Application of oscpk8 gene in regulating rice disease resistance

Abstract

The application discloses application of OsCPK8 gene in regulation of rice disease resistance and belongs to the technical field of molecular biology. The DNA sequence of the CPK8 gene is shown as SEQ ID NO. 1, and the coding region CDS sequence thereof is shown as SEQ ID NO. 2. The application mines and applies the rice CPK8 gene, and it is clear that the knockout mutant of the rice CPK8 gene significantly enhances the resistance of rice to rice blast and bacterial blight, and the active oxygen burst induced by chitin and flg22 is significantly enhanced. The gene function loss can significantly improve the rice resistance, indicating that the gene negatively regulates the immunity of rice, and the gene can be used as a target gene for improving the resistance of rice to rice blast and bacterial blight. The application provides a new and efficient target for rice molecular breeding, and has important significance for guaranteeing safe production of rice and promoting development of green agriculture.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the OsCPK8 gene in regulating disease resistance in rice. Background Technology

[0002] Rice blast is one of the most serious fungal diseases affecting rice production, caused by the fungus *Pyrrosia lingua*, which can infect rice throughout its entire growth cycle. Bacterial blight is the most widespread bacterial disease affecting rice, caused by the pathogenic strain of the Gram-negative bacterium *Xanthomonas*, and is a vascular disease. Both diseases severely impact rice growth and production. Currently, rice disease control mainly relies on chemical pesticides, but long-term and excessive use not only pollutes the environment but also easily leads to pesticide resistance in pathogens, posing a risk to food security. Utilizing plant immune mechanisms to cultivate 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 blight resistance genes have been cloned, less than 20% have actually been applied to breeding. This is mainly because existing resistance genes are mostly race-specific resistances, effective only against specific physiological races, while the rapid variation of pathogen populations in the field leads to poor resistance persistence and limited application. The plant innate immune system comprises two levels: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI is a basic immune response activated by plants through the perception of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) on the cell surface, possessing the potential for broad-spectrum resistance. However, although the plant PTI immune pathway can induce 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 kinases (CPKs) serve as key signaling nodes, rapidly activating downstream defense responses through phosphorylation modification after sensing immune signals. However, current functional studies of rice CPK family genes remain unsystematic, particularly regarding their potential in regulating broad-spectrum disease resistance. Existing technologies lack PTI pathway genes that can simultaneously enhance resistance to rice blast and bacterial blight and possess non-race-specific characteristics. This is primarily due to the complexity of the PTI signaling network, making it difficult to screen key genes with significant disease resistance phenotypes, and the difficulty of distinguishing the specificity of different CPK members in disease resistance function using traditional methods.

[0004] Therefore, mining CPK genes from the PTI signaling pathway that can trigger strong immune responses and have broad-spectrum disease resistance has become an important direction for breaking through the current bottleneck in disease-resistant breeding. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OsCPK8 gene in regulating rice disease resistance, thereby addressing the problems existing in the prior art. This invention discovers that the rice CPK8 gene can be used as a target gene to improve rice resistance to rice blast and bacterial blight. This invention provides a novel and highly efficient target for rice molecular breeding, which is of great significance for ensuring safe rice production and promoting the development of green agriculture.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an application of the rice CPK8 gene in regulating rice resistance to rice blast and / or bacterial blight. 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 in SEQ ID NO.1, and its coding region CDS sequence is shown in SEQ ID NO.2.

[0008] This invention also provides the application of biological materials related to the rice CPK8 gene in regulating the resistance of rice to rice blast and / or bacterial blight. 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 in SEQ ID NO.1, and its coding region CDS sequence is shown in 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.

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

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

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

[0012] The present invention also provides the application of biological materials related to the rice CPK8 gene in the breeding of rice with high resistance to rice blast and / or bacterial blight, wherein the DNA sequence of the CPK8 gene is shown in SEQ ID NO.1 and its coding region CDS sequence is shown in 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.

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

[0014] The present invention also provides a method for breeding rice with high resistance to rice blast and / or bacterial blight, characterized by 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 blight.

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

[0016] This invention also provides a method for identifying the CPK8 knockout genotype in rice, comprising the following steps:

[0017] Using the genomic DNA of the plant to be tested as a template, PCR amplification was performed using primers shown in SEQ ID NO.14-16 to obtain the PCR product of the sample to be tested.

[0018] Wild-type probes were obtained by PCR amplification using wild-type rice genomic DNA as a template and primers as shown in SEQ ID NO.15-16.

[0019] The PCR product of the sample to be tested is hybridized with an equal amount of the wild-type probe to obtain a hybrid product. The hybrid product is then detected to analyze the genotype of the plant to be tested.

