Rice tandem repeat paralogous gene pair GNK2_21 / 22 and application of the encoded protein thereof

Abstract

This invention discloses a pair of paralogous genes in rice with tandem repeats. GNK2_21 / 22 This invention, along with the application of its encoded proteins, belongs to the fields of plant biotechnology and crop disease resistance breeding technology. This invention is the first to disclose the application of rice... GNK2_21 and GNK2_22 The application of tandemly repeated paralogous genes in non-race-specific resistance to rice blast. CRISPR / Cas9 knockout experiments showed that single gene deletion had no significant effect on plant immunity, while simultaneous deletion of two genes led to a significant decrease in immunity, demonstrating that the two genes functionally form an immune-robust unit and synergistically maintain basic plant immune homeostasis through functional redundancy. The results of non-race-specific resistance validation showed that... GNK2_21 / 22 The mediated resistance is effective against different physiological races of rice blast fungus. This invention provides new molecular targets and breeding strategies for developing green immune inducers and breeding durable disease-resistant crop varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and crop disease resistance breeding technology, specifically relating to a pair of rice tandem repeat homologous genes. GNK2_21 / 22 And the applications of its encoded proteins. Background Technology

[0002] In agricultural production, plant disease control primarily relies on chemical pesticides and disease-resistant breeding using single resistance genes (R genes). However, both of these traditional strategies have inherent drawbacks. The extensive use of chemical pesticides not only causes environmental pollution but also leads to increasing pesticide resistance in pathogens, resulting in a year-on-year decline in control efficacy. On the other hand, resistance mediated by a single R gene is race-specific; pathogens can easily overcome this resistance by evolving virulence effectors, causing resistant varieties to lose their resistance after only a few years of promotion. Therefore, developing novel disease control strategies that can activate the plant's own immune system and possess broad-spectrum and durable resistance has become an important research direction in the field of green plant protection.

[0003] Pattern-triggered immunity (PTI) in plants is the first line of defense against pathogen invasion. PTIs activate a series of immune responses, including reactive oxygen species (ROS) bursts, defense gene expression, and callose deposition, by recognizing conserved molecular patterns (such as flagellin and chitin) in pathogens, thereby limiting pathogen infection. Unlike effector-triggered immunity (ETI) mediated by R genes, PTIs recognize conserved molecules essential for pathogen survival, thus exhibiting non-race-specificity and being less susceptible to evolutionary changes in pathogens. This makes them ideal targets for achieving durable, broad-spectrum resistance.

[0004] In recent years, plant apoplasts have received widespread attention as a frontline site for pathogen infection. Apoplasts contain various damage-associated molecular patterns (DAMPs). When plants are infected by pathogens or suffer mechanical damage, these endogenous signaling molecules are released or activated, triggering immune responses by recognizing their own receptors and enhancing the plant's disease resistance.

