Application of OsCPK8 gene in regulation and control of rice disease resistance

Abstract

The invention discloses application of an OsCPK8 gene in regulation and control of rice disease resistance, and belongs to the technical field of molecular biology. The DNA (Deoxyribose Nucleic Acid) sequence of the CPK8 gene is shown as SEQ ID NO.1, and the CDS (Coding Sequence) sequence of a coding region of the CPK8 gene is shown as SEQ ID NO.2. By excavating and applying the rice CPK8 gene, the resistance of the knockout mutant to rice blast and bacterial blight is obviously enhanced, and active oxygen outbreak induced by chitin and flg22 is obviously enhanced. The function deletion of the gene can significantly improve the rice resistance, which indicates that the gene can negatively regulate the rice immunity, and the gene can be used as a target gene for improving the resistance of rice to rice blast and bacterial leaf blight. The invention provides a brand-new high-efficiency target for rice molecular breeding, and has important significance for guaranteeing the safe production of rice and promoting the development of green agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to the application of OsCPK8 gene in regulating rice disease resistance. BACKGROUND

[0002] Rice blast is one of the most serious fungal diseases affecting rice production, caused by the fungus Pyricularia grisea, which can infect rice at all growth stages. Bacterial leaf blight is the most widespread bacterial disease of rice, caused by the Gram-negative bacterium Xanthomonas oryzae pv. oryzae, and belongs to vascular diseases. These two diseases seriously affect the growth and production of rice. At present, the prevention and control of rice diseases mainly relies on chemical pesticides, but long-term and large-scale use not only pollutes the environment, but also easily leads to drug resistance of pathogenic bacteria and causes food safety risks. Using plant immune mechanisms to breed disease-resistant varieties is considered a more economical and environmentally friendly approach. Although more than 200 rice blast resistance genes and more than 40 bacterial leaf blight resistance genes have been cloned, less than 20% of them have been applied to breeding. This is mainly because most of the existing resistance genes belong to small race-specific resistance, which is only effective against specific physiological races, while the pathogen population in the field varies rapidly, leading to poor resistance durability and limited application range. The plant innate immune system includes two levels of PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI is a basic immune response activated by plants through cell surface pattern recognition receptors (PRRs) to perceive pathogen-associated molecular patterns (PAMPs), which has the potential for broad-spectrum resistance. However, although the plant PTI immune pathway can trigger broad-spectrum resistance by recognizing conserved molecular patterns of pathogens, its immune response intensity is usually weak, making it difficult to directly provide effective field disease resistance.

[0003] In the PTI signaling pathway, calcium-dependent protein kinase (CPK) as a key signaling node can rapidly activate downstream defense responses through phosphorylation modification after sensing immune signals. However, the functional research on rice CPK family genes is still not systematic, especially the potential of broad-spectrum disease resistance has not been fully explored. There is a lack of PTI pathway genes that can simultaneously enhance resistance to rice blast and bacterial leaf blight and have non-small race-specific characteristics in the prior art, which is mainly due to the complexity of the PTI signaling network, making it difficult to screen key genes with significant disease resistance phenotype, and traditional methods are difficult to distinguish the specificity of different CPK members in disease resistance function.

[0004] Therefore, it is an important direction to break through the current disease resistance breeding bottleneck by mining CPK genes from the PTI signaling pathway that can trigger strong immune response and have broad-spectrum disease resistance characteristics. SUMMARY

[0005] The application aims to provide an application of OsCPK8 gene in regulating rice disease resistance, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the application provides the following solutions.

[0007] The application provides an application of a rice CPK8 gene in regulating rice resistance to rice blast and / or bacterial leaf blight, and knocking out or inhibiting the expression of the CPK8 gene can enhance the resistance of rice to rice blast and / or bacterial leaf blight; the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2.

[0008] The application also provides an application of a related biomaterial of a rice CPK8 gene in regulating rice resistance to rice blast and / or bacterial leaf blight, and knocking out or inhibiting the expression of the CPK8 gene can enhance the resistance of rice to rice blast and / or bacterial leaf blight; the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2; the related biomaterials include a protein encoded by the CPK8 gene, a recombinant plasmid containing the CPK8 gene or a recombinant microorganism containing the recombinant plasmid.

