be the Fo21 strain.

[0051] In any of the above applications or methods, the plant can be a dicotyledonous plant or a monocotyledonous plant. Further, the monocotyledonous plant can be rice. More specifically, the rice is the wild-type rice Zhonghua 11.

[0052] The present invention provides an OsCYP51H4 protein derived from rice. By overexpressing the OsCYP51H4 protein in the wild-type rice Zhonghua 11, an OsCYP51H4 overexpression line was obtained. By knocking out the OsCYP51H4 gene in the wild-type rice, two OsCYP51H4 knockout mutant materials, Oscyp51h4 - 2 and Oscyp51h4 - 4, were obtained. Through the analysis of the disease resistance of the OsCYP51H4 overexpression line and the OsCYP51H4 knockout mutant materials, it was found that after the OsCYP51H4 gene mutated, the plants were more susceptible to infection by Fusarium oxysporum and developed diseases, while overexpressing this gene could improve the resistance of rice to Fusarium oxysporum. It shows that the OsCYP51H4 protein can regulate the disease resistance of rice. Description of the Drawings

[0053] Figure 1 It shows the expression of OsCYP51H4 in different tissues at different stages. G: Germination stage; S: Seedling stage; B: Booting stage; M: Maturity stage.

[0054] Figure 2 It shows the subcellular localization of OsCYP51H4.

[0055] Figure 3 It shows the structural schematic diagram of the pCAMBIA1302 - OsCYP51H4 vector.

[0056] Figure 4 The nucleotide (A) and amino acid (B) sequences of the OsCYP51H4 gene knockout mutant.

[0057] Figure 5 qRT-PCR analysis of the OsCYP51H4 gene.

[0058] Figure 6 Disease resistance analysis of the OsCYP51H4 mutant and overexpression plants (Bar = 1 cm). Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0060] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0061] The media and their formulations involved in the following embodiments are specifically as follows:

[0062] NB2 callus induction medium: N6 medium + 2 mg / L 2,4-D + 30 g / L sucrose + 300 mg / L casein hydrolysate, 7 g / L agar powder, pH 5.8.

[0063] NB1 callus subculture medium: N6 medium + 0.5 mg / L 2,4-D + 30 g / L sucrose + 300 mg / L casein hydrolysate, 7 g / L agar powder, pH 5.8.

[0064] NB2C co-culture medium: NB2 medium + 10 g / L glucose + 100 μmol / L acetosyringone (AS), pH 5.2.

[0065] NB1S1 screening medium I: NB1 medium + 50 mg / L hygromycin + 250 mg / L ticarcillin, 7 g / L agar powder, pH 5.8.

[0066] NB1S2 screening medium II: NB1 medium + 100 mg / L hygromycin + 200 mg / L ticarcillin, 7 g / L agar powder, pH 5.8.

[0067] AAM-AS medium: AAM salts and amino acids + MS vitamins + 100 μmol / L acetosyringone (AS), pH 5.2.

[0068] RE1 pre-differentiation medium: MS salts and vitamins + 300 mg / L casein hydrolysate + 1 mg / L 6-BA + 0.5 mg / L KT + 0.2 mg / L ZT + 0.25 mg / L NAA + 100 mg / L hygromycin + 250 mg / L ticarcillin + 30 g / L sucrose + 30 g / L sorbitol, 10 g / L agar powder, pH 5.8.

[0069] RE2 pre-differentiation medium: MS salts and vitamins + 300 mg / L casein hydrolysate + 1 mg / L 6-BA + 0.5 mg / L KT + 0.2 mg / L ZT + 0.25 mg / L NAA + 50 mg / L hygromycin + 30 g / L sucrose + 30 g / L sorbitol, 10 g / L agar powder, pH 5.8.

[0070] Rooting medium: 1 / 2 MS medium + 0.5 mg / L NAA, 7 g / L agar powder, pH = 5.8.

[0071] The pCambia1302 vector in the following examples is described in the literature "Liu L, Zheng C, Kuang B, Wei L, Yan L, Wang T (2016), Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice. PLoS Genet 12(7): e1006085".

[0072] The pOs-sgRNA and pH-Ubi-cas9-7 vector systems in the following examples are both described in the literature "Jin Miao et al., Targeted mutagenesis in rice using CRISPR-Cas system. Cell Research (2013)

[0073] 23: 1233 - 1236.".