[0005] Current research indicates that GNK2 proteins derived from Ginkgo biloba possess direct in vitro antifungal activity, inhibiting fungal growth and classifying them as typical antifungal proteins. However, the existence of GNK2 homologs in gramineous crops, and whether their functional mechanisms are identical to those of Ginkgo GNK2, remains unclear. Of particular note is the widespread presence of tandem repeat paralogous gene pairs in the genomes of gramineous crops. These gene pairs often function synergistically through functional redundancy, yet no current research elucidates the function of GNK2 family tandem repeat pairs in plant immunity. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a rice tandem repeat homologous gene pair. GNK2_21 / 22 The invention also relates to the application of the encoded proteins. This invention has significant theoretical and practical value for revealing the synergistic effect of tandem repeat paralogous pairs in plant immunity, as well as for developing novel green immune inducers and cultivating durable disease-resistant crop varieties.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this invention provides a method to improve the efficiency of tandem repeat paralogous gene pairs in rice. GNK2_21 / 22 The expression is used in any of the following (1)-(4): (1) Improve rice resistance to non-race-specific rice blast; (2) Increase the reactive oxygen content in rice; (3) Develop rice varieties resistant to non-race-specific rice blast; (4) Preparation of plant immune inducers; The tandem repeat paralogous gene pair GNK2_21 / 22 Depend on GNK2_21 Genes and GNK2_22 Genome composition; The GNK2_21 The nucleotide sequence of the gene is shown in SEQ ID NO.1, as follows: SEQ ID NO: 1: ATGCAGCCGCGCTGTACGCTCATCCACGCCGTCGCCGTCGTCGCGGCGGCGGCGGCGGCCGCGCTGCTGCTTCCTCCGCTCGCCGCCGGCCAGCCGTGGCCAACGTGCGACACCAGCGCCGGCACGTACAAGGCCGGGAGCGCCTACGAGTCCAACCTCCGTGACCTCGCCGCCGCGCTCCGCGCGGACGCCGCCGCCTCGCCGTCGGCGCTCTTCGCCACGGGCAACCGCGGCGGCGCGCCCGACGCCGTCTACGGCCTCCTCCTCTGCCGCGGCGACCTCAGCGTCTCCGACTGCTTCGACTGCGGCACCCGCGTCCTCGCCGACGTCGGCCGCGTCTGCGGCGGCCGCCACGGCCGCGCCAAGGACGTGGCCCTCGTGTACAACCAGTGCTACGCCCGATTCTCCAACAAGGGCGACTTCCTCGCCGCCACCGACAACGCCGGCGGCGAGACGCTGCTCATCAGCGGCACCAACATCACCGGAGGCGCCGGCGTCGTGGCGGCGTACGACCGCGCGGTGACCGAGCTGCTCGCGGCCACCGTGCGGTACGCGGTGGAGGAGAACCCGGCGAGGCTGTTCGCCACGGGGCAGCGCGTGGGGGACGACGCCCGCGACCCGGGGTTCCGCAACATCTACTCCATGGCGCAGTGCTCGCCGGACCTGCCGCCGGCGTCGTGCCGCAGGTGCCTCGACGGCGTCTTGGCGCGGTGGTGGCAGGTGTTCCCGCTCAACGGCGAGGGCGCGAGGGTCGCCGGAGCGAGGTGCTACCTGAGGTCTGAGCTGGGCGTCGGCCCGTTCTACACCGGAGCTCCCATGGTGGTGCTGCGGGCGGACAAGGTCTAG。

[0008] The GNK2_22 nucleotide sequence of the gene is as shown in SEQ ID NO.3; SEQ ID NO: 3: ATGCAGCCGCGCTGTACGCTCATCCACGCCGTCGCCGTCGTCGCGGCGGCGGCGGCGGCCGCGCTGCTGCTTCCTCCGCTCGCCGCCGGCCAGCCGTGGCCGACGTGCGACACCAGCGCCGGCACGTACAAGGCCGGGAGCGCCTACGAGTCCAACCTCCGTGACCTCGCCGCCGCGCTCCGCGCGGGCGCCGCCGCCTCGCCGTCGGCGCTCTTCGCCACGGGCATCCGCGGCGGCGCGCCCGACGCCGTCTACGGCCTCCTCCTCTGCCGCGGCGACCTCAGCGTCTCCGACTGCTTCGACTGCGGCACCCGCGTCCTCGCCGACGTCGGCCGCGTCTGCGGCGGCCGCCACGGCCGCGCCAAGGACGTGGCCCTCGTGTACAACCAGTGCTACGCCCGATTCTCCAACAAGGGCGACTTCCTCGCCGCCACCGACAACGCCGGCGGCGAGACGCTGCTCATCAGCGGCACCAACATCACCGGAGGCGCCGGCGTCGTGGCGGCGTACGACCGCGCGGTGACCGAGCTGCTCGCGGCCACCGTGCGGTACGCGGTGGAGGAGAACCCGGCGAGGCTGTTCGCCACGGGGCAGCGCGTGGGGGACGACGCCCGCGACCCGGGGTTCCGCAACATCTACTCCATGGCGCAGTGCTCGCCGGACCTGCCGCCGGCGTCGTGCCGCAGGTGCCTCGACGGCGTCTTGGCGCGGTGGTGGCAGGTGTTCCCGCTCAACGGCGAGGGCGCGAGGGTCGCCGGCGCGAGGTGCTACCTGAGGTCTGAGCTGGGCGTCGGCCCGTTCTACACCGGAGCTCCCATGGTGGTGCTGCGGGCGGACAAGGTCTAG。

[0009] The increase in the expression of tandem repeat paralogous gene pairs in rice GNK2_21 / 22 refers to: increasing the expression of GNK2_21 and / or GNK2_22 genes in rice.

[0010] The term "tandem repeat paralogous gene pair" refers to two or more genes in a plant genome that are located on the same chromosome, produced through gene duplication, and are highly homologous in sequence and functionally related. These genes are arranged in tandem on the chromosome, adjacent to each other or spaced close together. In this invention, GNK2_21 Gene (SEQ ID NO.1) and GNK2_22 The gene (SEQ ID NO.3) is a typical pair of paralogous genes with tandem repeats, both of which are located in the tandem repeat region of chromosome 4 of rice.