[0009] Further, the amino acid sequence of the protein is shown as SEQ ID NO. 3.

[0010] The application also provides a method for improving the resistance of rice to rice blast and / or bacterial leaf blight, which comprises the step of knocking out or inhibiting the expression of the CPK8 gene in rice.

[0011] The application also provides an application of a rice CPK8 gene in cultivating rice with high resistance to rice blast and / or bacterial leaf blight, and the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2.

[0012] The application also provides an application of a related biomaterial of a rice CPK8 gene in cultivating rice with high resistance to rice blast and / or bacterial leaf blight, and the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2; the related biomaterials include a protein encoded by the CPK8 gene, a recombinant plasmid containing the CPK8 gene or a recombinant microorganism containing the recombinant plasmid.

[0013] Further, the amino acid sequence of the protein is shown as SEQ ID NO. 3.

[0014] The application also provides a method for cultivating rice with high resistance to rice blast and / or bacterial leaf blight, characterized in that the method comprises the step of knocking out or inhibiting the expression of the CPK8 gene in rice to obtain transgenic rice with high resistance to rice blast and bacterial leaf blight.

[0015] The application also provides an iHDA molecular marker for identifying the knockout genotype of rice CPK8, comprising an upstream iHDA-CPK8-F of a nucleotide sequence shown as SEQ ID NO. 14, an upstream iHDA-CPK8-probe-F of a nucleotide sequence shown as SEQ ID NO. 15, and a downstream iHDA-CPK8-R of a nucleotide sequence shown as SEQ ID NO. 16.

[0016] The application also provides a method for identifying the knockout genotype of rice CPK8, comprising the following steps:

[0017] PCR amplification is performed on the genomic DNA of the plant to be tested using the primers shown as SEQ ID NO. 14-16 to obtain a PCR product of the sample to be tested;

[0018] PCR amplification is performed on the genomic DNA of the wild-type rice using the primers shown as SEQ ID NO. 15-16 to obtain a wild-type probe;

[0019] The PCR product of the sample to be tested and the wild-type probe are hybridized in equal amounts to obtain a hybridization product, and the genotype of the plant to be tested is analyzed by detecting the hybridization product.

[0020] The application discloses the following technical effects:

[0021] The application mines and applies the rice CPK8 gene, and finds that the knockout mutant of the gene significantly enhances the resistance of rice to rice blast and bacterial leaf blight and the burst of reactive oxygen induced by chitin and flg22. The loss of function of the gene can significantly improve the resistance of rice, indicating that the gene negatively regulates the immunity of rice, and the gene can be used as a target gene to improve the resistance of rice to rice blast and bacterial leaf blight. The application provides a new and efficient target for rice molecular breeding, and has important significance for guaranteeing the safe production of rice and promoting the development of green agriculture. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only show some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 Figure 5 is a genotype detection result of a CPK8 gene knockout strategy; wherein, A is a schematic diagram of two knockout targets in the CPK8 gene and editing modes of three knockout mutants; B is identification of the CPK8 genotype by iHDA molecular markers, wherein M represents a DNA marker, No. 1 sample is a wild type, and No. 2-7 samples are mutant samples to be detected; it is found by combining sequencing result analysis that No. 5 sample has the same band type as the wild type, No. 2 sample is a 1 bp deletion homozygous mutation, No. 7 sample is a 1 bp insertion mutation, and No. 3, 4 and 6 samples are all heterozygous genotypes;

[0024] Figure 2 Figure 6 is a chitin and flg22 induced reactive oxygen burst of the CPK8 gene knockout mutant and the wild type; wherein, A and C are chitin and flg22 induced reactive oxygen burst, respectively; B and D are statistical results of chitin and flg22 induced reactive oxygen burst total values, respectively;

[0025] Figure 3 Figure 7 is a resistance identification result of the CPK8 gene knockout mutant to rice blast, wherein, A is a typical lesion symptom of the CPK8 gene knockout mutant and the wild type after being punched and inoculated with the rice blast RB22 strain, recorded on the 14th day after inoculation; B is a relative lesion area statistics of the inoculated leaves in (A) (***p < 0.001, **p < 0.01); C is a relative fungal biomass detection based on the typical leaves in (A), the expression level of the MoPot2 gene of the rice blast is quantified by RT-qPCR, and the OsUBQ gene is used as an internal reference (***p < 0.001);