[0074] The Fusarium oxysporum Fo21 strain in the following examples is described in the literature "Zhang Junhua et al., Screening and identification of insertion sites of T-DNA mutants with reduced pathogenicity of Fusarium oxysporum, the pathogen of rice damping-off".

[0075] Example 1. Expression pattern analysis of OsCYP51H4

[0076] To study the function of OsCYP51H4, in this example, the expression of this gene in 16 tissue parts of the wild-type japonica rice variety Zhonghua 11 (ZH11) was detected, including: radicle, plumule and endosperm at the germination stage; roots, stems, leaves and stubby stems at the seedling stage; hulls, rice nodes, pistils, leaf sheaths, stamens and leaves at the booting stage; hulls, embryos and endosperms at the maturity stage. The specific steps are as follows:

[0077] Extract the total RNA of different tissue parts of rice at different developmental stages using the Ultra-fast New Plant RNA Extraction Kit (Quick RNA Isolation Kit) produced by Huayueyang Biotechnology Co., Ltd. Perform reverse transcription using the reverse transcription kit FastQuant RT Kit (with gDNase) (Tiangen, KR106). Amplify this gene from the cDNA obtained by reverse transcription, and use the expression of the OsActin (Os03g50885) gene as an internal reference.

[0078] The primer sequences of OsCYP51H4 are as follows:

[0079] qPCR-OsCYP51H4-F: 5'-GACACAAGGAACGAGCAGCAT-3';

[0080] qPCR-OsCYP51H4-R: 5'-TCAGATCAACCACGCCACACT-3'.

[0081] The primers for the internal reference gene are:

[0082] OsActin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0083] OsActin-R: 5'-AATGAGTAACCACGCTCCGTCA-3'.

[0084] The PCR reaction system is as follows: 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.4 μL of each upstream and downstream primer, 3 μL of cDNA, and 6.2 μL of ddH2O.

[0085] The PCR reaction program is as follows: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, 40 cycles; 95°C for 15 s, 60°C for 60 s, 95°C for 15 s.

[0086] Use the Delta Delta CT relative quantitation method (2 -ΔΔCT(Method) Analyze the expression levels of this gene in different tissue parts.

[0087] The results are as Figure 1 shown. OsCYP51H4 is expressed in different tissue parts at different selected time periods. Among them, the expression level in the leaves at the booting stage is the highest, followed by the pistils at the booting stage and the leaves at the seedling stage. It is speculated that it may be involved in tissue-specific biological functions.

[0088] Example 2. Subcellular localization analysis of OsCYP51H4

[0089] Plant P450 is a membrane-bound protein, mainly associated with the endoplasmic reticulum (ER) and occasionally with plastids. Using the protein localization prediction software (http: / / psort.hgc.jp / ), it is predicted that CYP51H4 is mainly localized in the ER.

[0090] 1. Construct a subcellular localization vector

[0091] (1) Download the coding region sequence of OsCYP51H4 from the rice gene annotation website Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ). Use the software Primer Premier6.0 to design primers for the coding region of the target gene. The designed primers are respectively added with homologous recombination sequences for the BP reaction and commissioned Shanghai Majorbio Bio-pharm Technology Co., Ltd. to synthesize. The primer sequences are as follows:

[0092] PGWB505-OsCYP51H4-GFP-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATCACATATTCTCC-3';

[0093] PGWB505-OsCYP51H4-GFP-R:

[0094] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTAGGAAAGCGTCCGTCTCTT-3'.

[0095] (2) Use the primers in step (1) to perform PCR amplification with cDNA as the template.

[0096] The PCR reaction system is as follows: 5×KAPA Buffer A 5 μL, dNTP (10 mM) 0.5 μL, F (10 μm) 1.25 μL, R (10 μm) 1.25 μL, plasmids 100 ng, KAPA 0.1 μL, supplemented with ddH2O to 25 μL.

[0097] The PCR reaction procedure is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 2 min, followed by 35 cycles; final extension at 72°C for 5 min. Electrophoresis was performed on 1% agarose gel at 80 V for 1 h 30 min, and the gel was cut and recovered.

