Application of methyltransferase genes ATXR5 and ATXR6 in regulation and control of plant disease resistance

Abstract

The invention discloses application of methyltransferase genes ATXR5 and ATXR6 in regulation and control of plant disease resistance, and belongs to the technical field of molecular plant pathology. The ATXR5 gene and the ATXR6 gene are separated from arabidopsis thaliana, the full length of the ATXR5 gene is 1140 bp, and the full length of the ATXR6 gene is 1050 bp. After ATXR5 and ATXR6 in arabidopsis thaliana are knocked out, in-vivo disease resistance related genes of a weak mutant or a strong-process heterozygous mutant are greatly activated and expressed, and after the mutant is infected with the pseudomonas syringae, the disease resistance of plants to the pseudomonas syringae is remarkably improved. Besides, after mutants are infected by pseudomonas syringae, a large amount of antibacterial genes in weak mutants and strong sudden mutants can be detected to be activated and expressed, so that the gene can be applied to plant disease resistance and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular plant pathology, and in particular to application of methyltransferase genes ATXR5 and ATXR6 in regulating plant disease resistance. Background Art

[0002] ATXR5 and ATXR6 are specific histone H3K27 methyltransferase genes in Arabidopsis thaliana, which have three functional domains: Proliferating Cell Nuclear Antigen (PCNA) Interacting Protein (PIP), PHD and SET domains, respectively, which are mainly responsible for the catalysis of histone modification H3K27me1 in Arabidopsis thaliana.

[0003] However, there has been no report in the prior art on the use of methyltransferase genes ATXR5 and ATXR6 for regulating plant disease resistance. Summary of the Invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of methyltransferase genes ATXR5 and ATXR6 in regulating plant disease resistance.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: providing an application of methyltransferase genes ATXR5 and ATXR6 in regulating plant disease resistance; wherein the nucleotide sequence of the methyltransferase gene ATXR5 is shown in SEQ ID NO: 1, and the nucleotide sequence of the methyltransferase gene ATXR6 is shown in SEQ ID NO: 2.

[0006] Furthermore, the above-mentioned regulation of plant disease resistance is to improve plant disease resistance by knocking out the methyltransferase genes ATXR5 and ATXR6.

[0007] Furthermore, the expression of plant antibacterial genes was increased by knocking out the methyltransferase genes ATXR5 and ATXR6.

[0008] Furthermore, the pathogen to which the plant is resistant is Pseudomonas syringae.

[0009] A preparation for regulating plant disease resistance, comprising the methyltransferase genes ATXR5 and ATXR6 according to claim 1, or proteins encoded by the methyltransferase genes ATXR5 and ATXR6 according to claim 1.

[0010] A method for regulating plant disease resistance comprises the following steps: improving plant disease resistance by inhibiting methyltransferase genes ATXR5 and ATXR6 in plants.

[0011] The present invention has the following beneficial effects:

[0012] The ATXR5 and ATXR6 genes in the present invention are isolated from Arabidopsis thaliana. The full-length CDS of ATXR5 is 1140bp, the specific nucleotide sequence is shown in SEQ ID NO: 1, and the protein it encodes has 379 amino acids, the specific amino acid sequence is shown in SEQ ID NO: 3; the full-length CDS of ATXR6 is 1050bp, the specific nucleotide sequence is shown in SEQ ID NO: 2, and the protein it encodes has 349 amino acids, the specific amino acid sequence is shown in SEQ ID NO: 4.

[0013] In order to study the functions of ATXR5 and ATXR6, based on the atxr5; atxr6 weak mutants (mutant numbers are SALK_130607 and SAIL_240H01, respectively), we further used CRISPR to knock out the ATXR6 gene, thereby obtaining the atxr5; atxr6 strong mutant. Because the strong mutant homozygous mutant is lethal, we used the strong mutant heterozygous mutant in the experiment.

[0014] Knockout of ATXR5 and ATXR6 in Arabidopsis thaliana significantly activated the expression of disease-resistance genes in both weak and strong heterozygous mutants. Infection of these mutants with Pseudomonas syringae significantly enhanced the plants' resistance to the bacterium. Furthermore, infection with Pseudomonas syringae also revealed significant activation of antimicrobial genes in both weak and strong mutants. Therefore, this gene has potential applications in plant disease resistance and other applications.

