Rice blast resistance related gene osrlk6 and application thereof

Abstract

The present application relates to the technical field of plant genetic engineering, and in particular to a cloning, function verification and application of a rice blast resistance related gene OsRLK6 mediation, the negative regulation of OsRLK6 gene in rice blast resistance reaction is first discovered and proved, the function loss of OsRLK6 increases the resistance of rice to rice blast, and the expression level of OsRLK6 is improved, which can significantly reduce the resistance of rice to rice blast, and the function loss of OsRLK6 does not affect the agronomic traits of rice, and the present application can be applied to genetic improvement of related agricultural crops such as rice.

Description

Technical Field

[0001] This invention relates to the field of rice disease resistance breeding, specifically to a rice blast resistance-related gene. OsRLK6 And its applications. Background Technology

[0002] Rice is one of the world's most important food crops. Rice blast, caused by the rice blast fungus, is a global disease that has been on the rise in recent years. Rice blast not only causes significant yield reductions (10%-35%), and in severe cases, complete crop failure, but also affects rice quality. Practice has proven that breeding and promoting disease-resistant varieties is one of the most effective methods for controlling crop diseases, and understanding plant immune mechanisms is the theoretical basis for crop disease-resistant breeding. Research on the mechanisms by which plants utilize key regulatory proteins of the immune system to resist pathogen invasion will deepen our understanding of the interaction mechanisms between pathogens and plant hosts, providing important guidance and targets for breeders to develop novel biological breeding strategies, and accelerating the process of molecular breeding for crop disease resistance.

[0003] The plant's innate immune system is composed of a vast and complex regulatory system. In the process of interacting with pathogens, plants have evolved two levels of immune systems: PTI (PAMP / MAMPs-triggered immunity) and ETI (effector-triggered immunity) to recognize and defend against pathogen infection. The PTI response is broad-spectrum and persistent, a primary immune response induced by pattern recognition receptors (PRRs) on the host surface recognizing pathogen-associated molecular patterns (PAMPs / MAMPs). The ETI response is specialized, a secondary immune response triggered by the nucleotide-binding (leucine-rich repeat) receptor (NLR) within plant cells, directly or indirectly recognizing effector proteins secreted by pathogens, thereby effectively controlling diseases. Recent studies have shown that PTI and ETI can manipulate plant defense responses by synergistically regulating core components of immune signaling pathways.

[0004] Preliminary findings OsRLK6 The gene was upregulated after being induced by rice blast fungus, and knocking out this gene significantly improved rice blast resistance, indicating that... OsRLK6 Genes negatively regulate the resistance response of rice to rice blast. Summary of the Invention

[0005] To improve the genetic resistance of rice and other related agricultural crops, the interaction expression profiles of rice and rice blast fungus were analyzed and screened, revealing the expression of RLK-type receptor kinases induced by rice blast fungus infection. OsRLK6 Functional identification was conducted, and the discovery and proof were made for the first time. OsRLK6 The negative regulatory role of genes in rice resistance to rice blast was statistically revealed through field experiments. OsRLK6 The loss of function does not affect the agronomic traits of rice.

[0006] The primary objective of this invention is to provide a gene related to rice blast resistance. OsRLK6 , OsRLK6 The CDS sequence of the gene is shown in SEQ ID NO.1, and the full length of the CDS sequence is 2793 bp.

[0007] Another object of the present invention is to provide a method based on rice blast resistance-related genes. OsRLK6 Encoded protein, OsRLK6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, encoding 930 amino acids.

[0008] Another objective of this invention is to provide a rice blast resistance-related gene. OsRLK6 Or a rice blast resistance-related gene OsRLK6 Application of encoded proteins in improving plant disease resistance.

[0009] Another objective of this invention is to provide a rice blast resistance-related gene. OsRLK6 Or a rice blast resistance-related gene OsRLK6 Application of encoded proteins in improving rice blast resistance.

[0010] The beneficial effects of this invention are as follows:

[0011] (1) This invention is the first to discover OsRLK6 The negative regulatory effect of genes on rice resistance to rice blast was demonstrated by the identification method of spray inoculation. OsRLK6 Gene deletion improves rice's resistance to rice blast; through OsRLK6 Overexpression experiments proved OsRLK6 Gene overexpression reduces rice's resistance to rice blast, by... OsRLK6 The function of genes was investigated and confirmed. OsRLK6 Genes play an important role in rice resistance to rice blast, indicating that... OsRLK6 It is an excellent candidate gene for creating transgenic crops resistant to rice blast, and has important theoretical value and broad application prospects.

