Application of rice atl119 gene and rice atl120 gene in regulating rice blast resistance

Abstract

The present application relates to rice ATL119 gene and rice ATL120 The application of the gene in regulating rice blast resistance belongs to the technical field of molecular genetic breeding. Through functional mining and application verification of the rice ATL119 gene and rice ATL120 gene, it is found that the knock-out mutant of the gene has significantly enhanced resistance to rice blast, and the level of chitin-induced reactive oxygen burst is significantly increased. The above results show that the ATL119 protein and the ATL120 protein as immune negative regulatory factors, the genes encoding the same can be used to improve the resistance of plants to pathogenic bacteria. The present application provides a new and efficient target for rice disease resistance molecular breeding, and has important application value and broad popularization prospect in improving the disease control ability of rice and promoting the construction of green agricultural system.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding technology, and particularly to rice. ATL119 Genes and rice ATL120 Application of genes in regulating rice blast resistance. Background Technology

[0002] Rice blast, one of the most damaging diseases, often leads to significant yield reductions or even total crop failure. Among numerous control measures, breeding and promoting disease-resistant varieties is considered the most economical and environmentally friendly strategy, and advancements in molecular breeding technology have provided strong support for the rapid creation of resistant varieties.

[0003] Plant immune defense relies on a complex signal regulation system. Among these, the ubiquitin-proteasome system plays a crucial role in maintaining immune homeostasis by controlling the degradation of immune-related proteins. E3 ubiquitin ligases are the core components of this system responsible for substrate recognition, determining whether target proteins are degraded or functionally altered by adding ubiquitin tags. In recent years, although some E3 ligases have been reported to participate in rice immune regulation, the behavioral patterns of negative regulators in disease resistance signals and their synergistic mechanisms with positive regulators remain poorly understood. Due to the lack of molecular tools capable of precisely regulating immune strength, breeding practices have long faced the dilemma of "high yield but low disease resistance, and disease resistance but low yield."

[0004] Currently, some negative regulatory genes have been attempted to improve plant disease resistance, such as by inactivating them through gene editing to enhance resistance. However, this approach faces two main obstacles in practical application: firstly, most negative regulatory genes are involved in multiple life activities, and their deletion often leads to slow plant development, reduced yield, or other undesirable phenotypes; secondly, targets that can simultaneously improve disease resistance and good agronomic traits are extremely scarce. Therefore, how to screen key genes from the rice immune network that can effectively improve resistance without significantly affecting growth and development has become a core problem that urgently needs to be solved in the field of molecular breeding.

[0005] In the improvement of rice blast resistance, although several resistance-related genes have been identified, these genes are mostly variety-specific, with limited resistance ranges and are easily overcome by pathogens. In contrast, targeting negative regulators with broad-spectrum regulatory potential in immune pathways for modification holds promise for obtaining more durable and broader resistance. However, current molecular breeding strategies based on E3 ubiquitin ligases still face practical challenges such as a scarcity of available targets, unclear mechanisms of action, and the difficulty in achieving both disease resistance and yield, which greatly limits the promotion and application of this technology. Summary of the Invention

[0006] The purpose of this invention is to provide rice ATL119 Genes and rice ATL120This invention addresses the application of genes in regulating rice blast resistance, overcoming the technical shortcomings of existing technologies such as a lack of disease resistance improvement targets and the difficulty in simultaneously achieving disease resistance and growth. Through systematic mining of the rice immune negative regulatory network, this invention reveals for the first time the role of genes in regulating rice blast resistance. ATL119 Genes and rice ATL120 The biological function of genes in rice disease resistance response. Experiments show that rice ATL119 Genes and rice ATL120 This gene negatively regulates rice resistance to rice blast fungus; knocking out this gene significantly enhances rice resistance to rice blast, and simultaneously, the level of chitin-induced reactive oxygen species (ROS) burst is also significantly increased. This invention provides a new technical approach to resolving the long-standing contradiction between disease resistance and high yield in crop breeding, and offers a novel, highly efficient target gene for rice molecular breeding. Rice ATL119 Genes and rice ATL120 The discovery and application of genes have significant application value and broad prospects for promoting the development of green and sustainable agriculture, including improving the ability to control rice diseases, reducing dependence on chemical pesticides, and promoting the development of green and sustainable agriculture.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of rice ATL119 protein and / or rice ATL120 protein in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The amino acid sequence of the rice ATL119 protein is shown in SEQ ID NO.2; the amino acid sequence of the rice ATL120 protein is shown in SEQ ID NO.4.

