Rice blast resistance related gene OsSAPK10 and application thereof

Abstract

The invention relates to the technical field of rice disease-resistant breeding, in particular to a rice blast-resistant related gene OsSAPK10 of rice and application of the rice blast-resistant related gene OsSAPK10. The nucleotide sequence of the rice blast resistance related gene OsSAPK10 is shown as SEQ ID NO: 1, and the amino acid sequence of the protein coded by the gene OsSAPK10 is shown as SEQ ID NO: 2. It is found and proved for the first time that the rice blast resistance related gene OsSAPK10 positively regulates the resistance of rice to rice blast, the resistance of rice to rice blast can be reduced by knocking out the rice blast resistance related gene OsSAPK10 through a CRISPR / Cas9 method, and the resistance of rice to rice blast can be remarkably enhanced by improving the expression level of the rice blast resistance related gene OsSAPK10; normal growth and development of the rice are not affected by knockout or overexpression of the rice blast-resistant related gene OsSAPK10 of the rice. The invention can be applied to genetic improvement for improving resistance and yield of related agricultural crops such as rice.

Description

Technical Field

[0001] The present invention relates to the technical field of rice disease resistance breeding, and in particular to a rice blast resistance-related gene OsSAPK10 and its applications. Background Art

[0002] Rice blast, caused by rice blast fungi, is one of the main diseases that threaten the safe production of rice. Rice blast fungi mainly infect the leaf sheaths, leaves and panicles of rice. When the disease is severe, it will affect the photosynthesis of the leaves, thereby reducing the yield and quality of rice. The yield loss caused by rice blast infection is generally 10%~20%, and in severe cases it can reach 40%~50%. In particularly severe cases, it may even cause the entire rice field to have no harvest. The disease occurs in rice-growing areas in more than 80 countries around the world, causing an average annual reduction of about 10% in rice production. The lost rice can feed 60 million people. Studies have shown that cultivating rice blast-resistant varieties has become the most economical and effective means of prevention and control, so the discovery of new genes related to rice blast resistance is of great significance.

[0003] Plant sucrose non-fermentation-1-related protein kinase (SnRK) belongs to the Ser / Thr class of protein kinases. It regulates downstream proteins through phosphorylation and participates in the transduction of multiple physiological signaling pathways. It is widely involved in abiotic and biotic stresses, sugar signaling, seed germination and growth, and is considered to be the hub of the stress response signal connection network, nutrient metabolism and energy balance in plants. Based on sequence homology and protein structural characteristics, the SnRK gene family is divided into the SnRK1 subfamily, the SnRK2 subfamily and the SnRK3 subfamily. Among them, the SnRK2 subfamily has been identified and cloned in many plants and has the most complete functional research among the three subfamilies. As key components of the ABA signal transduction pathway, they play an important role in the ABA signaling pathway, and thus not only participate in the growth and development of plants, but also play a key role in plant response to stress. There are 10 SnRK2 genes in rice, named OsSAPK1-10 , plays an important role in the biological and abiotic stress of rice. OsSAPK8 , OsSAPK9 and OsSAPK10The three genes are sensitive to ABA, but the expression activated by high osmotic stress can activate all SAPK members. OsSAPK10 activated by ABA can directly interact with a rice ABF protein TRAB1. The results of in vitro kinase experiments showed that OsSAPK10 can phosphorylate the Ser102 site of TRAB1 protein in response to high osmotic stress. In addition to participating in the ABA signaling pathway, SAPK is also involved in other signaling pathways. For example, Lou.D et al. found that OsSAPK1 and OsSAPK2 improve salt tolerance by promoting the production of metabolites and the degradation of reactive oxygen species. In addition, OsSAPK4 can regulate genes with oxidative stress response functions and ion homeostasis, and play a role in responding to salt stress. OsSAPK2 also plays a role in the drought stress response of rice. When subjected to drought, high salt and PEG treatments, OsSAPK2 The expression level increased significantly, and ossapk2 The mutant is more sensitive to drought stress and reactive oxygen than the wild type. Aishek et al. also found that OsSAPK9 can play a role in increasing rice yield and responding to drought stress by regulating the opening and closing of stomata, cell permeability, and the expression of stress response genes. Recently, it has also been discovered that SAPK also plays an important role in biological stress. For example, OsSAPK10-mediated phosphorylation weakens the DNA binding ability of WRKY72, thereby enhancing resistance to bacterial blight.

