Rice blast and bacterial blight resistance related gene OsRLK3 of rice and application of rice blast and bacterial blight resistance related gene OsRLK3

Abstract

The invention relates to the technical field of rice disease-resistant breeding, in particular to a rice blast and bacterial leaf blight resistant related gene OsRLK3 of rice and application of the rice blast and bacterial leaf blight resistant related gene OsRLK3. The CDS sequence of the OsRLK3 gene is 3885 bp, and 1294 amino acids are encoded; the invention discovers and proves that the OsRLK3 gene positively regulates the resistance of rice to rice blast and bacterial leaf blight for the first time, the resistance of rice to rice blast and bacterial leaf blight can be reduced by knocking out the OsRLK3 gene through a CRISPR / Cas9 method, and the resistance of rice to rice blast and bacterial leaf blight can be remarkably enhanced by improving the expression level of the OsRLK3 gene; in addition, knockout and overexpression of the OsRLK3 gene do not affect normal growth and development of rice. Therefore, the invention can be applied to genetic improvement for improving resistance and yield of related agricultural crops such as rice and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of rice disease resistance breeding, and in particular to a rice blast and bacterial blight resistance-related gene OsRLK3 and an application thereof. Background Art

[0002] Plant innate immunity is primarily composed of two layers of defense. The first layer is composed of surface pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). Since PAMPs are not unique to pathogenic microorganisms but are widely present in microorganisms, they are also called microbe-associated molecular patterns (MAMPs). They activate immune signaling in plants and induce the expression of defense-related genes to limit the invasion of pathogens. This is the first layer of PTI response (PAMP-triggered immunity), also known as basal resistance. To further infect host plants, pathogens evolve effector proteins (effectors) to suppress the host plant's PTI response, making the plant susceptible to disease (effector-triggered susceptibility, ETS). In order to further inhibit the invasion of pathogens, plants have evolved resistance proteins (R), which are intracellular immune receptors. These immune receptors recognize effector proteins secreted by pathogens in different ways, thereby activating the immune response. This immune response is called ETI response (effector-triggered immunity).

[0003] In PTI, PAMPs / MAMPAs are primarily recognized by pattern recognition receptors (PRRs) on the host cell membrane. Through the PRR complex, they activate downstream signaling pathways such as MAPKs (Mitogen-Activated Protein Kinases) or calcium-dependent protein kinases (CDPKs). Ultimately, these signals are transmitted to the nucleus, activating downstream immune signaling and triggering the plant's disease resistance response, thereby resisting pathogen invasion. PRRs are primarily divided into two categories: RLKs (receptor-like kinases) and RLPs (receptor-like proteins). RLKs are primarily composed of an extracellular domain, a transmembrane domain, and an intracellular kinase domain. Depending on the structure of the extracellular domain, RLKs can be divided into LRR (Leucine rich repeat) type, LysM type, and Lectin type, with LRR type receptors being the most common. There are many RLK members in plants. There are approximately 1,131 RLK proteins in rice, but the functions of most of them are unknown. Summary of the Invention

[0004] The present invention aims to provide a rice gene, OsRLK3, associated with resistance to rice blast and bacterial blight, and its application. The inventors previously conducted transcriptome sequencing analysis on rice infected with the rice blast fungus and discovered a new receptor-like kinase gene, named OsRLK3, whose expression is induced by infection with the fungus. Knockout of this gene using CRISPR / Cas9 technology significantly reduced rice resistance to blast and bacterial blight, while increasing its expression significantly enhanced resistance to these diseases. Furthermore, neither knockout nor overexpression of this gene affected rice growth and development. Therefore, the OsRLK3 gene has a practical application in improving resistance to rice blast and bacterial blight. The OsRLK3 gene, associated with resistance to rice blast and bacterial blight, provided by the present invention can be used to genetically improve resistance in rice and other related agricultural crops.

[0005] The technical solution adopted in the present invention is as follows: A rice blast and bacterial blight resistance-related gene OsRLK3, whose CDS sequence is shown in SEQ ID NO.1.

[0006] The amino acid sequence of the protein encoded by the rice blast and bacterial blight resistance-related gene OsRLK3 is shown in SEQ ID NO.2.

[0007] Use of the above-mentioned rice blast and bacterial blight resistance-related gene OsRLK3 or the above-mentioned protein in regulating plant disease resistance; Furthermore, the application is to improve the resistance of rice to rice blast and bacterial blight by increasing the expression level of OsRLK3, a gene related to rice blast and bacterial blight resistance; Furthermore, the application can also be: knocking out the rice blast and bacterial blight resistance-related gene OsRLK3, thereby reducing the rice's resistance to rice blast and bacterial blight.