[0008] Optionally, the application can improve the rice's resistance to rice blast or cultivate rice blast-resistant varieties by reducing the expression levels of the rice ATL119 protein and / or the rice ATL120 protein.

[0009] This invention provides rice ATL119 Genes and / or rice ATL120 The application of genes in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0010] Optionally, the application knocks out the rice ATL119 Genes and / or the rice ATL120 Genes can be used to improve rice's resistance to rice blast or to cultivate rice varieties resistant to rice blast.

[0011] This invention provides knockout rice ATL119 Genes and / or rice ATL120 The application of gene-based biomaterials in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0012] Optionally, the biomaterial includes a recombinant vector and recombinant bacteria.

[0013] This invention provides a method for improving the resistance of rice to rice blast, comprising knocking out rice germ cells in rice plants. ATL119 Genes and / or rice ATL120 The steps of gene generation; the rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0014] This invention provides a method for cultivating rice resistant to rice blast, comprising knocking out rice germ cells in rice. ATL119 Genes and / or rice ATL120 The steps of gene generation; the rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0015] The present invention discloses the following technical effects: This invention utilizes rice... ATL119 Genes and rice ATL120 The gene was analyzed, and it was found that the knockout mutant of ATL119 significantly enhanced resistance to rice blast fungus, the most destructive pathogen in rice, while chitin-induced reactive oxygen species production was significantly increased. These results indicate that the ATL119 and ATL120 proteins negatively regulate plant disease resistance, and their encoding genes... ATL119 and ATL120 This invention provides a novel and highly efficient target for rice molecular breeding, which can be used as a target gene to improve plant disease resistance. It is of great significance for ensuring safe rice production and promoting the development of green agriculture. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 for atl119 , atl120 Single mutant and atl119 atl120 A schematic diagram of CRISPR / Cas9 gene editing with double mutants; where A represents... ATL119 Gene structure and knockout mutant editing methods; B is ATL120 Gene structure and knockout mutant editing methods; C represents atl119 atl120 Editing methods for double mutants; Figure 2 To identify rice using hole-punching inoculation ATL119 Genes and rice ATL120 Gene knockout single mutants and rice ATL119 Genes and rice ATL120 Resistance of gene knockout double mutants to rice blast; where A represents rice ATL119 Genes and rice ATL120 Gene knockout single mutant, rice ATL119 Genes and rice ATL120 Typical lesion symptoms of *Strombus oryzae* strain RB22 after inoculation with gene knockout double mutants and wild-type plants were recorded on day 14 post-inoculation; B shows the relative lesion area (mean ± SE) of the inoculated leaves corresponding to (A); C shows the relative fungal biomass of *Strombus oryzae* based on typical leaves in (A), quantified by RT-qPCR. MoPot2 Gene expression levels, in OsUBQ Genes were used as internal controls (mean ± SE); significant differences were determined by one-way ANOVA; different lowercase letters indicate significant differences. p <0.05; Figure 3 For rice ATL119 Genes and rice ATL120 Gene knockout single mutant, rice ATL119 Genes and rice ATL120 PAMP-induced reactive oxygen species (ROS) bursts in gene knockout double mutants and wild-type rice are shown. The curves represent the mean ROS production ± SE in real time; where A represents rice. ATL119 Genes and rice ATL120 Gene knockout single mutant, rice ATL119 Genes and rice ATL120Chitin-induced reactive oxygen species (ROS) bursts in gene knockout double mutants and wild-type chitin; B represents the quantitative analysis of total ROS production based on the area under the curve (AUC) in A; (mean ± SE); significant differences were determined by one-way ANOVA; different lowercase letters indicate significant differences. p <0.05. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention identified two novel rice homologous genes, LOC_Os12g24490 and LOC_Os12g24530, which were named respectively. ATL119 Genes and ATL120 Gene, ATL119The CDS sequence of the gene is shown in SEQ ID NO.1, which is 558 bp in length and encodes the ATL119 protein containing 185 amino acids. The amino acid sequence of the ATL119 protein is shown in SEQ ID NO.2. ATL120 The CDS sequence of the gene is shown in SEQ ID NO.3, with a total length of 639 bp, encoding the ATL120 protein containing 212 amino acids. The amino acid sequence of the ATL120 protein is shown in SEQ ID NO.4.