[0004] Previous studies have shown that the Arabidopsis SnRK2 protein SnRK2.6 (OST1) plays a very important role in plant immunity. In the early stage of the experiment, the inventors searched for homologous proteins of SnRK2.6 in rice and found that the rice SnRK2 protein OsSAPK10 had the highest homology with SnRK2.6 and was induced to express by rice blast infection. Further studies found that OsSAPK10 was involved in the regulation of rice blast resistance, and knocking out OsSAPK10 The gene significantly reduced rice blast resistance and increased OsSAPK10 The expression level of the gene enhances the resistance of rice to rice blast disease, and OsSAPK10 The gene is not involved in the regulation of rice growth and development; therefore, OsSAPK10 The existence of genes provides an applied basis for improving rice resistance to rice blast. Summary of the invention

[0005] The purpose of the present invention is to provide a rice blast resistance related gene OsSAPK10 The invention provides a rice blast resistance-related gene OsSAPK10 It has a positive regulatory effect on rice blast resistance. OsSAPK10 Gene deletion reduces rice's resistance to rice blast, while OsSAPK10 Overexpression of the gene can enhance rice's resistance to rice blast. OsSAPK10 The genes can be applied to genetic improvement to enhance the resistance of rice and other related agricultural crops.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides a rice blast resistance-related gene OsSAPK10 , the rice blast resistance related gene OsSAPK10 The nucleotide sequence is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a rice blast resistance related gene OsSAPK10 The encoded protein has an amino acid sequence as shown in SEQ ID NO: 2.

[0008] The third aspect of the present invention provides the above-mentioned rice blast resistance related gene OsSAPK10 Application in regulating rice resistance to rice blast; Further, the regulation is positive regulation; Furthermore, the positive regulation is: overexpression of rice blast resistance related genes OsSAPK10 Improving rice resistance to rice blast or knocking out genes related to rice blast resistance OsSAPK10 Reduce rice resistance to rice blast.

[0009] The fourth aspect of the present invention provides the above-mentioned rice blast resistance related gene OsSAPK10 Application in breeding rice blast-resistant varieties.

[0010] The fifth aspect of the present invention provides a method for regulating the resistance of rice to rice blast fungus. When it is necessary to reduce the resistance of rice to rice blast fungus, OsSAPK10 Gene knockout; when it is necessary to improve the resistance of rice to rice blast fungus, OsSAPK10 Gene overexpression; OsSAPK10 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0011] The significant advantages of the present invention are: 1. The present invention searches for the homologous protein OsSAPK10 of Arabidopsis immune protein SnRK2.6 in rice and identifies the function of the protein. It first discovers and proves the positive regulatory role of OsSAPK10 in rice resistance to rice blast. OsSAPK10 Gene reduces rice resistance to rice blast disease, while overexpression OsSAPK10 The gene can enhance rice's resistance to rice blast, indicating that OsSAPK10 The gene positively regulates rice resistance to rice blast; the above results all indicate OsSAPK10The gene is an excellent candidate gene for creating transgenic rice with enhanced resistance to rice blast and for crop molecular design, and has important theoretical value and broad application prospects; 2. The present invention knocks out the rice variety Zhonghua 11 (ZH11) by genetic engineering method OsSAPK10 The gene reduces rice's resistance to rice blast disease, compared with the wild type. ossapk10 The mutant had more lesions and more rice blast fungus growth, larger lesion area, and less H2O2 accumulation; while in ZH11, the OsSAPK10 The expression level of the gene can significantly enhance the resistance of rice to rice blast. Compared with the wild type, the OsSAPK10 The lesion area and growth of rice blast fungus in the overexpressing plants were both smaller than those in the wild type; the rice blast resistance gene provided by the present invention OsSAPK10 It can be applied to genetic improvement to enhance the resistance of rice and other related agricultural crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 In Example 2 of the present invention ossapk10 Growth and development phenotypes of the mutants.