[0026] Figure 4 Figure 8 is a resistance identification result of the CPK8 gene knockout mutant to white leaf blight, wherein, A is a typical lesion symptom of the CPK8 gene knockout mutant and the wild type after being cut and inoculated with the white leaf blight PXO99A strain, recorded on the 10th day after inoculation; B is a b lesion length statistics of the inoculated leaves in (A) (***p < 0.001). DETAILED DESCRIPTION

[0027] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentration, intensity, and time, and the like, every intermediate value of the

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0030] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all other such modifications and variations that come within the scope of the application are to be included within the scope thereof. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0032] The inventors identified a new rice gene LOC_Os03g59390, which is named as CPK8 gene, the genomic sequence of which is shown as SEQ ID NO. 1, with a total length of 4300 bp, the CDS sequence of which is shown as SEQ ID NO. 2, with a total length of 1617 bp, encoding a CPK8 protein containing 539 amino acids, the amino acid sequence of the CPK8 protein is shown as SEQ ID NO. 3.

[0033] SEQ ID NO. 1 (genomic sequence of CPK8 gene):

[0034]

[0035] SEQ ID NO. 2 (CDS sequence of CPK8 gene):

[0036]

[0037] SEQ ID NO. 3 (amino acid sequence of CPK8 protein):

[0038] MGNCCGTPATAEEGGKRRRRGKQKKANPFTVAYNRAPSSAGAAAGRPGLMVLRDPTGRDLGARYELGGELGRGEFGITYLCTEAETGDRYACKSISKRKLRTPVDVEDVRREVEIMRHMPSHPNIVSLRAAYEDEDNVHLVMELCEGGELFDRIVARGHYTERAAAAVTRTIVEVVQMCHRHGVMHRDLKPENFLYANKKDSSPLKAIDFGLSVFFRPGERFTEIVGSPYYMAPEVLKRHYGPEVDVWSAGVILYILLCGVPPFWAETEQGVAQAIIRSVVDFKREPWPRVSEPAKDLVKRMLDPNPMTRLTAEQVLEHPWLHDSKKMPDIPLGDAVRARLQQFAAMNKLKKKALKVIAEHLSAEEAADIKDMFDKMDVSKNGQLTFEDFKAGIRKLGNQMPDSDLKILMDAADIDKNGILDYQEFVAVSIHVRKIGNDEHIQKAFSYFDQNKSGYIEIEELREALVDEIDGNDEDIINSIIRDVDTDKDGKISYDEFAVMMKAGTDWRKASRQYSRQRFSNLSLKLQKDGSISDDTQ*.

[0039] The present application discloses a CPK8 gene knockout mutant of rice, and the resistance of the mutant to rice blast and bacterial blight is significantly enhanced, and the active oxygen burst induced by chitin and flg22 is significantly enhanced.

[0040] Example 1 Construction and identification of CPK8 gene knockout mutant

[0041] I. Construction of CPK8 gene knockout mutant

[0042] In this embodiment, the CRISPR / Cas9 gene editing technology is used to create the CPK8 gene knockout mutant. The specific implementation steps are as follows:

[0043] 1. Construction of sgRNA expression vector

[0044] Two specific sgRNA target sites were designed for the coding region of rice CPK8 gene (LOC_Os03g59390):

[0045] Target 1 sequence: 5'-GATCACGTACCTGTGCACGG-3', SEQ ID NO. 4;

[0046] Target 2 sequence: 5'-TCTTCGACAGGATCGTCGCG-3', SEQ ID NO. 5.

[0047] (1) Target 1 vector construction

[0048] Synthetic oligonucleotide chain for cloning:

[0049] Forward primer: 5'-GGCAGATCACGTACCTGTGCACGG-3', SEQ ID NO. 6;

[0050] Reverse primer: 5'-AAACCCGTGCACAGGTACGTGATC-3', SEQ ID NO. 7.

[0051] The above primers were mixed in equal amounts, heat denatured at 95°C for 5 min, and then slowly cooled to room temperature (25°C) to anneal and form double-stranded DNA fragments.

[0052] The pRGEB32 vector plasmid was digested with BsaI restriction enzyme at 37°C for 1 h, and the linearized vector fragment was recovered.

