Rice brown planthopper-resistant gene Bph56, molecular marker closely linked to rice brown planthopper-resistant gene Bph56 and application

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a rice brown planthopper resistant gene Bph56, a molecular marker closely linked with the same and application of the rice brown planthopper resistant gene Bph56. The rice Bph56 gene provided by the invention is of great significance for cultivating a new variety of brown planthopper resistant rice. The rice Bph56 gene is successfully cloned through a map-based cloning method and is subjected to function annotation and identification, the gene is completely dominant, the homozygous and heterozygous genotypes endow rice with equal brown planthopper resistance, and the characteristic is beneficial to application of the gene in breeding. On the basis, the Bph56 gene can be introduced into a plant variety susceptible to brown planthopper through traditional breeding technologies such as hybridization, backcross and the like or genetic engineering means, and a novel brown planthopper-resistant plant variety with stable hereditary characters is obtained through cultivation. The invention provides important germplasm resources for effectively reducing insect pests and realizing yield increase and stable yield of crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering, in particular, to a brown planthopper resistance gene of rice Bph56 and a closely linked molecular marker thereof and application. BACKGROUND

[0002] The brown planthopper (Nilaparvata lugens) is a monophagous pest of rice, which causes plant growth potential to weaken, yield to decline, and even whole plant to die and absolute loss by sucking phloem sap through stylet. Controlling the development and damage of the brown planthopper is of great significance to ensure the safety of rice production.

[0003] For a long time, the control of the brown planthopper mainly relies on chemical pesticides. However, the significant increase in resistance of the brown planthopper to pesticides due to the large-scale use of pesticides year after year leads to a decline in control effect year by year. For example, the brown planthopper has developed strong resistance to imidacloprid, which was once the main effective pesticide. At the same time, chemical control also kills a large number of natural enemies in the rice field, which easily leads to the re-rampage of the brown planthopper population, further increasing the difficulty of control. Therefore, it is difficult to achieve sustainable management of the brown planthopper by relying solely on chemical means (Lou Yonggen and Cheng Jia'an, 2011).

[0004] It has been proven that using crop resistance genes to breed pest-resistant varieties is the most economical, effective and environmentally friendly strategy in pest control, and is also an important direction of agricultural green development (Zhang Jie et al., 2019; Deng Yiwu et al., 2021). The revised National Rice Variety Approval Standards in 2021 further highlight the green direction and safety of varieties, and clearly require the identification of brown planthopper resistance for rice varieties in southern rice-growing areas, and the inclusion of pest-resistant varieties in the classification and approval system of green and high-quality varieties.

[0005] Research on rice resistance to the brown planthopper began in the early 1970s, and more than 40 brown planthopper resistance genes have been identified from rice germplasm resources through molecular marker linkage analysis. Since the Wuhan University team first isolated the first rice brown planthopper resistance gene Bph14 using map-based cloning in 2009 (Du et al. 2009), more than ten brown planthopper resistance genes represented by Bph3, Bph6, Bph9 and Bph30 have been cloned, laying a foundation for the breeding of new rice varieties resistant to the brown planthopper.

[0006] Under the selection pressure of resistance genes, Nilaparvata lugens can cause harmful mutations to form new biotypes that can overcome the original resistance genes, which can lead to the loss of varietal resistance to pests. Such harmful mutations are one of the main challenges in the continuous prevention and control of Nilaparvata lugens. Therefore, it is of great significance to continue to explore new genes or excellent allelic variations in rice germplasm resources for resistance to Nilaparvata lugens, which can further enrich the resistance gene resources and develop new rice varieties with high resistance level. SUMMARY

[0007] The purpose of the present application is to provide a rice resistance gene Bph56 to Nilaparvata lugens and its closely linked molecular marker and application.

[0008] The present application adopts the method of forward genetics to construct a separation population of rice resistant to Nilaparvata lugens, and uses the method of map-based cloning to isolate the rice resistance gene Bph56 to Nilaparvata lugens. Bph56 Through genetic transformation of the genomic complementation vector, the susceptible rice plants exhibit the phenotype of resistance to Nilaparvata lugens, and the CRISPR / Cas9 technology is used to knockout the gene in the resistant near-isogenic line material containing the gene, which leads to a significant down-regulation of the resistance of the plant to Nilaparvata lugens, thereby confirming the function of the gene Bph56 to Nilaparvata lugens. Bph56 Bph56 Bph56 Bph56

[0009] In order to achieve the purpose of the present application, in the first aspect, the present application provides a rice Bph56 protein, which is: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4; or (b) a protein derived from (a) by substitution, deletion or addition of one or more amino acids and having equivalent function.

[0010] In the second aspect, the present application provides a rice resistance gene Bph56 to Nilaparvata lugens or a biological material containing the gene, wherein the gene encodes the Bph56 protein. Bph56 Bph56 Bph56 i) the nucleotide sequence shown in SEQ ID NO: 1-2 in series, the nucleotide sequence shown in SEQ ID NO: 3 or the nucleotide sequence shown in SEQ ID NO: 5; ii) the nucleotide sequence of i) which is substituted, deleted and / or increased by one or more nucleotides and expresses the same functional protein; ​​​​​​​​iii) A nucleotide sequence that hybridizes with the nucleotide sequence of i) under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. iv) Nucleotide sequences that have more than 90% homology with nucleotide sequences i), ii), or iii) and express the same functional protein; or, v) A nucleotide sequence that is completely complementary to the nucleotide sequence of i), ii), iii) or iv); The biological materials include, but are not limited to, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0011] Thirdly, the present invention provides the Bph56 protein, the gene... Bph56 Or any of the following applications of the biomaterial: (1) Improve plant resistance to brown planthoppers; (2) Cultivating transgenic plants; (3) Plant variety improvement; (4) Improvement of plant germplasm resources; (5) Used to prepare any one or more of the products in (1) to (4).