[0098] (3) Activate and extract the plasmid of Escherichia coli DB3.1 carrying the entry vector pDONR207 stored at -80°C. Integrate the target sequence into pDONR207 by homologous recombination (BP reaction). The BP reaction system is as follows: 1 μL of PCR recovery product (10 - 20 ng / μL), 3 μL of pDONR207 plasmid (50 ng / μL), and 1 μL of BP Mix. Gently pipette and mix evenly with a pipette tip, and incubate at 25°C for 1 h. Take 2 μL of the reaction solution and add it to 50 μL of Escherichia coli DH5α competent cells, pipette and mix well, quickly insert it into ice and let it stand for 20 min, heat shock at 42°C for 90 s, quickly insert it into ice and let it stand for 2 min, add 750 μL of LB liquid medium (without any antibiotics) in a laminar flow hood, and culture it in a shaker at 37°C and 200 rpm for 45 min; Take 50 μL of the cultured bacterial solution and spread it on LB + Strep (streptomycin, 100 μg / mL) solid medium, and culture it inverted in a 37°C constant temperature incubator for 12 - 16 h. After single colonies grow, pick a single colony into 400 μL of LB + Strep (100 μg / mL) liquid medium, and culture it in a shaker at 37°C and 200 rpm for 4 h; Use vector primers (pDONR-F: 5'-TCGCGTTAACGCTAGCATGGATCTC-3'; pDONR-R: 5'-GTAACATCAGAGATTTTGAGACAC-3') for positive clone identification; Extract the plasmid of the positive clone, take 1 μL of the plasmid for agarose gel electrophoresis to detect the concentration, and store it at -20°C for later use. After the recovery is completed, take 1 μL to detect the plasmid quality and concentration. Integrate the target fragment into the expression vector PGWB505 by homologous recombination (LR reaction). The LR reaction system is as follows: 1 μL of PGWB505 plasmid (40 ng / μL), 2 μL of linearized recombinant vector (5 - 20 ng / μL), and 0.5 μL of LR Mix. Incubate overnight at 25°C. The transformation steps are the same as above. When single colonies grow, pick a single colony into 400 μL of LB + Strep (100 μg / mL) liquid medium, and culture it in a shaker at 37°C and 200 rpm for 4 h; Use primers (PGWB505-F: 5'-CATTTGGAGAGAACACGGG-3' and PGWB505-R: 5'-CCGGACACGCTGAACTTGTGG-3') for positive clone identification; Extract the plasmid of the positive clone, take 1 μL of the plasmid to detect the plasmid quality and concentration, and store it at -20°C for later use.

[0099] 2. Introduce the prepared expression vector into Agrobacterium tumefaciens EHA105.

[0100] (1) Rinse the electroporation cuvette soaked in 75% alcohol preservation solution once with absolute ethanol, and place it in the laminar flow hood to dry.

[0101] (2) Thoroughly ice-bath the electroporation cuvette in advance for about 10 min; add 100 ng of plasmid into 100 μL of Agrobacterium tumefaciens EHA105 competent cells, gently pipette and mix 2 - 3 times, then transfer it into the electroporation cuvette, prevent the generation of bubbles, and cover the cuvette lid.

[0102] (3) Set the electroporator to 1800 V, place the electroporation cuvette into the electroporator, take out the electroporation cuvette after hearing two "beeps", quickly add 750 μL of LB liquid medium, and culture at 28 °C and 200 rpm for 2 - 3 h.

[0103] (4) Spread 50 - 200 μL of the cultured bacterial solution on the LB + Rif + Strep (both antibiotic concentrations are 100 μg / mL) solid medium, and culture it upside down at 28 °C for 36 h.

[0104] (5) After single colonies grow, pick single colonies and inoculate them into the LB + Rif + Strep (both antibiotic concentrations are 100 μg / mL) liquid medium, and culture at 28 °C and 200 rpm for 36 h.

[0105] 3. Tobacco transformation

[0106] (1) Prepare Nicotiana benthamiana that is 3 - 4 weeks old and in good growth condition.

[0107] (2) Transfer 10 μL of the positive clone bacterial solution into 30 mL of fresh LB + Rif + Strep (both antibiotic concentrations are 100 μg / mL) medium, place it on a shaker, and culture at 28 °C and 200 rpm for 20 - 24 h.

[0108] (3) Centrifuge at 2000 g for 15 min to collect the bacterial cells.