[0008] Application of the above rice blast and bacterial blight resistance-related gene OsRLK3 or the above protein in rice genetic breeding; Furthermore, the application is to improve the resistance of rice to rice blast and bacterial blight by increasing the expression level of OsRLK3, a gene related to rice blast and bacterial blight resistance; Furthermore, the application can also be: knocking out the rice blast and bacterial blight resistance-related gene OsRLK3, thereby reducing the rice's resistance to rice blast and bacterial blight.

[0009] The beneficial effects of the present invention are: 1. To improve the genetic resistance of rice and other related agricultural crops, the present invention discovered a new receptor-like kinase gene, OsRLK3, whose expression is induced by rice blast infection through transcriptome sequencing analysis of rice infected with the rice blast fungus. Functional characterization of the gene revealed and demonstrated for the first time the positive regulatory role of OsRLK3 in rice resistance to rice blast and bacterial blight. The present invention demonstrated that deletion of the OsRLK3 gene reduced rice resistance to these diseases, while overexpression of the OsRLK3 gene enhanced their resistance, demonstrating that OsRLK3 positively regulates rice resistance to these diseases. These results indicate that OsRLK3 is an excellent candidate gene for creating transgenic rice with enhanced resistance to rice blast and bacterial blight and for crop molecular design, and has important theoretical value and broad application prospects. 2. The present invention knocks out the OsRLK3 gene in the rice variety Zhonghua 11 (ZH11) through genetic engineering methods to reduce the rice's resistance to rice blast. Compared with the wild type, the osrlk3 mutant has more rice blast lesions and rice blast fungus growth, and longer bacterial blight lesions. Increasing the expression level of the OsRLK3 gene can significantly enhance the rice's resistance to rice blast and bacterial blight, as manifested by less rice blast lesions and rice blast fungus growth, and shorter bacterial blight lesions. The rice blast-resistance gene OsRLK3 provided by the present invention can be used for genetic improvement to enhance the resistance of rice and other related agricultural crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the expression pattern of the OsRLK3 gene after infection with rice blast fungus.

[0011] Figure 2 Diagram showing the identification of resistance to rice blast and bacterial blight in osrlk3 knockout mutants and OsRLK3 overexpressing plants.

[0012] Figure 3 Figure 2 shows the growth and development phenotypes of OsRLK3 knockout mutants and OsRLK3 overexpressing plants. DETAILED DESCRIPTION

[0013] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples, but the present invention is not limited thereto. In the present invention, rice variety ZH11, rice blast pathogen Guy11 and rice bacterial blight pathogen PXO99 are used as research objects.

[0014] Example 1 This example provides a rice blast and bacterial blight resistance-related gene OsRLK3, whose CDS sequence is shown in SEQ ID NO. 1 and has a full length of 3885 bp. Also provided is a protein encoded by the rice blast and bacterial blight resistance-related gene OsRLK3, whose amino acid sequence is shown in SEQ ID NO. 2 and has a full length of 1294 amino acids.

[0015] Example 2 The OsRLK3 gene is induced to express when infected by rice blast fungus; (1) Materials and methods Rice seedling soil planting: Prepare a 90 cm diameter sterile petri dish and place a piece of absorbent filter paper. Place shelled rice variety ZH11 seeds in the dish and add sterile water to cover the seeds. Soak the seeds at 37°C for approximately 2 to 3 days until they turn white. Then, grow them in a light incubator (28°C, 16 hours of light, 8 hours of darkness, 70% humidity) for 5 to 7 days. Mix nutrient soil and vermiculite in a 1:1 ratio to make soil. Transplant the rice seedlings into the mixed soil and continue growing. Culture of rice blast fungus: Inoculate laboratory-stored rice blast fungus Guy11 onto CM medium and culture in the dark at 28°C for 3-7 days. Use a sterile scalpel to scrape the fungus from the CM medium and transfer it to rice bran medium. Culture in the dark at 28°C for 7-10 days. After the hyphae have grown all over the medium, gently scrape off the hyphae on the surface of the medium with a glass slide. Transfer the culture to a 26°C light incubator with alternating cycles of 14 hours light and 10 hours dark to stimulate sporulation. A large number of spores will be produced after 3-7 days. Spray inoculation method: suspend the bacteria in 0.02% Tween-20 solution to a concentration of 1×10 5Spray the spores of the rice blast pathogen Guy11 at a concentration of 100 μg / ml evenly on the leaves of 2-3 week-old rice seedlings to ensure that all the rice leaves are covered with the spores. After 24 hours of dark treatment at 26°C and high humidity, continue to incubate at 26°C with 12 hours of light and 12 hours of darkness, maintaining a high humidity during this period. Observe the disease phenotype after 3-5 days. Transcriptome sequencing analysis: Three-week-old wild-type ZH11 rice seedlings were spray-inoculated with the rice blast pathogen Guy11. Rice leaves were sampled at 0 and 24 hours after inoculation. Rice leaf samples sprayed with sterile water served as the control (CK), and transcriptome analysis was performed on these samples. Quantitative detection of OsRLK3 gene expression after rice blast infection: Three-week-old wild-type ZH11 rice seedlings were spray-inoculated with the rice blast pathogen Guy11. Rice leaves were sampled at 0, 24, 36, 48, and 72 hours. Rice leaf samples sprayed with sterile water served as a control (CK). Total RNA was extracted from the samples and reverse-transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect the expression of the OsRLK3 gene, whose CDS sequence is shown in SEQ ID NO. 1, in each sample using Ubiquitin as an internal reference gene.

