Rice brown planthopper resistant gene BPH50 and application thereof

Abstract

The invention discloses a brown planthopper resistant gene BPH50 of rice and application of the brown planthopper resistant gene BPH50. The method comprises the following steps: carrying out GWAS analysis on brown planthopper resistance characters of a large number of rice varieties in the seedling stage by utilizing miniature core germplasm resources in a rice germplasm bank of the Ministry of Agriculture of America, screening out significant sites which are distributed on a No.6 chromosome and have the maximum insect-resistant effect, and comparing candidate genes in an NLR gene cluster where the sites are located, finally, the novel NLR gene ORF9 of Pi9, 920 and the NLR gene ORF9 of which no allele is detected in a Nipponbare reference genome are determined as candidate genes. A CRISPR-Cas9 gene knockout technology is utilized to find that an insect-resistant variety 551 can lose brown planthopper resistance by knocking out the ORF9 gene, so that the function of the ORF9 in brown planthopper resistance of rice is verified, and the ORF9 is named as BPH50. The invention provides a novel functional gene resource and a molecular tool for insect-resistant breeding of rice, and has important agricultural application value.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to a rice brown planthopper resistance gene BPH50 and an application thereof. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's three major staple crops, and its yield and cultivated area are directly related to global food security. China, one of the world's largest rice producers, cultivates an average of 30 million hectares of rice annually, accounting for nearly a quarter of the global rice-cultivated area. Therefore, ensuring stable and high rice yields is crucial for both national and global food security.

[0003] However, in rice production, brown planthopper (Nilaparvatalugens Brown planthoppers (BPH) are one of the most significant and destructive insect pests. Using their sharp stylets, they suck the phloem sap of rice plants, causing stunted plant growth, wilting and lodging, severe grain yield reductions, and even complete plant death in some areas, resulting in significant losses in rice yield and quality. Furthermore, long-term reliance on chemical pesticides for pest control not only significantly increases production costs but also leads to a range of problems, including environmental pollution, ecological imbalance, and increased pest resistance. Therefore, developing an efficient, green, and sustainable insect-resistant breeding strategy is particularly urgent.

[0004] In recent years, with the advancement of molecular biology and genomics, the use of molecular breeding methods to discover and clone genes that confer resistance to the brown planthopper (NLP) in rice has become an increasingly important approach for combating this pest. Genome-wide association studies (GWAS) provide a highly efficient means of uncovering the genetic basis of complex traits, enabling researchers to rapidly locate significant loci associated with insect resistance within large-scale germplasm collections. Previous studies have shown that NLP resistance in rice is typically controlled by multiple loci, with most resistance genes occurring as clusters and exhibiting significant inter-variety diversity. This opens the door to further discovering novel resistance genes. Summary of the Invention

[0005] The present invention aims to provide a novel rice brown planthopper resistance gene, BPH50, and to clarify its application value in insect-resistant breeding through forward genetics and functional validation. The specific technical solutions include: A rice brown planthopper resistance gene BPH50, the nucleotide sequence of which is shown in SEQ ID NO.1; Furthermore, the cDNA sequence of the rice brown planthopper-resistance gene BPH50 is shown as SEQ ID NO.2, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.3.

[0006] Use of the above rice brown planthopper-resistance gene BPH50 in reducing the ability of rice to resist brown planthoppers; Furthermore, CRISPR-Cas9 technology was used to knock out the rice brown planthopper-resistant gene BPH50. After knocking out the rice brown planthopper-resistant gene BPH50, the rice lost its brown planthopper-resistant phenotype.

[0007] Use of the rice brown planthopper-resistance gene BPH50 in breeding transgenic rice with reduced brown planthopper resistance; Furthermore, by knocking out the rice brown planthopper resistance gene BPH50, transgenic rice with reduced brown planthopper resistance was cultivated.