[0053] The double-stranded DNA fragments formed by annealing were connected with the linearized pRGEB32 vector as follows: linearized vector 100 ng, T4 ligase 0.5 μL, T4 ligase buffer 1 μL, and the above DNA double-stranded fragments were added to make the reaction system to 10 μL.

[0054] The connection reaction was carried out at 16°C for 10 h. The connection product was transformed into E. coli DH5α competent cells, and coated on LB solid plate containing 50 mg / L kanamycin for screening. Single colony was picked for colony PCR identification, and positive clone was extracted for DNA sequencing verification to ensure correct insertion of sgRNA expression cassette.

[0055] (2) Target 2 vector construction

[0056] Synthetic oligonucleotide chain for cloning:

[0057] Forward primer: 5'-GGCATCTTCGACAGGATCGTCGCG-3', SEQ ID NO. 8;

[0058] Reverse primer: 5'-AAACCGCGACGATCCTGTCGAAGA-3', SEQ ID NO. 9.

[0059] The subsequent steps, including annealing, vector digestion, ligation, transformation and identification, are the same as the target 1 vector construction method.

[0060] 2. Genetic transformation and mutant acquisition of rice

[0061] The sgRNA recombinant plasmid verified by sequencing was transformed into the embryogenic callus of japonica rice variety 'Zhonghua 11' by Agrobacterium-mediated method (completed by Unchirbio Technology Co., Ltd.). After the steps of hygromycin resistance screening, callus differentiation, rooting culture and the like, the T0 generation transgenic rice plants were finally obtained. The transformation steps are as follows:

[0062] Disinfection: the shelled rice was washed with 75% alcohol for 1 min, then disinfected with sodium hypochlorite with a final concentration of 7% for 30 min, and finally washed with sterile water for 10 times;

[0063] Callus induction: the disinfected seeds were placed in NB medium, and dark culture was carried out at 28°C for about 14 d. The freshly induced callus was transferred to a new induction medium for dark culture for about 14 d, at which time a large amount of callus was obtained.

[0064] Agrobacterium infection: fresh EHA105 Agrobacterium colonies were selected and cultured in LB medium containing resistance for 16 h until OD = 0.6. 1 mL was added to 100 mL of non-resistant LB, and 250 rpm was shaken for 8 h. The OD was about 0.1 to 0.2. The bacteria were collected by centrifugation at 4000 rpm and 4°C for 10 min. After removing the supernatant, the bacteria were washed once with AAM-AS medium, and the OD was adjusted to about 0.6. The callus was infected with the bacterial solution for 20 to 30 min, and occasionally shaken. The callus was placed on sterilized filter paper to absorb the residual bacterial solution, but not completely blown dry. The callus was transferred to NB-AS medium with filter paper, and dark culture was carried out at 28°C for 2-3 d.

[0065] Water washing and screening: the callus was transferred to a sterilized conical flask and washed with sterile water for 10 times, and the last washing was for 30 min. The sterile water was removed, and sterile water with hygromycin resistance was added again, and shaken for 30 min. The callus was collected and the water was absorbed on the filter paper. The callus was transferred to new NB-THA medium, and dark culture was carried out at 28°C for 14 d. It was transferred to new NB-THA medium again, and the screening step was repeated once after 14 d.

[0066] Pre-differentiation: the healthy callus was transferred to pre-differentiation medium, and dark culture was carried out at 28°C for 14 d.

[0067] Differentiation: transfer the healthy callus to differentiation medium, 2-3 weeks light culture at 28℃;

[0068] Rooting: transfer the callus with leaf bud to rooting medium, 2-3 weeks light culture at 28℃;

[0069] Transplanting and identification: transplant the seedling-shaped small plants to water for 3 days of seedling raising, and take a small amount of leaf to extract DNA to identify whether the transformation is successful, and transplant the positive seedlings.

[0070] II. Identification of CPK8 gene knockout mutants

[0071] 1. Genomic DNA extraction

[0072] The CTAB method was used to extract the genomic DNA of the leaves of wild type (WT) rice and T0 generation transgenic rice plants.

