Rice planthopper-resistant gene rph715 and application thereof

Abstract

This invention discloses a rice planthopper resistance gene, RPH715, and its application. Using a planthopper-resistant indica rice restorer line, Shuhui 715 (R715), as experimental material, resistance studies were conducted on R715 through resistance identification, genetic analysis, and gene mapping. Furthermore, gene editing technology was used to discover the novel planthopper resistance gene, RPH715. The nucleotide sequence of this rice planthopper resistance gene, RPH715, is shown in SEQ ID NO: 1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 2. This invention also protects a method for breeding planthopper-resistant rice based on this gene. The rice material containing this resistance gene provided by this invention and the molecular marker for this resistance gene developed by the inventors can be used to create new planthopper-resistant rice varieties. Additionally, this gene can also be used to produce transgenic planthopper-resistant rice. This planthopper resistance gene provides a necessary component for constructing novel rice planthopper-resistant breeding systems and has significant implications for production practice.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and plant insect-resistant breeding. Specifically, it relates to a rice planthopper-resistant gene RPH715 and its application. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's three major food crops. Currently, nearly half of the world's population relies on rice for sustenance, and the global rice cultivation area is approximately 167 million hectares. 2 China's rice planting area in 2020 was approximately 30 million hectares. 2 Rice not only meets the basic needs of the Chinese people, but also, through international trade, is widely exported, providing crucial support for my country's economic development and growth. Since the 20th century, the global rice industry has seen simultaneous growth in acreage, total output, yield per unit area, consumption, and imports and exports. Therefore, rice production plays an extremely important role in ensuring food security and improving people's living standards.

[0003] Crop diseases and pests have always been a significant threat to food security. Global warming and adjustments in agricultural planting structures have led to more frequent outbreaks and epidemics of these pests. Long-term practice has proven that utilizing disease- and pest-resistant genes to breed resistant varieties and improve crop resistance is the most economical, effective, and sustainable development strategy for controlling pests and diseases.

[0004] The rice planthopper, also known as the rice leafhopper, is a species of insect belonging to the family Delphacidae within the order Homoptera. It is an insect with piercing-sucking mouthparts and is one of the most serious pests in rice production. On September 15, 2020, the brown planthopper was listed as a Class A crop pest by the Ministry of Agriculture and Rural Affairs of China. Research indicates that the rice planthopper migrated from *Leymus chinensis* to *Oryza* plants approximately 250,000 years ago, and rice has since become its primary host. Historically, the rice planthopper has been a secondary pest in rice production. In recent decades, due to changes in rice varieties and production methods, it has developed into one of the most serious pests affecting rice production. Furthermore, it has been found that rice planthoppers can exhibit pathogenic variations, forming populations—biotypes—that adapt to resistant varieties. Therefore, durable resistance is a major challenge in global rice breeding for resistance to rice planthoppers.

[0005] Currently, my country's control strategies for rice planthoppers include chemical control, biological control, agricultural control, and breeding resistant varieties. Biological control refers to strengthening the protection and utilization of natural enemies and other beneficial organisms of rice planthoppers to achieve control, such as protecting natural enemies like frogs and spiders, and raising ducks in rice paddies. However, these measures are difficult for farmers to accept due to their operational complexity and cost. Agricultural control involves scientific water and fertilizer management in rice paddies, timely field drying, avoiding excessive nitrogen fertilizer application, preventing excessive vegetative growth in the later stages of rice growth, and creating ecological conditions unfavorable to the breeding and reproduction of rice planthoppers, thereby controlling them. However, this method is not very effective in controlling rice planthoppers. Breeding resistant varieties aims to enable rice to resist rice planthoppers itself. Currently, most rice varieties planted have poor resistance to rice planthoppers, and control mainly relies on chemical insecticides. However, the overuse of chemical pesticides not only severely pollutes the environment, leading to pesticide residues in rice and seriously affecting rice quality, but also kills off a large number of natural enemies of rice planthoppers and easily induces resistance in rice planthoppers, resulting in even more rampant infestations. Therefore, utilizing the inherent insect resistance of rice varieties is the safest method for controlling rice planthoppers, without affecting rice quality or the environment. The application of insect-resistant varieties is the most economical and effective control measure. Cloning rice planthopper-resistant genes and elucidating the resistance mechanism are of great significance for the genetic improvement of rice planthopper resistance.