[0013] Figure 2 In Example 3 of the present invention ossapk10 Identification of mutants resistant to rice blast.

[0014] Figure 3 In Example 4 of the present invention OsSAPK10 Detection of OsSAPK10-HA protein in overexpressing plants and identification of their resistance to rice blast.

[0015] Note: ossapk10 express OsSAPK10 Knockout mutants, OsSAPK10 -OE means OsSAPK10 Overexpression plants. * p < 0.05, ** p < 0.01. DETAILED DESCRIPTION

[0016] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0017] In the present invention, the rice variety Zhonghua 11 (ZH11) and the rice blast fungus Guy11 were used as research objects.

[0018] Embodiment 1: This embodiment provides rice OsSAPK10 Gene, the rice OsSAPK10 The CDS sequence of the gene is shown in SEQ ID NO: 1, and the full length of the sequence is 1089 bp; the rice OsSAPK10The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, encoding 362 amino acids.

[0019] Embodiment 2: Knockout rice OsSAPK10 The gene does not affect the growth and development of rice: (1) Materials and methods ossapk10 Construction of mutants: Using CRISPR-Cas9 technology to generate mutants in rice under ZH11 background OsSAPK10 The gene was knocked out, and the knockout target site was 5'-TGTCCCGCACCAGCTCGTAC-3' to obtain ossapk10 Mutants. Among them, mutants ossapk10-1 For rice OsSAPK10 A T base was inserted at position 69 of the gene CDS, resulting in a frameshift mutation after amino acid position 23 and premature termination after amino acid position 141. ossapk10-2 For rice OsSAPK10 An A base was inserted at position 69 of the gene CDS, resulting in premature termination after amino acid position 22.

[0020] Investigation of main agronomic traits: wild type ZH11 and ossapk10 The mutants were soaked, germinated and sown at the same time, and transplanted into the same field at the same time and managed normally. Their main agronomic traits were then investigated.

[0021] (2) Results and analysis See also Figure 1 , Figure 1 A is ossapk10-1 and ossapk10-2 genotype of the mutant; Figure 1 B is wild-type ZH11, and ossapk10-1 and ossapk10-2 Plant morphology of mutants, bar = 10 cm; Figure 1 C is wild-type ZH11, and ossapk10-1 and ossapk10-2 Histogram of plant height of mutants; Figure 1 D is wild-type ZH11, and ossapk10-1 and ossapk10-2 The ear morphology of the mutant, bar = 1 cm; Figure 1 E is wild-type ZH11, and ossapk10-1 and ossapk10-2 Histogram of spike length of mutants; Figure 1 F is wild-type ZH11, and ossapk10-1 and ossapk10-2 The mutant grain length was photographed, bar = 0.5 cm; Figure 1 G is wild-type ZH11, and ossapk10-1 and ossapk10-2 Histogram of grain length of mutants; Figure 1 H is wild-type ZH11, and ossapk10-1 ossapk10-2 The grain width of the mutant was photographed, bar = 0.5 cm; Figure 1 I is wild-type ZH11, and ossapk10-1 and ossapk10-2 Mutant grain width histogram. Figure 1 The results showed that knockout rice OsSAPK10 The gene does not affect the normal growth and development of rice.

