Cloning of rice OsCPK13 gene and application of rice OsCPK13 gene in prevention and treatment of rice blast

Abstract

The invention relates to the technical field of plant genetic engineering. The invention particularly relates to cloning of the rice OsCPK13 gene and application of the rice OsCPK13 gene in prevention and treatment of rice blast. The positive regulation and control effect of the rice OsCPK13 gene in rice blast resistance reaction is found and proved for the first time, the resistance of rice to rice blast can be remarkably enhanced by improving the expression level of the rice OsCPK13 gene, and the resistance of rice to rice blast can be reduced by knocking out the rice OsCPK13 gene. The method can be applied to genetic improvement for improving the resistance of related crops such as rice.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rice disease-resistant breeding, and particularly relates to cloning of rice OsCPK13 gene and application thereof in rice blast prevention. BACKGROUND

[0002] Rice (Oryza sativa) is one of the most important food crops in China, and rice blast caused by Magnaporthe oryzae is one of the most important fungal diseases threatening rice production in China. Rice blast can occur in all rice-growing regions of the world, and the average annual yield loss of rice caused by rice blast is up to 10%-30%, and in severe cases, up to 40%-50%, and in extreme cases, even no harvest, which can meet the food demand of several tens of millions of people every year. Therefore, prevention and control of rice blast is of great significance to improve rice yield and ensure food security.

[0003] Plants can produce nutrients and energy required for their own growth by photosynthesis, which is the basis for many organisms to survive in nature. Similarly, pathogenic bacteria have evolved various strategies to infect plants to absorb nutrients from plants for survival and reproduction, while plants have evolved a strong innate immune system to defend against pathogenic bacterial attacks. They struggle with each other and co-evolve. The immune system of plants first recognizes the conserved components PAMPs / MAMPs (pathogen-associated molecular patterns or microbe-associated molecular patterns) of pathogenic bacteria by pattern recognition receptors (PRRs) located on the cytoplasmic membrane, which initiates the first layer of defense response PTI (PAMP-triggered immunity) of plants. Pathogenic bacteria further evolve effectors to enter plant cells to inhibit the PTI response of plants to achieve the purpose of continuous infection, while plants evolve R (resistance) proteins, also known as NB-LRR (nucleotide binding and leucine-rich repeat) proteins, which can directly or indirectly recognize effectors to activate the second layer of defense response ETI (effector-triggered immunity) of plants to achieve the effect of plant defense.

[0004] In PTI reaction, plants recognize PAMPs or MAMPs through PRRs, and then initiate a series of immune responses, such as the burst of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK), and the influx of calcium ions (Ca 2+ ) into cells. Among them, Ca 2+ is an important second messenger in plants. Both PTI and ETI lead to the influx of Ca 2+ , thereby initiating intracellular Ca 2+ signaling. In plants, calcium-dependent protein kinases (CPKs / CDPKs) contain C-terminal Ca 2+ binding EF-hand domains and N-terminal kinase domains, so they act as both Ca 2+ sensors and decoders, and through their Ca 2+ dependent kinase activity, they transduce Ca 2+ signals into phosphorylation marks on their substrates. SUMMARY

[0005] In order to improve the genetic improvement of rice and other related crops, the present application provides a rice OsCPK13 gene and functionally identifies the gene, and for the first time discovers and proves the positive regulation of OsCPK13 gene in the prevention and treatment of rice blast.

[0006] The technical solutions adopted by the present application are as follows: OsCPK13 gene, the CDS sequence of which is shown as SEQ ID NO. 1.

[0007] The protein encoded by the above-mentioned OsCPK13 gene, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0008] The above-mentioned OsCPK13 gene or the protein encoded by the OsCPK13 gene is applied in the prevention and treatment of rice blast; Further, the application is the application of OsCPK13 gene in the genetic improvement or molecular breeding of rice resistance to blast; Further, the expression of OsCPK13 gene in rice is improved, and a transgenic rice with overexpression of OsCPK13 gene is obtained, and the transgenic rice has increased resistance to blast.