[0008] A method for reducing rice's resistance to brown planthoppers comprises the steps of knocking out the rice brown planthopper resistance gene BPH50 to reduce the rice's resistance to brown planthoppers. The nucleotide sequence of the rice brown planthopper resistance gene BPH50 is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.2.

[0009] A method for cultivating transgenic rice with reduced brown planthopper resistance comprises the steps of knocking out the rice brown planthopper resistance gene BPH50 to reduce the rice's brown planthopper resistance ability; the nucleotide sequence of the rice brown planthopper resistance gene BPH50 is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.2.

[0010] The present invention utilizes the USDA Mini Core Germplasm Bank, which covers representative varieties from more than 18,000 rice materials in 75 countries around the world, fully reflecting the genetic diversity and rich phenotypic information of rice. By conducting GWAS analysis of the brown planthopper resistance traits of these materials at the seedling stage, a significant locus with the greatest insect resistance effect was discovered at 10.39Mb on chromosome 6 (preliminarily named qBPH6). Further fine alignment and candidate gene screening of the NLR (nucleotide binding-leucine repeat) gene cluster in this region were performed, and finally a new NLR gene ORF9 (BPH50) was identified, for which no alleles were detected in the conventional reference genome - Nipponbare.

[0011] As core components of the plant immune system, NLR genes are widely involved in pathogen recognition and defense responses. Their diverse genetic structure and function make them valuable for resistance breeding. Although multiple brown planthopper resistance genes have been reported, some potential resistance genes remain underutilized. The discovery of BPH50 not only enriches the rice brown planthopper resistance gene pool but also provides new molecular tools and research directions for further understanding the interaction mechanism between rice and brown planthoppers.

[0012] Further validation of the candidate gene ORF9 by knockout using CRISPR-Cas9 technology revealed that knocking out ORF9 eliminated the brown planthopper-resistant phenotype in rice variety 551, which previously exhibited high resistance. This confirmed the key role of ORF9 in regulating brown planthopper resistance in rice and resulted in the gene being named BPH50. This discovery provides an important molecular target for developing highly insect-resistant rice varieties using molecular breeding methods, and also lays the foundation for reducing the use of chemical pesticides and promoting sustainable, green agriculture.

[0013] By comprehensively utilizing advanced whole-genome association analysis technology, large-scale germplasm resources, and gene editing tools, this field is gradually establishing a complete system from gene mining to functional verification to resistance breeding, and the cloning and functional verification of BPH50 in the present invention is an important part of this system.

[0014] The advantages of the present invention are: 1. For the first time, the large-scale and diverse germplasm resources in the USDA mini-core germplasm bank were used to accurately locate a significant brown planthopper resistance locus located at 10.39Mb on chromosome 6 through genome-wide association analysis (GWAS), providing a strong basis for subsequent candidate gene screening.

[0015] 2. A new brown planthopper-resistant gene BPH50 was successfully cloned in the NLR gene cluster. Its complete genome, cDNA sequence and encoded protein sequence were identified, greatly enriching the rice insect-resistant gene library.

[0016] 3. CRISPR-Cas9 technology was used to verify the knockout function of BPH50. The experimental results showed that after knocking out BPH50, the insect-resistant varieties lost their resistance, which fully demonstrated the key regulatory role of BPH50 in rice resistance to brown planthoppers and provided a clear molecular target for resistance breeding.

[0017] 4. The discovery of BPH50 provides a new technical means for constructing insect-resistant transgenic rice and developing molecular marker-based resistance breeding technology, which will help accelerate the cultivation of new highly resistant rice varieties, reduce the use of chemical pesticides, and achieve green agricultural production.

[0018] 5. This invention not only has significant practical application value, but also provides an important research tool and theoretical basis for further analyzing the molecular mechanism of the interaction between rice and brown planthoppers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Schematic diagram of the location of candidate genes in the qBPH6 region on rice chromosome 6.

[0020] Figure 2: Diagram of the CRISPR-Cas9 knockout vector structure corresponding to the candidate gene (yellow box indicates the exon region).