[0012] In this invention, the plant is a grass, preferably a rice plant, and more preferably rice.

[0013] Fourthly, the present invention provides a method for improving plant resistance to brown planthoppers or a method for creating brown planthopper-resistant plants, the method comprising: 1) incorporating the gene into the plant. Bph56 ;or, 2) To enable plants to express the Bph56 protein.

[0014] Fifthly, the present invention provides a method for improving rice resistance to brown planthoppers or a method for creating brown planthopper-resistant rice, the method comprising: overexpressing the gene in rice. Bph56 ; The overexpression method can be selected from the following 1) to 5), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By importing enhancers.

[0015] Expression vectors carrying the target gene can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2). nd Edition).

[0016] In a sixth aspect, the present invention provides the application of transgenic plants obtained according to the method in plant (particularly rice) breeding.

[0017] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0018] In a seventh aspect, the present invention provides a gene Bph56 A tightly linked molecular marker (denoted as K1956) contains a C / G polymorphic SNP at 19567713 bp on rice chromosome 11; the above physical location is based on the reference genome version number ZS97RS3 of the rice variety Zhenshan 97.

[0019] Furthermore, the genotype of the site exhibiting the aforementioned polymorphism is CC, and the corresponding rice material does not contain the aforementioned gene. Bph56 The genotype of the site exhibiting the aforementioned polymorphism is GG, and the corresponding rice material contains the gene described above. Bph56 The genotype of the site exhibiting the aforementioned polymorphism is CG, and the corresponding gene in the rice material is... Bph56 It is a heterozygous type.

[0020] This invention also provides KASP primers for amplifying the molecular markers, as follows (SEQ ID NO:6-8): K1956_FAM: 5'-gaaggtgaccaagttcatgctAGATTAAGCAGGCTTGATGTGTG-3'; K1956_HEX: 5'-gaaggtcggagtcaacggattAGATTAAGCAGGCTTGATGTGTC-3'; K1956_COM: 5'-GCATTTGAGCACGCAATTGC-3'.

[0021] Eighthly, the present invention provides any of the following applications of the said molecular marker or its detection reagents or kits: (1) Identification of rice phenotypes resistant to brown planthopper; (2) Identification and improvement of rice germplasm resources or molecular marker-assisted breeding of rice resistant to brown planthopper; (3) Early prediction of brown planthopper resistant rice materials; (4) Screening rice materials resistant to brown planthopper; (5) Regarding the gene Bph56 Genotyping was performed.

[0022] Ninthly, the present invention provides a method for screening rice resistant to brown planthoppers, comprising the following steps: 1) Extract genomic DNA from the rice plants to be tested; 2) KASP typing detection of the genomic DNA was performed using the KASP primers; 3) Determine whether the rice plant to be tested contains the gene based on the test results. Bph56 The detection of Hex fluorescence signal indicates that the plant contains the gene described above. Bph56 The rice plants were resistant to brown planthoppers; the detection of Fam fluorescence signal indicated that the plants did not contain the gene described. Bph56 The rice plants were not resistant to brown planthoppers; the detection of Fam and Hex fluorescence signals indicated that the gene in the plants was present. Bph56 The rice plant is a heterozygous variety and is resistant to brown planthoppers.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) This invention successfully cloned a rice brown planthopper resistance gene using map-based cloning technology in forward genetics. Bph56 It was also verified that it has the function of resisting brown planthopper in rice.

[0024] (two) Bph56 The gene exhibits complete dominance, and both homozygous and heterozygous genotypes confer equal resistance to brown planthoppers on rice. This characteristic is beneficial for the application of this gene in breeding. Based on this, traditional breeding techniques such as hybridization and backcrossing, or genetic engineering methods, can be used to... Bph56 This invention involves introducing genes into plant varieties susceptible to brown planthoppers to cultivate new varieties resistant to brown planthoppers with stable genetic traits. This provides important germplasm resources for effectively reducing pest damage and achieving increased and stable crop yields.

[0025] (three) Bph56 The gene encodes a novel LRR-like protein, which has important implications for enriching the diversity of brown planthopper resistance genes, elucidating the mechanism of brown planthopper resistance, and breeding applications.

[0026] (iv) The present invention provides and Bph56The tightly linked KASP molecular marker K1956 can be used for marker-assisted breeding of brown planthopper resistance. Its selection target is clearly defined, and genotyping can be performed at the rice seedling stage, providing a convenient, rapid, and efficient screening method to determine whether hybrid or backcross materials contain the brown planthopper resistance gene. Bph56 This allows for accurate prediction of whether rice plants possess resistance to brown planthoppers, which can be applied to the breeding of new brown planthopper-resistant rice varieties. Attached Figure Description

[0027] Figure 1 This is the fine mapping result of the Bph56 gene provided in Example 1 of the present invention. The black boxes represent chromosomal segments, with molecular markers above them, and the numbers below them representing the number of recombinant individuals screened between two adjacent molecular markers. n is the number of individuals in the segregating population used to screen for recombinant individuals. The Bph56 gene is finely mapped to the region between markers K1955 and InD1958, and is closely linked to K1956. The finely mapped region of Bph56 contains three genes, of which gene 3 encodes an LRR-like protein and serves as a candidate gene for Bph56.

