Gene LOCOs06g17970 for regulating and controlling brown planthopper resistance of rice and application thereof

Abstract

The invention provides a gene LOCOs06g17970 for regulating and controlling brown planthopper resistance of rice and application of the gene LOCOs06g17970, and belongs to the technical field of plant biotechnology and crop improvement. Through metabonomics genome association analysis, it is found that the gene LOCOs06g17970 is significantly related to the damascone level, the damascone content can be increased by knocking out or inhibiting the gene, and the resistance of rice to brown planthopper is significantly enhanced. The invention provides a method for improving the brown planthopper resistance of rice through gene editing or exogenous damascone application on the basis of the gene LOCOs06g17970, the transgenic rice after the gene LOCOs06g17970 is knocked out or inhibited shows excellent resistance to brown planthoppers, and the main agronomic traits of the rice are not obviously influenced. The invention provides a novel molecular tool and a novel breeding strategy for developing brown planthopper resistant rice varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant biotechnology and crop improvement, and in particular to the regulation of the brown planthopper resistance gene LOC_Os06g17970 and its application in improving the resistance of rice to the brown planthopper. BACKGROUND

[0002] Rice (Oryza sativa L.) is one of the most important food crops globally, particularly in Asia, where it sustains over half of the human population. However, rice production has long been threatened by various pests, among which the brown planthopper (Nilaparvata lugens, BPH) is one of the most devastating pests. BPH feeds on rice phloem sap by piercing and sucking, causing plant growth inhibition, yellowing, and even death, and can induce "hopper burn" leading to up to 30% yield loss, especially in Asian rice regions. Since the Green Revolution in the 1960s, BPH has become one of the major limiting factors for rice production, severely threatening global food security.

[0003] To combat the damage caused by BPH, traditional breeding methods have successfully identified several resistance genes, such as Bph6, Bph9, and Bph30, by screening resistant germplasm resources. These genes enhance rice resistance to BPH by strengthening physical barriers (such as monosaccharide content in hard tissue cell walls) or chemical defenses (such as secondary metabolite accumulation). For example, Bph30 significantly enhances rice resistance to BPH by regulating the monosaccharide composition of hard tissue cell walls. However, the rapid evolution of BPH biotypes enables it to break through the defense of a single resistance gene, limiting the durability of resistant varieties. In addition, traditional breeding relies on phenotypic screening, which is time-consuming and inefficient, making it difficult to meet the urgent demand for highly resistant varieties in modern agriculture.

[0004] In recent years, the development of molecular biology and omics technologies has provided new opportunities for revealing the resistance mechanisms of rice to pests. Metabolomics, as a method for studying all metabolites in an organism, can directly reflect the physiological and metabolic changes of plants under pest stress, revealing the correlation between genotype and phenotype. Previous studies have shown that secondary metabolites (such as phenylpropanoids, flavonoids, and terpenoids) play a key role in plant pest defense. For example, the phenylpropanoid pathway enhances rice resistance to BPH by synthesizing lignin and salicylic acid, while flavonoids such as quercetin derivatives can reduce the feeding preference of pests. However, there have been few systematic studies on BPH resistance-related metabolites, especially the identification of key metabolites and their regulatory genes based on metabolomics genome-wide association study (mGWAS).

[0005] mGWAS is an efficient method integrating metabolomics and genomics, which can mine the genetic basis of metabolic diversity by associating metabolite levels with single nucleotide polymorphism (SNP) sites. Previous studies have made progress in rice yield, quality, and stress resistance using mGWAS, but its application in brown planthopper resistance is still immature. At the same time, the potential of metabolites as biomarkers in predicting plant resistance gradually emerges. For example, metabolomics data showed higher accuracy than genomic data in predicting the outcrossing advantage of rice yield. However, existing prediction models rely heavily on post-infection metabolic data, ignoring the predictive potential of metabolic differences under non-infection conditions, which limits the practical application of metabolomics in breeding.

[0006] In addition, the progress of gene editing technology (such as CRISPR / Cas9) provides the possibility for precise improvement of rice resistance. By knocking out genes that negatively regulate resistance, the defense ability of plants can be significantly improved. For example, knocking out the transcription factor OsSPL10 that regulates phenylpropanoid metabolism can enhance the resistance of rice to brown planthopper. However, there is currently no report of identifying and utilizing brown planthopper resistance genes through mGWAS and gene editing technology. Therefore, it is urgent to develop new resistance genes and their regulated metabolites to cope with the continuous threat of brown planthopper and promote the molecular breeding of resistant rice varieties.