[0009] The above-mentioned OsCPK13 gene or the protein encoded by the OsCPK13 gene is applied in the reduction of rice blast resistance; Further, the OsCPK13 gene in rice is knocked out by using gene editing technology, and the resistance of rice to blast is reduced.

[0010] The application has the advantages that: (1) The application proves that overexpression of OsCPK13 gene can enhance the resistance of rice to rice blast through OsCPK13 overexpression experiment and hole punching inoculation experiment; compared with wild type ZH11, the leaf lesion area of OsCPK13 overexpression plant is smaller, the Magnaporthe oryzae biomass is lower, and the callose deposition is increased, which indicates that OsCPK13 positively regulates the disease resistance of rice to rice blast, and indicates that the OsCPK13 gene can be applied to genetic improvement of rice and other related agricultural crops to improve the resistance.

[0011] (2) The application proves that deletion mutation of OsCPK13 gene reduces the resistance of rice to rice blast through spray inoculation method; compared with wild type ZH11, the oscpk13 mutant has more lesions, higher Magnaporthe oryzae biomass, and reduced callose deposition; the above results show that the OsCPK13 gene is a potential resource for creating rice blast resistance enhanced transgenic rice and crop molecular design excellent candidate gene beneficial mutation improvement, and has important theoretical value and broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Figure for detection of OsCPK13 protein expression level of OsCPK13 overexpression plant, detection of OsCPK13 gene expression level and resistance identification of inoculation of Magnaporthe oryzae.

[0013] Figure 2 Figure for identification of knockout target of OsCPK13 knockout mutant plant.

[0014] Figure 3 Figure for resistance identification of inoculation of Magnaporthe oryzae of OsCPK13 knockout mutant plant. DETAILED DESCRIPTION

[0015] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited thereto.

[0016] In the application, rice variety ZH11 and rice blast physiological race Guy11 are used as research objects.

[0017] Example 1: Overexpression of OsCPK13 improves the resistance of rice to rice blast (1) Materials and methods Based on the rice OsCPK13 gene sequence (the CDS sequence of the rice OsCPK13 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2), the CDS sequence of the OsCPK13 gene was amplified by PCR and then inserted into the multiple cloning site after the HA tag of the pCXUN-HA vector using seamless cloning technology to construct an overexpression vector of the OsCPK13 gene. The OsCPK13 gene was overexpressed in the rice variety ZH11 using Agrobacterium EHA105-mediated genetic transformation, resulting in OsCPK13 overexpressing rice plants OE-OsCPK13-1 and OE-OsCPK13-2. RNA was extracted from ZH11, OE-OsCPK13-1 and OE-OsCPK13-2, respectively, and reverse transcribed into cDNA. The expression level of OsCPK13 gene in the plants was detected by real-time quantitative PCR. At the same time, total protein was extracted from the plants and the expression level of OsCPK13 protein was detected.