[0109] (4) Resuspend the bacterial cells with MMA solution (2-(N-morpholino)ethanesulfonic acid pH = 5.6 10 mM, MgCl2 10 mM, AS, 100 μM), adjust OD600 to 0.6, and let it stand at room temperature for 2 h.

[0110] (5) Use the needle of a syringe to pierce several holes on the back of the tobacco leaf, then remove the needle and inject with the syringe, mark the injected leaf, and perform fine fluorescence observation on the tobacco leaf 2 - 3 days later.

[0111] The results are asFigure 2 as shown. From Figure 2 it can be observed that the fluorescence patterns of the OsCYP51H4-GFP fusion protein and the ER marker HDEL:mCherry protein are almost similar, indicating that, like most plant CYP450 proteins, OsCYP51H4 is mainly localized on the endoplasmic reticulum (ER).

[0112] Example 3. Construction of the OsCYP51H4 Overexpression Vector and the CRISPR / Cas9 Gene Editing Vector

[0113] I. Construction of the OsCYP51H4 Overexpression Vector

[0114] 1. Amplification of the OsCYP51H4 Coding Region

[0115] (1) Download the coding region sequence of OsCYP51H4 from the rice gene annotation website Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ).

[0116] (2) Use the software Primer Premier 6.0 to design primers for the coding region of the target gene. The designed primers are each added with an 18bp expression vector sequence and entrusted to Shanghai Majorbio Bio-pharm Technology Co., Ltd. The primer sequences are as follows:

[0117] 51H4-SpeI-OE-GFP-F: 5'-CCACCATGGTAGATCTGAATGGATCACATATTCTCC-3';

[0118] 51H4-SpeI-OE-GFP-R: 5'-CCTTGCTCACCATACTAGAGGAAAGCGTCCGTCTCT-3'.

[0119] (3) Use the cDNA reverse transcribed from the total RNA of wild-type rice Zhonghua 11 as a template to amplify the target fragment.

[0120] The PCR reaction system is as follows: 2×PCR buffer for KOD FX 10μL, 2mM dNTP 2μL, KOD FX (1U / μL) 0.5μL, Forward primer (10μΜ) 1μL, Reverser primer (10μM) 1μL, cDNA 0.5μL, ddH2O 4μL, with a total volume of 20μL.

[0121] The PCR reaction program is as follows: 94°C for 2 min; 98°C for 10 s, 68°C for 30 s, 68°C for 2 min, 35 cycles; 68°C for 10 min.

[0122] After the PCR reaction, take 1 μL and detect the size of the target band by 1.0% agarose gel electrophoresis to see if it is correct. Use the StarPrep Gel Extraction Kit from GenStar to recover the above amplified products.

[0123] 2. Linearization of the overexpression vector pCambia1302

[0124] Digest the modified pCambia1302 vector with SpeⅠ to obtain a linearized vector. The digestion system is as follows: 20 μL of 10×H buffer, 2 μL of SpeⅠ (Takara), 10 μL of plasmid, 8 μL of ddH2O, with a total volume of 40 μL. The digestion reaction is carried out at 37°C for about 5 h, and the specific time is determined according to the digestion effect detected by electrophoresis. The target fragment obtained from the digestion reaction is recovered using the StarPrep Gel Extraction Kit from GenStar.

[0125] 3. Construction of the pCambia1302-OsCYP51H4 vector

[0126] Perform a recombination reaction on the linearized vector (digestion product) and the inserted fragment (amplified product) at a molar ratio of 1:2, react at 50°C for 5 min to obtain a recombinant product; cool it down to 4°C or immediately place it on ice. Take 5 - 10 μL of the recombinant product and add it to 100 μL of competent cells, gently flick the tube wall to mix evenly, and let it stand on ice for 30 min. After heat shock in a 42°C water bath for 45 sec, immediately place it on ice and cool for 2 - 3 min. Add 900 μL of LB liquid medium (without antibiotics), shake the bacteria at 37°C for 1 h (rotation speed 200 - 250 rpm). Centrifuge at 5,000 rpm (2,500×g) for 5 min, discard 900 μL of the supernatant. Resuspend the bacterial cells with the remaining medium, and gently spread it evenly on a plate containing kanamycin resistance with a sterile spreading rod. Incubate it upside down in a 37°C incubator for 12 - 16 h. Pick several colonies on the transformation plate of the recombination reaction for colony PCR identification. For the colonies identified as positive by colony PCR, the remaining bacterial liquid can be inoculated into LB liquid medium containing kanamycin and cultured overnight, then extract the plasmid for digestion identification, or directly perform first-generation sequencing. The correctly identified vector is named pCambia1302-OsCYP51H4( Figure 3 ).