[0073] 2. PCR amplification and sequencing identification

[0074] Specific identification primers were designed for PCR amplification for two different CRISPR target regions:

[0075] Target 1 region amplification:

[0076] Upstream primer CPK8-site1-CX-F: 5'-TCGCCTCGCCTCCTTTGT-3', SEQ ID NO. 10;

[0077] Downstream primer CPK8-site1-R: 5'-GGCGCATGATCTCCACCT-3', SEQ ID NO. 11.

[0078] Target 2 region amplification:

[0079] Upstream primer CPK8-site2-CX-F: 5'-CCTACGAGGACGAGGACAAC-3', SEQ ID NO. 12;

[0080] Downstream primer CPK8-site2-CX-R: 5'-TGACTCTAACAAGCACCAAGGT-3', SEQ ID NO. 13.

[0081] Due to the high GC content in the surrounding region of the target, GC enhancer was used in the PCR reaction system to improve the amplification efficiency. The high-fidelity PCR kit of Nanjing Novozyme Company was used, and the reaction program was as follows: 96℃ pre-denaturation for 5min, 96℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 15s, 35 cycles, 72℃ full extension for 5min, 4℃ storage of samples.

[0082] PCR amplification products were sent to Shanghai Genesee Biotech Co., Ltd. for DNA sequencing. The sequencing results were compared with the wild type CPK8 gene sequence to analyze whether there were insertion or deletion mutations in the target region. Through this method, three homozygous mutant strains with mutations in the target region were finally identified, named cpk8-2, cpk8-8 and cpk8-17 respectively. Figure 1 A).

[0083] 3. iHDA molecular marker analysis genotype

[0084] In order to establish a rapid and low-cost genotyping method, an iHDA molecular marker was designed for target 1:

[0085] iHDA-CPK8-F: 5'-TACGAGCTCGGCGGCGAGCTG-3', SEQ ID NO. 14;

[0086] iHDA-CPK8-probe-F: 5'-TACGAGCTCGGCGGCGAGCTGGGGCGCGGCGAGTTCGGGATCACGTACCTGTGCGGCGGAGACGGGGGACAGGTACGCGTGCAAG-3', SEQ ID NO. 15;

[0087] iHDA-CPK8-R: 5'-GGCGCATGATCTCCACCTCG-3', SEQ ID NO. 16.

[0088] The specific steps are as follows:

[0089] Sample amplification: using the genomic DNA of the plant to be tested as the template, using primer pair iHDA-CPK8-F / iHDA-CPK8-R for PCR amplification, obtaining the PCR product of the sample to be tested.

[0090] Probe preparation: using wild type rice genomic DNA as a template, using primer pair iHDA-CPK8-probe-F / iHDA-CPK8-R for PCR amplification, obtaining wild type probe.

[0091] Molecular hybridization: equal amounts of PCR product of sample to be tested and wild type probe were mixed, and hybridization program was run on PCR instrument: denaturation at 95℃ for 5 min, annealing at 72℃ for 10 min, then reduced to 4℃ for short-term storage or -20℃ for long-term storage.

[0092] Electrophoresis detection: Take 10 μL of hybridization product and mix it with 2 μL of premixed nucleic acid dye (Gel red) in 6× DNA loading buffer. Use an 8% non-denaturing polyacrylamide gel and electrophoresis at a constant voltage of 90V for 45 min. After electrophoresis, observe and photograph the gel using a gel imaging system.

[0093] like Figure 1 As shown in Figure B, sample 1 is wild-type, and samples 2-7 are mutant samples to be tested. Analysis of the sequencing results revealed that sample 5 has the same banding pattern as wild-type, sample 2 has a 1bp deletion homozygous mutation, sample 7 has a 1bp insertion mutation, and samples 3, 4, and 6 are all heterozygous genotypes. This indicates that the iHDA marker can clearly distinguish between different genotypes such as wild-type, heterozygous mutation, and homozygous mutation (including base insertions or deletions), providing an efficient tool for large-scale screening in subsequent breeding.

[0094] Example 2: PTI Immunoreaction - Detection of Reactive Oxygen Explosion

[0095] To assess the function of the CPK8 gene in rice model-triggered immune (PTI) responses, this embodiment used chemiluminescence to detect the level of reactive oxygen species (ROS) bursts induced by typical pathogen-associated molecular pattern (PAMP) flg22 and chitin.