[0011] This invention reveals for the first time that rice GNK2_21 / 22 As tandem repeat pairs, they work together to maintain basal immune homeostasis through functional redundancy. The term "functional redundancy" refers to two or more genes having the same or overlapping functions. When one gene fails, the other gene can take its place and continue to perform the function, so the plant does not show obvious abnormalities.

[0012] The CRISPR / Cas9 knockout experiments in this application showed that the basal immune levels of single-gene knockout lines (ko21, ko22) were not significantly different from those of the wild type, while the basal immune level of the double-gene knockout line (dko) was significantly reduced. This indicates that the two genes together constitute an immune robust unit, synergistically maintaining the plant's basal immune homeostasis through functional redundancy. The absence of a phenotype from single-gene knockout means that this immune system is not easily destroyed by pathogens attacking a single gene, exhibiting higher evolutionary stability. Protecting and applying the two genes as a single "unit" is the core innovation of this invention.

[0013] The aforementioned non-race-specific resistance to rice blast refers to resistance to different types of rice blast pathogens.

[0014] The plant immune inducer increases the content of reactive oxygen species in rice and promotes the expression of endogenous defense genes, thereby generating non-race-specific resistance to rice blast.

[0015] In a second aspect, this invention provides a method to improve the efficiency of tandem repeat paralogous gene pairs in rice. GNK2_21 / 22 The encoded protein activity is applied in any of the following (1)-(3): (1) Improve rice resistance to non-race-specific rice blast; (2) Increase the reactive oxygen content in rice; (3) Preparation of plant immune inducers; The improvement of tandem repeat paralogous genes in rice GNK2_21 / 22 The encoded protein activity refers to: increasing the activity of rice. GNK2_21 and / or GNK2_22 The activity of gene-encoded proteins; The GNK2_21 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, as follows: SEQ ID NO: 2: MQPRCTLIHAVAVVAAAAAAALLLPPLAAGQPWPTCDSAGTYKAGSAYESNLRDLAAALRADAAASPSALFATGNRGGAPDAVYGLLLCRGDLSVSDCFDCGTRVLADVGRVCGGRHGRAKDVALVYNQCYARFSNKGDF LAATDNAGGETLLISGTNITGGAGVVAAYDRAVTELLAATVRYAVEENPARLFATGQRVGDDARDPGFRNIYSMAQCSPDLPPASCRRCLDGVLARWWQVFPLNGEGARVAGARCYLRSELGVGPFYTGAPMVVLRADKV.

[0016] The GNK2_22 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4, as follows: SEQ ID NO: 4: MQPRCTLIHAVAVVAAAAAAALLLPPLAAGQPWPTCDSAGTYKAGSAYESNLRDLAAALRAGAAASPSALFATGIRGGAPDAVYGLLLCRGDLSVSDCFDCGTRVLADVGRVCGGRHGRAKDVALVYNQCYARFSNKGDF LAATDNAGGETLLISGTNITGGAGVVAAYDRAVTELLAATVRYAVEENPARLFATGQRVGDDARDPGFRNIYSMAQCSPDLPPASCRRCLDGVLARWWQVFPLNGEGARVAGARCYLRSELGVGPFYTGAPMVVLRADKV.

[0017] GNK2_21 / 22 Genetically encoded proteins exert their effects through pattern-triggered immunity (PTI). PTI recognizes conserved molecular patterns of pathogens (such as chitin in fungal cell walls and flagellin in bacteria). These molecular structures are crucial for the survival of pathogens and are not easily mutated. Therefore, regardless of how the pathogen mutates, as long as it belongs to the rice blast fungus, its conserved molecular patterns remain, and the PTI mechanism can recognize and activate the immune response.

[0018] The GNK2_21 Gene-encoded proteins and GNK2_22The proteins encoded by genes form immune-robust modules. The loss or dysfunction of a single protein has no significant effect on the basic immune level of plants, but the simultaneous loss or dysfunction of both proteins leads to a significant decrease in the basic immune level of rice.

[0019] In a third aspect, the present invention provides a method for improving non-race-specific resistance to rice blast, by increasing the number of tandem repeat paralogous genes in rice. GNK2_21 / 22 To increase the expression level of [a specific substance], thereby improving non-race-specific resistance to rice blast; The improvement of tandem repeat paralogous genes in rice GNK2_21 / 22 The expression level refers to: increasing the expression level of rice GNK2_21 and / or GNK2_22 Gene expression levels.

[0020] The improvement of rice GNK2_21 and / or GNK2_22 Gene expression levels include: constructing overexpression vectors for genetic transformation, or using gene editing technology to activate endogenous promoters.