[0006] Currently, 52 genes related to rice planthopper resistance have been reported, of which 48 have been mapped and 17 have been cloned, including 10 major genes. Chinese researchers, including Du et al., were the first in the world to clone the planthopper resistance gene Bph14. Bph14 activates the salicylic acid signaling pathway after brown planthopper infection, inducing callose deposition in phloem cells and the production of trypsin inhibitors, thus reducing the brown planthopper's feeding, growth rate, and lifespan. Ji et al. also successfully cloned the BPH18 gene located on the long arm of chromosome 12 using map-based cloning technology. BPH18 encodes a CC-NBS-NBS-LRR protein. Os12g37290 and Os12g37280 together constitute the BPH18 gene. Os12g37290 encodes the NBS domain, and Os12g37280 encodes the LRR domain. BPH18 and BPH26 are functionally distinct alleles. BPH18 participates in both anti-tropism and resistance mechanisms, conferring resistance to brown planthoppers on rice. Bph3 is the major gene for resistance to brown planthoppers in rice, identified from Rathu Heenati, and inherited independently of Bph1. Cloning revealed that Bph3 is a gene cluster composed of three plasma membrane lectin receptor kinase genes: OsLecRK1, OsLecRK2, and OsLecRK3. Zhou et al. identified 3502 associated SNPs and 59 associated loci through GWAS analysis and cloned and verified the novel planthopper resistance gene Bph37 via transgenic analysis. Ren et al. identified and cloned a new rice planthopper resistance gene in the rice variety Ptb33, naming it Bph32. Wang et al. located a rice planthopper resistance gene in the indica rice variety AC-1613, naming it Bph30. Shi et al. successfully cloned Bph30; in rice containing Bph30, the planthopper's mouthparts had difficulty penetrating the thick-walled tissue to reach the phloem. They also cloned another member of this family, Bph40, through genome-wide association analysis, which also exhibited good resistance. Wang et al. used map-based cloning to precisely locate a resistance gene locus within a 24kb region on the short arm of chromosome 6. They cloned BPH29, a resistance gene containing a B3 DNA-binding domain, through genetic analysis and transgenic experiments. Hu et al. discovered that the brown planthopper-inducing gene Bphi008a enhances rice resistance to brown planthoppers, acting downstream of the ethylene signaling pathway and located in the cell nucleus. Tong et al. identified and analyzed the function of the rice HPL3 gene, which encodes a lipid hydroperoxide lyase (HPL) OsHPL3 / CYP74B2. This HPL hydrolyzes linolenic acid (LA) to produce green leaf volatiles (GLVs). OsHPL3 regulates rice's specific defense responses to different intruders by affecting the levels of JA, GLVs, and other volatiles, positively regulating resistance to rice planthoppers. Guo et al. discovered the important role of OM64 in resistance to rice planthoppers and rice stem borers.Xin et al. cloned a xylanase inhibitory protein (XIP) gene, OsHI-XIP, which encodes a protein located in the endoplasmic reticulum (ER). Overexpression of OsHI-XIP reduced the feeding and oviposition behavior of rice planthoppers, but did not affect the growth and development of rice plants. Even though scientists have cloned some rice planthopper-resistant genes, this is far from sufficient to address the current state of planthopper infestation. Therefore, discovering and cloning new rice planthopper-resistant genes and realizing their breeding applications is urgently needed. Summary of the Invention

[0007] In view of this, the present invention addresses the aforementioned problems by locating and identifying a rice planthopper resistance gene, RPH715, along with its DNA sequence and the sequence of its encoded protein. The RPH715 gene has the function of regulating rice resistance to planthoppers. Utilizing this gene to generate new rice planthopper-resistant lines has significant application value in future planthopper-resistant rice breeding practices.

[0008] The use of the protein encoded by the gene DNA sequence or related DNA sequence provided by this invention is for:

[0009] The gene RPH715 provided by this invention is a rice resistance to rice planthopper-related gene. The DNA sequence of RPH715 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In rice, this gene is located on chromosome 4.

[0010] The present invention also includes any of the following DNA sequences a)-c): a) a DNA sequence having at least 90% (preferably at least 95%) sequence similarity to the above-described RPH715 gene sequence and having the same function; b) a DNA sequence capable of hybridizing with the DNA of the sequence described in (a) under stringent conditions; c) a DNA sequence complementary to any of the above-described sequences.

[0011] Those skilled in the art should understand that the rice planthopper resistance gene described in this invention comprises a highly homologous functional equivalent sequence that is highly homologous to the RPH715 gene and has the same resistance regulatory function. The highly homologous functional equivalent sequence comprises a DNA sequence capable of hybridizing with the nucleotide sequence of the RPH715 gene disclosed in this invention under stringent conditions. The “stringent conditions” used in this invention are well-known and include, for example, hybridization at 60°C for 12-16 hours in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA, followed by washing at 65°C for 15-60 minutes with a washing solution containing 0.1% SDS and 0.1% SSC.

[0012] The functional equivalent sequence also includes a DNA sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to the sequence shown in the RPH715 gene disclosed in this invention, and has a regulatory function on resistance to rice planthoppers, and can be isolated from any plant. The percentage of sequence similarity can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), the Needleman-Wunsch global alignment method (J.Mol.Biol., 48(3): 443-53, 1970), the Smith-Waterman local alignment method (J.Mol.Biol., 147: 195-197, 1981), the Pearson and Lipman similarity search method (PNAS, 85(8): 2444-2448, 1988), and the algorithm of Karlin and Altschul (Altschul et al., J.Mol.Biol., 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.

[0013] The gene sequences described in this invention can be isolated from any plant, including but not limited to Brassica, maize, wheat, sorghum, Capsella, white mustard, castor bean, sesame, cottonseed, flaxseed, soybean, Arabidopsis, Common bean, peanut, alfalfa, oats, rapeseed, barley, oats, rye, millet, sorghum, triticale, single wheat, spelt, double wheat, flax, gramma grass, frisbee, false sorghum, fescue, perennial wheatgrass, sugarcane, cranberry, papaya, banana, safflower, oil palm, cantaloupe, apple, cucumber, dendrobium, gladiolus, chrysanthemum, lily family, cotton, eucalyptus, sunflower, brassica, beet, coffee, ornamental plants, and pine trees. Preferably, the plants include maize, soybean, safflower, mustard, wheat, barley, rye, rice, cotton, and sorghum.