[0028] Figure 2 This is a schematic diagram of the genotyping results of the KASP marker K1956 for a sample provided in Embodiment 2 of the present invention.

[0029] Figure 3 This is the molecular marker-assisted selection culture belt provided in Example 3 of the present invention. Bph56 Brown planthopper resistance in the genetically modified strain Luoyang 56.

[0030] Figure 4 This is provided in Embodiment 5 of the present invention. Bph56 The results of resistance identification of transgenic plants with candidate gene genome complementation vectors to brown planthoppers. Among them, Luoyang 56 is a transgenic planthopper carrying the 9311 background. Bph56 Near-isogenic line materials were used as insect-resistant controls; 9311 was the transgenic recipient material, used as an insect-susceptible control; G56-1 and G56-2 were representative. Bph56 Transgenic plants with genomic complementation vectors for candidate genes.

[0031] Figure 5 This is the identification result of resistance to brown planthopper in CRISPR / Cas9 homozygous knockout plants of the Bph56 candidate gene in the genetic background of Luoyang 56, as provided in Example 5 of this invention. Luoyang 56 is a plant carrying the Bph56 candidate gene in the 9311 background. Bph56 Near-isogenic line material was used as an insect-resistant control; 9311 was used as an insect-susceptible control; Gene 3 KO was a CRISPR / Cas9 homozygous knockout plant of Bph56 candidate gene 3 in the genetic background of Luoyang 56. Detailed Implementation

[0032] This invention provides a rice brown planthopper resistance gene. Bph56 And its applications.

[0033] This invention also provides a gene related to the stated gene. Bph56 Tightly linked molecular marker (K1956).

[0034] The present invention adopts the following technical solution: In a first aspect, the present invention provides a rice Bph56 protein, the rice Bph56 protein comprising the amino acid sequence shown in SEQ ID NO:4.

[0035] It should be understood that, without affecting the activity of the Bph56 protein, those skilled in the art can obtain an amino acid sequence with the same function by substituting, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO:4, and thus apply it to the genetic improvement of rice brown planthopper resistance.

[0036] Secondly, the present invention provides a nucleic acid molecule that encodes the rice Bph56 protein.

[0037] According to the present invention, a nucleic acid molecule preferably comprises any one of the following nucleotide sequences: (1) Nucleotide sequences as shown in SEQ ID NO:1-2 in tandem, or SEQ ID NO:3 or SEQ ID NO:5; (2) The perfectly complementary sequence of the nucleotide sequence shown in (1); (3) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in (1). (4) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (1) under strict conditions and expresses the same functional protein; (5) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of (1), (2), (3) or (4) and expresses the same functional protein.

[0038] Thirdly, the present invention provides a biomaterial containing the aforementioned nucleic acid molecules.

[0039] Furthermore, the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism, or cell.

[0040] Preferably, the carrier uses pCAMBIA1300-Ubi1-LTP2-DsRed as its backbone.

[0041] Preferably, the microorganism includes Agrobacterium.

[0042] Fourthly, the present invention provides the application of the rice Bph56 protein, the nucleic acid molecule, or the biological material in any of the following: (1) Improve plant resistance to brown planthoppers; (2) Cultivating transgenic plants; (3) Plant variety improvement; (4) Improvement of plant germplasm resources; (5) Prepare products for any one or more of (1) to (4).

[0043] Preferably, the plant is a grass belonging to the Poaceae family.

[0044] More preferably, the plant is a member of the genus *Oryza*.

[0045] More preferably, the plant is rice.

[0046] Furthermore, methods for cultivating transgenic plants, improving plant varieties, or improving plant germplasm resources include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0047] Fifthly, the present invention provides a method for improving plant resistance to brown planthoppers, comprising: increasing the rice Bph56 protein in the plant and / or Bph56 Gene expression levels.

[0048] Preferably, the plant is a grass belonging to the Poaceae family.

[0049] More preferably, the plant is a member of the genus *Oryza*.

[0050] More preferably, the plant is rice.

[0051] Furthermore, methods for increasing the expression level of rice Bph56 protein and / or Bph56 gene in the plant include, but are not limited to, transgenic, hybrid, backcross, self-cross, or asexual reproduction.

[0052] More preferably, the method for increasing the expression level of rice Bph56 protein and / or Bph56 gene in the plant includes: hybridizing the plant carrying rice Bph56 protein and / or Bph56 gene with other plants, and retaining the offspring carrying rice Bph56 protein and / or Bph56 gene.

[0053] In a sixth aspect, the present invention provides a method for creating plants resistant to brown planthoppers, comprising: introducing rice Bph56 protein and / or... Bph56 Gene; In a seventh aspect, the present invention provides a method for improving the resistance of plants to brown planthoppers or a method for creating plants resistant to brown planthoppers.

[0054] Preferably, the plant is a grass belonging to the Poaceae family.

[0055] More preferably, the plant is a member of the genus *Oryza*.

[0056] More preferably, the plant is rice.

[0057] Preferably, the method includes: placing rice Bph56 protein and / or Bph56 When a plant carrying the gene is hybridized with other plants, it retains the rice Bph56 protein and / or Bph56 The offspring of the gene produce offspring plants resistant to brown planthoppers.