[0015] ATXR5_CDS:

[0016]

[0017] ATXR6_CDS:

[0018] ATGGTGGCTGTGAGGCGAAGGAGAACACAAGCGTCAAACCCTAGATCCGAACCACCGCAACACATGTCGGATCATGATTCCGATTCCGATTGGGATACAGTCTGCGAAGAATGCAGTTCCGGTAAACAACCAGCAAAGCTGCTTCTTTGCGACAAATGCGATAAAGGGTTTCATCTTTTCTGTCTCAGACCGATCCTCGTTTCAGTTCCCAAAGGCTCTTGGTTCTGCCCTTCTTGTTCCAAACATCAGATCCCTAAATCTTTCCCTCTTATTCAGACTAAAATTATAGATTTCTTCCGGATTAAGCGGTCTCCAGATTCATCTCAAATCTCAAGTTCTTCAGATAGTATTGGGAAGAAACGGAAAAAGACTAGCTTGGTGATGTCAAAGAAGAAGAGAAGGCTTCTTCCATACAATCCTAGCAATGATCCTCAAAGGAGGCTAGAGCAAATGGCGTCTCTGGCCACTGCGTTGAGAGCTTCCAACACCAAGTTCAGCAATGAGCTTACTTATGTATCTGGAAAGGCTCCAAGATCTGCAAACCAAGCTGCTTTTGAGAAAGGAGGCATGCAGGTTCTATCTAAAGAAGGCGTAGAGACCTTAGCCTTGTGCAAGAAAATGATGGACCTCGGTGAATGCCCGCCACTTATGGTCGTCTTCGATCCTTATGAAGGGTTCACAGTAGAGGCGGACAGGTTTATAAAAGACTGGACAATTATCACAGAGTATGTTGGAGATGTTGATTATCTGAGCAATAGAGAAGATGACTATGATGGAGACAGTATGATGACTCTACTTCATGCCTCTGATCCTTCGCAATGTCTCGTAATTTGCCCTGACAGACGCAGTAACATCGCCCGGTTCATCAGTGGCATCAACAATCACTCACCAGAAGGGAGGAAGAAGCAGAACCTGAAGTGTGTGAGGTTCAACATCAACGGAGAAGCTAGGGTTCTTCTCGTAGCTAATAGAGACATATCGAAAGGGGAAAGATTGTATTATGATTACAACGGATATGAACATGAGTATCCAACTGAACATTTTGTATAA(SEQ ID NO:2);

[0019] Amino acid sequence encoded by ATXR5:

[0020] MATWNASSPAASPCSSRRRTKAPARRPSSESPPPRKMKSMAEIMAKSVPVVEQEEEEDEDSYSNVTCEKCGSGEGDDELLLCDKCDRGFHMKCLRPIVVRVPIGTWLCVDCSDQRPVRRLSQKKILHFFRIEKHTHQTDKLELSQEETRKRRRSCSLTVKKRRRKLLPLVPSEDPDQRLAQMGTLASALTA LGIKYSDGLNYVPGMAPRSANQSKLEKGGMQVLCKEDLETLEQCQSMYRRGECPPLVVVFDPLEGYTVEADGPIKDLTFIAEYTGDVDYLKNREKDDCDSIMTLLLSEDPSKTLVICPDKFGNISRFINGINNHNPVAKKKQNCKCVRYSINGECRVLLVATRDISKGERLYYDYNGYEHEYPTHHFL(SEQ ID NO:3);

[0021] The amino acid sequence encoded by ATXR6 is:

[0022] MVAVRRRRTQASNPRSEPPQHMSDHDSDSDWDTVCEECSSGKQPAKLLLCDKCDKGFHLFCLRPILVSVPKGSWFCPSCSKHQIPKSFPLIQTKIIDFFRIKRSPDSSQISSSSDSIGKKRKKTSLVMSKKKRRLLPYNPSNDPQRRLEQMASLATALRASNTKFSNELTYVSGKA PRSANQAAFEKGGMQVLSKEGVETLALCKKMMDLGECPPLMVVFDPYEGFTVEADRFIKDWTIITEYVGDVDYLSNREDDYDGDSMMTLLHASDPSQCLVICPDRRSNIARFISGINNHSPEGRKKQNLKCVRFNINGEARVLLVANRDISKGERLYYDYNGYEHEYPTEHFV(SEQ ID NO:4). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Generate atxr6c-1 / 2 gene knockout mutation information for CRISPR-Cas9.

[0024] Figure 2 Transcriptome sequencing showed that a large number of disease resistance-related genes were activated in the atxr5;atxr6 mutant.

[0025] Figure 3 The phenotypes of the wild type and atxr5;atxr6 mutant infected with Pseudomonas syringae are shown.

[0026] Figure 4 The expression of antibacterial-related genes in the wild type and atxr5;atxr6 mutant after infection. DETAILED DESCRIPTION

[0027] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0028] The following experiments were performed after knocking out ATXR5 and ATXR6 in Arabidopsis thaliana. The information on the CRISPR-Cas9-generated atxr6c-1 / 2 gene knockout mutation is available at Figure 1 .