[0021] In a fourth aspect, the present invention provides a plant immune inducer, wherein the plant immune inducer has GNK2_21 protein and / or GNK2_22 protein as active ingredients.

[0022] The plant immune inducer does not have direct in vitro antibacterial activity. It is applied to plants through foliar spraying, seed soaking, or seed coating to induce resistance in plants.

[0023] In a fifth aspect, the present invention provides a method for breeding rice varieties resistant to non-race-specific rice blast, comprising the following steps: Using marker-assisted selection, we can screen for individuals in hybrid offspring who simultaneously possess... GNK2_21 Genes and GNK2_ 22 Gene-based plants.

[0024] The beneficial effects of this invention are: 1. Advanced Induction Mechanism: The unit involved in this invention does not directly kill pathogens, but rather achieves control by activating the plant's own immune switches. This "non-killing" induction strategy is less likely to exert selective pressure on pathogens, thus resulting in more durable broad-spectrum resistance.

[0025] 2. Robustness of the immune system: The role of tandem repeats in maintaining immune system homeostasis has been revealed for the first time. This "double insurance" mechanism ensures that plants can maintain a high baseline of induced resistance even if a single gene is damaged, providing a new target for improving crop immunity.

[0026] 3. Dual prevention and control approach: It can enhance the endogenous resistance of varieties through genetic engineering, and can also be used as a green immune inducer to achieve immediate prevention and control through exogenous spraying, which has extremely high application flexibility.

[0027] 4. Broad-spectrum resistance: This protein induces pattern-triggered immunity in rice itself, resulting in broad-spectrum resistance that is not race-specific and is unlikely to cause pathogens to develop drug resistance.

[0028] 5. Environmentally friendly: As a biological macromolecule, recombinant protein is easily degraded and leaves no residue in the natural environment, making it an ideal candidate module to replace traditional chemical pesticides and achieve green pest control. Attached Figure Description

[0029] Figure 1 This is a maximum likelihood phylogenetic tree for the GNK2 protein. The tree was constructed using the full-length sequence. Members are divided into seven branches (groups 1-7) and represented by different colors. Bootstrap values ​​are shown as circles on the branches.

[0030] Figure 2 Results of GNK2_21 / 22 expression level examination. The expression levels of GNK2_21 and GNK2_22 were detected using qRT-PCR. Actin was used as the internal reference gene. Three biological replicates were set up for each time point.

[0031] Figure 3 Subcellular localization analysis of GNK2_21 and GNK2_22 proteins.

[0032] Figure 4 For in vitro antibacterial activity assay.

[0033] Figure 5 To induce reactive oxygen species (ROS) bursts and defense gene expression in rice.

[0034] Figure 6 Verification of the enhancement of rice resistance to rice blast by exogenous application of GNK2_21 and / or GNK2_22 proteins.

[0035] Figure 7 GNK2_21 and / or GNK2_22 Detection of defense gene expression after single and double knockout and verification of rice resistance to rice blast.

[0036] Figure 8 GNK2_21 and / or GNK2_22 Resistance testing for different races. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0039] Specific information on the physiological races (JS23-7, JS23-25, JS23-26) used in the examples is described in the following literature: Zhao T, Ma S, Kong Z, Zhang H, Wang Y, Wang J, Liu J, Feng W, Liu T, Liu C, Liang S, Lu S, Li X, Zhao H, Lu C, Latif MZ, Yin Z, Li Y, Ding 2024 May 6;17(5):807-823. doi: 10.1016 / j.molp.2024.04.009. Epub 2024 Apr 25. PMID: 38664971. Example 1: GNK2 Genome-wide identification of gene families and pairs of tandem repeat paralogous genes GNK2_21 / 22 Localization and Induced Expression Analysis HMMER software (v3.3) was used to search for protein sequences containing the GNK2 conserved domain in the rice reference genome (Oryza sativa japonica cv. Nipponbare). The E-value threshold was set to 1e-10. Redundant sequences were removed by manual proofreading, resulting in 79 GNK2 family members. The chromosomal distribution of the GNK2 family members was analyzed using MCScanX software. The results showed that... GNK2_21 and GNK2_22Located in the tandem repeat region of chromosome 4, it exhibits a typical tandem repeat arrangement. According to the definition of "tandem repeat paralogous gene pair" in this invention, the two belong to the same chromosome, are generated through gene replication events, have highly homologous sequences, and are functionally related paralogous gene pairs.