[0024] SEQ ID NO.1 ( ATL119 (CDS sequence of the gene) .

[0025] SEQ ID NO.2 (Amino acid sequence of ATL119 protein): MDAPRRRSNGAWEINLVRRSPATARTDRCSRLLLLWSGFVGVVVVLYLFVGHVWASVATAVLLAAAGWFTWYYFGAAPAPPVLPDHHQPAAPVEARGLSQEDIEAIPAFEYRRGSSGSGVAQCAVCIAAVKDGDTVRRLPACGHAFHAPCVDGWLRDHATCPMCRADVVKVAGETTPATEEEPPV。

[0026] SEQ ID NO.3 ( ATL120 CDS sequence of the gene): ATGGAAGTCGTCGTCGCTCGCCGGGGCGTCGTCGTCCTGCGGCTGCGCCACGGCGAGCCGCCGCCCAGCGCCGCCGCGCGGTCTGGCGGGCGCTGCTCGAGGCTGCTCCTCCTGTGGACGTTCGCCGTCGGCTTCGCCGCCGGGCTGTTCATGTTCGCCAGCCACGTCTTGGCCGCCGTCGCCGCCGCCGTGCTCTTCGCCGCCATGTGCCTGTACACGTGCCTGTGCCTGAACAACGCGGCGCCGGAGGAGGAAGAGGAGCAGCAGCAGCAGCCGCCTGTTCTGCTGCTTCCCGGTCAGGCGCTGCGCGTCGCCGTGATGCCGCCATCGGTGGGGCGGCTGCAGCAGCAGGAGGTCAACGGCGGCGGCGGCGGCGGAGGCCTCAGGCAGGAGGACGTCGAGGCCGCCATCCCGGCGTTCGAGTACAGTAAGGGTTCGGGCGGCGCGGCGGAGGCGGAGCAGTGCGCCGTGTGCATCGGAGTCGTGCGGCGGGGGGAGACGGTGCGGCGGCTGCCGGCGTGCGGGCACGCGTTCCACGCGGCGTGCATCGACGGGTGGCTCCGCGCGCACGCCACGTGCCCCGTGTGCCGCGCCGACGTCAAGTCGCCGCCGGCGGCGGCGGCGGAGCGCCGGTGTAG。

[0027] SEQ ID NO.4 (Amino acid sequence of ATL120 protein): MEVVVARRGVVVLRLRHGEPPPSAAARSGGRCSRLLLLWTFAVGFAAGLFMFASHVLAAVAAAVLFAAMCLYTCLCLNNAAPEEEEEQQQQPPVLLLPGQALRVAVMPPSVGRLQQQEVNGGGGGGGLRQEDVEAAIPAFEYSKGSGGAAEAEQCAVCIGVVRRGETVRRLPACGHAFHAACIDGWLRAHATCPVCRADVKVAAGGGGGAPV.

[0028] The following is for ATL119 Genes and ATL120 The correlation between genes and rice disease resistance was verified, mainly through... ATL119 Genes and ATL120 The gene knockout mutants will be specifically verified and explained.

[0029] The target sequences and primers designed in this invention are shown in Table 1.