[0096] 1. Preparation of plant materials

[0097] Wild-type (WT, Zhonghua 11) and CPK8 gene knockout mutants (cpk8-2, cpk8-8, and cpk8-17) seeds were surface-sterilized and then sown in tissue culture flasks containing 1 / 2 MS solid medium. They were vertically cultured in a light incubator (12h light / 12h dark, 26℃) for 10-12 days. After culture, healthy seedlings with uniform growth were selected for experiments.

[0098] 2. Leaf sheath fragment treatment

[0099] Select the longest leaf sheath from each seedling and cut it evenly into segments approximately 3 mm in length using a sterile blade. Gently pick up the leaf sheath segments with sterile forceps and place them into 96-well microplates, with 4 leaf sheath segments in each well. Then, add 100 μL of sterile deionized water to each well and incubate the microplate in the dark for 10–16 h to deplete constitutive reactive oxygen species and restore tissue homeostasis.

[0100] 3. Preparation of detection solution and induction

[0101] Before testing, use a pipette to remove all sterile water from each well.

[0102] Immediately add 100 μL of freshly prepared test solution to each well. Test solution components:

[0103] 50 mM Tris-HCl buffer (pH 7.5), 10 µg / mL horseradish peroxidase (HRP, Bio-Rad), 10 µM L-012 chemiluminescent probe, 50 µM flg22 (Shanghai Sangon Biotech Co., Ltd.) or 40 µg / mL chitin (Sigma-Aldrich).

[0104] 4. Chemiluminescence signal detection

[0105] After adding the test solution, immediately place the 96-well plate into a Varioskan Flashmultireader (BioTek). Start measuring the chemiluminescence signal immediately at room temperature and monitor continuously for 45 minutes. The instrument is set to measure for 400 ms per well.

[0106] 5. Data processing and calculation of total reactive oxygen species

[0107] The total amount of reactive oxygen species (ROS) bursts is quantified by calculating the area under the chemiluminescence kinetic curve (AUC). The specific calculation formula is as follows:

[0108] Total reactive oxygen species = [∑(y1: y n )×2-y1-y n × Time interval / Measurement time per well / 2,

[0109] Among them, y1 to y n : Represents the chemiluminescence value readings at time points 1 to n; n: Total number of reading points, n=45;

[0110] Time interval: The time difference between two consecutive readings, which is 1 min 45 s;

[0111] Measurement time per well: The integration time of the instrument for a single measurement of each sample is 400 ms, or 0.4 s.

[0112] The formula essentially approximates the luminescence values ​​between consecutive time points as the sum of the areas of a trapezoid, thereby calculating the total area under the curve and accurately reflecting the total amount of reactive oxygen species.

[0113] 6. Results and Analysis

[0114] The final test results are as follows Figure 2 As shown. Compared with the wild type, the CPK8 gene knockout mutant exhibits higher levels of flg22 ( Figure 2 (A and B) and chitin ( Figure 2The peak of the kinetic curve of the active oxygen burst was higher, and the calculated total active oxygen burst was significantly increased. The results showed that the loss of function of CPK8 gene could significantly enhance the PTI immune response of rice, and proved that CPK8 was an important negative regulator of PTI signal pathway.

[0115] Example 3 Rice blast resistance identification

[0116] To clarify the function of CPK8 gene in rice resistance to fungal diseases, this example carried out a systematic analysis of disease resistance phenotype and pathogen growth quantification of CPK8 gene knockout mutants by artificial inoculation of Magnaporthe oryzae.

[0117] 1. Plant material and pathogen preparation

[0118] Plant material: Select wild type (WT, Zhonghua 11) and CPK8 gene knockout mutant (cpk8-2, cpk8-8 and cpk8-17) rice plants with consistent growth to 7 weeks old.

[0119] Pathogen activation and sporulation: Magnaporthe oryzae physiological race RB22 was inoculated on oatmeal medium plates and cultured at room temperature under continuous light for 14 days to induce massive sporulation.

[0120] Spore suspension preparation: The spores on the surface of the culture medium were gently scraped with sterile water containing 0.5% (v / v) Tween-20, and the concentration was adjusted to 5.0×10 5 spores / mL for standby.