[0020] The present invention discloses the following technical effects:

[0021] This invention, through the discovery and application of the rice CPK8 gene, clearly demonstrates that its knockout mutant exhibits significantly enhanced resistance to rice blast and bacterial blight, and a significantly enhanced reactive oxygen species (ROS) burst induced by chitin and flg22. The loss of function of this gene can significantly improve rice resistance, indicating its negative regulation of rice immunity. This gene can be used as a target gene to improve rice resistance to rice blast and bacterial blight. This invention provides a novel and highly efficient target for rice molecular breeding, which is of great significance for ensuring safe rice production and promoting the development of green agriculture. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This section presents the CPK8 gene knockout strategy and genotype detection results. A shows a schematic diagram of the two knockout target sites in the CPK8 gene and the editing methods of the three knockout mutants. B shows the iHDA molecular marker used to identify the CPK8 genotype. In the diagram, M represents the DNA marker. Sample 1 is wild-type, and samples 2-7 are mutant samples to be tested. Analysis of the sequencing results revealed that sample 5 had the same banding pattern as the wild-type, sample 2 had a 1bp deletion homozygous mutation, sample 7 had a 1bp insertion mutation, and samples 3, 4, and 6 were all heterozygous genotypes.

[0024] Figure 2 The reactive oxygen species (ROS) bursts induced by chitin and flg22 in CPK8 gene knockout mutants and wild-type individuals are shown. Among them, A and C are the ROS bursts induced by chitin and flg22, respectively; B and D are the statistical results of the total ROS bursts induced by chitin and flg22, respectively.

[0025] Figure 3 The results of resistance identification of CPK8 gene knockout mutants to rice blast are shown. A represents typical lesion symptoms of CPK8 gene knockout mutants and wild-type plants after perforation with *Bacillus oryzae* strain RB22, recorded on day 14 post-inoculation; B represents the relative lesion area statistics of the inoculated leaves corresponding to (A) (***p < 0.001, **p < 0.01); C represents the relative fungal biomass detection based on typical leaves in (A), quantifying the expression level of the *Bacillus oryzae* MoPot2 gene by RT-qPCR, with the OsUBQ gene as an internal control (***p < 0.001).

[0026] Figure 4 The results of resistance identification of CPK8 gene knockout mutant to bacterial blight are shown. In this paper, A shows the typical lesion symptoms of CPK8 gene knockout mutant and wild-type leaf cuttings inoculated with bacterial blight pathogen PXO99A strain, and the data were recorded on the 10th day after inoculation. B shows the statistical data of the length of lesion b on the inoculated leaves corresponding to (A) (***p < 0.001). Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The inventors identified a new rice gene LOC_Os03g59390, which they named the CPK8 gene. Its genome sequence is shown in SEQ ID NO.1, with a total length of 4300 bp. Its CDS sequence is shown in 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 in SEQ ID NO.3.

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

[0034]

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

[0036]

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

[0038] *

[0039] This invention demonstrates that by knocking out the rice CPK8 gene, the knockout mutant significantly enhances resistance to rice blast and bacterial blight, and significantly enhances the reactive oxygen species burst induced by chitin and flg22. The invention is illustrated in detail below with specific embodiments.

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

[0041] I. Construction of CPK8 gene knockout mutant

[0042] This embodiment uses CRISPR / Cas9 gene editing technology to create a 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 targeting the coding region of the 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) Construction of target 1 vector

[0048] Synthesize oligonucleotide chains for cloning:

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

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

[0051] The primers were mixed in equal amounts and subjected to heat denaturation at 95°C for 5 minutes. The mixture was then slowly cooled to room temperature (25°C) to anneal and form a double-stranded DNA fragment.

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

[0053] The annealed double-stranded DNA fragment was ligated with the linearized pRGEB32 vector according to the following system: 100 ng of linearized vector, 0.5 μL of T4 ligase, 1 μL of T4 ligase buffer, and the above-mentioned DNA double-stranded fragment was added to bring the reaction system to 10 μL.

[0054] The ligation reaction was carried out at 16°C for 10 h. The ligation product was transformed into E. coli DH5α competent cells and screened on LB agar plates containing 50 mg / L kanamycin. Single colonies were picked for colony PCR identification, and plasmids were extracted from positive clones and their DNA was sequenced to verify that the sgRNA expression cassette was correctly inserted.

[0055] (2) Construction of target 2 vector

[0056] Synthesize oligonucleotide chains for cloning:

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

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

[0059] Subsequent steps, including annealing, vector digestion, ligation, transformation, and identification, are the same as those for constructing the vector for target 1.