[0014] The application described in this invention is as follows: This invention provides the nucleotide sequence of a rice planthopper resistance gene RPH715 and the protein encoded by its gene, and also provides a rice material containing this resistance gene, Shuhui 715 (R715). This invention relates to the use of a planthopper-resistant rice material in rice seed production, specifically, including but not limited to, in certain application embodiments, using the rice material R715 containing the planthopper resistance gene RPH715 as a parent for hybridization breeding, and combining the molecular marker of this resistance gene to create new planthopper-resistant rice. Furthermore, the RPH715 gene provided by this invention can be used to create transgenic plants to obtain new planthopper-resistant rice.

[0015] Compared with the prior art, the present invention can achieve the following technical effects:

[0016] 1) This invention discovers and identifies the rice planthopper resistance gene RPH715 by combining resistance identification, genetic analysis and gene mapping of materials resistant to rice planthoppers with molecular techniques.

[0017] 2) This invention utilizes the rice planthopper resistance gene RPH715, combined with molecular marker-assisted breeding methods, to create a new rice material that also possesses resistance to rice planthoppers. This is of great significance for achieving rice resistance to rice planthopper damage and producing high-yield and high-quality rice, and has very important application value in rice disease and pest resistance breeding.

[0018] Of course, any product implementing this invention does not necessarily need to achieve all of the technical effects described above at the same time. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 This invention relates to the in vitro identification of single-plant rice planthopper-resistant material R715. (A1), (B1), and (C1) represent the growth of R715, TN1, and MH86 before planthopper infestation, respectively; (A2), (B2), and (C2) represent the growth of R715, TN1, and MH86 6 days after planthopper infestation, respectively.

[0021] Figure 2 This invention relates to the seedling group identification of rice material R715, which is resistant to rice planthoppers, and rice material TN1, which is susceptible to rice planthoppers. (A) shows the growth of R715 (left) and TN1 (right) before rice planthopper infection; (B) shows a comparison of the growth of TN1 (left) and R715 (right) 7 days after rice planthopper infection.

[0022] Figure 3 This invention relates to the field group resistance identification of rice materials R715 (resistant to rice planthoppers), TN1 (susceptible to rice planthoppers), and MH86 (moderately resistant to rice planthoppers). The comparison shows the growth of rice after planthopper infestation of R715 (left), TN1 (middle), and MH86 (right).

[0023] Figure 4 This is a comparison of individual plants of the mature rice materials R715 (resistant to rice planthoppers), TN1 (susceptible to rice planthoppers), and MH86 (moderately resistant to rice planthoppers) after being infested by rice planthoppers. Scale bar: 20cm.

[0024] Figure 5This invention utilizes SSR molecular markers to locate the chromosome 4 of rice.

[0025] Figure 6 This invention utilizes BSA resequencing technology to analyze the distribution of two offspring and their Δindex association values ​​on chromosomes;

[0026] Figure 7 This is the fine localization of the resistance gene of R715 in this invention on chromosome 4;

[0027] Figure 8 This invention describes the specific mutation details of the resistance gene knockout target sequence and the target sequence of the knockout mutant plant.

[0028] Figure 9 This invention relates to the seedling survival rate of a single gene knockout plant in vitro for insect resistance identification.

[0029] Figure 10 This invention relates to the in vitro insect resistance identification of single plants with the R715 resistance gene knocked out. (A), (B), (C), (D), (E), and (F) represent the growth of TN1, R715, KO-1, KO-21, KO-28, and KO-31 before rice planthopper infestation, respectively; (G), (H), (I), (J), (K), and (L) represent the growth of TN1, R715, KO-1, KO-21, KO-28, and KO-31 seven days after rice planthopper infestation, respectively.

[0030] Figure 11 This invention compares the phenotypes of R715 resistance gene knockout plants, R715 wild-type plants, and TN1 plants after 7 days of infestation by rice planthoppers. (A), (B), (C), (D), (E), and (F) represent the phenotypes of TN1, R715, KO-1, KO-21, KO-28, and KO-31 after 7 days of rice planthopper infestation, respectively.

[0031] Figure 12 It is a cross-sectional view of the leaf sheath tissue of rice seedlings and a magnified view of the vascular bundles and sclerenchyma tissues.

[0032] Figure 13 These are cross-sections of leaf sheath sclereidium from resistant and knockout plants. Scale bar: 20 μm.

[0033] Figure 14 This is an electrophoresis image of recombinant inbred lines containing resistance genes screened using molecular markers according to the present invention;

[0034] Figure 15This invention presents the in vitro insect resistance identification of single plants of recombinant inbred lines containing the resistance gene RPH715 screened out in this invention. Among them, (A), (B), (C), (D), (E), (F), (G), (H), (I), and (J) represent the growth status of TN1, R715, R238, R600, MaS, Qianlidao, 1, 2, 3, and 4 before rice planthopper infestation, respectively; (K), (L), (M), (N), (O), (P), (Q), (R), (S), and (T) represent the growth status of TN1, R715, R238, R600, MaS, Qianlidao, 1, 2, 3, and 4 6 days after rice planthopper infestation, respectively. Detailed Implementation

[0035] The following will describe the implementation of the present invention in detail with reference to the embodiments, so as to fully understand how the present invention uses technical means to solve technical problems and achieve technical effects and to implement it accordingly.