[0021] Figure 3 : Sequencing identification of BPH50 knockout transgenic seedlings, showing target site deletion or mutation.

[0022] Figure 4 : Phenotypic comparison between the BPH50 homozygous mutant and the wild type after inoculation with brown planthoppers. DETAILED DESCRIPTION

[0023] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0024] Example 1: Candidate gene analysis and construction of BPH50 knockout mutants This example describes in detail how to screen candidate regions from a rice mini-core collection using GWAS, further identify candidate genes within the qBPH6 region, and construct knockout mutants using CRISPR-Cas9 technology to verify their function. The specific steps are as follows: 1. Candidate Gene Analysis ①GWAS analysis and candidate region identification The seedling resistance data of brown planthoppers covering more than 18,000 materials in the USDA mini core germplasm bank were used, and the genome-wide association study (GWAS) method was used to perform statistical analysis on the phenotypic and genotypic data of each variety.

[0025] The analysis results showed that there is a single nucleotide polymorphism (SNP) at 10.39 Mb on rice chromosome 6, tentatively named qBPH6, that is significantly associated with brown planthopper resistance. This locus has the most significant resistance effect among all the loci tested.

[0026] ②Comparison and screening of NLR gene clusters in the candidate gene region Based on the Nipponbare reference genome annotation, the region where qBPH6 is located was finely mapped, and an NLR gene cluster was found in the region.

[0027] The sequences, structures, and expression levels of candidate NLR genes in this region were compared between the insect-resistant variety 551 (Minicore number W4194) and the susceptible variety. These candidate genes included the known Pi9 and 920 genes, as well as a novel candidate gene, ORF9 (also known as BPH50), for which no alleles were detected in Nipponbare. The nucleotide sequence of BPH50 is shown in SEQ ID NO. 1; the cDNA sequence of BPH50 is shown in SEQ ID NO. 2; and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 3.

[0028] 2. Construction of knockout mutants ① CRISPR-Cas9 system design Targeting the coding region of the candidate gene BPH50, two target regions with high specificity and low off-target risk were selected using online CRISPR design tools (such as CRISPR-P) to ensure that their functions could be simultaneously disrupted.

[0029] Design two pairs of gRNA sequences and splice the standard gRNA framework sequence at the end of the gRNA to ensure the formation of the correct secondary structure. The sample primer design is as follows:

Targeting BPH50 Target 1

[0030] PCR system (20 μL) configuration: template DNA 1 μL, upstream primer 1 μL, downstream primer 1 μL, 2×Phanta Mix 10 μL, ddH2O 7 μL.

[0031] PCR program: pre-denaturation at 95°C for 3 minutes; 35 cycles (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds); extension at 72°C for 5 minutes.

[0032] After confirming that the gRNA expression module of the expected size is obtained by agarose gel electrophoresis, gel recovery and purification are performed.

[0033] ③Construction of knockout vector The recovered gRNA module is digested with BsaI and then spliced ​​with the Cas9 expression vector (such as pC1300-Cas9).

[0034] The ligation reaction system (15 μL) comprises: about 70 ng of gRNA module, 100 ng of Cas9 carrier, 0.5 μL of BsaI-HF, 1.5 μL of 10×Cutsmart Buffer, 0.5 μL of T4 DNA ligase, 0.5 μL of T4 DNA ligase buffer, and ddH2O to 15 μL.

[0035] The ligation reaction conditions are as follows: reaction at 37°C for 15 min, then 12 cycles (5 min at 37°C, 5 min at 10°C, and 5 min at 20°C), and finally storage at 12°C.

[0036] The constructed vector is transformed into E. coli, and after PCR detection and sequencing verification of the correct insertion of gRNA and the correct structure of the vector, the vector is transformed into Agrobacterium (such as EHA105).

[0037] (4) Rice transformation and mutant screening The verified BPH50 knockout vector is introduced into the pest-resistant variety 551 (Minicore number W4194) by using Agrobacterium-mediated transformation technology.