[0030] Table 1. Designed target sequences and primer sequences Example 1 I. Experimental Methods and Procedures 1. ATL119 Genes and ATL120 Construction of gene knockout mutants ATL119 Genes and ATL120 The gene knockout mutant was developed by Weimi Biotechnology Co., Ltd. The steps are as follows: Primers (SEQ ID NO. 8 and SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13) were denatured at 95℃ and then annealed to form DNA double-stranded fragments. The pRGEB32 vector was digested with BsaI and the linearized vector was recovered. The above DNA double strands and linearized vector were mixed and subjected to T4 DNA ligase ligation reaction (ligation system: 100 ng linearized vector, 0.5 μL T4 ligase, 1 μL T4 ligase buffer, and up to 10 μL DNA double-stranded fragments). After overnight ligation at 16℃, the mixture was transformed into DH5α competent cells. After kanamycin selection, single colonies were picked for colony PCR identification. Plasmids were extracted from positive colonies and sequenced correctly. These colonies were then handed over to Weimi Biotechnology Co., Ltd. for genetic transformation of rice Zhonghua 11 (ZH11), and finally obtained... ATL119 Genes and ATL120 Gene knockout mutant.

[0031] 2. ATL119 Genes and ATL120 Identification of gene knockout mutants Genomic DNA of each mutant was extracted using the TPS method and used as a template for PCR amplification targeting the knockout site.

[0032] The amplification primers for knocking out the target site are: upstream primer ATL119-T1-F, upstream primer ATL120-T1-F, downstream primer ATL119-T1-R, and downstream primer ATL120-T1-R.

[0033] Amplification was performed using 2×NG PCR MasterMix (Shanghai Huiling), and the components in the kit were prepared according to the reaction system in Table 2.

[0034] Table 2 Reaction System The PCR amplification procedure is shown in Table 3.

[0035] Table 3 PCR amplification program The obtained product was sent to Shanghai Qingke Company for DNA sequencing, and two strains were eventually detected. ATL119 Gene knockout single mutant — atl119 #6 and atl119 #7; 2 strains ATL120 Gene knockout single mutant — atl120 #6 and atl120 #11; 2 rice plants ATL119 Genes and rice ATL120 Gene knockout double mutant — atl119 atl120 #3 and atl119 atl120 #13, ATL119 Genes and ATL120 The mutation status of gene knockout mutants is as follows: Figure 1 As shown.

[0036] 3. ATL119 Genes and ATL120 Identification of resistance to rice blast in gene knockout mutants Rice fungus ( Magnaporthe oryzae abbreviation M. oryzae The physiological race RB22 is disclosed in the literature "Selection of Differential Isolates of Magnaporthe oryzae In the application for Postulation of Blast Resistance Genes, the applicant committed to distributing the gene for 20 years from the date of application.

[0037] Oatmeal culture medium: 20 g / L oat flour, 15 g / L agar.

[0038] For identification ATL119 Genes and ATL120 The function of the gene in rice disease resistance was investigated, and its knockout mutant and the corresponding wild type (ZH11) were used as controls to study the fungal rice blast fungus (ZH11). Magnaporthe oryzae abbreviation M. oryzae Physiological race RB22 inoculation: (1) When using the perforation method, the physiological race RB22 of rice blast fungus was inoculated onto oat flake medium and cultured at room temperature for 10 days to induce sporulation. Fresh spores were collected using sterile water containing 0.5% (v / v) Tween-20 and the concentration was adjusted to 5 × 10⁻⁶. 5 1 spore / mL. Six-week-old rice seedlings were selected, and inoculation was performed by punching holes in the middle of the second leaf from the top. Phenotypic characteristics were observed two weeks after inoculation. The relative lesion area was measured using ImageJ software, and the transposon elements of the rice blast fungus were quantified by qPCR. MoPot2 To determine the relative fungal biomass, rice OsUBQ As an internal reference gene. The specific two pairs of qRT-PCR primers are: MoPot2 -qRT-F、 MoPot2 -qRT-R、 UBQ -qRT-F and UBQ -qRT-R.