[0036] Example 1: Insect resistance identification of rice material Shuhui 715 (R715) resistant to rice planthoppers

[0037] 1. Seedling stage single-plant resistance identification

[0038] The inventors used R715, a rice material with high resistance to rice planthoppers bred in their laboratory (Biotechnology Laboratory of the Rice Research Institute, Sichuan Agricultural University), to identify its resistance to rice planthoppers. The experimental materials also used included the susceptible material TN1 (also known as Taichung No. 1), a commonly used rice material highly susceptible to rice planthoppers; and the control material MH86 (also known as Minghui 86), a moderately resistant rice material to rice planthoppers. Traditional seedling resistance identification involves one seedling per pot, with each pot covered by a transparent plastic bag. The method used in this experiment was an improvement on the traditional method. First, hydroponically cultured seedlings were cultivated until they reached the two-leaf stage. The roots of the seedlings were wrapped with cotton soaked in rice nutrient solution and placed in flat-bottomed, round-mouthed straight tubes (height: 150 mm, diameter: 28 mm). Ten tubes were used for each material to be tested, one seedling per tube, constituting one replicate. Three replicates were performed for each material. Five to six 2nd-3rd instar rice planthopper nymphs were introduced into each straight-row tube, and the tubes were sealed with rubber stoppers with ventilation holes. The tubes were placed in a culture room at 28℃ and 75% humidity, with rice nutrient solution added as needed. Each tube was observed daily, and the growth of the seedlings and rice planthoppers was recorded by observation and photography. When the mortality rate of TN1 seedlings reached 95% or higher, the resistance phenotype of each seedling was evaluated according to the rice insect resistance rating standards established by the International Rice Research Institute (Table 1). Then, a weighted average was calculated to evaluate the resistance level of each line, and the resistance level of each line was determined based on the resistance level. Resistance level 0 represents immunity; resistance levels 1-3 represent insect resistance; resistance levels 3.1-5 represent moderate resistance; resistance levels 5.1-7 represent moderate susceptibility; and resistance levels 7.1-9 represent high susceptibility. The experimental results are as follows: Figure 1 As shown, the results indicated that 6 days after rice planthopper infection, the leaves of susceptible TN1 seedlings turned yellow and withered, with over 95% dying; the mortality rate of control MH86 seedlings was 48.35%, with surviving seedlings showing some yellowing leaves, indicating that rice planthopper damage was more severe; while the mortality rate of resistant R715 seedlings was only 17.25%, with surviving seedlings still having bright green leaves and few yellowing leaves, indicating that rice planthoppers did not cause significant harm to their growth and development. Through statistical analysis of the phenotypic characteristics of individual plants in multiple experiments and the statistical analysis of seedling survival rates, it was concluded that the average resistance level of R715 seedlings was 2.5, classifying it as a resistant material; the average resistance level of MH86 seedlings was 5, classifying it as a moderately resistant material; and the average resistance level of TN1 seedlings was 9, classifying it as a susceptible material.

[0039] Table 1. Rice Seedling Stage Resistance to Rice Planthopper Rating Table

[0040]

[0041] The inventors, using a hydroponic seedling method, sowed the sprouting seeds into 96-cell hydroponic boxes, with one box each of R715 and TN1 forming one group, for a total of four groups. Three groups were used for experimental replicates, and one group was kept as a backup. After sowing, the seedling growth was observed daily. If any seedlings were diseased, weak, or damaged, they were replaced from the backup hydroponic boxes to ensure consistent and robust growth of the tested seedlings. From 14 to 18 days after sowing, one box each of R715 and TN1 were placed in a dedicated rice planthopper rearing cage with 100-mesh mesh, inoculated with an average of 3-5 nymphs (2nd-3rd instar) per seedling. During the experiment, conditions suitable for the normal growth of rice planthoppers, such as an indoor temperature of 28℃ and a relative humidity of 75%, were maintained. Infection lasts approximately 10-14 days. When over 95% of TN1 seedlings have died, the resistance phenotype of each seedling is evaluated and the mortality rate is statistically analyzed, referring to the standards in Table 1. Then, a weighted average is calculated to evaluate the resistance level of each family. The resulting resistance score for each family is its phenotypic value. Results are as follows... Figure 2 As shown, TN1 plants almost completely withered and died 7 days after infestation by rice planthoppers, while most R715 plants grew healthily with no leaf damage; a few had one yellow leaf, and very few had one to two yellow leaves (or one leaf withered). Phenotypic observation and statistical analysis of each seedling used in the experiment showed that the average resistance rating of R715 at the seedling stage was 2.2, classifying it as a resistant material; while the average resistance rating of TN1 at the seedling stage was 9, classifying it as a susceptible material.