[0012] (2) The present invention also involves the following:OsRLK6 The main agronomic traits of rice after gene mutation, such as plant height, panicle length, number of effective tillers, grain length, and grain width, were investigated, showing that... OsRLK6 Gene deletion does not affect rice yield and quality. OsRLK6 Genes are relatively conserved in rice, and OsRLK6 To negatively regulate rice blast resistance, gene editing or mutation methods can be used to... OsRLK6 Gene editing or site-directed mutation can create new rice materials resistant to rice blast, thus providing excellent intermediate materials for rice disease resistance breeding. Attached Figure Description

[0013] Figure 1 In Embodiment 1 of the present invention OsRLK6 Schematic diagram of phenotypic identification and detection of mutant rice blast fungus;

[0014] Figure 2 In Embodiment 2 of the present invention OsRLK6 Schematic diagram of rice blast resistance identification in overexpressing plants;

[0015] Figure 3 In Embodiment 3 of the present invention OsRLK6 A schematic diagram of subcellular localization;

[0016] Figure 4 In Embodiment 4 of the present invention OsRLK6 A schematic diagram of the conservative analysis. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the present invention; the present invention focuses on the rice variety ZH11 and the rice blast fungus Guy11.

[0018] Example 1

[0019] OsRLK6 Identification of disease resistance phenotypes

[0020] (1) Materials and Methods

[0021] Rice cultivation method: Remove the husks from the rice plants and soak them in 75% ethanol for 2 minutes. Discard the ethanol and continue soaking in 50% pasteurized solution for 40 minutes, inverting the plants several times during this process. Perform this operation in a clean bench. Discard the pasteurized solution and rinse the seeds 5-6 times with sterile water. Discard the sterile water and evenly spread the seeds on MS medium, generally 15-20 seeds / plate. Cultivate the seeds in a greenhouse at 28℃ for one week before transplanting. Place the seedlings in a long-day incubator and then move them outdoors for another week to continue cultivation, ensuring suitable light and moisture during this period.

[0022] Preparation of *Magnapordica oryzae* spore suspension: Place preserved *Magnapordica oryzae* filter paper discs in CM solid medium and incubate at 28℃ to allow *Magnapordica oryzae* to grow. Once the *Magnapordica oryzae* growth area reaches more than half the area of ​​the culture dish, cut the medium into small pieces using a sterile scalpel in a laminar flow hood and transfer to rice bran medium for further cultivation. After the *Magnapordica oryzae* hyphae cover more than 3 / 4 of the rice bran medium, gently scrape off the surface hyphae using a sterile scraper in a laminar flow hood. Continue cultivation in a light incubator for 3-5 days to promote sporulation. Prepare a 0.2% Tween-20 solution, pour it into the rice bran medium, and gently scrape the rice bran medium with a scraper to wash off the spores. Filter the spores and count them using a hemocytometer. The spore concentration should reach 1×10⁻⁶. 5 pcs / ml;

[0023] Spray inoculation method: Prepare a solution with a concentration of 1×10 5 The bacterial solution of 1000 cells / ml was evenly sprayed onto rice plants that had been growing for 2-3 weeks, ensuring that all rice leaves were covered with the bacterial solution. After 24 hours of dark treatment in a high-humidity environment, the plants were cultured under 12 hours of light and 12 hours of darkness, while maintaining high humidity. The disease phenotype was observed after 3-5 days.

[0024] Rice blast fungus biomass detection: Equal amounts of leaves from mutant and wild-type plants with consistent disease trends were selected, and rice genomic DNA was extracted and its concentration was determined. Ubiquitin and rice blast fungus MoPot2 As an internal reference gene, the growth of rice blast fungus in rice leaves was detected by DNA-level qPCR.

[0025] (2) Results and Analysis

[0026] See Figure 1 , Figure 1 a) is to use the spray inoculation method for ZH1. osrlk6-1 and o srlk6-2 Inoculation with *Magnapordica oryzae* strain Guy11 was performed, and observations and photographs were taken 3 days after inoculation. Results showed that compared to ZH1, osrlk6-1 and o srlk6-2 All showed increased resistance to rice blast fungus, reduced lesion area, and Bar=1 cm. Figure 1 b is the detection of ZH11 by qPCR using the spray inoculation method. osrlk6-1 and o srlk6-2 The biomass of *Oryza sativa* in diseased leaves showed that the biomass of *Oryza sativa* in the mutant was significantly lower than that in the wild type. In summary, this indicates... OsRLK6 Gene mutations enhance rice's resistance to rice blast.