[0039] The reaction system was prepared using a kit from Nanjing Novizan Pharmaceutical Co., Ltd.: 10 μL of 2 × ChamQ Universal SYBR qPCRMaster Mix; 0.4 μL each of 10 μM upstream and downstream primers; 2 μL of cDNA template; and water to a final volume of 20 μL.

[0040] PCR amplification was performed using a three-step method on a real-time quantitative PCR instrument. The PCR amplification program is shown in Table 4.

[0041] Table 4 PCR Amplification Program The results are as follows Figure 2 As shown in B and C, ATL119 Genes and ATL120 The lesion area and blast fungus biomass of the gene knockout mutant lines were significantly lower than those of the wild type, indicating that... ATL119 Genes and ATL120 Gene knockout mutants exhibit enhanced resistance to rice blast fungus.

[0042] (2) When spraying, prepare a spore suspension containing 0.025% (v / v) Tween-20 (1×10⁻⁶ spores). 6The entire plant of 3-week-old seedlings was sprayed with a spore / mL solution. Leaf symptom images were taken on day 7 post-inoculation. Results are as follows: Figure 2 As shown in A, ATL119 Genes and ATL120 The lesion area of ​​the gene knockout mutant line was significantly smaller than that of the wild type, indicating that... ATL119 Genes and ATL120 Gene knockout mutants exhibit enhanced resistance to rice blast fungus. In summary, ATL119 Genes and ATL120 Genes negatively regulate rice resistance to rice blast fungus.

[0043] 4. Determination of reactive oxygen species in rice leaf sheaths Select tissue culture flasks containing 1 / 2 MS solid medium that have grown for 10-12 days with consistent growth. ATL119 Genes and ATL120 Leaf sheaths from gene knockout mutants and wild-type rice ZH11 were uniformly cut into segments approximately 3 mm long. These segments were gently transferred using tweezers into 96-well microplates containing 100 μL of sterile water, with three leaf sheath segments per well. After overnight incubation in the dark, the sterile water was washed off, and the solution was replaced with chitin (50 mM Tris-HCl, pH 7.5, 10 µg / mL horseradish peroxidase (HRP, Bio-Rad), 10 µM L-012 chemiluminescent probe, 50 µM chitin). The plates were then quickly placed into a Varioskan Flash multireader (BioTek) for 45 min. The final results are shown below. Figure 3 The results showed that ATL119 Genes and ATL120 Chitin-induced reactive oxygen species bursts were significantly increased in gene knockout mutants.

[0044] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of rice ATL119 protein and / or rice ATL120 protein in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The amino acid sequence of the rice ATL119 protein is shown in SEQ ID NO.2; the amino acid sequence of the rice ATL120 protein is shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that, The application achieves the effect of improving rice resistance to rice blast or cultivating rice blast-resistant varieties by reducing the expression levels of the rice ATL119 protein and / or the rice ATL120 protein.

3. Rice ATL119 Genes and / or rice ATL120 The application of genes in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

4. The application according to claim 3, characterized in that, The application involves knocking out the rice. ATL119 Genes and / or the rice ATL120 Genes can be used to improve rice's resistance to rice blast or to cultivate rice varieties resistant to rice blast.

5. Knock out rice plants ATL119 Genes and / or rice ATL120 The application of gene-based biomaterials in any of the following: (1) Improve the rice's resistance to rice blast; (2) Cultivating rice varieties resistant to rice blast; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

6. The application according to claim 5, characterized in that, The biomaterials include recombinant vectors and recombinant bacteria.

7. A method for improving the resistance of rice to rice blast, characterized in that, Including knockout rice ATL119 Genes and / or rice ATL120 The steps of gene generation; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

8. A method for cultivating rice resistant to rice blast, characterized in that, Including knockout rice ATL119 Genes and / or rice ATL120 The steps of gene generation; The rice ATL119 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the rice ATL120 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

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