[0060] 2. Rice genetic transformation and mutant acquisition

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

[0062] Disinfection: Wash the hulled rice with 75% alcohol for 1 minute, then disinfect with sodium hypochlorite at a final concentration of 7% for 30 minutes, and finally wash with sterile water 10 times.

[0063] Callus induction: Sterilized seeds were placed in NB medium and cultured in the dark at 28°C for about 14 days. The freshly induced callus tissue was then transferred to a new induction medium and cultured in the dark for another 14 days. At this time, a large amount of callus tissue was obtained.

[0064] Agrobacterium infection: Fresh EHA105 Agrobacterium colonies were picked and cultured in LB medium containing antibiotics for 16 h until OD = 0.6. 1 mL of the culture was then transferred to 100 mL of antibiotic-free LB medium and shaken at 250 rpm for 8 h until OD was approximately 0.1 to 0.2. The cells were collected by centrifugation at 4000 rpm, 4°C for 10 min. After removing the supernatant, the cells were washed once with AAM-AS medium, and the OD was adjusted to approximately 0.6. Callus was then immersed in the bacterial solution for 20 to 30 min, with occasional shaking. The callus was spread on sterile filter paper to absorb any remaining bacterial solution, but not completely dried. The callus was then transferred to NB-AS medium lined with filter paper and incubated in the dark at 28°C for 2-3 days.

[0065] Washing and screening: Transfer the callus to a sterile Erlenmeyer flask and wash 10 times with sterile water, the last wash lasting 30 minutes. Remove the sterile water, add fresh sterile water containing hygromycin resistance, and shake for 30 minutes. Collect the callus and blot off excess water on filter paper. Transfer the callus to fresh NB-THA medium and incubate in the dark at 28°C for 14 days. Transfer it again to fresh NB-THA medium and repeat the screening process after 14 days.

[0066] Predifferentiation: Healthy callus tissue was transferred to predifferentiation medium and cultured in the dark at 28°C for 14 days;

[0067] Differentiation: Transfer healthy callus to differentiation medium and incubate at 28°C for 2 to 3 weeks;

[0068] Rooting: Transfer the callus that has produced leaf buds to a rooting medium and culture at 28°C for 2 to 3 weeks under light;

[0069] Transplanting and identification: Transplanted seedlings that have achieved morphological development into clean water for 3 days to harden off, and a small amount of leaves were taken to extract DNA to determine whether the transformation was successful. Positive seedlings were transplanted.

[0070] II. Identification of CPK8 gene knockout mutants

[0071] 1. Genomic DNA extraction

[0072] Genomic DNA was extracted from the leaves of wild-type (WT) rice and T0 generation transgenic rice plants using the CTAB method.

[0073] 2. PCR amplification and sequencing identification

[0074] Specific identification primers were designed for PCR amplification targeting 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 area surrounding the target site, a GC enhancer was used in the PCR reaction system to improve amplification efficiency. A high-fidelity PCR kit from Novizan Pharmaceuticals (Nanjing) was used, with the following reaction program: 96℃ pre-denaturation for 5 min, 96℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 15 s, 35 cycles, followed by a final extension at 72℃ for 5 min, and sample storage at 4℃.

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

[0083] 3. iHDA molecular marker analysis of genotype

[0084] To establish a rapid and low-cost genotyping method, a set of iHDA molecular markers was designed targeting 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 a template, PCR amplification was performed using primer pair iHDA-CPK8-F / iHDA-CPK8-R to obtain the PCR product of the sample to be tested.

[0090] Probe preparation: Wild-type probes were obtained by PCR amplification using wild-type rice genomic DNA as a template and primer pair iHDA-CPK8-probe-F / iHDA-CPK8-R.

[0091] Molecular hybridization: Mix equal amounts of the PCR product of the sample to be tested with the wild-type probe, and run the hybridization program on the PCR instrument: denature at 95℃ for 5 min, anneal at 72℃ for 10 min, and then cool 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 2Under the induction of C and D, the peak value of the reactive oxygen species (ROS) burst kinetic curve was higher, and the calculated total ROS burst amount was significantly increased. This result indicates that loss of function of the CPK8 gene can significantly enhance the PTI immune response in rice, demonstrating that CPK8 is an important negative regulator of the PTI signaling pathway.

[0115] Example 3: Identification of rice blast resistance

[0116] To clarify the function of the CPK8 gene in rice resistance to fungal diseases, this example uses artificial inoculation with rice blast fungus to systematically analyze the disease resistance phenotype and quantitative growth of the CPK8 gene knockout mutant.