[0007] Based on this, the present application systematically studies the resistance mechanism of rice to brown planthopper through metabolomics and mGWAS technology, and for the first time identifies the gene LOC_Os06g17970 significantly associated with the level of β-damascenone, and verifies its function through gene knockout and exogenous metabolite application. The present application fills the gap of metabolomics in the study of brown planthopper resistance and provides a new theoretical basis and technical tool for the development of efficient and sustainable resistant rice varieties. SUMMARY

[0008] To overcome the limitations of brown planthopper resistance genes in existing technologies, the low efficiency of traditional breeding, and the shortcomings of metabolomics in resistance mechanism research, the present application provides a gene LOC_Os06g17970 regulating rice resistance to brown planthopper and its application in improving rice resistance to brown planthopper, as well as a method and system for enhancing the resistance of rice to brown planthopper by regulating the gene LOC_Os06g17970 and β-damascenone. The present application identifies the association between the gene LOC_Os06g17970 and β-damascenone through metabolomics genome-wide association study (mGWAS), significantly improves the resistance of rice to brown planthopper using gene editing and exogenous metabolite application, and maintains the stability of major agronomic traits, providing precise and efficient tools for rice resistance breeding and pest control.

[0009] To achieve the above-mentioned purpose, the specific technical solutions of the present application are as follows:

[0010] In a first aspect, the present application provides a gene LOC_Os06g17970 for regulating resistance to brown planthopper in rice, wherein the nucleotide sequence of the gene LOC_Os06g17970 is shown in SEQ ID NO. 1, or a homologous sequence having at least 90% identity with the sequence shown in SEQ ID NO. 1. The sequence of the gene LOC_Os06g17970 is located on the sixth chromosome of rice, and is associated with the linkage disequilibrium region of SNPrs6_191562334.

[0011] In a second aspect, the present application provides an application of the gene LOC_Os06g17970 in improving the resistance of rice to brown planthopper, wherein the level of damascenone in rice is increased by knocking out or inhibiting the gene LOC_Os06g17970, and the resistance of rice to brown planthopper is enhanced.

[0012] In a third aspect, the present application provides an expression vector comprising the gene LOC_Os06g17970 or an antisense sequence thereof, for knocking out or inhibiting the gene LOC_Os06g17970.

[0013] In a fourth aspect, the present application provides a method for improving the resistance of rice to brown planthopper, wherein the level of damascenone in rice is increased by knocking out or inhibiting the gene LOC_Os06g17970, and the resistance of rice to brown planthopper is enhanced.

[0014] Further, the gene LOC_Os06g17970 is knocked out or inhibited by CRISPR / Cas9 or RNAi technology.

[0015] Further, damascenone is applied to rice seedlings to enhance the early resistance of rice to brown planthopper.

[0016] In a fifth aspect, the present application provides a method for preparing transgenic rice with resistance to brown planthopper, wherein the transgenic rice with resistance to brown planthopper is obtained by the method for improving the resistance of rice to brown planthopper.

[0017] Further, the agronomic traits of the transgenic rice with resistance to brown planthopper obtained by the method include seed setting rate, ear number per plant, thousand-grain weight, and plant height, which have no significant difference from wild type.

[0018] In a sixth aspect, the present application provides a kit for rice breeding, comprising primers or probes for detecting the expression of the gene LOC_Os06g17970 or the level of damascenone.

[0019] In a seventh aspect, the present application provides an application of the kit in breeding rice with resistance to brown planthopper.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] The present application finds that gene LOC_Os06g17970 is significantly related to damascenone level through metabolomics genome-wide association analysis, and knocking out or inhibiting the gene can increase the damascenone content and significantly enhance the resistance of rice to brown planthopper. The present application provides a method for improving the resistance of rice to brown planthopper based on gene LOC_Os06g17970 through gene editing or exogenous application of damascenone, and transgenic rice with knocked out or inhibited gene LOC_Os06g17970 shows excellent resistance to brown planthopper, and the main agronomic traits of the rice are not significantly affected. The present application provides a new molecular tool and breeding strategy for developing brown planthopper-resistant rice varieties. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Distribution of brown planthopper resistance levels of 168 rice varieties;

[0023] Figure 2 Manhattan plot based on metabolite genome-wide association analysis;

[0024] Figure 3 Trend scatter plot of damascenone content in rice under non-brown planthopper infestation conditions;

[0025] Figure 4 Trend scatter plot of damascenone content in rice under brown planthopper infestation conditions;

[0026] Figure 5 Body weight and honeydew secretion results of brown planthopper after feeding on rice with exogenously applied damascenone for 48 h;