[0058] Eighthly, the present invention provides a KASP molecular marker K1956 that is closely linked to the rice brown planthopper resistance gene Bph56, and its application in the breeding of brown planthopper resistant rice.

[0059] The corresponding marker primers are as follows: K1956_FAM: 5'-gaaggtgaccaagttcatgctAGATTAAGCAGGCTTGATGTGTG-3'; K1956_HEX: 5'-gaaggtcggagtcaacggattAGATTAAGCAGGCTTGATGTGTC-3'; K1956_COM: 5'-GCATTTGAGCACGCAATTGC-3'.

[0060] This invention also provides a method for identifying whether rice varieties contain a gene for resistance to brown planthopper using molecular markers. Bph56 The method involves amplifying the rice genomic DNA to be tested using the aforementioned primers. If the K1956 PCR product detects a Fam fluorescence signal, the SNP is labeled as a C base and the rice sample does not contain the brown planthopper resistance gene Bph56. If a Hex fluorescence signal is detected, the SNP is labeled as a G base and the rice sample contains the brown planthopper resistance gene Bph56. If both Fam and Hex fluorescence are detected simultaneously, the SNP is labeled as heterozygous C / G, and the rice sample contains... Bph56 The gene is heterozygous.

[0061] This invention also provides a method for screening rice varieties resistant to brown planthoppers. The method involves amplifying the genomic DNA of the rice sample using the primer pair described in K1956 and detecting the amplification products. If the K1956 PCR product detects a Hex fluorescence signal, the rice sample contains the homozygous brown planthopper resistance gene Bph56. If both Fam and Hex fluorescence are detected simultaneously, the rice sample contains heterozygous Bph56. Both methods indicate that the rice variety possesses resistance to brown planthoppers.

[0062] Those skilled in the art will understand that molecular markers designed or generated based on the rice Bph56 protein and / or Bph56 gene sequence disclosed in this invention can be used for the breeding of rice resistant to brown planthoppers.

[0063] Ninth aspect, the present invention provides a method for cultivating plants resistant to brown planthoppers using transgenic technology, comprising: (1) transforming plant callus cells with a polynucleotide containing the Bph56 gene; wherein the polynucleotide sequence is shown in SEQ ID NO:1-2 in series or in SEQ ID NO:3 or SEQ ID NO:5; (2) regenerating the transformed plant cells into plants; (3) culturing the regenerated plants and expressing the above polynucleotide, and harvesting T1 generation seeds; (4) sowing the T1 generation seeds, using the Bph56 gene detected by K1956, and harvesting T2 generation seeds with homozygous gene.

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0065] The rice material used in this invention is as follows: Bph56 The parental line IRGC 55210 was obtained from the International Rice Research Institute (IRRI), and the brown planthopper-susceptible variety Yangdao 6 (9311) was obtained from the National Germplasm Bank of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The above biological materials are available to the public from the applicant.

[0066] Example 1 Bph56 Fine localization and molecular marker development The rice variety IRGC 55210, introduced from the International Rice Research Institute, was used to evaluate its resistance to brown planthoppers using the seedling group method (Huang et al. 2001 Identification and mapping of two brown planthopper resistance genes in rice. Theor Appl Genet 102, 929-934). The brown planthopper source used was a common brown planthopper population. The results showed that IRGC 55210 exhibited high resistance to brown planthoppers, with a resistance level of 1.14. IRGC 55210 was crossed with the brown planthopper-susceptible rice variety 9311 to construct a backcross population. The resulting BC1F1 strain was tested for resistance to brown planthoppers, and the resistant BC1F1 strain was backcrossed with 9311. The resulting BC2F1 strain was also tested for resistance to brown planthoppers, and the resistant BC2F1 strain was backcrossed with 9311. The resulting BC3F1 strain was also tested for resistance to brown planthoppers, and the resistant BC3F1 strain was selected. Genomic DNA was extracted using the CTAB method. Genotypic genetic background analysis was performed on the parents IRGC 55210 and 9311, as well as five BC3F1 genes resistant to brown planthopper, using the rice green gene chip GSR40K. The results showed that the 13-21 Mb region of rice chromosome 11 was linked to the brown planthopper resistance phenotype, indicating that the brown planthopper resistance gene in the resistant parent IRGC 55210 is located in this region. This gene was named Bph56.

[0067] This invention performed 30X second-generation genome sequencing on the parents IRGC 55210 and 9311, respectively. The sequencing results were compared to identify polymorphic KASP and InDel markers for the Bph56 localization region between the two parents. Using genotypic data from 120 BC3F2:3 accessions and corresponding brown planthopper resistance phenotypes, linkage recombination was used to locate Bph56 between KASP markers K1929 and K1978. The physical location corresponds to the region from 19290059 bp to 19784304 bp on chromosome 11 of the Zhenshan 97 reference genome (version ZS97RS3).

[0068] Using the KASP markers K1929 and K1978 of this invention, 865 BC3F2 single plants were screened, yielding 9 recombinant single plants. By internal marker encryption within the recombinant single plants and combining this with their brown planthopper resistance phenotype, Bph56 was located within the region defined by markers K1946 and K1972. Further screening of 5954 BC4F2 single plants using the KASP markers K1946 and K1972 flanking the Bph56 location region yielded 36 recombinant single plants. KASP markers K1955 and K1956, as well as the InDel marker InD1958, were developed for use within the location region. Genotypic analysis of the recombinant single plants was performed using these molecular markers, and combined with the brown planthopper resistance phenotype analysis of key recombinant single plants, ultimately locating Bph56 within the region defined by markers K1955 and InD1958, closely linked to K1956. Figure 1 The physical location corresponds to the interval from 19556369 bp to 19589872 bp on chromosome 11 of the Zhenshan 97 reference genome (version number ZS97RS3).