[0038] After tissue culture and screening (using the corresponding resistant screening medium), T0 generation transgenic plants are obtained.

[0039] The CTAB method is used to extract the genomic DNA of the T0 generation plants, and PCR amplification is performed on the target region.

[0040] The PCR amplification product is sequenced to confirm the occurrence of deletion or frameshift mutation in the target region, thereby obtaining a functional knockout BPH50 mutant.

[0041] Through the above detailed candidate gene analysis and CRISPR-Cas9 knockout construction process, the BPH50 knockout mutant is successfully constructed in the pest-resistant variety 551 (Minicore number W4194) in this embodiment, which provides a solid experimental basis for further studying the molecular mechanism of resistance to brown planthopper in the qBPH6 region and the function of the resistance gene. After screening and obtaining homozygous mutant materials, subsequent propagation and functional verification experiments are carried out.

[0042] Figure 2 The CRISPR-Cas9 vector schematic diagram of BPH50 is shown.

[0043] Example 2: Obtaining of homozygous material of BPH50 knockout mutant This embodiment describes in detail the whole process of screening from T0 generation transgenic plants to obtaining stable homozygous mutant, and the specific steps are as follows: 1. Preliminary screening of T0 generation transgenic plants ① Sample collection and genomic DNA extraction Fresh leaves (about 0.1 g / plant) were collected from T0 plants transformed by CRISPR-Cas9, placed in a 1.5 mL centrifuge tube, and pre-sterilized 6 mm grinding beads were added.

[0044] After rapid freezing using liquid nitrogen, the sample was fully ground into powder in a fully automatic sample grinder.

[0045] Extract genomic DNA using the CTAB method: a. Add 600 μL of 2% CTAB lysis buffer (containing 5 M NaCl, 1 M Tris-HCl (pH 8.0), and 0.5 M EDTA) preheated to 65°C, mix thoroughly, and place in a water bath at 65°C for 30 minutes (gently invert every 10 minutes to mix).

[0046] b. After cooling, add 600 μL of chloroform:isoamyl alcohol (24:1) mixture, mix gently and then centrifuge at high speed (12000 rpm, room temperature for 10 minutes).

[0047] c. Carefully take the supernatant (about 400 μL), add an equal volume of anhydrous ethanol, mix well, and place at -20°C for 30 minutes, then centrifuge (12000 rpm, 10 minutes) and wash the precipitate with 75% ethanol.

[0048] d. Finally, air-dry the precipitate and dissolve it in 50 μL ddH2O to obtain the genomic DNA sample.

[0049] ②PCR amplification and preliminary identification The target region was amplified by PCR using pre-designed identification primers.

[0050] The PCR system (20 μL) was as follows: 1 μL of template DNA, 1 μL of each primer (10 μM), 10 μL of 2× Phanta Mix, and 7 μL of ddH2O. The PCR program was as follows: pre-denaturation at 95°C for 3 min; 35 cycles (95°C for 30 s, 55°C for 30 s, and 72°C for 30 s); and extension at 72°C for 5 min.

[0051] The amplified products were detected by agarose gel electrophoresis and sent for Sanger sequencing to confirm whether the target region contained the expected deletion, insertion, or frameshift mutation.

[0052] According to the sequencing results, T0 plants containing the expected mutation and presenting a heterozygous state were recorded.

[0053] 2. T1 Generation Planting and Genotype Screening ① Self-pollination and seed collection The preliminarily identified T0 mutants were self-pollinated, seeds were collected, and sown and cultured in a greenhouse or on a culture medium to ensure uniform growth of each plant.

[0054] ②T1 generation sample collection and DNA extraction At the T1 seedling stage, new leaves (about 0.1 g) were collected from each plant, and genomic DNA was extracted again using the aforementioned CTAB method to ensure stable sample quality.

[0055] ③PCR amplification and genotype identification The target region of T1 generation plants was amplified by PCR using the same set of identification primers, and the amplified products were subjected to Sanger sequencing.