[0029] Example 1: Transcriptome analysis of strong mutants

[0030] The transcriptome analysis process of the strong mutant is as follows:

[0031] 1. Disinfect Arabidopsis seeds with 75% ethanol for 10 minutes, air-dry on filter paper, and spread on 1 / 2 MS culture medium. After 3 days in a 4°C cold room, place in a 21°C long-day incubator. After 5 days of germination, transfer the seedlings to soil and continue incubation under long-day conditions. Sample leaves after two weeks of growth.

[0032] 2. Using Promega The super total RNA extraction kit instructions were used to extract RNA, followed by transcriptome sequencing. The specific steps are as follows:

[0033] (1) Add 2% (V / V) 1-thioglycerol to the RNA lysis buffer.

[0034] (2) Prepare DNAase I incubation solution.

[0035] (3) Ensure that anhydrous ethanol has been added to the RNA wash solution in the appropriate proportion.

[0036] (4) Take 0.1 g of plant material and freeze it quickly in liquid nitrogen. Use a proofing machine to proof for 45 seconds and then put it into liquid nitrogen for quick freezing.

[0037] (5) Use the proofing machine to make proofs again.

[0038] (6) Add 500 μL of the prepared RNA lysis buffer to each sample, then add 500 μL of RNA diluent, mix well with a pipette, and let it stand at room temperature for 5 minutes.

[0039] (7) Centrifuge at 12,000 × g for 5 min at room temperature, carefully aspirate the supernatant, add 0.5 times the volume of anhydrous ethanol, and mix by pipetting up and down 20 times.

[0040] (8) The mixed liquid was added to the centrifuge column twice, centrifuged at 12,000 × g for 1 min, and the filtrate was discarded.

[0041] (9) Add 600 μL RNA wash buffer, centrifuge at 12,000 × g for 45 s, and discard the filtrate.

[0042] (10) Add 50 μL of DNAase I incubation solution to the center of the adsorption membrane and incubate at room temperature for 15 min.

[0043] (11) Repeat step 7 twice.

[0044] (12) Replace the centrifuge column in the collection tube and centrifuge at 12,000 × g for 2 min at room temperature.

[0045] (13) Place the centrifuge column in a new nuclease-free collection tube, open the lid and let it air dry for 3 minutes, add 50 μL of nuclease-free water, and let it stand at room temperature for 2 minutes.

[0046] (14) Centrifuge at 12,000 × g for 2 min at room temperature, take 5 μg of RNA and send it to Annouda Gene Technology Co., Ltd. for sequencing, and store the rest in a -80°C refrigerator.

[0047] The RNA extracted above was subjected to transcriptome sequencing. The specific results are shown in Figure 2 .Depend on Figure 2 It can be seen that both in the weak mutant atxr5; atxr5; atxr6 hyp ;atxr6 hyp or the strong mutant atxr5;atxr5;atxr6 hyp ;atxr6 c-1 In the experiment, antibacterial related genes were activated and expressed in large quantities.

[0048] Example 2: Leaf treatment test using Pst DC3000

[0049] The specific process of using Pst DC3000 to treat blades is as follows:

[0050] 1. Detect bacterial counts after treating leaves with Pst DC3000, including the following steps:

[0051] (1) Take out the frozen Pseudomonas syringae Pst DC3000 strain from a -80°C freezer, inoculate it into KB liquid medium containing rifampicin, and culture it at 30°C and 200 rpm on a shaker for about 16 hours.

[0052] (2) An appropriate amount of the activated strain was inoculated into KB liquid culture medium containing rifampicin and cultured on a shaker at 30°C and 200 rpm for about 12 hours.

[0053] (3) Take 1 mL of culture medium and centrifuge at 4,000 × g for 3 minutes at room temperature. Discard the supernatant and resuspend the bacterial pellet in 1 mL of sterile ddH2O. Dilute 10-fold and measure the OD value using a spectrophotometer. The target activity should be between 0.3 and 0.5. Further dilute with sterile ddH2O to an OD value of 0.005.

[0054] (4) Select healthy Arabidopsis leaves grown under short-day conditions for 4 weeks, mark the leaves, and inject the diluted bacterial suspension into the lower surface of the leaves using a syringe until fully saturated. Gently wipe off excess liquid with absorbent paper, and then return the plants to short-day conditions for continued growth.

[0055] (5) Three hours after injection, samples were taken using a 6 mm perforator. The leaves were placed in a pre-prepared centrifuge tube containing 1 mL of sterile ddH2O. The leaf surface was washed with a pipette and the liquid was discarded.