[0042] 3. Field group resistance identification

[0043] The inventors planted TN1, R715, and MH86 rice varieties in two rows each, with 10 plants per row (plant spacing 16.5 cm, row spacing 23 cm). One row of TN1 was planted after each material to attract planthoppers. Ten replicates were set up, with the planting order of each material randomized. Normal field management was implemented, but no pesticides were sprayed. After each rice material entered the jointing stage, TN1 stubble infested with numerous planthoppers was manually and evenly placed in the center of each plot, and observations were conducted daily. After TN1 plants began to wither and die, the growth of other materials was recorded daily. When the mortality rate of TN1 reached approximately 95%, the number of surviving individual plants in other materials was counted, and the plant mortality rate for each material was calculated (complete wilting and chlorosis were considered death). The results showed that TN1 plants began to die 34 days after planthopper infestation (when all materials had entered the mature stage), and after 7 days, a large proportion (80%) of TN1 plants had died. When over 95% of TN1 plants had died, the base of almost all MH86 rice stubble turned yellow and was covered in honeydew and sooty mold, but the upper leaves remained partially green, indicating a moderate level of resistance to rice planthoppers. In contrast, the base of R715 rice stubble was clean and free of planthoppers, honeydew, and sooty mold. Only some older leaves withered due to natural growth, and the upper leaves showed only normal physiological aging and yellowing; the other leaves remained a normal green. Therefore, it is evident that rice planthoppers caused almost no significant damage to R715. Figure 3 By the time the grains matured, all TN1 plants had died. R715 plants showed no obvious signs of damage, with only a few individual plants dying. MH86 plants suffered moderate damage, with most turning yellow and dying. Both R715 and the surviving MH86 plants produced grains, but most MH86 grains were empty, while R715 grains developed normally with few empty grains. Figure 4 Statistical analysis of these three materials revealed that MH86 has an average resistance rating of 5.8, classifying it as a medium-resistance material; while R715 has an average resistance rating of 2.0, classifying it as a high-resistance material (rating criteria are shown in Table 2).

[0044] Table 2. Field Rice Planthopper Resistance Identification and Rating Standards

[0045]

[0046] Example 2: Genetic analysis of resistance of Shuhui 715 (R715) to rice planthopper

[0047] The inventors conducted reciprocal crosses (R715×TN1 and TN1×R715) between R715 and the highly susceptible rice planthopper material TN1 to obtain F1 generation seeds. After sowing some of the F1 generation seeds from the reciprocal crosses, the dominant and recessive genetic relationships were observed through resistance testing. The remaining F1 generation seeds were either bagged and self-crossed or partially backcrossed with TN1 to obtain F2 generation seeds. R715, TN1, backcrosses of F1 generation seeds with TN1, and self-crossed F2 generation seeds were planted in the field to construct a genetic population. R715, TN1, and the F1 generation seeds from the reciprocal crosses were planted in 4 rows of 80 plants each; the backcrosses of F1 generation seeds with TN1 were planted in 15 rows of 300 plants each; and the F2 generation seeds from the reciprocal crosses were planted in 20 rows of 400 plants each, all planted individually. During planting, one row of TN1 was planted every four rows of material to attract rice planthoppers, ensuring the uniformity of planthopper distribution during the resistance testing. Starting from the jointing stage, TN1 seedlings carrying rice planthopper larvae were evenly placed in the field. After 14 days, the resistance of the reciprocal cross F1 generation was investigated and recorded, along with the number of plants in the F2 segregating generation exhibiting and not exhibiting rice planthopper resistance. The chi-square test was then used to verify whether the rice planthopper resistance trait exhibited a typical dominant-recessive inheritance relationship and cytoplasmic inheritance characteristics. Results showed that both R715 and TN1 reciprocal cross F1 generations showed high resistance to rice planthoppers, indicating that high resistance to rice planthoppers is a trait controlled by a dominant nuclear gene and is unrelated to cytoplasmic inheritance. The backcross offspring of the reciprocal cross F1 generation and TN1 showed the high resistance phenotype of R715 and the non-resistance phenotype of TN1 (Table 3). Out of a total of 300 plants, 160 plants showed the non-resistance phenotype and 140 plants showed the high resistance phenotype. The chi-square test showed that X0... 2 =1.2033 < X 2 0.05,1 =3.84, and its segregation ratio conforms to the theoretical ratio of 1:1. The backcross progeny of the reciprocal cross F1 generation and TN1 also showed the highly resistant rice planthopper phenotype of R715 and the highly susceptible rice planthopper phenotype of TN1 (Table 3). Among the total number of 298 plants, 141 plants showed the non-resistant rice planthopper phenotype and 157 plants showed the highly resistant rice planthopper phenotype. After chi-square test, X 2 =0.7550 < X 2 0.05,1 =3.84, and its segregation ratio also conforms to the theoretical ratio of 1:1. The F2 generation of the orthogonal F1 generation, after self-pollination, also exhibited both non-resistant and highly resistant phenotypes of rice planthoppers. Out of a total of 400 plants, 312 plants showed the highly resistant phenotype, and 88 plants showed the non-resistant phenotype. The chi-square test showed that Xb = 3.84, and its segregation ratio also conformed to the theoretical ratio of 1:1. 2 =1.7633 < X 2 0.05,1=3.84, and its segregation ratio conforms to the theoretical ratio of 3:1; the self-pollinated offspring of the reciprocal cross F1 also showed two phenotypes: one resistant to rice planthoppers and the other highly resistant to rice planthoppers. Out of a total of 399 plants, 309 plants exhibited the highly resistant phenotype, and 90 plants exhibited the resistant phenotype. The chi-square test showed that Xb = 3.84, which is consistent with the theoretical ratio of 3:1. 2 =1.1433 < X 2 0.05,1 =3.84, and its segregation ratio also conforms to the theoretical ratio of 3:1. This indicates that the inheritance of the high resistance to rice planthopper trait in the rice planthopper-resistant material R715 used in this study is controlled by a pair of dominant nuclear genes.