[0040] Take rice seedlings at the four-leaf stage and spray them with a suspension of rice blast fungus spores (1×10⁻⁶). 5 Leaves were collected at 0 h, 6 h, 12 h, 24 h, and 48 h post-inoculation. Total RNA was extracted and reverse transcribed into cDNA. The expression levels of GNK2_21 and GNK2_22 were detected by qRT-PCR. Actin was used as the internal reference gene. Three biological replicates were set up at each time point. The qRT-PCR results showed that the expression of GNK2_21 and GNK2_22 began to be significantly upregulated 12 h after inoculation with rice blast fungus. This result indicates that the expression of GNK2_21 and GNK2_22 is induced by the pathogen, and the two are arranged in tandem repeats in the genome, suggesting that they may participate as a functional whole in the plant immune response process. Figure 2 ).

[0041] Example 2: Subcellular localization analysis of GNK2_21 and GNK2_22 proteins Using rice leaf cDNA as a template, the full-length coding sequences of GNK2_21 and GNK2_22 were amplified, respectively, and cloned into the pCAMBIA1300-GFP vector via homologous recombination to obtain the GNK2_21::GFP and GNK2_22::GFP fusion expression vectors. The empty vector pCAMBIA1300-GFP was used as a control.

[0042] The above vectors were transformed into Agrobacterium GV3101 strain and resuspended in OD240. 600 =0.6% was injected into the leaves of Nicotiana benthamiana. 48 h after injection, leaf samples from the injected area were prepared into slides, and the GFP fluorescence signal was observed under a laser confocal microscope.

[0043] Laser confocal microscopy revealed that the fluorescence signals of both GNK2_21::GFP and GNK2_22::GFP were mainly distributed at the cell periphery, overlapping with the fluorescence signals at the cell wall site after plasmolysis, indicating that both proteins are located in the apoplast space. This result suggests that both GNK2_21 and GNK2_22 are secreted proteins capable of entering the apoplast space to exert their functions, consistent with their functional characteristics as endogenous immune activators. Figure 3 ).

[0044] Example 3: Expression, purification and in vitro antibacterial activity assay of recombinant GNK2_21 and GNK2_22 proteins (1) Expression and purification of recombinant proteins The coding sequences of GNK2_21 and GNK2_22 were cloned into the pMAL vector (MBP fusion expression system) and transformed into Escherichia coli BL21(DE3) strain. The cultures were incubated at 37°C until OD200. 600 =0.6, add IPTG to a final concentration of 0.5 mM, and induce expression at 25℃ for 8 h. Collect bacterial cells by centrifugation, lyse by sonication, and collect the supernatant. Purify the target protein using a Ni-NTA affinity chromatography column. The purity of the purified protein was determined by SDS-PAGE, and the concentration was determined by the BCA method.

[0045] (2) In vitro antibacterial activity detection The direct inhibitory effect of recombinant proteins on *Strombus oryzae* was detected using a mycelial growth inhibition method. Purified GNK2_21 and GNK2_22 proteins, mixed with equal masses of each (final concentration 10 μM), were added to PDA medium. An equal volume of PBS buffer was used as a negative control. *Strombus oryzae* mycelial cakes with a diameter of 5 mm were inoculated, and after incubation at 28℃ for 5 days, the colony diameter was measured, and the inhibition rate was calculated.

[0046] 2. Experimental Results SDS-PAGE results showed that the purified GNK2_21 and GNK2_22 proteins both showed a single electrophoretic band, with a molecular weight of approximately 35 kDa and a purity of >95%.

[0047] In vitro antibacterial assays showed that the inhibition rates of GNK2_21 protein, GNK2_22 protein, and their synergistic combination were 2.1%±1.5%, 1.8%±1.2%, and 2.5%±1.8%, respectively, with no significant difference compared to the PBS control (0%) (P>0.05). These results indicate that GNK2_21 and GNK2_22 proteins do not possess direct in vitro antibacterial activity. Figure 4 ).

[0048] Example 4: Exogenous application of GNK2_21 and / or GNK2_22 proteins induces reactive oxygen species bursts and defense gene expression in rice. The GNK2_21 and GNK2_22 recombinant proteins used in this example were expressed and purified using the same methods as in Example 3. The stored proteins were removed from -80°C, thawed on ice, and then adjusted to the required concentration (10 μM) with PBS buffer before use.