[0056] (6) Add 100 μL of sterile ddH2O to the centrifuge tube containing the sample, grind the leaves thoroughly in a mortar to release the pathogens, and then add 900 μL of sterile ddH2O to a total volume of 1 mL.

[0057] (7) Perform 10-fold or 100-fold dilution and spread 50 μL of the liquid on a TSA agar plate containing rifampicin.

[0058] (8) Culture at 30°C for 48 hours and count the colony-forming units as a control.

[0059] (9) Three days after infection, samples were taken again using a 6 mm perforator. The leaves were placed in a pre-prepared centrifuge tube containing 1 mL of sterile ddH2O. The leaf surface was washed with a pipette and the liquid was discarded.

[0060] (10) Add 100 μL of sterile ddH2O to the centrifuge tube containing the sample, grind the leaves thoroughly in a mortar to release the pathogens, and then add 900 μL of sterile ddH2O to a total volume of 1 mL.

[0061] (11) Perform 1,000-fold and 10,000-fold dilutions and apply 50 μL of the liquid to a TSA agar plate containing rifampicin. Incubate at 30°C for 48 hours and count the colonies. Figure 3 .

[0062] Depend on Figure 3 It can be seen that when the wild type and mutants were treated with Pseudomonas syringae Pst DC3000, atxr5; atxr5; atxr6 hyp ;atxr6 hyp 、atxr5;atxr5;atxr6 hyp ;atxr6 c -1 and atxr5; atxr5; atxr6 hyp ;atxr6 c -2 showed enhanced resistance to Pst DC3000.

[0063] 2. Detecting gene expression after treating leaves with Pst DC3000 includes the following steps:

[0064] (1) Take out the frozen Pseudomonas syringae Pst DC3000 strain from a -80°C freezer, inoculate it into KB liquid medium containing rifampicin, and culture it at 30°C and 200 rpm on a shaker for about 16 hours.

[0065] (2) An appropriate amount of the activated strain was inoculated into KB liquid culture medium containing rifampicin and cultured on a shaker at 30°C and 200 rpm for about 12 hours.

[0066] (3) Take 1 mL of culture medium and centrifuge at 4,000 × g for 3 min at room temperature. Discard the supernatant and resuspend the bacterial pellet in 1 mL of sterile ddH2O. Dilute 10-fold and measure the OD value using a spectrophotometer. The target activity state is between 0.3 and 0.5.

[0067] (4) Arabidopsis leaves grown under long-day conditions for 2-3 weeks were infected with diluted Pst DC3000 culture medium. Samples were collected 48 hours later for RNA extraction to evaluate gene expression. For the control group, Arabidopsis seedlings were inoculated with 10 mmol / LMgCl2. Specific results are shown in Figure 4 .

[0068] Pst DC3000 infection induces the expression of multiple CRKs genes, including CRK4, CRK6, or CRK36, and Arabidopsis plants overexpressing CRK4, CRK6, or CRK36 show enhanced resistance to Pst DC3000. Figure 4It can be seen that in Col, the expression of these genes is induced by Pst DC3000 infection. Under normal circumstances, the expression of these genes has been upregulated in the strong mutant atxr5; atxr6. When infected with Pst DC3000, the expression levels of these genes are even higher. The above results indicate that the upregulation of the expression of disease resistance-related genes may lead to the weak mutant atxr5; atxr5; atxr6 hyp ;atxr6 hyp or the strong mutant atxr5;atxr5;atxr6 hyp ;atxr6 c-1 Shows enhanced resistance to pathogens.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of methyltransferase genes ATXR5 and ATXR6 in regulating plant disease resistance; The nucleotide sequence of the methyltransferase gene ATXR5 is shown in SEQ ID NO: 1, and the nucleotide sequence of the methyltransferase gene ATXR6 is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that Improve plant disease resistance by knocking out the methyltransferase genes ATXR5 and ATXR6.

3. The use according to claim 1, characterized in that The expression of plant antibacterial genes was improved by knocking out the methyltransferase genes ATXR5 and ATXR6.

4. The use according to any one of claims 1 to 3, characterized in that The pathogen to which the plant is resistant is Pseudomonas syringae.

5. A preparation for regulating plant disease resistance, characterized in that: Comprising the methyltransferase genes ATXR5 and ATXR6 according to claim 1, or proteins encoded by the methyltransferase genes ATXR5 and ATXR6 according to claim 1.

6. A method for regulating plant disease resistance, characterized in that: The following steps are involved: Improve plant disease resistance by inhibiting the methyltransferase genes ATXR5 and ATXR6 in plants.

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