[0069] The primers used to amplify the above-mentioned markers are as follows: The KASP primers used to amplify marker K1929 (a SNP site with polymorphism A (IRGC 55210) / T (9311) at chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO: 9-11): K1929_FAM 5'-gaaggtgaccaagttcatgctCCTGACGCCTTCCAAGCA-3'; K1929_HEX 5'-gaaggtcggagtcaacggattTTCCTGACGCCTTCCAAGCT -3'; K1929_COM 5'-CCACTCCAGTTAGCCAAGCA -3'; The KASP primers used to amplify marker K1945 (a SNP site with polymorphism C(IRGC 55210) / G(9311) at chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO:12-14): K1945_FAM 5'-gaaggtgaccaagttcatgctACAGCACAAAGAGTTCATTAGTCG-3'; K1945_HEX 5'-gaaggtcggagtcaacggattACAGCACAAAGAGTTCATTAGTCC -3'; K1945_COM 5'- ATGCCGTGTCTCCAAATCG-3'; The KASP primers used to amplify marker K1946 (a SNP site with polymorphism G (IRGC 55210) / A (9311) at 19467418 bp on chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO:15-17): K1946_FAM 5'- GAAGGTGACCAAGTTCATGCTTCCATGCATGACTATCAACAATGG-3'; K1946_HEX 5'-GAAGGTCGGAGTCAACGGATTTCCATGCATGACTATCAACAATGA-3'; K1946_COM 5'- GCCTGCTATATTGTTTTTGTGGGT-3'; The KASP primers used to amplify marker K1955 (a SNP site with polymorphism A (IRGC 55210) / C (9311) at 19556394 bp on chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO:18-20): K1955_FAM 5'-GAAGGTGACCAAGTTCATGCTGTACCACATGTTGCTCTACAAGTTA -3'; K1955_HEX 5'-GAAGGTCGGAGTCAACGGATTGTACCACATGTTGCTCTACAAGTTC -3'; K1955_COM 5'-GAAACTTGTCCCGTTTTAGGAAGG-3'; The KASP primers used to amplify marker K1972 (a SNP site with polymorphism T(IRGC 55210) / A(9311) at 19723144 bp on chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO:21-23): K1972_FAM 5'-gaaggtgaccaagttcatgctCCTCAGCTACAACAGGTCAAGTA-3'; K1972_HEX 5'-gaaggtcggagtcaacggattCCTCAGCTACAACAGGTCAAGTT-3'; K1972_COM 5'-GAATGGCCCTTTGGATTGGC -3'; The KASP primers used to amplify marker K1978 (a SNP site with polymorphism G (IRGC 55210) / A (9311) at chromosome 11 of the ZS97 reference genome (version ZS97RS3)) are (SEQ ID NO:24-26): K1978_FAM 5'-gaaggtgaccaagttcatgctGAACTCTAAGACATGCTCACATACG-3'; K1978_HEX 5'-gaaggtcggagtcaacggattGAACTCTAAGACATGCTCACATACA-3'; K1978_COM 5'- CTTGGGCATAGAGGTCAGATT-3'; The primers used to amplify the marker InD1958 (an insertion / deletion site located at 19589697-19589872 bp on chromosome 11 of the Zhenshan 97 reference genome (version ZS97RS3)) are (SEQ ID NO:27-28): InD1958 F 5'- TCCTCACCCCATTACTGGGTTTATC-3'; InD1958 R 5'-GTAGTATGCATGTTACTTGAATCAGATG -3'; The product size of IRGC 55210, amplified by the InD1958 marker, was 141 bp, and the product size of 9311, a rice variety susceptible to brown planthopper, was 176 bp.

[0070] Example 2: Application of Bph56 tightly linked KASP markers in genotyping of segregating populations This invention selected 92 individuals from a BC2F2 backcross population constructed from the insect-resistant parent IRGC 55210 and the superior restorer line Huahui 8612 (R8612). Genotyping analysis was performed using the Bph56 tightly linked KASP marker K1956 to determine its efficiency in screening for the Bph56 gene. The results showed that the ratios of the three different genotypes—G:G (Bph56 homozygous), G:C (Bph56 heterozygous), and C:C (without Bph56)—were 22:47:23, conforming to the Mendelian single-gene segregation ratio of 1:2:1. This further validated that K1956 is a codominant marker capable of distinguishing between two different homozygous and heterozygous individuals, with the detection site simultaneously exhibiting single-gene segregation. Genotyping results for some samples are shown below.Figure 2 As shown.

[0071] The KASP primers used to amplify the molecular marker K1956 are as follows (SEQ ID NO:6-8): K1956_FAM: 5'-gaaggtgaccaagttcatgctAGATTAAGCAGGCTTGATGTGTG-3'; K1956_HEX: 5'-gaaggtcggagtcaacggattAGATTAAGCAGGCTTGATGTGTC-3'; K1956_COM: 5'-GCATTTGAGCACGCAATTGC-3'.