[0049] Four-leaf stage rice seedlings were subjected to the following treatments: Negative control: Spray with PBS buffer Positive control: Spray with 10 μM flg22 (a known PTI inducer) GNK2_21 protein treatment: Spray with 10 μM GNK2_21 protein. GNK2_22 protein treatment: Spray with 10 μM GNK2_22 protein. Synergistic composition treatment: Spray with a mixture of 10 μM GNK2_21 and GNK2_22 proteins of equal mass (total protein concentration 10 μM). Each treatment was set up with 3 biological replicates, each replicate containing 10 seedlings.

[0050] (1) Detection of reactive oxygen species Thirty minutes after spraying, leaves were collected for DAB (3,3′-diaminobenzidine) staining and NBT (nitroblue tetrazolium) staining to observe the accumulation of reactive oxygen species. DAB staining was used to detect hydrogen peroxide (H2O2) accumulation, and NBT staining was used to detect superoxide anion (O2). 2- )accumulation.

[0051] DAB staining results showed that leaves treated with GNK2_21 and GNK2_22 proteins alone exhibited light brown staining, while leaves treated with the synergistic combination showed distinct dark brown staining, with a color depth comparable to the flg22 positive control group. The PBS negative control group showed no staining. NBT staining results were consistent with DAB staining, with the synergistic combination treatment group showing distinct blue-purple staining. These results indicate that both GNK2_21 and GNK2_22 proteins can induce reactive oxygen species (ROS) bursts, and that they have a synergistic effect.

[0052] (2) Detection of defense gene expression Leaves were collected at 0 h, 1 h, 3 h, 6 h, and 12 h after spraying and immediately frozen in liquid nitrogen. Total RNA was extracted, reverse transcribed into cDNA, and the expression levels of defense marker genes were detected by qRT-PCR. The genes detected included PR1a, PR1b, and WRKY45. Actin was used as the internal control gene. Three replicates were performed at each time point.

[0053] qRT-PCR results showed that GNK2_21 and GNK2_22 proteins could induce the expression of defense genes, and the synergistic use of the two significantly enhanced the induction effect. Figure 5 ).

[0054] Example 5: Verification of the enhancement of rice resistance to rice blast by exogenous application of GNK2_21 and / or GNK2_22 proteins Rice seedlings were treated according to the method in Example 4 (PBS negative control, GNK2_21 protein, GNK2_22 protein, synergistic composition). After 24 h of treatment, rice blast fungus spore suspension (1×10⁻⁶) was spray-inoculated. 5(number of lesions per mL). Five days after inoculation, the percentage of lesion area on each leaf was counted.

[0055] The results showed that 5 days after inoculation, typical rice blast lesions appeared on the leaves of the PBS negative control group, with a lesion area ratio of 25.6% ± 3.2%. The lesion area ratios in the GNK2_21 protein and GNK2_22 protein treatment groups were 15.3% ± 2.1% and 16.1% ± 2.4%, respectively, significantly lower than the negative control group (P < 0.05). The lesion area ratio in the synergistic combination treatment group was 4.8% ± 1.2%, significantly lower than the single treatment groups (P < 0.01). The pathogen biomass detection results were consistent with the lesion area trend. These results indicate that exogenous application of GNK2_21 and / or GNK2_22 proteins can significantly enhance rice resistance to rice blast, and the synergistic effect is optimal. Figure 6 ).

[0056] Example 6: Verification of functional redundancy and non-race-specific resistance of GNK2_21 / 22 tandem repeat pairs 1. Experimental Materials Using conventional methods, we constructed a single knockout line (ko21) of GNK2_21, a single knockout line (ko22) of GNK2_22, and a double knockout line (dko). Simultaneously, we constructed overexpression lines (OE21) of GNK2_21, overexpression line (OE22) of GNK2_22, and overexpression lines of both GNK2_21 and GNK2_22 (OE21 / 22).

[0057] (1) Construction of knockout vector Using CRISPR / Cas9 technology, specific targets were designed for the exon regions of GNK2_21 and GNK2_22, and complementary sgRNA oligonucleotides were designed for the BsaI restriction site of the pYLCRISPR / Cas9-MH vector.

[0058] GNK2_21 Target: 5'-[TACGCCCGATTCTCCAACAAGGG]-3' GNK2_22 Target: 5'-[ GGCGAGACGCTGCCTCATCAGCGG]-3' The paired oligonucleotides were denatured and annealed by heating at 95°C for 5 minutes and then slowly cooling to room temperature to form double-stranded sgRNA fragments. The intermediate vector U3 was then digested with BsaI restriction endonuclease, and the annealed double-stranded fragments were ligated into the vector to construct an intermediate plasmid containing an sgRNA expression cassette driven by the rice U3 promoter. Sequencing confirmed the correct insertion sequence.