[0127] Sequencing results showed that the pCambia1302-OsCYP51H4 vector was obtained by inserting the DNA molecule shown in Sequence 3 into the SpeⅠ restriction site of the pCambia1302 vector while keeping other sequences of the pCambia1302 vector unchanged. The pCambia1302-OsCYP51H4 vector expresses the OsCYP51H4 protein, and the amino acid sequence of the OsCYP51H4 protein is as shown in Sequence 2.

[0128] II. Construction of CRISPR / Cas9 gene editing vector

[0129] The CRISPR / Cas9 gene editing vector for knocking out OsCYP51H4 was constructed using the pOs-sgRNA and pH-Ubi-cas9-7 vector systems. The specific construction steps are as follows:

[0130] 1. Design of target sequence and synthesis of primer pairs

[0131] The gRNA target sequence was designed using the CRISPOR website (http: / / crispor.tefor.net / ), and the off-target situation was evaluated using the Cas-offinder website (http: / / www.rgenome.net / cas-offinder / ). The characteristic sequence after this target sequence is the NGG sequence (i.e., the PAM sequence), the length of the target sequence is 20 bp, and to improve the cleavage efficiency of the Cas9 protein, the first position at the 5' end was set as G.

[0132] The OsCYP51H4-gRNA target sequence is as follows: 5'-GAAGACGAGCAAGCCGCGGC-3' (Sequence 4).

[0133] The primer pair for the OsCYP51H4 target sequence was synthesized as follows:

[0134] OsCYP51H4-gRNA-F: 5'-TGTGTGAAGACGAGCAAGCCGCGGC-3';

[0135] OsCYP51H4-gRNA-R: 5'-AAACGCCGCGGCTTGCTCGTCTTCA-3'.

[0136] 2. Construction of intermediate vector

[0137] The corresponding two single-stranded primers formed a dimer structure through an annealing reaction, and after digestion with Bsa I, they were ligated to the pOs-sgRNA vector to form the pOs-sgRNA-OsCYP51H4-spacer recombinant plasmid containing the target sequence of the rice OsCYP51H4 gene.

[0138] 3. Construction of CRISPR / Cas9 Gene Editing Vector

[0139] Using invitrogen LR Clonase II to catalyze the LR reaction between the cloning vector pOs-pOs-sgRNA-OsCYP51H4-spacer and the expression vector pH-Ubi-cas9-7 to form the recombinant vector pH-Ubi-cas9-7-OsCYP51H4-spacer, which is the CRISPR / Cas9 gene editing vector. The recombinant plasmid was transformed into Escherichia coli DH5α by heat shock method and then subjected to sequencing identification and plasmid extraction.

[0140] Example 4. Creation and Identification of Transgenic Rice Plants

[0141] 1. Creation of Transgenic Rice Plants

[0142] The pCambia1302-OsCYP51H4 vector or the CRISPR / Cas9 gene editing vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation, and then the wild-type rice Zhonghua 11 mature embryo-derived embryogenic callus was infected with the Agrobacterium containing the target vector to obtain transgenic rice plants. The specific steps are as follows:

[0143] Select plump and mature rice seeds, remove the glumes, place them in 70% ethanol for surface disinfection for 5 min, and then disinfect them in 0.1% HgCl for 10 min (or in 20% NaClO for 20 min). After disinfection, rinse them several times with sterile water to minimize the residual disinfectant on the seed surface. Place the treated seeds in sterile water and soak them for 5 - 15 h. In a laminar flow hood, excise the seed embryos and place them on the NB2 induction medium, with the cut surface in contact with the medium. Place about 20 embryos in each petri dish and incubate them in the dark at 25°C for 4 - 5 weeks. Transfer the induced callus to the subculture medium NB1 and culture it in the dark at 25°C for 2 - 3 weeks; select pale yellow and relatively dense embryogenic callus and transfer it to a new NB1 subculture medium for continued culture for 2 - 3 weeks. Select well-grown yellow and dense embryogenic callus, cut it into pieces about 2 mm in size, transfer it to a new NB1 medium, and continue to culture it in the dark at 25°C for 4 - 6 days for transformation. Immerse the callus in freshly prepared AAM-AS resuspended at OD 600For about 20 minutes in Agrobacterium at around 0.5, shake it from time to time during this period. Pour out the bacterial liquid and suck it dry with a pipette. Then place the callus pieces on a petri dish lined with filter paper for 5 - 10 minutes to suck dry the residual liquid on the surface of the callus. Then transfer it to the NB2C co - culture medium covered with a layer of filter paper and culture it at 25°C for 3 - 4 days. Transfer the co - cultured callus to the NB1S1 medium for screening culture for two weeks. After two weeks, transfer it to the NB1S2 screening medium for dark culture for two generations, about 15 days for each generation. Most of the callus tissues turn brown about 10 days after screening, and then milky white resistant callus tissues regrow at the edge of the browned tissues. Transfer the bright yellow resistant callus tissues to the differentiation medium RE1 for differentiation culture. First, culture it in the dark for 1 week, and then culture it under light for 2 - 3 weeks. Transfer the callus tissues with adventitious buds differentiated to the differentiation medium RE2 for light culture for 2 weeks. If the state is not good, it can be continuously induced to differentiate on RE2. When the differentiated plants grow to 2 - 3 cm, transfer them to the 1 / 2MS rooting medium and culture for about two weeks. When the small seedlings grow to about 10 cm, open the sealing film of the container and harden the seedlings for 5 - 7 days, then they can be transplanted to the culture room or the field.

[0144] 2. Identification of transgenic rice plants

[0145] (1) Identification of mutant positive plants

[0146] Take the leaves of well - growing T0 generation plants and extract DNA using the CTAB method. Screen positive plants with the detection primers of the hygromycin resistance gene. The primer sequences are as follows:

[0147] Hyg - F: 5'-CGAGAGCCTGACCTATTGCAT - 3';

[0148] Hyg - R: 5'-CTGCTCCATACAAGCCAACCAC - 3'.

[0149] Conduct PCR identification on the screened positive plants, and analyze the changes of the target sequence by Sanger sequencing. If a homozygous mutation occurs, it is an effective gene - knockout plant, and the phenotype can be directly observed in the T0 generation; if it is a heterozygous mutation, the phenotype of the homozygous mutant still needs to be observed after segregation in the T1 generation.

[0150] The primer pair for identifying the target sequence of OsCYP51H4 is as follows:

[0151] gRNA - F: 5'-TAAAGCAGAGGCAGCAGAGG - 3';

[0152] gRNA - R: 5'-CCCAAGTGACCTGAGCGTAA - 3'.

[0153] Two positive OsCYP51H4 knockout mutant materials were selected and denoted as Oscyp51h4-2 and Oscyp51h4-4. The nucleotide and amino acid sequence mutations are as follows Figure 4 shown.

[0154] Compared with the sequence of the wild-type rice Zhonghua 11, Oscyp51h4-2 only differs in that a base A insertion occurred on both homologous chromosomes of the OsCYP51H4 gene (Sequence 3). The position of this base A insertion is between the 117th and 118th positions of Sequence 1, resulting in premature termination of protein translation.

[0155] Compared with the sequence of the wild-type rice Zhonghua 11, Oscyp51h4-4 only differs in that a base deletion occurred on both homologous chromosomes of the OsCYP51H4 gene (Sequence 3). The deleted base is at the 117th position of Sequence 1, resulting in premature termination of protein translation.

[0156] (2) Identification of overexpressing positive plants

[0157] After the tissue culture seedlings were transferred to the culture room and waited for 3 - 4 weeks until the plants grew stably with good growth status, a small amount of leaves were taken to extract DNA using the CTAB method or young leaves were taken for direct amplification. Positive plants were screened using the detection primers for the hygromycin resistance gene. The primer sequences are as follows:

[0158] Hyg-F: 5'-CGAGAGCCTGACCTATTGCAT-3';

[0159] Hyg-R: 5'-CTGCTCCATACAAGCCAACCAC-3'. With negative control and positive control, the plants that amplified the target fragment are positive transgenic plants, named OsCYP51H4-OE. And the gene expression levels of the positive transgenic plants and the wild-type rice Zhonghua 11 were verified.