[0027] Figure 6 Expression pattern diagram of gene LOC_Os06g17970;

[0028] Figure 7 Mutant forms of mutant strains #7, #21, and #43;

[0029] Figure 8 Seedling stage resistance test results of gene LOC_Os06g17970 knockout strains;

[0030] Figure 9 Resistance scores of gene LOC_Os06g17970 knockout strains and wild type C521 at the seedling stage;

[0031] Figure 10 Body weight and honeydew secretion results of brown planthopper after feeding on gene LOC_Os06g17970 knockout strains and wild type C521 for 48 h;

[0032] Figure 11The damascenone content of the gene LOC_Os06g17970 knockout strain and wild type C521;

[0033] Figure 12 The results of the agronomic trait evaluation of the gene LOC_Os06g17970 knockout strain and wild type C521. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0035] The present application provides a gene LOC_Os06g17970 for regulating resistance of rice to brown planthopper, the nucleotide sequence of the gene LOC_Os06g17970 is the sequence shown in SEQ ID NO. 1, or a homologous sequence having at least 90% or more of the sequence shown in SEQ ID NO. 1. The sequence of the gene LOC_Os06g17970 is located on the sixth chromosome of rice, and is related to the linkage disequilibrium region of SNP rs6_191562334. By knocking out or inhibiting the gene LOC_Os06g17970, the damascenone level in rice is increased, and the resistance of rice to brown planthopper is enhanced.

[0036] Example 1: Identification of the association between LOC_Os06g17970 and damascenone by mGWAS

[0037] Rice samples from different geographical regions, ecological environments and genetic backgrounds were collected and grouped according to the resistance phenotype to brown planthopper. The resistance phenotype was indicated by the weight gain rate (WGR) of brown planthopper, and was divided into four categories: resistance (R, WGR < 0.3), medium resistance (MR, 0.3 ≤ WGR < 0.6), medium sensitivity (MS, 0.6 ≤ WGR < 0.9) and sensitivity (S, WGR > 0.9). The present application takes 168 rice varieties (127 indica rice, 24 intermediate type, 11 japonica rice and 6 javanica rice) as the research object, and evaluates the resistance level by 48 h brown planthopper feeding experiment (the distribution of the resistance level of brown planthopper of 168 rice varieties is shown in Figure 1 ), to ensure the reliability of the phenotype data

[0038] Metabolites from rice leaf sheath tissues (including normal growth and 48 h after brown planthopper feeding) were detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS) in both positive and negative ion modes, yielding 3,341 metabolic features. Metabolite data were standardized (logarithmic transformation, mean centering, and standard deviation normalization). Metabolomics-related genome association analysis (mGWAS) was used to screen for resistance-related metabolites and genes. Results are shown in [Figure missing]. Figure 2 The figure shows that the single nucleotide polymorphism (SNP) site rs6_191562334 was significantly associated with the level of damascone (NEG-656) (r = -0.23, p = 0.003 under non-infected conditions, and r = -0.19, p = 0.012 under infected conditions).

[0039] Linkage disequilibrium (LD) analysis identified 30 genes within a 10 kb range upstream and downstream of rs6_191562334. Among them, the gene LOC_Os06g17970, located on rice chromosome 6 and encoding the protein CC-NBS-LRR, was selected as a candidate gene. LASSO regression analysis further confirmed the negative correlation between SNPs near this gene and damascone levels, indicating that LOC_Os06g17970 can act as a negative regulator affecting rice resistance to brown planthoppers (scatter plots showing the trends of damascone content in rice under non-brown planthopper infestation and brown planthopper infestation conditions are shown below). Figure 3 and Figure 4 (As shown).

[0040] Example 2: Exogenous application of damastones enhances rice resistance to brown planthopper.

[0041] A 5 μM damascone ethanol solution (the control group received the same amount of ethanol) was applied to the leaf sheaths of the susceptible rice variety TN1, once every 30 minutes for a total of three times. After 48 hours, the body weight and honeydew secretion of brown planthoppers were measured. The results are shown below. Figure 5 . Figure 5 The results showed that, compared with the control group, the brown planthopper weight gain was significantly reduced in the damascone-treated group (p<0.05) and the honeydew secretion was reduced (p<0.01), indicating that exogenous application of damascone can significantly enhance the resistance of rice to brown planthopper.

[0042] Example 3: CRISPR / Cas9 knockout of LOC_Os06g17970 enhances rice resistance to brown planthopper.