[0059] The sgRNA expression cassettes that were verified by sequencing were transferred from the intermediate vector to the binary vector pYLCRISPR / Cas9-MH using restriction endonucleases. This vector already carries the Cas9 protein expression cassette. For the GNK2_21 single knockout vector (ko21) and the GNK2_22 single knockout vector (ko22), only the corresponding single sgRNA expression cassette was ligated. For the double knockout vector (dko), the two sgRNA expression cassettes of GNK2_21 and GNK2_22 were tandemly cloned into the same pYLCRISPR / Cas9-MH vector, which can be done using a one-step Golden Gate reaction or sequential ligation.

[0060] After vector construction, the ko21, ko22, and dko plasmids were transformed into Agrobacterium (EHA105 strain), and rice callus was transformed using Agrobacterium-mediated rice genetic transformation. Regenerated plants were obtained after hygromycin resistance screening. The target region was amplified by PCR and the mutation was identified by sequencing. Finally, homozygous GNK2_21 single knockout, GNK2_22 single knockout, and double knockout rice lines were obtained.

[0061] (2) Construction of overexpression vector The full-length coding sequences of GNK2_21 and GNK2_22 were amplified using rice cDNA as a template using universal primers 5'-ATGCAGCCGCGC-3' and 5'-CTAGACCTTGTCCGCC-3'. Since the coding regions of the two genes are highly similar, both can be amplified simultaneously using the same primers. The amplified products were purified and ready for use.

[0062] The amplified products were ligated into the entry vector pDONR221 using Gateway technology. Specifically, the purified PCR product was subjected to a BP recombination reaction with the pDONR221 vector, transformed into E. coli, and multiple clones were selected for sequencing. Based on the sequencing results, GNK2_21 and GNK2_22 were distinguished and stored as entry clones pENTR-GNK2_21 and pENTR-GNK2_22, respectively.

[0063] The pENTR-GNK2_21 vector was subjected to a logistic regression (LR) recombination reaction with the overexpression final vector pUN-35S-1300 (containing the 35S promoter, hygromycin resistance gene, and Gateway attR site) to obtain the OE21 vector, which expresses GNK2_21 driven by the 35S promoter. Similarly, pENTR-GNK2_22 was subjected to LR recombination with pUN-35S-1300 to obtain the OE22 vector. To construct the OE21 / 22 vector co-expressing GNK2_21 and GNK2_22, a tandem expression cassette strategy was adopted: the complete 35S::GNK2_22 expression cassette was excised from the OE22 vector using restriction endonucleases (HindIII and EcoRI) and inserted into the OE21 vector, thus forming a structure with two independent expression cassettes tandemly on one vector.

[0064] Finally, the OE21, OE22, and OE21 / 22 vectors were transformed into Agrobacterium-mediated transformation of rice callus tissue. Regenerated plants were obtained through hygromycin resistance screening. The expression levels of the target genes were detected using qRT-PCR or Western blot, and rice lines with high expression of GNK2_21, GNK2_22, and both genes were screened for overexpression.

[0065] 2. Experimental Methods (1) Basic immune level detection Four-leaf stage seedlings of wild type, ko21, ko22 and dko lines were collected and extracted directly from leaves without inoculation with pathogens. The basal expression levels of defense marker genes PR1a, PR1b and WRKY45 were detected by qRT-PCR.

[0066] qRT-PCR results showed that the basal expression levels of PR1a, PR1b, and WRKY45 in the ko21 and ko22 single knockout lines were not significantly different from those in the wild type (P>0.05); however, the basal expression levels of PR1a, PR1b, and WRKY45 in the dko double knockout lines were significantly lower than those in the wild type. These results indicate that GNK2_21 and GNK2_22 have functional redundancy in maintaining basal plant immunity, and the simultaneous loss of both leads to the collapse of basal immunity. Figure 7 ).

[0067] (2) Verification of disease resistance Wild-type, ko21, ko22, dko, OE21, and OE22 seedlings at the four-leaf stage were respectively sprayed with a suspension of rice blast fungus spores (1×10⁻⁶). 5 (number of lesions per mL), and the lesion area was counted after 5 days.

[0068] The results showed that 5 days after inoculation, the proportion of lesions in wild-type strains was 24.3%±2.8%; the lesion areas of single knockout lines of ko21 and ko22 were 22.1%±2.5% and 23.5%±2.7%, respectively, with no significant difference from wild-type; the lesion area of ​​double knockout lines of dko significantly increased to 58.6%±4.2% (P<0.01); while the lesion areas of overexpressing lines of OE21, OE22, and OE21 / 22 significantly decreased, to 13.2%±1.5%, 14.1%±1.8%, and 2.8±0.9%, respectively (P<0.01). These results further validated the synergy and redundancy of GNK2_21 and GNK2_22 in disease resistance, which together constitute an immune robust unit. Figure 7 ).