[0018] Identification of rice blast resistance in plants overexpressing OsCPK13: The perforation method was used: Rice seedlings approximately one month old were used. A perforator was used to gently press a hole in the rice leaf to create a wound, but not to penetrate it. 10 μL of a 5×10⁻⁶ concentration was then inoculated. 5 A suspension of Guy11 spores per mL of *Magnapordica oryzae* was dropped onto the surface of perforated leaves, and the perforations were sealed with tape. Inoculated rice was placed in a 26℃ greenhouse in the dark for 24 hours, followed by 5–7 days of culture under 12 hours of light and 12 hours of darkness and high humidity. Disease phenotype was observed, leaf lesion size was recorded, and *Magnapordica oryzae* biomass and callus deposition experiments were performed. *Magnapordica oryzae* biomass detection: Equal amounts of leaves from ZH11, OE-OsCPK13-1, and OE-OsCPK13-2 plants with consistent disease trends were collected. Rice genomic DNA was extracted, and its concentration was determined. Using the rice Ubiquitin gene and the *Magnapordica oryzae* MoPot2 gene as internal reference genes, the growth of *Magnapordica oryzae* in diseased rice leaves was detected at the DNA level using qPCR. Callus deposition experiment: The youngest leaves of rice seedlings cultured in an incubator for 5-7 days were cut with scissors and placed in 2mL EP tubes. Chitin solution was added to a final concentration of 400nM, and the tubes were vacuum-treated for 30min. A fixative solution of ethanol:acetic acid = 3:1 was added and the tubes were fixed at room temperature for 5h, with the fixative solution being changed an average of 3 times. The tubes were washed 3 times with distilled water, then 70% alcohol was added and the tubes were left at room temperature for 2h. The tubes were washed 3 times with distilled water, then 50% alcohol was added and the tubes were left at room temperature for 2h. The tubes were washed 3 times with distilled water, then distilled water was added and the tubes were left overnight. The next day, 10% NaOH solution was added and the tubes were left at room temperature for 1h to make the rice leaf tissue transparent. The tubes were washed 4 times with distilled water, then 0.01% aniline blue was dissolved in K2HPO4 (pH 9.5) for staining. The tubes were stained on a low-speed shaker in the dark for 4h. The number of calluses was observed and counted using a laser confocal microscope.

[0019] (2) Results and Analysis See Figure 1 , Figure 1 A represents the detection of OsCPK13 protein expression levels in plants; Figure 1 B represents the detection of the OsCPK13 gene expression level in plants; Figure 1 C represents the phenotypic observation 7 days after inoculation with a suspension of blast fungus Guy11 spores using the perforation inoculation method. Bar = 1 cm. Figure 1 D represents the area of ​​diseased leaves, statistically analyzed using ImageJ software; Figure 1 E represents the biomass of rice blast fungus in diseased leaves. The relative gene expression level was calculated using a 2-1T / T ratio. -△△CT Law; Figure 1 F represents the detection of corpus callosum deposition, observed under bright-field confocal microscopy; Figure 1 G represents the statistical analysis of callus deposition in plants using ImageJ software.

[0020] Compared with the ZH11 control, OsCPK13 protein expression was successfully detected in plants overexpressing OsCPK13, and qPCR results showed that the OsCPK13 gene expression level was significantly increased. Figure 1 A-1B). Phenotypic analysis of the inoculated lesions showed that plants overexpressing OsCPK13 had smaller lesion areas compared to wild-type ZH11 after inoculation. Figure 1 C-1D), and the biomass of rice blast fungus in diseased leaves also showed that the biomass of rice blast fungus in plants with OsCPK13 overexpression was less than that in wild-type plants (C-1D). Figure 1 E), and further observation and statistical analysis of corpus callosum deposition revealed that, compared with wild-type ZH11, plants overexpressing OsCPK13 had greater corpus callosum deposition. Figure 1 F-1G). This indicates that overexpression of the OsCPK13 gene can enhance the resistance of rice to rice blast.

[0021] Example 2: Knockout of OsCPK13 gene and obtaining of knockout mutants (1) Materials and methods According to the sequence of the rice OsCPK13 gene (the CDS sequence of the rice OsCPK13 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2), two target sites for knockout were selected, and the OsCPK13 gene was knocked out in the background of rice variety ZH11 by CRISPR / Cas9 technology. T0 generation rice was obtained by Agrobacterium-mediated genetic transformation of rice callus, and independent genetic lines were obtained by genotyping.

[0022] (2) Results and analysis Referring to Figure 2 , two target sites for knockout were selected according to the sequence of the rice OsCPK13 gene, which were 5'-TTAGATCCAAGTACCTGAG-3' and 5'-GACCACCCAGTCTCCGCGT-3'. Genotyping found that two knockout mutant lines, oscpk13-1 and oscpk13-2, were obtained; oscpk13-1 had a deletion of two bases at the CDS 27bp of the OsCPK13 gene; oscpk13-2 had an insertion of one base at the CDS 169bp of the OsCPK13 gene. Both types of mutations cause frame shift mutations, resulting in the premature appearance of a stop codon, which are OsCPK13 gene function loss mutants.