[0022] Embodiment 3: Knockout rice OsSAPK10 Gene reduces rice's resistance to rice blast: (1) Materials and methods Wild-type ZH11, and ossapk10-1 and ossapk10-2 Planting of mutants: After soaking and germinating, the seeds are planted in small flower pots filled with seedling soil and placed in an artificial climate chamber for cultivation; Culture of rice blast spores: Place the filter paper with rice blast strain Guy11 spores on solid complete culture medium and culture it in the dark at 28℃ for 8 days. Then use a sterile blade to take the culture medium with rice blast mycelium and transfer it to rice bran culture medium. After culturing in the dark at 28℃ for 10 days, scrape off the mycelium and culture it in a light incubator at 26℃ for 3 days. The rice blast spores can be used for subsequent inoculation experiments. Spray inoculation method: suspend the spores of rice blast fungus in 0.02% Tween-20 solution and filter them through double-layer lens paper, then adjust the spore concentration to 1×10 5 Then, use a sprayer to evenly spray the suspension of rice blast fungus spores on the leaves of rice seedlings grown for 15 to 20 days, and place them in a dark environment at 26°C and high humidity for 24 hours. Then continue to culture at 26°C, 12 hours of light / 12 hours of darkness, and keep the environment high and humid during the period. Observe the disease phenotype after 3 to 5 days. Punch hole inoculation method: The preparation of rice blast fungus spore solution is the same as above. When the seedlings to be inoculated grow to about 40 days, use a mouse ear puncher to make wounds at 1 / 3 of the distance from the tip of the new leaf and the second leaf, then add 10μL of rice blast fungus spore solution to the wound, and seal the wound and spore solution with tape. After dark treatment at 26℃ high humidity environment for 24 hours, continue to culture at 26℃, 12h light / 12h dark conditions, keep the environment high humidity during the period, and observe the disease phenotype after 8-10 days; OsSAPK10Analysis of gene expression after rice blast infection: ZH11 seedlings grown for 20 days were spray-inoculated with rice blast fungus, and samples were taken at 0h, 24h, and 48h after inoculation. Samples treated with sterile water were used as controls. Total RNA was extracted from the samples using the TRIzol method, and DNA chains were synthesized using a reverse transcription kit. OsSAPK10 Quantitative PCR analysis was performed using gene-specific primers and a quantitative PCR kit; DAB staining analysis of H2O2: Wild-type ZH11 and ossapk10 The mutants were spray-inoculated with rice blast fungus. Two days after inoculation, the inoculated leaves were immersed in the prepared 1 mg / mL DAB solution, and then vacuumed in a vacuum pump for 3 minutes in the dark (air pressure was 0.8 MPa), and then left to stand for 30 minutes. After that, the sample was wrapped with tin foil to avoid light and placed on a horizontal shaker at low speed and room temperature overnight. The next day, the DAB liquid in the sample was poured out, anhydrous ethanol was added, and decolorization was carried out in a 95°C water bath. During this period, the decolorization solution was changed until the leaf tissue was transparent. After complete decolorization, it was scanned with a scanner and the accumulation of hydrogen peroxide was observed.

[0023] Determination of relative rice blast fungus biomass: Total DNA of diseased leaves was extracted, and then quantitative PCR analysis of DNA level was performed using a quantitative PCR kit. The relative rice blast fungus biomass was the Pot2 The amount of gene relative to rice ubiquitin The amount of genes.