[0072] To further clarify whether the genotypes of BC2F2 individual plants detected by the KASP marker K1956 of this invention are consistent with their brown planthopper resistance phenotypes, 22 homozygous Bph56 genotypes, 47 heterozygous Bph56 genotypes, and 23 BC2F2 individual plants without Bph56, determined by K1956 of this invention, were self-crossed to obtain the corresponding BC2F23 materials. The resistance performance of these BC2F23 materials was examined using the seedling group method (the resistance level of these BC2F23 families represents the brown planthopper resistance phenotype of BC2F2 individual plants). The results showed that the genotypes of these BC2F2 individual plants (reflected by the detection results of the KASP marker K1956 of this invention) were completely consistent with their brown planthopper resistance phenotypes, with a consistency of 100%: the average resistance value of the 22 BC2F2 materials with the Bph56 homozygous genotype was around 2.5, showing homozygous resistance to brown planthoppers; the average resistance value of the 47 BC2F2 materials with the Bph56 heterozygous genotype was between 4.1 and 4.5, showing heterozygous resistance to brown planthoppers; and the 23 BC2F2 materials without Bph56 all showed homozygous susceptibility to brown planthoppers, with resistance values ​​of around 9.

[0073] The above results further demonstrate that the tightly linked KASP marker K1956 of the Bph56 gene developed in this invention co-segregates with the brown planthopper resistance phenotype, and can be applied to marker-assisted selection breeding practices for Bph56 to cultivate brown planthopper-resistant rice varieties containing Bph56. It also shows that the molecular marker method provided by this invention can accurately screen rice materials containing the brown planthopper resistance gene Bph56, thereby improving the efficiency of brown planthopper resistance breeding.

[0074] Example 3: Molecular marker-assisted selection for cultivation Bph56 Genetically improved strains For BC3F1 obtained by continuous backcrossing of IRGC 55210 with 9311, apply Bph56 Genotypic analysis was performed on the linked KASP marker K1956 to screen for...Bph56 Heterozygous single plants were crossed with the recurrent parent 9311 to obtain BC4F1. Screening was then performed as described above. Bph56 The heterozygous BC4F1 was crossed with the recurrent parent 9311 to obtain BC5F1. The same selection process was then performed. Bph56 The heterozygous BC5F1 was crossed with the recurrent parent 9311 to obtain BC6F1. The same selection process was then performed. Bph56 Five heterozygous BC6F1 plants were analyzed for genotypic genetic background using the rice green gene chip GSR40K to screen for carriers. Bph56 Furthermore, the BC6F1 plant with the genetic background most closely related to the recurrent parent 9311 was self-crossed to obtain BC6F2. From this, the BC6F2 plant with the homozygous Bph56 genotype and the genetic background most closely related to the recurrent parent 9311 was selected and self-crossed to obtain BC6F23, thus obtaining the near-isogenic line Luoyang 56 containing Bph56. The results of brown planthopper seedling resistance identification showed that the Bph56 improved line Luoyang 56 is highly resistant to brown planthoppers. Figure 3 This indicates that Bph56 has a promising future in breeding rice varieties resistant to brown planthoppers.

[0075] Example 4 Bph56 Location interval sequence determination To determine the genomic sequence of the Bph56 fine mapping region, a Fosmid genome library of the Bph56 insect-resistant parent was constructed by Takara. The Fosmid genome library was screened using molecular markers specific to the Bph56 fine mapping region. End sequencing of the selected positive clones determined their overlap, ultimately identifying two positive clones, 12H1 and 2C10, that covered the entire Bph56 fine mapping region. Sequencing analysis of the full sequences of Fosmid clones 12H1 and 2C10 yielded the complete sequence of the Bph56 mapping region, specifically the sequence between molecular markers K1955 and InD1958, approximately 53.4 kb in length. Gene prediction and annotation were performed using FGENESH, revealing… Bph56 Candidate gene 3 within the localization region encodes an LRR-like protein, which is considered as... Bph56 The candidate gene. This gene corresponds to the gene OsZS97_11G0272900 in Zhenshan 97.

[0076] Example 5 Bph56 Transgenic functional verification of candidate genes 1. Construction of genome complementation vector (1) Vector double enzyme digestion The transgenic backbone vector used in this study was the pCAMBIA1300-Ubi1-LTP2-DsRed vector (see CN117701589A) constructed in the inventor's laboratory. The aleurone-specific promoter LTP2 drives the expression of the DsRed gene in the mature rice seed coat, resulting in a red seed coat color, which facilitates the identification of positive transgenic plants. pCAMBIA1300-Ubi1-LTP2-DsRed was double-digested with HindIII and EcoRI to obtain linearized pCAMBIA1300-Ubi1-LTP2-DsRed for later use.

[0077] (2) Full-length sequence amplification PCR amplification was successfully performed using KOD Plus Neo (Toyobo) high-fidelity polymerase to obtain... Bph56 The full-length genome sequence of the candidate gene (nucleotide sequence shown in tandem in SEQ ID NO:1-2), including the corresponding promoter, expression cassette and terminator of the candidate gene.

[0078] (3) Connection Using the ClonExpress II One Step Cloning Kit (catalog number: C112-01) from Novizan, homologous recombination was performed to... Bph56 The full-length genomic sequence of the candidate gene (nucleotide sequence shown in SEQ ID NO:1-2) was ligated into pCAMBIA1300-Ubi1-LTP2-DsRed after double enzyme digestion and linearization. The resulting recombinant product was transformed into E. coli TOP10 via heat shock, then plated on LB agar plates containing 50 mg / L kanamycin and grown for 16 hours. Positive clones were selected, and plasmids were sent to Beijing Aoke Biotechnology Co., Ltd. for whole-plasmid sequencing. The plasmids with correct sequencing were retained as the genomic complementation vector for the candidate gene (denoted as pCAMBIA1300-G56-LTP2-DsRed).