[0117] 1. Preparation of plant materials and pathogens

[0118] 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.

[0119] Pathogen activation and sporulation: The physiological race RB22 of rice blast fungus (Magnaporthe oryzae) was inoculated on oat flakes agar plates and cultured under continuous light at room temperature for 14 days to induce a large number of sporulations.

[0120] Preparation of spore suspension: Gently scrape the spores on the surface of the culture medium with sterile water containing 0.5% (v / v) Tween-20, and adjust the concentration to 5.0 × 10⁻⁶. 5 1 spore / mL, for later use.

[0121] 2. Inoculation treatment and disease development

[0122] Inoculation was performed using the hole-punching method. Tiny wounds were made in the middle of the second-to-last leaf of the plant, and a suspension of spores from the physiological race RB22 of rice blast fungus was dripped onto the wounds to form inoculation sites. After inoculation, the plants were placed in darkness and high humidity for 24 hours to promote spore germination and infection. They were then transferred to a greenhouse with normal light and humidity for cultivation, and disease development was continuously observed.

[0123] 3. Disease resistance phenotype analysis

[0124] Fourteen days after inoculation, phenotypic observation and image acquisition were performed on the inoculated leaves. The lesion area at the inoculation site was measured and the relative lesion area was calculated.

[0125] Relative lesion area (%) = (average lesion area of ​​mutants / average lesion area of ​​wild type) × 100%.

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

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

[0128] qPCR primers:

[0129] MoPot2 transposable element for rice blast fungus (used for quantifying fungal 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 (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] The ChamQ Universal SYBR qPCR Master Mix kit from Nanjing Novizan Pharmaceutical Co., Ltd. was used. The reaction system (20 μL) was as follows: 10 μL of 2 × ChamQ Universal SYBR qPCR Master Mix; 0.4 μL each of 10 μM upstream and downstream primers; 2 μL of cDNA template; and water to a final volume of 20 μL. The reaction was performed in two steps on a real-time quantitative PCR instrument.

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

[0137] 5. Results and Analysis

[0138] The final result is as follows Figure 3 As shown. Compared with the wild type, the CPK8 gene knockout mutant (cpk8) exhibits significantly enhanced resistance to rice blast, specifically in the following ways:

[0139] Lesion phenotype: The lesion area on the leaves of the mutant 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 result is as follows Figure 4 As shown.

[0153] Lesion phenotype: The extent of lesion expansion on leaves of the CPK8 gene knockout mutant was significantly less than that of the wild type. Figure 4 A).

[0154] Lesion length statistics: Quantitative statistical analysis of lesion length showed that the average lesion length of the CPK8 knockout mutant was significantly shorter than that of the wild type. Figure 4 B).

[0155] The results of this experiment demonstrate that knocking out the CPK8 gene can significantly enhance rice's resistance to bacterial blight and limit the spread of the pathogen in leaf tissues.

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

[0157] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a rice CPK8 gene in regulating rice resistance to rice blast and / or bacterial blight, characterized in that, Knocking out the CPK8 gene enhances the resistance of rice to rice blast and / or bacterial blight; the DNA sequence of the CPK8 gene is shown in SEQ ID NO.1, and its coding region CDS sequence is shown in SEQ ID NO.

2.

2. The application of a rice CPK8 gene-related biomaterial in regulating rice resistance to rice blast and / or bacterial blight, characterized in that, Knocking out the CPK8 gene enhances the resistance of rice to rice blast and / or bacterial blight; the DNA sequence of the CPK8 gene is shown in SEQ ID NO.1, and its coding region CDS sequence is shown in SEQ ID NO.2; the related biological materials include recombinant plasmids with the CPK8 gene knocked out or recombinant microorganisms containing the recombinant plasmids.

3. A method for improving the resistance of rice to rice blast and / or bacterial blight, characterized in that, The step includes knocking out the CPK8 gene as described in claim 1 in rice.

4. The application of a biological material related to the rice CPK8 gene in the breeding of rice with high resistance to rice blast and / or bacterial blight, characterized in that, The DNA sequence of the CPK8 gene is shown in SEQ ID NO.1, and its coding region CDS sequence is shown in SEQ ID NO.2; the related biological materials include recombinant plasmids that knock out the CPK8 gene or recombinant microorganisms containing the recombinant plasmids.

5. A method for cultivating rice with high resistance to rice blast and / or bacterial blight, characterized in that, The method includes the step of knocking out the CPK8 gene as described in claim 1 in rice to obtain transgenic rice with high resistance to rice blast and bacterial blight.

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