[0056] Based on the sequencing results, the genotype of the target region of each plant was determined to distinguish between wild type, heterozygous type and homozygous mutant types.

[0057] Homozygous mutants with the expected mutations in all alleles were screened, and the mutation type of each plant (e.g., frameshift caused by a specific small deletion or insertion) was recorded in detail.

[0058] 3. Acquisition and confirmation of T2 generation stable materials ①Self-pollination purification A batch of homozygous T1 mutant plants were selected for self-pollination, and T2 generation seeds were collected to ensure that the mutants were genetically stable in subsequent generations.

[0059] ② Repeat detection of T2 generation DNA extraction, PCR amplification, and sequencing were repeated on the T2 generation plants to verify whether the homozygous mutation in the target region remained stable.

[0060] At the same time, phenotypic observations were performed on the T2 generation plants to record whether growth or disease phenotypes associated with resistance changes appeared, so as to verify the results of molecular testing.

[0061] 4. Data recording and statistical analysis During the screening process of each generation, the seed germination rate, PCR test results, sequencing information and resistance phenotypic data of each plant were recorded in detail.

[0062] Statistical software was used to analyze the data of each group to determine the proportion of homozygous mutants and their correlation with the brown planthopper resistance phenotype, providing reliable data support for subsequent functional verification.

[0063] This example begins with preliminary screening of T0-generation mutants and describes in detail the complete process of genomic DNA extraction, PCR amplification, Sanger sequencing, and self-pollination screening of T1 and T2 generations. Through rigorous molecular biological testing and statistical analysis, stable homozygous BPH50 knockout mutants were obtained, laying a solid foundation for subsequent functional verification of brown planthopper resistance and resistance breeding experiments.

[0064] Figure 3 Shown is the sequence analysis of homozygous BPH50 knockout mutants. The reference sequence is the genome of the insect-resistant variety 551 (Minicore number W4194). ORF9_KO#1 has a one-base insertion at 79 bp of the BPH50 nucleotide sequence, while ORF9_KO#6 has a three-base deletion at 83 bp of the BPH50 nucleotide sequence.

[0065] Example 3: Functional verification of BPH50 knockout mutant homozygous material against brown planthopper This example describes in detail how to functionally verify the brown planthopper resistance of homozygous BPH50 knockout mutant materials through field and laboratory experiments. The steps include material preparation, brown planthopper inoculation, phenotypic observation, data recording and statistical analysis, and molecular detection and verification. The specific steps are as follows: 1. Material Preparation ①Seed treatment and sowing a. Select the obtained BPH50 knockout homozygous mutant materials ORF9_KO#1 and ORF9_KO#6 (confirmed by the aforementioned T2 generation screening) and the control variety (insect-resistant material 551).

[0066] b. Surface disinfection of seeds: first soak in 70% ethanol for 10 minutes, then treat with 0.1% mercuric chloride solution for 30 minutes, and then rinse with plenty of sterile water.

[0067] c. Sow the sterilized seeds evenly in pre-sterilized nutrient soil or standard culture medium, ensuring that the number of seeds sown in each group is consistent (e.g., 20 seeds per group), and set up at least three replicates.

[0068] ② Seedling cultivation Place the seed container in a constant temperature growth chamber, control the temperature at around 28°C, and the photoperiod is 16 hours light / 8 hours dark.

[0069] b. Water regularly and keep the soil moist until the plant reaches the three-leaf stage (usually about 10 to 14 days). This is the best time to inoculate with brown planthoppers.

[0070] 2. Brown planthopper inoculation ① Source and pretreatment of brown planthopper Collect healthy brown planthopper nymphs from an insect-free room or a dedicated rearing room.

[0071] ②Inoculation method a. Evenly distribute each group of plants in an inoculation container (such as a seedling tray or seedling box) and cover the container with a 200-mesh fine gauze to prevent brown planthoppers from escaping.