[0079] 2. Obtaining the full-length cDNA sequence by Bph56 Using the reverse transcription product of total RNA from the leaf sheath of parental IRGC 55210 as a template, according to Bph56 Primers were designed based on the predicted cDNA sequence of candidate gene 3, and the results were obtained through PCR amplification and sequencing. Bph56 Partial cDNA sequence information of candidate gene 3.

[0080] Primers were then designed based on the obtained cDNA sequence, and the TaKaRa 5' and 3' Full RACE kits were used to obtain... Bph56 The 5' and 3' end sequences of candidate gene 3.

[0081] Based on the amplification products described above, and through comparison, B was determined. ph56 The transcription start and termination sites of candidate gene 3 were determined, ultimately yielding B. ph56 The full-length cDNA sequence of candidate gene 3 (SEQ ID NO:3), the amino acid sequence of the protein it encodes is shown in SEQ ID NO:4, and its ORF sequence is shown in SEQ ID NO:5.

[0082] 3. Construction of CRISPR / Cas9 vectors A CRISPR / Cas9 knockout vector for candidate gene 3 of Bph56 was constructed using the pYLCRISPR / Cas9 multi-target vector developed by Academician Yaoguang Liu's research group at South China Agricultural University (see Ma et al 2015. A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant. 2015;8:1274–1284. doi: 10.1016 / j.molp.2015.04.007). Based on the CDS sequence of candidate gene 3 of Bph56, the knockout site was analyzed using the online software CRISPR-GE (http: / / skl.scau.edu.cn / home / ). The specific target sequences sgRNA1 (5'-ACATCTCCAAGGCTGTCGTC-3', SEQ ID NO:29) and sgRNA2 (5'-ATGACGCGCGGTTTGACGAA-3', SEQ ID NO:30) of the Bph56 candidate gene were selected. Primers were designed to amplify the promoters of sgRNA1 and U6a, and sgRNA2 and U6b, respectively. The primer sequences are as follows (SEQ ID NO:31-34): gRT1-910:ACATCTCCAAGGCTGTCGTCgttttagagctagaaat OsU6aT1-910: GACGACAGCCTTGGAGATGTCggcagccaagccagca gRT2-910:ATGACGCGCGGTTTGACGAAgttttagagctagaaat OsU6bT2-910: TTCGTCAAACCGCGCGTCATCaacacaagcggcagc Overlap PCR was used to amplify sgRNA1 and U6a fragments carrying the target fragments together, and sgRNA2 and U6b fragments together, with the fragments at both ends being able to pair in reverse complementary directions. The recovered fragments are the sgRNA expression cassettes.

[0083] Enzyme digestion and ligation were performed using variable-temperature cycling for approximately 10-15 cycles (37℃ 5 min; 10℃ 5 min, 20℃ 5 min) to ligate the sgRNA expression cassette into the pYLCRISPR / Cas9 plasmid. The plasmid was extracted from correctly sequenced positive clones, named Cas910, and used for subsequent genetic transformation experiments.

[0084] 4. Construction of transgenic plants Using Agrobacterium-mediated genetic transformation, Bph56 The genome complementation vector of candidate gene 3 (pCAMBIA1300-G56-LTP2-DsRed) was transferred into the insect-susceptible recipient material 9311. The corresponding transgenic plants with genome complementation vectors of candidate genes (represented by G56) were obtained. After harvesting the seeds, the corresponding T1 generation transgenic plants with genome complementation vectors were obtained.

[0085] Using Agrobacterium-mediated genetic transformation, Bph56 Candidate gene 3 was carried in the CRISPR / Cas9 vector Cas910 transfected into the 9311 background. Bph56 The near-isogenic line Luoyang 56 was used. Genomic DNA was extracted from T0 generation CRISPR / Cas9 knockout plants using the CTAB method. Primers were designed based on the sequences at both ends of the target site to perform PCR amplification and sequencing identification of these transgenic plants, clarifying their genetic makeup. Bph56 The knockout mutation type of candidate gene 3 and whether it is a homozygous knockout. For the identified... Bph56 Candidate gene 3 was transduced into homozygous knockout plants of Luoyang 56 using a CRISPR / Cas9 vector. After harvesting the seeds of the progeny, the corresponding T1 generation was obtained. Bph56 Candidate gene 3 knockout plants.

[0086] 5. Identification of resistance to brown planthopper in transgenic plants Applying the seedling group method to Bph56 Candidate gene 3 T1 generation genome-complemented transgenic plants (represented by G56) were used to identify resistance to brown planthoppers, with the transgenic recipient 9311 as a susceptible control. Bph56 The near-isogenic line Luoyang 56 was used as an insect-resistant control. For example... Figure 4As shown, 5 days after inoculation with brown planthoppers, the entire susceptible recipient 9311 plant died. Transgenic plants with candidate gene complementation vectors represented by G56-1 and G56-2, along with the resistant control material Luoyang 56, all grew healthily without leaf damage, exhibiting strong resistance to brown planthoppers. The above brown planthopper resistance identification results indicate that candidate gene 3 is... Bph56 .