[0048] Table 3. Statistical table of planthopper resistance results in different generations of segregating populations.

[0049]

[0050] Example 3: Gene mapping of the rice planthopper resistance gene RPH715

[0051] 1. SSR-based gene mapping

[0052] First, polymorphic screening was performed on 600 pairs of SSR markers on the 12 chromosomes of rice synthesized in our laboratory (Biotechnology Laboratory, Rice Research Institute, Sichuan Agricultural University) for the resistant and susceptible parents R715 and TN1. A total of 121 pairs of SSR primers showed polymorphism, with a polymorphism rate of 20.2%. From the F2 generation population constructed from R715 and TN1, 30 leaf samples each of highly susceptible and highly resistant phenotypes were randomly selected. Rice DNA was extracted using the CTAB method to form resistance and susceptibility gene pools, respectively. DNA amplification was performed between the two pools using the 121 selected SSR markers. The results showed polymorphism at the RM551 and RM261 markers. PCR linkage analysis was then performed on 120 highly susceptible individuals from the F2 population using primers RM551 and RM261 and their upstream and downstream adjacent primers. The results showed that the resistance gene of R715 is located between the RM335 and RM261 markers on the short arm of chromosome 4. The mapping population was expanded, and RM335 and RM261 were placed in a large segregating population of 528 F2 individuals exhibiting high susceptibility for validation. Simultaneously, new polymorphic markers upstream and downstream of these two markers were designed for validation (Table 4). The results showed 29 recombinant single plants at RM261 and 25 at RM335. This indicates that markers upstream of RM335 and downstream of RM261 yielded more recombinant single plants, suggesting that the gene is located between RM335 and RM261, rather than at either end. Further marker screening revealed that the number of recombinant single plants decreased to 17 at RM3892, 20 at RM16478, and 13 at RM16465. Finally, 6 and 5 recombinant single plants were found at RM518 and RM8213, respectively. Therefore, we preliminarily determined that the resistance gene for R715 should be located between the two markers RM518 and RM8213, with corresponding physical locations of 2,021,760 bp and 4,441,848 bp on chromosome 4, respectively, and the total length of this region is approximately 2.42 M. Figure 5 ).

[0053] 2. Gene mapping based on BSA

[0054] Leaves were taken from individual plants of the parental lines R715 and TN1, as well as leaves from 40 highly resistant and 40 highly susceptible individual plants in the R715 / TN1 F2 population. These were mixed thoroughly and sent to Beijing Biomarker Biotechnology Co., Ltd. for phenotypic mapping studies based on BSA resequencing technology. Sequencing was performed using the Nipponbare genome as a reference. The distribution of SNP-index and InDel-index on chromosomes in the progeny was analyzed and mapped. The results showed clusters of variant sites between 2M and 6M on chromosome 4. Figure 6 ).

[0055] 3. Develop InDel markers for fine-grained positioning

[0056] Based on the results of map-based cloning, the gene was located between 2M and 4.4M on chromosome 4. Using BSA sequencing, the gene was further located between 2M and 6M. Combining these two methods, the localization interval was narrowed down to 2M-4.4M on chromosome 4. We developed InDel markers (Table 4) based on the BSA sequencing results for fine mapping. According to the rice genome sequence published on Rice Genome Browser (http: / / rice.plantbiology.msu.edu / cgi-bin / gbrowse / rice / #search), we downloaded 1000bp sequences (500bp before and after the mutation site). Using Primer Premier 5 software, we designed InDel primers and performed PCR amplification on the DNA of the parents and 538 highly susceptible F2 generation plants. The results were then detected by electrophoresis on a 3.5% TBE agarose gel. Five reciprocal colonies were found at marker indel4358, four at indel3946, four at indel3794, and two at indel2672. Finally, one reciprocal colony was found at position 2812347 bp using the developed marker indel2812, and two reciprocal colonies were found at position 3572644 bp using the developed marker indel3572. Therefore, we located this gene in the 760 kb region between markers indel2812 and indel3572 on chromosome 4. Figure 7 ).

[0057] Table 4 SSR and InDel-labeled primers

[0058]

[0059] Example 4: Functional verification of the rice planthopper resistance gene RPH715

[0060] 1. Analysis and identification of candidate genes

[0061] The inventors searched for all genes within the localized region (a 760kb region between indel2812 and indel3572 on the short arm of chromosome 4) and analyzed their functional annotations. They found that these genes were broadly classified into three categories: kinase genes, expressed protein genes, and transposon and retrotransposon protein genes. Based on the functional analysis of previously reported genes related to rice planthopper resistance, and combined with gene editing technology, the inventors identified the Nipponbare allele LOC_Os04g05580 (a kinase gene containing a lectin domain) in R715 as the rice planthopper resistance gene, naming it RPH715. Using bioinformatics techniques well-known to those skilled in the art, the inventors identified the complete rice planthopper resistance gene RPH715 from the rice R715 genome. After cDNA amplification and software analysis, its CDS was 1944bp, as shown in SEQ ID NO: 1, encoding a 648-amino acid rice planthopper resistance control protein, the sequence of which is shown in SEQ ID NO: 2. The results of whole nucleotide sequence analysis showed that the RPH715 gene is 4235 bp in length (SEQ ID NO: 3, including the 5'UTR and part of the 3'UTR).