[0024] (2) Results and analysis See also Figure 2 , Figure 2 A is OsSAPK10 Expression analysis in wild-type ZH11 samples at different time points after treatment with rice blast fungus and sterile water; Figure 2 B is wild type ZH11 and ossapk10 Leaf phenotype of the mutant 4 days after spray inoculation, bar = 1 cm; Figure 2 C is wild-type ZH11 and ossapk10 The number of lesions on the diseased leaves of mutants was counted 4 days after spray inoculation; Figure 2 D is wild type ZH11 and ossapk10 The diseased leaf phenotype of the mutant 8 days after punching and inoculation, bar=1cm; Figure 2 E is wild-type ZH11 and ossapk10 The lesion area of ​​the mutant leaves was counted 8 days after the mutant was inoculated with fungi; Figure 2 F is wild type ZH11 and ossapk10 Statistics of the relative growth of rice blast fungi in diseased leaves 8 days after the mutants were inoculated with fungi; Figure 2 G is the wild type ZH11 and ossapk10DAB staining analysis of H2O2 in mutant leaves, bar=1cm. The results showed that OsSAPK10 The expression of the gene is induced by rice blast infection ( Figure 2 A); After spray inoculation, compared with the wild type, ossapk10 The mutant had more lesions on its leaves ( Figure 2 B and Figure 2 C); After punching and inoculation, compared with the wild type, ossapk10 The mutant had a larger lesion area and more rice blast fungus growth ( Figure 2 D to Figure 2 F); After spray inoculation with rice blast fungus, ossapk10 The mutant accumulated less H2O2 than the wild type ( Figure 2 G); the above results show that knockout OsSAPK10 The gene reduced rice's resistance to rice blast.

[0025] Embodiment 4: Overexpression rice OsSAPK10 Gene improves rice blast resistance: (1) Materials and methods OsSAPK10 Construction of overexpression plants: By constructing 35S promoter-driven OsSAPK10-HA Overexpression vector was transformed into ZH11 to obtain OsSAPK10 Overexpressing plants.

[0026] OsSAPK10 Detection of OsSAPK10-HA protein in overexpressing plants: Extraction of T2 generation OsSAPK10 Total proteins in leaves of overexpressing plants were then detected by immunoblotting using HA antibody for OsSAPK10-HA protein.

[0027] The methods for punching and inoculating rice blast fungus and detecting relative rice blast fungus biomass are the same as those in Example 3.

[0028] (2) Results and analysis See also Figure 3 , Figure 3 A is OsSAPK10 Detection of OsSAPK10-HA protein in overexpressing plants; Figure 3 B is wild type ZH11 and OsSAPK10 Diseased leaf phenotype of overexpressing plants 8 days after punching and inoculation, bar = 1 cm; Figure 3 C is wild-type ZH11 and OsSAPK10 Statistics of lesion lengths on diseased leaves of overexpression plants 8 days after punching and inoculation; Figure 3 D is wild type ZH11 and OsSAPK10The relative growth of rice blast fungi in the diseased leaves of overexpressing plants 8 days after punching and inoculation. The results showed that OsSAPK10-HA protein OsSAPK10 Overexpression plants can express normally ( Figure 3 A); After punching and inoculation with rice blast fungus, compared with the wild type, OsSAPK10 The lesions on diseased leaves of overexpressing plants were shorter ( Figure 3 B and Figure 3 C, and the growth of rice blast fungus is less ( Figure 3 D); The above results indicate that overexpression OsSAPK10 The results showed that the rice had better resistance to rice blast.

[0029] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A rice blast resistance-related gene OsSAPK10 , characterized in that: The rice blast resistance related gene OsSAPK10 The nucleotide sequence is shown in SEQ ID NO:

1.

2. A rice blast resistance-related gene according to claim 1 OsSAPK10 The encoded protein is characterized by: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. Use of the rice blast resistance-related gene OsSAPK10 as claimed in claim 1 in regulating rice resistance to rice blast.

4. The use according to claim 3, characterized in that: The regulation is positive regulation.

5. The use according to claim 4, characterized in that: The positive regulation is: overexpression of rice blast resistance related genes OsSAPK10 Improving rice resistance to rice blast or knocking out genes related to rice blast resistance OsSAPK10 Reduce rice resistance to rice blast.

6. The rice blast resistance-related gene according to claim 1 OsSAPK10 Application in breeding rice blast-resistant varieties.

7. A method for regulating rice resistance to rice blast fungus, characterized in that: When it is necessary to reduce the resistance of rice to rice blast fungus, OsSAPK10 Gene knockout; when it is necessary to improve the resistance of rice to rice blast fungus, OsSAPK10 Gene overexpression; OsSAPK10 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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