[0069] (3) Verification of non-race-specific resistance To verify whether GNK2_21 / 22-mediated resistance is non-race-specific, inoculation experiments were conducted using different physiological races of rice blast fungus (JS23-7, JS23-25, JS23-26). Four-leaf stage seedlings of wild-type, OE21 / 22 double-overexpression, and dko double-knockout lines were sprayed with spore suspensions of different races (1×10⁻⁶). 5 (Number of lesions per mL), and the lesion area was counted after 5 days. The small races were preserved in our laboratory.

[0070] The results showed that wild-type resistance varied among different races (lesion area 15.3%–28.6%); the OE21 / 22 double-overexpression lines showed high resistance to all tested races (lesion area 2.1%–3.5%), while the dko double-knockout lines showed high susceptibility to all tested races (lesion area 52.3%–63.8%). These results indicate that GNK2_21 / 22-mediated resistance is not race-specific and is effective against all tested races. Figure 8 ).

[0071] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Enhance the pairing of tandem repeat paralogous genes in rice GNK2_21 / 22 The expression is used in any of the following (1)-(4): (1) Improve rice resistance to non-race-specific rice blast; (2) Increase the reactive oxygen content in rice; (3) Develop rice varieties resistant to non-race-specific rice blast; (4) Preparation of plant immune inducers; The tandem repeat paralogous gene pair GNK2_21 / 22 Depend on GNK2_21 Genes and GNK2_22 Genome composition; The GNK2_21 The nucleotide sequence of the gene is shown in SEQ ID NO.1; GNK2_22 The nucleotide sequence of the gene is shown in SEQ ID NO.3; The improvement of tandem repeat paralogous genes in rice GNK2_21 / 22 The expression refers to: improving the rice GNK2_21 and / or GNK2_22 Gene expression.

2. The application according to claim 1, characterized in that, The aforementioned non-race-specific resistance to rice blast refers to resistance to different types of rice blast pathogens.

3. The application according to claim 1, characterized in that, The plant immune inducer increases the content of reactive oxygen species in rice and promotes the expression of endogenous defense genes, thereby generating non-race-specific resistance to rice blast.

4. Improve the pairing of tandem repeat paralogous genes in rice GNK2_21 / 22 The encoded protein activity is applied in any of the following (1)-(3): (1) Improve rice resistance to non-race-specific rice blast; (2) Increase the reactive oxygen content in rice; (3) Preparation of plant immune inducers; The improvement of tandem repeat paralogous genes in rice GNK2_21 / 22 The encoded protein activity refers to: increasing the activity of rice. GNK2_21 and / or GNK2_22 The activity of gene-encoded proteins; The GNK2_21 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; GNK2_22 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

4.

5. The application according to claim 4, characterized in that, The GNK2_21 Gene-encoded proteins and GNK2_22 The proteins encoded by genes form immune-robust modules. The loss or dysfunction of a single protein has no significant effect on the basic immune level of plants, but the simultaneous loss or dysfunction of both proteins leads to a significant decrease in the basic immune level of rice.

6. A method for improving non-race-specific resistance to rice blast, characterized in that, By increasing the number of tandem repeat paralogous genes in rice GNK2_21 / 22 To increase the expression level of [a specific substance], thereby improving non-race-specific resistance to rice blast; The improvement of tandem repeat paralogous genes in rice GNK2_21 / 22 The expression level refers to: increasing the expression level of rice GNK2_ 21 and / or GNK2_22 Gene expression levels.

7. The method according to claim 6, characterized in that, Improve rice GNK2_21 and / or GNK2_22 Gene expression levels include: constructing overexpression vectors for genetic transformation, or using gene editing technology to activate endogenous promoters.

8. A plant immune inducer, characterized in that, The plant immune inducer uses GNK2_21 protein and / or GNK2_22 protein as its active ingredients.

9. The plant immune inducer according to claim 8, characterized in that, The plant immune inducer does not have direct in vitro antibacterial activity. It is applied to plants through foliar spraying, seed soaking, or seed coating to induce resistance in plants.

10. A method for breeding rice varieties resistant to non-race-specific rice blast, characterized in that, Includes the following steps: Using marker-assisted selection, we can screen for individuals in hybrid offspring who simultaneously possess... GNK2_21 Genes and GNK2_22 Gene-based plants.

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