[0062] 2. Functional verification of candidate genes

[0063] The inventors utilized gene editing technology, using R715 as a background, to knock out the resistance gene RPH715 to verify whether it was a gene for resistance to rice planthoppers. Specifically, using CRISPR / Cas9 technology, they first designed knockout target sites for the RPH715 gene (SEQ ID NO: 4, SEQ ID NO: 5), then constructed a knockout vector, and identified the recombinant vector. The knockout vector was then introduced into the genome of wild-type rice R715 using a rice transgenic method. After the regenerated plants were transplanted and survived, they were screened for transformation using hygromycin. Total DNA was extracted from leaves of 11 T0 generation positive plants. Target site 1 of the RPH715 gene in the R715 genome was amplified using primer pairs RPH715-gRNA-seq-F1 (SEQ ID NO: 6) and RPH715-gRNA-seq-R1 (SEQ ID NO: 7), and target site 2 was amplified using primer pairs RPH715-gRNA-seq-F2 (SEQ ID NO: 8) and RPH715-gRNA-seq-R2 (SEQ ID NO: 9). Sequencing verification was then performed. Homozygous mutant plants were screened, and seeds were retained after fruiting. The T1 generation plants from these mutants were used for further resistance identification against rice planthoppers, thus verifying the function of the RPH715 gene. Sequencing results showed that 5 plants were homozygous mutants and 6 plants were heterozygous mutants. The specific mutation details of the target sequence in the homozygous mutant plants are as follows: Figure 8As shown, plant KO-1 has a deletion of 13 bases; plant KO-21 has a deletion of 1 C base; plant KO-22 has a deletion of 1 C base; plant KO-28 has an insertion of 1 C base; and plant KO-31 has a deletion of 2 bases. The mutations in each plant number resulted in premature termination of the gene encoding the protein.

[0064] Single-plant in vitro insect resistance was assessed for four T1 generation homozygous mutant lines (KO-1, KO-21, KO-28, and KO-31) using the same method as described in Example 1. Experimental results ( Figure 9 The results showed that 6 days after rice planthopper infestation, the resistant material R715 showed a few dead plants, with a survival rate as high as 96.67% by the 7th day. In contrast, the susceptible material TN1 began to show dead plants 3 days after infestation, and almost all plants died after 7 days, with a survival rate of only 3.33%. Similarly, the knockout lines KO-1, KO-28, and KO31 also began to show dead plants 3 days after infestation, but the mortality trend was slower than that of TN1, with final seedling survival rates of 26.67%, 43.33%, and 50%, respectively. The knockout line KO-21 began to show dead plants 4 days after infestation, with a final seedling survival rate of 40%. This indicates that knocking out the resistance gene RPH715 significantly reduces the survival rate of plants after rice planthopper infestation. Furthermore, phenotypic observations were conducted on the various lines 7 days after infestation. Figure 10 , Figure 11 The resistance levels of each material were determined. The results showed that the insect-resistant material R715 only had one leaf that turned yellow, and its overall growth was not significantly affected. Based on the method described in Example 1, R715's resistance level was determined to be 1.87, classifying it as a resistant material. The susceptible material TN1 almost entirely withered and died, with a resistance level of 9, classifying it as a highly susceptible material. Most plants in the gene knockout lines KO-1 and KO-21 withered and died; surviving plants had 1-2 leaves that shrank, and the plants began to wilt, with growth significantly inhibited. Their resistance levels were 7.5 and 7.1, respectively, classifying them as highly susceptible materials. The surviving plants in the knockout lines KO-28 and KO-31 had yellowing leaves; in severe cases, the leaves curled and gradually began to wilt, with some plants withering and dying. Their resistance levels were 6.6 and 6.4, respectively, classifying them as moderately susceptible materials. In conclusion, plants with the RPH715 resistance gene knocked out have significantly reduced or even lost their resistance to rice planthoppers. Therefore, the gene RPH715 in the resistant rice material R715 is a rice resistance gene against rice planthoppers.

[0065] Example 5: Cytological observation of resistant materials and RPH715 knockout plants

[0066] To investigate whether the RPH715 gene induces resistance to rice planthoppers by affecting the development of rice tissue cells, the inventors conducted cytological observations of the leaf sheaths of susceptible (TN1) and resistant (R715) rice plants, as well as gene knockout plants in this context. Rice planthoppers are typical insects that feed on the phloem sap of vascular bundles, primarily damaging rice plants by probing and sucking sap from the phloem of the leaf sheaths. During the process of the stylet piercing the plant, saliva is secreted to form a salivary sheath, which seals the stylet within the phloem sieve tubes, serving as a feeding channel to extract sap from the phloem sieve tubes. Therefore, the inventors selected leaf sheaths from two-week-old rice seedlings as material for semi-thin sections. Figure 12 The image shows a cross-section and a magnified view of a rice leaf sheath. The magnified view shows the vascular bundles and sclerenchyma of the leaf sheath. The plant's vascular bundle system, composed of phloem and xylem, runs throughout the entire rice plant and is its main transport tissue, responsible for long-distance transport within the plant. The xylem, composed of xylem vessels, mainly transports water and dissolved inorganic salts, while the phloem, composed of sieve tubes and companion cells, mainly transports dissolved assimilates. The vascular bundles in the stem serve as transport channels for water, minerals, and organic nutrients, functioning as the "flow" in the "source-sink flow." The sclerenchyma contains multiple layers of cells with rigid secondary walls, forming a thick region to protect and mechanically support the internal tissues. It is located between the leaf sheath epidermis and the underlying vascular system.