[0023] Example 3: Identification of disease resistance phenotype of OsCPK13 knockout mutants (1) Materials and methods Spray inoculation method was used to uniformly spray the prepared rice blast fungus Guy11 spore suspension with a concentration of 1x10 5 The spore suspension was uniformly sprayed on 14-day-old ZH11, oscpk13-1 and oscpk13-2 seedlings, ensuring that the rice leaves were completely covered with the spore suspension. After 24h of dark treatment in a high-humidity environment, the plants were further cultured under 12h light and 12h dark conditions, and the environment was kept humid during the culture. The disease phenotype was observed after 3-5 days, and the biomass of the rice blast fungus was detected. Rice blast fungus biomass detection: equal amounts of leaves with consistent disease trends were selected from the OsCPK13 knockout mutants and wild type, respectively, and the rice genomic DNA was extracted. The concentration was determined using rice Ubiquitin and rice blast fungus MoPot2 as internal reference genes. The growth of the rice blast fungus in the diseased leaves was detected by qPCR at the DNA level.

[0024] (2) Results and analysis Referring to Figure 3, Figure 3 A shows the phenotypic observation 4 days after inoculation with spore suspension of rice blast fungus Guy11 using the spray inoculation method. ZH11 is the wild type, and oscpk13-1 and oscpk13-2 are two allelic mutants created by CRISPR / Cas9 technology. Bar = 1 cm. Figure 3 B represents the biomass of rice blast fungus in diseased leaves. The relative gene expression level was calculated using a 2-1 method. -△△CT Law; Figure 3 C represents the deposition of plant callosities, observed under a bright-field confocal microscope; Figure 3 D represents the statistical analysis of callus deposition in plants using ImageJ software.

[0025] Based on the phenotype of spray inoculation, the number of lesions in rice significantly increased after the OsCPK13 gene was lost. Figure 3 A) The biomass of rice blast fungus increased ( Figure 3 B), reduced deposition of the corpus callosum ( Figure 3 C-3D) indicates that the OsCPK13 gene knockout mutation reduces the resistance of rice to rice blast; among which, Figure 3 A shows that compared to ZH11, both oscpk13-1 and oscpk13-2 exhibited reduced resistance to rice blast fungus and increased lesions; Figure 3 B shows that the biomass of rice blast fungus in the knockout mutant is significantly higher than that in the wild type; ​ C and 3D images show reduced callus deposition in the oscpk13 mutant, further illustrating that the OsCPK13 gene positively regulates rice resistance to rice blast.

Claims

1. OsCPK13 Genes are characterized by: The CDS sequence is shown in SEQ ID NO.

1.

2. The one as described in claim 1 OsCPK13 Gene-encoded proteins are characterized by: The amino acid sequence is shown in SEQ ID NO.

2.

3. The claim 1 OsCPK13 Gene or as described in claim 2 OsCPK13 Application of gene-encoded proteins in the prevention and control of rice blast.

4. The application according to claim 3, characterized in that: OsCPK13 Application of genes in the genetic improvement or molecular breeding of rice resistance to rice blast.

5. The application according to claim 4, characterized in that: Improve rice OsCPK13 Gene expression, acquisition OsCPK13 Transgenic rice with overexpressed genes, which exhibits increased resistance to rice blast.

6. The claim 1 OsCPK13 Gene or as described in claim 2 OsCPK13 Application of gene-encoded proteins in reducing rice blast resistance.

7. The application according to claim 6, characterized in that: Using gene editing technology to knock out rice OsCPK13 Genes that reduce rice's resistance to rice blast.

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