[0016] (2) Results and analysis See also Figure 1 , Figure 1 A shows the rice variety ZH11 inoculated with the rice blast pathogen Guy11 by spray inoculation. Samples were taken at 0 h and 24 h after inoculation, and transcriptome analysis was performed on the samples. The transcriptome analysis revealed that the OsRLK3 gene was significantly induced after inoculation, while there was no significant difference in the control (CK) treatment. Figure 1 B shows that the rice variety ZH11 was inoculated with the rice blast fungus Guy11 by spray inoculation. Samples were taken at 0h and 24h, 36h, 48h and 72h after inoculation, and then the expression of the OsRLK3 gene in these samples was detected by quantitative PCR. The results were consistent with the transcriptome analysis, and the expression of the OsRLK3 gene was significantly induced after inoculation. The above results suggest that the OsRLK3 gene may be involved in the disease resistance immune response of rice.

[0017] Example 3 The blast and bacterial blight resistance of the osrlk3 mutant was significantly weakened compared with the wild type, while the blast and bacterial blight resistance of the OsRLK3 overexpressing plants was significantly enhanced compared with the wild type. (1) Materials and methods Obtaining osrlk3 knockout mutants: The OsRLK3 gene was knocked out in the rice cultivar ZH11 using the Crisper / Cas9 method to generate osrlk3 mutants. Two different 20-nt sequences (gRNAs-OsRLK3-1: 5'-GCAATAGACCTCGCATCTGA-3'; gRNAs-OsRLK3-2: 5'-CCTTTGCTTCAAGTCTCAGC-3') were selected as target sites for Cas9 cleavage. After generating knockout lines, the target sites were sequenced and analyzed. The results showed that the knockout line osrlk3-1 had a T deletion at the target site, while the knockout line osrlk3-2 had a C deletion at the target site.

[0018] Construction and detection of OsRLK3 overexpressing plants: The full-length CDS of the OsRLK3 gene was ligated downstream of the 35S promoter of the pCAMBIA-1300 vector to obtain an OsRLK3 overexpression vector; the constructed OsRLK3 overexpression vector was transformed into the wild-type rice variety ZH11 through Agrobacterium-mediated genetic transformation; the expression levels of the OsRLK3 gene in the different T0 generation strains obtained were detected by fluorescence quantitative PCR; the progeny of two strains (OE-OsRLK3-4 and OE-OsRLK3-9) with the highest T0 generation OsRLK3 gene expression levels and significantly higher than those of the wild-type rice variety ZH11 were used to identify resistance to rice blast and bacterial blight.

[0019] The rice culture and the spray inoculation method for rice blast fungus were the same as those in Example 2. The growth period of the spray-inoculated material was 3 weeks, and the disease phenotype was observed 3-4 days after the spray inoculation. Determination of rice blast fungus biomass: Total DNA was extracted from diseased leaves, and then quantitative PCR analysis was performed at the DNA level using a quantitative PCR kit. The relative rice blast fungus biomass was calculated as the amount of the Pot2 gene in rice blast fungus relative to the amount of the ubiquitin gene in rice. Leaf cutting method for inoculating rice bacterial blight pathogen: soak the rice seeds and germinate them in the field, then plant them in the field 25 days later. When the rice materials grow to the tillering stage, use scissors to dip the OD 600 The culture medium of bacterial blight pathogen of rice was 1%, and then the leaves were cut 1 cm away from the tip of the leaves for inoculation. The length of the bacterial blight spots was measured 14 days after inoculation to determine the extent of the disease.