[0067] After observing the slices of each material ( Figure 13 We found that the thick-walled tissue of the susceptible material TN1 consisted of two cell layers, with relatively large and few cells. In contrast, the thick-walled tissue of the resistant material R715 consisted of three cell layers, with a denser arrangement of smaller but more numerous cells. This undoubtedly enhanced the effectiveness of the secondary cell wall, making the thick-walled tissue stronger. Similarly, the robust thick-walled tissue made it more difficult for the rice planthopper's stylet to penetrate, thus preventing the planthopper from sucking large amounts of phloem sap. Observation of sections of the RPH715 gene knockout material revealed that, compared to the background material R715, the knockout lines KO-1, KO-21, and KO-28 had fewer than two cell layers in their thick-walled tissue, significantly reduced cell number, and lower density, greatly weakening the strength of the thick-walled tissue. This made it easier for the rice planthopper's stylet to penetrate the thick-walled tissue and suck phloem, exacerbating the damage caused by the rice planthopper. In conclusion, we believe that the RPH715 gene in the planthopper-resistant material R715 has a certain function in regulating the development of thick-walled tissue cells in rice leaf sheaths.

[0068] Example 6: Utilization of the rice planthopper resistance gene RPH715

[0069] The inventors used rice material R715, which contains the rice planthopper resistance gene RPH715, to create new rice materials resistant to rice planthoppers. They developed a pair of molecular markers, RPH715-M-F1 (SEQ ID NO: 10) and RPH715-M-R1 (SEQ ID NO: 11), to screen for rice varieties containing the resistance gene, utilizing the nucleotide sequence differences between the planthopper resistance gene and allele sequences in other rice materials. Specifically, R715 was used as one of the parents and crossed with four other rice materials—R238, R600, MaS, and Qianli Rice—that are not resistant to rice planthoppers but have significant breeding potential. The hybrid offspring were then self-pollinated over multiple generations to form recombinant inbred lines. After screening for recombinant inbred lines containing the rice planthopper resistance gene RPH715 using marker-assisted breeding, insect resistance was assessed.

[0070] The inventors screened recombinant inbred lines resistant to rice planthoppers based on the molecular markers RPH715-M-F1 (SEQ ID NO: 10) and RPH715-M-R1 (SEQ ID NO: 11). The experimental results ( Figure 14 The results showed that, except for the DNA electrophoresis bands of R238, R600, MaS, Qianli Rice, and TN1, which were inconsistent in size with the bands of the insect-resistant material R715, the DNA electrophoresis bands of the remaining lines numbered 1 (recombinant inbred line of R715 and R238), 2 (recombinant inbred line of R715 and R600), 3 (recombinant inbred line of R715 and MaS), and 4 (recombinant inbred line of R715 and Qianli Rice) were all consistent with the band pattern of the parental R715. This indicates that the rice planthopper resistance gene RPH715 has been successfully introduced into these recombinant inbred lines. Next, single-plant in vitro insect identification was performed on these materials containing the resistance gene. Figure 15 The resistance determination method was the same as described in Example 1. The results showed that the susceptible material TN1 had a resistance level of 9, classifying it as highly susceptible; the parental material R715 had a resistance level of 1.2, classifying it as resistant; the other four parental materials R238, R600, MaS, and Qianlidao had resistance levels of 5.4, 5.2, 6, and 5.8, respectively, all classifying them as moderately susceptible; and the recombinant inbred lines numbered 1, 2, 3, and 4 had resistance levels of 4, 3.6, 2.8, and 2.6, respectively, classifying them as resistant. These results indicate that the recombinant inbred lines incorporating the rice planthopper-resistant gene RPH715 exhibit significantly improved resistance to rice planthoppers compared to their respective non-resistant parents.

[0071] In summary, this invention, through resistance identification, genetic analysis, and gene mapping of resistant materials, combined with gene editing technology, discovered a new rice planthopper resistance gene, RPH715, and determined that it has the function of regulating rice resistance to rice planthoppers. The new recombinant inbred lines obtained by this invention using the rice planthopper resistance gene RPH715 also have resistance to rice planthoppers, which has very important applications in agricultural production.

[0072] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0073] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

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Claims

1. A rice planthopper resistance regulatory gene RPH715, characterized in that, The DNA sequence of the gene is shown in SEQ ID NO:

1.

2. A protein encoded by the rice planthopper resistance regulatory gene RPH715, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. An expression cassette, expression vector, or engineered bacteria containing the rice planthopper resistance regulatory gene RPH715 as described in claim 1.

4. The application of the rice planthopper resistance regulatory gene RPH715 as described in claim 1 in breeding to improve rice resistance to planthoppers.

5. A molecular marker primer pair for identifying whether rice contains the rice planthopper resistance regulatory gene RPH715 as described in claim 1, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively.

6. The application of the rice planthopper resistance regulatory gene RPH715 as described in claim 1 in the preparation of transgenic rice plants with planthopper resistance.

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