[0087] Applying the seedling group method to the genetic background of Luoyang 56 Bph56 Homozygous knockout plants of candidate gene 3 (represented by Gene 3KO) were used to identify resistance to brown planthoppers, with the transgenic receptor 9311 serving as a susceptible control. Bph56 The near-isogenic line Luoyang 56 was used as a resistant control. Five days after inoculation with brown planthoppers, the susceptible recipient line 9311 died completely, while the resistant control line Luoyang 56 survived and grew well, demonstrating strong resistance to brown planthoppers. Bph56 The CRISPR / Cas9 vector of candidate gene 3 transfected homozygous knockout plants of Luoyang 56 showed susceptibility to brown planthoppers and wilted and died. Figure 5 The experimental results further confirm that candidate gene 3 is... Bph56 Gene.

[0088] The above insect resistance identification results fully demonstrate that candidate gene 3 is... Bph56 Gene. Bph56 The genomic nucleotide sequence of the gene is shown in SEQ ID NO:1-2 in tandem, the full-length cDNA sequence is shown in SEQ ID NO:3, the amino acid sequence encoding the protein is shown in SEQ ID NO:4, and its ORF sequence is shown in SEQ ID NO:5.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Rice Bph56 protein, characterized in that, It is: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:4; or (b) A protein derived from (a) with the sequence shown in SEQ ID NO:4 substituted, deleted or added with one or more amino acids and having the same function.

2. Rice brown planthopper resistance gene Bph56 Or biological materials containing the said gene, characterized in that, The gene Bph56 Encoding the protein of claim 1; Among them, the gene Bph56 for: i) The nucleotide sequences shown in SEQ ID NO:1-2 in tandem, or the nucleotide sequences shown in SEQ ID NO:3 or SEQ ID NO:5; ii) nucleotide sequences of i) that have been substituted, deleted and / or added with one or more nucleotides and express the same functional protein; iii) A nucleotide sequence that hybridizes with the nucleotide sequence of i) under stringent conditions and expresses the same functional protein, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE or 0.1×SSC solution containing 0.1% SDS, followed by washing the membrane with the solution. iv) Nucleotide sequences that have more than 90% homology with nucleotide sequences i), ii), or iii) and express the same functional protein; or, v) A nucleotide sequence that is completely complementary to the nucleotide sequence of i), ii), iii) or iv); The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

3. Any of the following applications of the protein of claim 1, the gene of claim 2, or the biological material: (1) Improve plant resistance to brown planthoppers; (2) Cultivating transgenic plants; (3) Plant variety improvement; (4) Improvement of plant germplasm resources; (5) Used to prepare any one or more of the products in (1) to (4).

4. The application according to claim 3, characterized in that, The plant in question is rice.

5. A method for improving plant resistance to brown planthoppers or for creating brown planthopper-resistant plants, characterized in that, The method includes: 1) incorporating the gene of claim 2 into a plant; or, 2) To enable plants to express the protein of claim 1.

6. A method for improving rice resistance to brown planthoppers or for creating brown planthopper-resistant rice, characterized in that, The method includes: overexpressing the gene of claim 2 in rice; The overexpression method is selected from the following 1) to 5), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By importing enhancers.

7. The application of the transgenic plant obtained according to the method of claim 5 or 6 in plant breeding; Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

8. The gene according to claim 2 Bph56 Closely linked molecular markers, characterized by, The molecular marker contains a C / G polymorphism SNP at 19567713 bp on rice chromosome 11; the physical location of the SNP is based on the reference genome version number ZS97RS3 of the rice variety Zhenshan 97. The genotype of the site exhibiting the aforementioned polymorphism is CC, and the corresponding rice material does not contain the aforementioned gene. Bph56 ; The genotype of the site exhibiting the aforementioned polymorphism is GG, and the corresponding rice material contains the gene described above. Bph56 ; The genotype of the site exhibiting the aforementioned polymorphism is CG, and the corresponding gene in the rice material is... Bph56 It is a hybrid type; The KASP primers used to amplify the molecular marker are as follows: K1956_FAM: 5'-gaaggtgaccaagttcatgctAGATTAAGCAGGCTTGATGTGTG-3'; K1956_HEX: 5'-gaaggtcggagtcaacggattAGATTAAGCAGGCTTGATGTGTC-3'; K1956_COM: 5'-GCATTTGAGCACGCAATTGC-3'.

9. Any of the following applications of the molecular marker of claim 8 or its detection reagent or kit: (1) Identification of rice phenotypes resistant to brown planthopper; (2) Identification and improvement of rice germplasm resources or molecular marker-assisted breeding of rice resistant to brown planthopper; (3) Early prediction of brown planthopper resistant rice materials; (4) Screening rice materials resistant to brown planthopper; (5) Regarding the gene Bph56 Genotyping was performed.

10. A method for screening rice varieties resistant to brown planthopper, characterized in that, Includes the following steps: 1) Extract genomic DNA from the rice plants to be tested; 2) Perform KASP typing detection on the genomic DNA using the KASP primers described in claim 8; 3) Determine whether the rice plant to be tested contains the gene described in claim 2 based on the test results. Bph56 The detection of Hex fluorescence signal indicates that the plant contains the gene described above. Bph56 The rice plants described are resistant to brown planthoppers; The detection of Fam fluorescence signal indicates that the plant does not contain the gene described. Bph56 The rice plants were not resistant to brown planthoppers; the detection of Fam and Hex fluorescence signals indicated that the gene in the plants was present. Bph56 The rice plant is a heterozygous variety and is resistant to brown planthoppers.

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