[0121] 2. Inoculation treatment and disease development

[0122] Punch inoculation method was used for inoculation. A small wound was made in the middle of the second leaf from the bottom of the plant, and spore suspension of Magnaporthe oryzae physiological race RB22 was added to the wound to form inoculation points. After inoculation, the plants were placed in dark and high humidity conditions for 24 h to promote spore germination and infection. Then they were transferred to a normal light and humidity greenhouse for cultivation, and the disease development was continuously observed.

[0123] 3. Disease resistance phenotype analysis

[0124] After 14 days of inoculation, the phenotype of the inoculated leaves was observed and images were collected. The lesion area of the inoculation point was measured and the relative lesion area was calculated.

[0125] Relative lesion area (%) = (mutant average lesion area / wild type average lesion area) x 100%.

[0126] 4. Quantitative analysis of pathogen biomass (qPCR)

[0127] To accurately quantify the pathogen content in the plant body, real-time fluorescent quantitative PCR (qPCR) technology was used to detect the specific genes of rice blast fungus.

[0128] qPCR primers:

[0129] Rice blast fungus transposable element MoPot2 (used for quantifying pathogen biomass):

[0130] Upstream primer (MoPot2-qRT-F): 5'-ACGACCCGTCTTTACTTATTTGG-3', SEQ ID NO. 17;

[0131] Downstream primer (MoPot2-qRT-R): 5'-AAGTAGCGTTGGTTTTGTTGGAT-3', SEQ ID NO. 18.

[0132] Rice internal reference gene OsUBQ (used for standardization):

[0133] Upstream primer (UBQ-qRT-F): 5'-TTCTGGTCCTTCCACTTTCAG-3', SEQ ID NO. 19;

[0134] Downstream primer (UBQ-qRT-R): 5'-ACGATTGATTTAACCAGTCCATGA-3', SEQ ID NO. 20.

[0135] ChamQ Universal SYBR qPCR Master Mix kit from Nanjing Novozyme was used. The reaction system (20 μL) was as follows: 2 × ChamQ Universal SYBR qPCR Master Mix 10 μL; 10 μM upstream and downstream primers 0.4 μL each; cDNA template 2 μL; water to 20 μL. The reaction was performed on a real-time fluorescent quantitative PCR instrument according to the two-step method.

[0136] Data analysis: the relative biomass of rice blast fungus in the mutants was calculated using the 2 -ΔΔCt method, with the pathogen biomass in the wild type sample as the reference (set to 1).

[0137] 5. Results and analysis

[0138] The final results are shown in Figure 3 Compared with the wild type, the CPK8 gene knockout mutant (cpk8) showed significantly enhanced resistance to rice blast, which was manifested in:

[0139] Lesion phenotype: the lesion area on the mutant leaf was significantly smaller than that of the wild type (Figure 3 (A and B).

[0140] Pathogen growth: Quantitative analysis by qPCR showed that the relative expression level (i.e., relative fungal biomass) of the MoPot2 gene in the leaves of the mutant was significantly lower than that of the wild type. Figure 3 (C).

[0141] The above results indicate that knocking out the CPK8 gene can effectively inhibit the growth and spread of rice blast fungus in rice tissues, thereby significantly enhancing rice's resistance to rice blast.

[0142] Example 4: Identification of resistance to bacterial blight

[0143] To elucidate the function of the CPK8 gene in rice resistance to bacterial diseases, this embodiment uses the leaf-cutting inoculation method to identify the resistance of CPK8 gene knockout mutants to bacterial blight.

[0144] 1. Preparation of plant materials and pathogens

[0145] Plant materials: Wild-type (WT, Zhonghua 11) and CPK8 gene knockout mutants (cpk8-2, cpk8-8 and cpk8-17) rice plants with uniform growth at 7 weeks of age were selected.

[0146] Pathogen activation and culture: The highly virulent strain PXO99A of *Xanthomonas oryzae* pv. oryzae, the causal agent of rice bacterial blight, was used. Single colonies were picked from fresh NA agar plates and inoculated into sterile test tubes containing 2 mL of NA liquid medium. The inoculated tubes were placed in a shaker at 28°C and incubated overnight (approximately 12-16 hours) with shaking at 200 rpm to allow the bacterial culture to reach the logarithmic growth phase.

[0147] 2. Preparation of inoculum solution

[0148] Take an appropriate amount of the activated bacterial solution and measure its optical density (OD) at a wavelength of 600 nm using a spectrophotometer. 600 Adjust the bacterial concentration to OD using sterile NA liquid medium or physiological saline. 600 =0.8, reserved.