[0043] The gene LOC_Os06g17970 was knocked out in the rice variety C521 using CRISPR / Cas9 technology (see the expression pattern diagram of gene LOC_Os06g17970). Figure 6). A 23 bp sequence (5'-GCTTCATAGTCAGTGCCCCTAGG-3') targeting the third exon was designed, and pYLCRISPR / Cas9Pubi-H vector was constructed and transformed into C521. Three T2 generation mutants (#7, #21, #43) were obtained, and the mutant forms of #7, #21, #43 are shown in Figure 7 , #7, #21, #43 carried 13 base deletions, 1 base deletion and 1 base insertion, respectively, resulting in premature termination of the gene. The results of the rice seedling stage resistance test are shown in Figure 8 , Figure 8 , showing that the wild type C521 died after 10 days of BPH infection, while the knockout strains #7, #21, #43 all survived, and the resistance score (see Figure 9 ) decreased, indicating that the plant resistance was significantly enhanced (p<0.05). After BPH feeding on knockout strains for 48 h, the body weight and honeydew secretion of BPH are shown in Figure 10 , Figure 10 , showing that the body weight and honeydew secretion of BPH were significantly reduced (p<0.05). LC-MS analysis showed that the results are shown in Figure 11 , Figure 11 , showing that the damascenone level in knockout strains was significantly increased, and further increased after BPH infection, indicating that the resistance of rice to BPH can be enhanced by knocking out the gene LOC_Os06g17970.

[0044] Example 4: Agronomic trait evaluation

[0045] The T2 generation knockout strains (#7, #21, #43) obtained in Example 3 were evaluated for agronomic traits, including percent of filled grain, no. panicles per plant, 1000-grain weight and plant height. The evaluation results are shown in Figure 12 , Figure 12 , showing that compared with the wild type C521, the plant height of the knockout strains was slightly reduced (p<0.05), but there was no significant difference in the percent of filled grain, no. panicles per plant and 1000-grain weight, indicating that knocking out the gene LOC_Os06g17970 did not significantly affect the main yield traits of rice.

[0046] In summary, the transgenic rice with knocked out or inhibited gene LOC_Os06g17970 according to the present application showed excellent resistance to BPH, and the main agronomic traits of the rice were not significantly affected. The present application provides a new molecular tool and breeding strategy for developing BPH-resistant rice varieties.

[0047] The above detailed description describes the implementation of the present application, but the present application is not limited to the specific details in the above implementation. Within the scope of the claims and technical concepts of the present application, various simple modifications and changes can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for modulating the brown planthopper resistance gene LOC_Os06g17970 in rice, characterized in that, The nucleotide sequence of the gene LOC_Os06g17970 is the sequence shown in SEQ ID NO. 1, or a homologous sequence having at least 90% identity with the sequence shown in SEQ ID NO.

1.

2. The use of the gene LOC_Os06g17970 in improving the resistance of rice to Nilaparvata lugens according to claim 1, characterized in that, The resistance of rice to Nilaparvata lugens is improved by knocking out or inhibiting the gene LOC_Os06g17970.

3. An expression vector, characterized by, The gene LOC_Os06g17970 or its antisense sequence of claim 1 is included.

4. A method for improving the resistance of rice to Nilaparvata lugens, characterized by, The level of damascenone in rice is improved by knocking out or inhibiting the gene LOC_Os06g17970 of claim 1, and the resistance of rice to Nilaparvata lugens is enhanced.

5. The method for improving rice resistance to brown planthopper according to claim 4, characterized in that, The gene LOC_Os06g17970 is knocked out or inhibited by CRISPR / Cas9 or RNAi technology.

6. A method for improving rice resistance to brown planthopper according to claim 4, characterized in that, Damascenone is applied to rice seedlings to enhance the early resistance of rice to Nilaparvata lugens.

7. A method for preparing transgenic rice resistant to brown planthoppers, characterized in that, The transgenic rice with resistance to Nilaparvata lugens is obtained by the method of any one of claims 4-6.

8. The method of claim 7, wherein the transgenic rice having resistance to the brown planthopper is prepared by introducing into the rice genome a DNA construct comprising a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having 95% or more identity to the nucleotide sequence of SEQ ID NO:

1. The agronomic traits of the obtained transgenic rice with resistance to Nilaparvata lugens include seed setting rate, panicle number per plant, 1000-grain weight, and plant height, which have no significant difference with wild type.

9. A kit for breeding of rice, characterized by, The primers or probes for detecting the expression of the gene LOC_Os06g17970 of claim 1 or the level of damascenone are included.

10. The kit of claim 9 is used in the breeding of rice with resistance to Nilaparvata lugens.

Images