[0020] (2) Results and analysis See also Figure 2 , Figure 2A-2C shows the osrlk3 knockout mutant and wild-type ZH11 inoculated with the rice blast pathogen Guy11 by spray inoculation. Four days after inoculation, phenotypic observations and photos were taken (A), lesion counts (B), and the biomass of the rice blast pathogen was measured (C). Bar = 1 cm. Figure 2 D and 2E show the osrlk3 knockout mutant and wild type ZH11 inoculated with rice bacterial blight pathogen PXO99 using the leaf cutting method. 14 days after inoculation, the diseased leaves were cut off and photographed (D) and the lesion length was counted (E); Figure 2 Figures F-2H show OsRLK3-overexpressing plants and wild-type ZH11 plants inoculated with the rice blast pathogen Guy11 by spray inoculation. Four days after inoculation, phenotypes were observed and photographed (F), lesion numbers were counted (G), and blast biomass was measured (H). Bar = 1 cm. Figures I and J show OsRLK3-overexpressing plants and wild-type ZH11 plants inoculated with the rice bacterial blight pathogen PXO99 by leaf clipping. Fourteen days after inoculation, diseased leaves were cut and photographed (I) and lesion lengths were counted (J). from Figure 2 As can be seen from the figure, compared with the wild type, the osrlk3 knockout mutant has more blast lesions ( Figure 2 A and B), the growth of rice blast fungus is greater ( Figure 2 C), the length of bacterial blight spots is longer ( Figure 2 D and 2E), indicating that after the OsRLK3 gene knockout mutation, the resistance of rice to rice blast and bacterial blight was reduced; while the OsRLK3 overexpression plants had fewer rice blast lesions than the wild type ( Figure 2 F and 2G), the growth of rice blast fungus was less ( Figure 2 H), the length of bacterial blight lesions is shorter ( Figure 2 I and 2J), indicating that overexpression of the OsRLK3 gene improved rice resistance to rice blast and bacterial blight. These results indicate that the OsRLK3 gene positively regulates rice resistance to rice blast and bacterial blight.

[0021] Example 4 Knockout and overexpression of the OsRLK3 gene did not affect rice growth and development; (1) Materials and methods The osrlk3 knockout mutant, OsRLK3 overexpressing plants and wild-type rice variety ZH11 were sown and transplanted simultaneously in normal paddy fields. Agronomic traits were investigated and analyzed under normal growth conditions until the rice matured.

[0022] (2) Results and analysis See also Figure 3 and Table 1, Figure 3 A: The osrlk3 knockout mutant and wild-type ZH11 plants were photographed at maturity, bar = 15 cm; Figure 3 B shows the photos of OsRLK3-overexpressing plants and wild-type ZH11 plants at maturity, bar = 15 cm; Table 1 shows the statistical results of the main agronomic traits of osrlk3 knockout mutants, OsRLK3-overexpressing plants and wild-type ZH11 at maturity.

[0023] from Figure 3 As shown in Table 1, there were no significant differences in plant morphology and main agronomic traits between the osrlk3 knockout mutant and OsRLK3 overexpressing plants and the wild type ZH11.

[0024] Table 1 Statistics of main agronomic traits of osrlk3 knockout mutant, OsRLK3 overexpression plants and wild type ZH11 The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A gene related to rice resistance to rice blast and bacterial blight OsRLK3 , characterized in that: The CDS sequence is shown in SEQ ID NO.

1.

2. The rice blast and bacterial blight resistance-related gene according to claim 1 OsRLK3 The encoded protein is characterized by: The amino acid sequence is shown in SEQ ID NO.

2.

3. The rice blast and bacterial blight resistance-related gene according to claim 1 OsRLK3 Or the use of the protein according to claim 2 in regulating plant disease resistance.

4. The use according to claim 3, characterized in that: By improving the genes related to rice resistance to rice blast and bacterial blight OsRLK3 The expression level of the gene was increased to improve the resistance of rice to rice blast and bacterial blight.

5. The use according to claim 3, characterized in that: Knocking out genes related to rice blast and bacterial blight resistance OsRLK3 , reducing rice's resistance to rice blast and bacterial blight.

6. The rice blast and bacterial blight resistance-related gene according to claim 1 OsRLK3 Or the use of the protein according to claim 2 in rice genetic breeding.

7. The use according to claim 6, characterized in that: By improving the genes related to rice resistance to rice blast and bacterial blight OsRLK3 The expression level of the gene was increased to improve the resistance of rice to rice blast and bacterial blight.

8. The use according to claim 6, characterized in that: Knocking out genes related to rice blast and bacterial blight resistance OsRLK3 , reducing rice's resistance to rice blast and bacterial blight.

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