[0072] b. When rice seedlings grow to the two-leaf, one-heart stage in soil or hydroponic culture, remove weak seedlings and inoculate with 2- to 3-year-old brown planthopper nymphs at a ratio of 1:10 between rice seedlings and brown planthoppers, ensuring that each plant is inoculated with the same number of nymphs.

[0073] c. Place the inoculated container in an artificial climate chamber, maintain the temperature at around 30°C, the humidity at around 70%, and the lighting conditions similar to those for seedling cultivation.

[0074] 3. Phenotypic Observation and Data Recording ① Phenotypic observation a. After inoculation, observe the growth of the plants daily.

[0075] b. Record the main phenotypic changes of each plant, such as leaf yellowing, wilting, lodging, and changes in plant height.

[0076] c. Compare the phenotypic differences between the BPH50 knockout mutant and the wild type under brown planthopper infestation, and take representative images for subsequent statistical analysis.

[0077] ② Resistance Scoring and Data Recording a. When the mortality rate of the susceptible control group exceeds 90%, the resistance level of the rice seedlings is counted and evaluated using the seedling group method. A score of 0 to 9 is assigned based on the degree of plant damage, where 0 indicates healthy plant growth with no damaged leaves and 9 indicates complete plant death.

[0078] b. Count the resistance scores of each group of plants and record the detailed data.

[0079] This example establishes a comprehensive experimental workflow for validating the brown planthopper resistance of homozygous BPH50 knockout mutants, from seed treatment and seedling culture to standardized brown planthopper inoculation and detailed phenotypic documentation. The results confirm the key role of BPH50 in brown planthopper resistance in rice and provide a solid foundation for subsequent resistance breeding and molecular mechanism research.

[0080] The results are as follows Figure 4 As shown in the figure, the brown planthopper resistance of two knockout mutant strains, ORF9_KO#1 and ORF9_KO#6, was identified at the seedling stage. The results showed that the leaves of the knockout mutant plants dried up, while the leaves of the wild-type insect-resistant material 551 (Minicore No. W4194) plants mostly remained green and grew normally, indicating that after the BPH50 gene was knocked out, the brown planthopper resistance of the insect-resistant material 551 (Minicore No. W4194) was significantly reduced, and the resistant phenotype was transformed into a highly susceptible phenotype, which fully demonstrated that the BPH50 gene plays a key role in brown planthopper resistance.

Claims

1. A rice brown planthopper resistance gene BPH50 , characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The rice brown planthopper-resistant gene according to claim 1 BPH50 , characterized in that: Its cDNA sequence is shown in SEQ ID NO.

2.

3. The rice brown planthopper-resistant gene according to claim 2 BPH50 , characterized in that: The amino acid sequence of the protein encoded by it is shown in SEQ ID NO.

3.

4. The rice brown planthopper-resistant gene according to any one of claims 1 to 3 BPH50 Application in reducing rice resistance to brown planthoppers.

5. The use according to claim 4, characterized in that: By knocking out the brown planthopper resistance gene in rice BPH50 , reducing rice's resistance to brown planthoppers.

6. The rice brown planthopper-resistant gene according to any one of claims 1 to 3 BPH50 Application in breeding transgenic rice with reduced resistance to brown planthopper.

7. The use according to claim 6, characterized in that: By knocking out the brown planthopper resistance gene in rice BPH50 , cultivating transgenic rice with reduced resistance to brown planthoppers.

8. A method for reducing the resistance of rice to brown planthoppers, characterized by: Including knocking out the rice brown planthopper resistance gene BPH50 , steps to reduce rice resistance to brown planthoppers; The rice brown planthopper resistance gene BPH50 The nucleotide sequence is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.

2.

9. A method for cultivating transgenic rice with reduced resistance to brown planthoppers, characterized by: Including knocking out the rice brown planthopper resistance gene BPH50 , steps to reduce rice resistance to brown planthoppers; The rice brown planthopper resistance gene BPH50 The nucleotide sequence is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.2.

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