[0160] The primer sequences for fluorescence quantitative PCR are as follows:

[0161] qPCR-OsCYP51H4-F: 5'-GACACAAGGAACGAGCAGCAT-3';

[0162] qPCR-OsCYP51H4-R: 5'-TCAGATCAACCACGCCACACT-3'.

[0163] The primer sequences for the internal reference gene are as follows:

[0164] OsActin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0165] OsActin-R: 5'-AATGAGTAACCACGCTCCGTCA-3'.

[0166] Positive transgenic plants with significantly higher gene expression levels than negative controls were used to obtain T1 generation transgenic positive materials of OsCYP51H4-OE after field multiplication for disease resistance analysis. Compared with the wild-type rice Zhonghua 11, the expression level of the OsCYP51H4 gene in the leaves of the T1 generation transgenic positive materials of OsCYP51H4-OE was increased by about 40-fold ( Figure 5 ).

[0167] Example 5. Disease Resistance Analysis of OsCYP51H4 Mutants and Overexpressing Plants

[0168] 1. Preparation of Fusarium oxysporum Spore Suspension

[0169] Before the experiment, the Fusarium oxysporum Fo21 strain was activated on PDA (without antibiotics) medium and cultured in an incubator at 28 °C for 7 d until the colonies covered the culture dish. The mycelial plugs with a diameter of 5 mm were punched out and put into PDA (without antibiotics) liquid medium, and after shaking culture at 28 °C for 72 h, it could be used, with a concentration of about 10 7 cells / mL.

[0170] 2. Inoculation of Pathogenic Bacteria on Rice Seedlings

[0171] Seeds of wild-type rice Zhonghua 11, the OsCYP51H4 overexpressing positive line OsCYP51H4-OE prepared in Example 4, and the OsCYP51H4 knockout mutant lines Oscyp51h4-2 and Oscyp51h4-4 were used as experimental materials. About 80 - 100 seeds of each material were disinfected with 10% NaClO for 10 min, rinsed with water, soaked in the incubator, and germinated for seedling raising after showing white. At the 2 - 3 leaf stage (about 12 days), the pathogenic bacteria were inoculated; a distilled water treatment group was set as the control. After rinsing, they were immersed in a culture bottle containing 100 mL of spore suspension with a concentration of 10 7 cells / mL, about 10 plants per bottle, and each treatment was repeated 3 times. The culture conditions of rice were 25 °C, normal photoperiod, and the phenotypes were observed about 5 days later.

[0172] The results are as Figure 6As shown in the figure. On the 5th day of water treatment, the basal parts of the stems of the wild type, mutant lines, and overexpressing rice lines grew normally without the appearance of disease spots. On the 5th day after inoculation with Fusarium oxysporum (Fo21), compared with the wild-type control plants, the disease spot area at the basal part of the stem of the mutant materials was larger and the color was darker, indicating that the disease occurred more severely than that of the wild type. In the overexpressing materials on the 5th day after inoculation, compared with the wild type, almost no disease spots appeared, and even if the disease symptoms appeared, they were significantly lighter than the control. It can be seen that after the OsCYP51H4 gene mutates, the plants are more susceptible to infection by Fusarium oxysporum and develop diseases, while overexpressing this gene can improve the resistance of rice to Fusarium oxysporum.

[0173] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. Use of a protein, a nucleic acid molecule encoding the protein, an expression cassette containing the nucleic acid molecule, a recombinant vector or a recombinant microorganism in the following (c1) or (c2): (c1) Regulating plant disease resistance; (c2) Cultivating a transgenic plant with improved disease resistance; The protein is as follows (a1) or (a2): (a1) The protein shown in SEQ ID NO: 2 in the sequence listing; (a2) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein described in (a1); The disease resistance is resistance to diseases caused by Fusarium oxysporum; The plant is rice.

2. The application according to claim 1, wherein: The nucleic acid molecule is the DNA molecule shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing.

3. A method for cultivating a transgenic plant with improved disease resistance, comprising the step of increasing the expression level of the protein described in claim 1 in a recipient plant to obtain a transgenic plant; the disease resistance of the transgenic plant is higher than that of the recipient plant; The disease resistance is resistance to diseases caused by Fusarium oxysporum; The plant is rice.

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