[0149] 3. Inoculation treatment and disease development

[0150] The leaf-cutting inoculation method was used. Sterilized scissors were dipped in bacterial suspension and 2 cm of the tips of the top three leaves of rice plants during the booting stage were cut off. The plants were then treated in darkness for one day, followed by normal light incubation. Ten days after inoculation, when disease symptoms had fully developed, an investigation was conducted. The length of the lesion below each cut was precisely measured using calipers or a ruler. At least three leaves were measured per plant, and at least six plants were investigated per line.

[0151] 4. Results and analysis

[0152] The final results are shown in Figure 4 .

[0153] Lesion phenotype: The lesion expansion on the leaves of CPK8 knockout mutants is significantly lighter than that of wild type (A). Figure 4

[0154] Lesion length statistics: Quantitative statistical analysis of lesion length shows that the average lesion length of CPK8 knockout mutants is significantly shorter than that of wild type (B). Figure 4

[0155] The experimental results prove that knocking out CPK8 gene can significantly enhance the resistance of rice to bacterial leaf blight and limit the expansion of pathogenic bacteria in leaf tissue.

[0156] Combined with the resistance identification results of rice blast, it is fully shown that CPK8 is an important broad-spectrum disease resistance negative regulator, and the loss of function can simultaneously improve the resistance of rice to fungal diseases (rice blast) and bacterial diseases (bacterial leaf blight).

[0157] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.​​

Claims

1. Application of rice CPK8 gene in regulating resistance of rice to rice blast and / or bacterial leaf blight, characterized in that, Knocking out or inhibiting the expression of the CPK8 gene can enhance the resistance of rice to rice blast and / or bacterial blight; the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO.

2.

2. The application of the biological material related to the rice CPK8 gene in regulating the resistance of rice to rice blast and / or bacterial leaf blight, characterized in that, Knocking out or inhibiting the expression of the CPK8 gene can enhance the resistance of rice to rice blast and / or bacterial blight; the DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2; the related biological materials include the protein encoded by the CPK8 gene, a recombinant plasmid containing the CPK8 gene, or a recombinant microorganism containing the recombinant plasmid.

3. Use according to claim 2, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

3.

4. A method for improving the resistance of rice to rice blast and / or bacterial leaf blight, characterized by, The step of knocking out or inhibiting the expression of the CPK8 gene in rice.

5. The use of a rice CPK8 gene in breeding rice with high resistance to rice blast and / or bacterial leaf blight, characterized in that, The DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO.

2.

6. The use of a biological material related to the CPK8 gene of rice in the breeding of rice with high resistance to rice blast and / or bacterial leaf blight, characterized in that, The DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence is shown as SEQ ID NO. 2; the related biological materials include the protein encoded by the CPK8 gene, a recombinant plasmid containing the CPK8 gene, or a recombinant microorganism containing the recombinant plasmid.

7. Use according to claim 6, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

3.

8. A method of breeding rice plants with high resistance to blast and / or bacterial leaf blight, characterized in that, The step of knocking out or inhibiting the expression of the CPK8 gene in rice to obtain a transgenic rice with high resistance to rice blast and bacterial blight.

9. An iHDA molecular marker for identifying a knockout genotype of rice CPK8, characterized in that, The upstream iHDA-CPK8-F includes a nucleotide sequence shown as SEQ ID NO. 14, the upstream iHDA-CPK8-probe-F includes a nucleotide sequence shown as SEQ ID NO. 15, and the downstream iHDA-CPK8-R includes a nucleotide sequence shown as SEQ ID NO.

16.

10. A method for identifying a knockout genotype of rice CPK8, characterized in that, The steps include: Using the genomic DNA of the plant to be tested as a template and using the primers shown as SEQ ID NO. 14-16 to perform PCR amplification to obtain a PCR product of the sample to be tested; Using the genomic DNA of wild-type rice as a template and using the primers shown as SEQ ID NO. 15-16 to perform PCR amplification to obtain a wild-type probe; Hybridizing the PCR product of the sample to be tested with the wild-type probe to obtain a hybridization product, detecting the hybridization product, and analyzing the genotype of the plant to be tested.

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