[0027] Example 2

[0028] OsRLK6 Gene overexpression reduces rice resistance to rice blast.

[0029] (1) Materials and Methods

[0030] OsRLK6 Detection of gene overexpression levels in plants: ZH11, OE-OsRLK6-8 , OE-OsRLK6- 12 RNA was extracted from three plants from each group, reverse transcribed into cDNA, and the expression level of OsRLK6 in the overexpressing plants was detected using real-time quantitative PCR. OsRLK6 The gene overexpression level was detected in materials with a growth cycle of 1 month.

[0031] OsRLK6 Identification of rice blast resistance in overexpressing gene plants: wild-type rice material ZH11, overexpressing gene... OE-OsRLK6-8 , OE-OsRLK6-12 The rice cultivation and inoculation methods are the same as in Example 1. The growth cycle of the inoculated material is 2-3 weeks. The disease phenotype is observed 3-5 days after spray inoculation, and quantitative analysis of rice blast bacteria biomass is performed.

[0032] (2) Results and Analysis

[0033] Compared to the wild type, overexpression of the plant OsRLK6 Gene expression levels were significantly upregulated, and the overexpressed material met the experimental requirements.

[0034] right OsRLK6 Gene-overexpressing plants were inoculated with Guy11 rice blast fungus using a spray method. See [link to relevant documentation]. Figure 2 a, the results showed that the lesion area of ​​ZH11 was smaller than OE-OsRLK6 This indicates overexpression OE-OsRLK6 It can reduce the resistance of rice to rice blast, Bar=1cm; qPCR detection of ZH11, OE-OsRLK6-8 , OE-OsRLK6-12 Rice blast biomass in diseased leaves, see Figure 2 b. The results showed that the biomass of rice blast fungus in the overexpression lines was higher than that in the wild-type ZH11.

[0035] In summary, this demonstrates the effect of overexpression. OsRLK6 The gene can reduce rice's resistance to rice blast, and the above phenotype further proves this. OsRLK6 Negative gene regulation of rice resistance to rice blast.

[0036] Example 3

[0037] OsRLK6 Subcellular localization

[0038] (1) Materials and Methods

[0039] The 35S:OsRLK6-EGFP vector was constructed and transformed into Agrobacterium strain GV3101. Two days after injection into tobacco, the bacteria were observed in tobacco leaves using laser confocal microscopy. OsRLK6 Cellular location of genes.

[0040] (2) Results and Analysis

[0041] The expression of OsRLK6-GFP in tobacco cells was observed using laser confocal microscopy, such as... Figure 3 As shown, OsRLK6-GFP is mainly expressed on the cell membrane, Bar=20 μm.

[0042] Example 4

[0043] Osnac29 Statistical analysis and conservation analysis of agronomic traits of mutants

[0044] (1) Materials and Methods

[0045] Wild-type ZH11 and osrlk6 Fifty seeds were sown from each mutant strain for germination. Three days after germination, seedlings were raised in paddy fields. After 25 to 30 days of growth, the seedlings were transplanted into paddy fields, maintaining consistent row and plant spacing. Plants were photographed during the heading stage, and then, at maturity, ZH11 strains with uniform growth were selected. osrlk6-1 and osrlk6-2 Twenty plants from each group were used to collect statistics on agronomic traits, including plant height, panicle length, number of effective tillers, average number of grains per panicle, grain length, and grain width.

[0046] A preliminary analysis of the conservation of LOC_Os06g38780 was conducted based on a pan-genome analysis database of 33 resequencing rice genetic diversity materials (https: / / ricerc.sicau.edu.cn / RiceRC / tools / blastBefore?seq=). Figure 4 As shown, OsRLK6 The gene sequence is consistent with the sequences of 33 resequencing rice varieties, therefore OsRLK6 It is relatively conserved in the rice genome.

[0047] (2) Results and Analysis

[0048] Statistical results show osrlk6 The mutant and ZH11 showed no difference from the wild type in major agronomic traits, including plant height, ear length, number of effective tillers, grain length, and grain width (Table 1), indicating that... OsRLK6 The loss of gene function does not affect the growth and development of rice.

[0049] Table 1 osrlk6Statistical survey of agronomic traits of mutants

[0050]

[0051] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A rice blast resistance-related gene, OsRLK6, for negative regulation of rice blast resistance, characterized in that: The CDS sequence of the OsRLK6 gene is shown in SEQ ID NO.1, with a full length of 2793 bp.

2. A rice blast resistance-related gene according to claim 1 OsRLK6 Used for negative regulation of rice blast resistance, characterized by: OsRLK6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

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