A rice gene Bph54 for resistance to brown planthopper and its application

Abstract

This invention provides a rice gene Bph54 for resistance to brown planthopper and its application. The rice provided by this invention... Bph54 Genes are of great significance for breeding new rice varieties resistant to brown planthoppers. This invention successfully cloned the rice Bph54 gene using map-based cloning, and performed functional annotation and identification. This gene exhibits complete dominance, and both homozygous and heterozygous genotypes confer equal resistance to brown planthoppers in rice. This characteristic is beneficial for the application of this gene in breeding. Based on this, the Bph54 gene can be introduced into brown planthopper-susceptible plant varieties through traditional breeding techniques such as hybridization and backcrossing, or through genetic engineering, to cultivate new varieties of plants resistant to brown planthoppers with stable genetic traits. This invention provides important germplasm resources for effectively reducing insect damage and achieving increased and stable crop yields.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a rice gene Bph54 for resistance to brown planthopper and its applications. Background Technology

[0002] Insects are among the most complex and diverse groups of organisms on Earth. Of the one million insect species on Earth, about half feed on plants, making agricultural pests one of the most serious biological hazards in agricultural production. Rice planthoppers (brown planthoppers, white-backed planthoppers, and gray planthoppers) rank first among various rice diseases and pests, with the brown planthopper being the most prevalent. Controlling the development and damage caused by brown planthoppers is a crucial requirement for ensuring the safety of rice production.

[0003] Traditional methods of controlling brown planthoppers rely excessively on chemical pesticides. The continuous and extensive use of chemical insecticides has led to a significant increase in the planthoppers' resistance, gradually diminishing the effectiveness of pesticide control. Furthermore, the use of insecticides has eliminated natural enemies of brown planthoppers in rice paddies, making control even more difficult. Relying solely on pesticides can no longer achieve the goal of sustainable management of rice planthoppers.

[0004] Practice has proven that the foundation of integrated pest management and sustainable control of crops lies in resistant varieties. Utilizing the crop's own disease and pest resistance genes to improve and breed new varieties is the most economical, effective, and environmentally friendly measure for pest and disease control, and it is also the inevitable path for future green agricultural development. In the long-term co-evolution of rice and brown planthopper, rice has developed resistance genes, while brown planthoppers, under the selection pressure of these resistance genes, can undergo harmful mutations, forming new populations that can overcome existing resistance, i.e., new biotypes. Currently, those skilled in the art have identified more than 40 brown planthopper resistance genes from cultivated and wild rice resources, among which… Bph1, Bph2, Bph3, Bph7, Bph9, Bph10, Bph14, Bph15, Bph18, Bph21, Bph26, Bph29, Bph30, Bph32, Bph37 and Bph40 The gene has been cloned using map-based cloning. Currently, more than 10 biotypes of the brown planthopper have been reported, and the loss of resistance due to variations in the planthopper's pathogenicity poses a potential challenge to its sustainable control. Therefore, it is necessary to further explore new brown planthopper-resistant genes or superior alleles with specific resistance spectra in rice germplasm resources to address the biotype variations in the brown planthopper. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a rice brown planthopper resistance gene Bph54 and its applications.

[0006] This invention employs forward genetics to construct a segregating population of rice resistant to brown planthoppers. Using map-based cloning, the brown planthopper resistance gene Bph54 was isolated. Genetic transformation of the Bph54 gene induced a brown planthopper-resistant phenotype in susceptible rice, confirming the brown planthopper-resistant function of the Bph54 gene.

[0007] In a first aspect, the present invention provides a rice Bph54 protein, the rice Bph54 protein comprising any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein having the same function obtained by substituting, inserting or deleting one or more amino acids from the amino acid sequence shown in SEQ ID NO.3.

[0008] Secondly, the present invention also provides a nucleic acid molecule that encodes the rice Bph54 protein.

[0009] According to the present invention, a nucleic acid molecule preferably comprises any one of the following nucleotide sequences: (1) A nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4; (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 as 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.

[0010] Thirdly, the present invention also provides a biomaterial containing the aforementioned nucleic acid molecule, wherein the biomaterial is recombinant DNA, expression cassette, transposon, vector, microorganism, or cell.

[0011] According to a biomaterial provided by the present invention, preferably, the carrier uses pCAMBIA1300-Ubi1-LTP2-DsRed as a backbone.

[0012] According to a biological material provided by the present invention, preferably, the microorganism includes Agrobacterium.

[0013] Fourthly, the present invention also provides the use of the rice Bph54 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).

[0014] In accordance with the application of the rice Bph54 protein, the nucleic acid molecule, or the biomaterial provided by the present invention, preferably, the plant is a grass.

[0015] More preferably, the plant is a member of the genus *Oryza*.

[0016] More preferably, the plant is rice.

[0017] According to the application of the rice Bph54 protein, the nucleic acid molecule, or the biological material provided by the present invention, preferably, the 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.

[0018] Fifthly, the present invention also provides a method for improving plant resistance to brown planthopper, comprising: increasing the expression level of rice Bph54 protein and / or Bph54 gene in the plant; The rice Bph54 protein includes any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.3.

[0019] According to a method for improving plant resistance to brown planthopper provided by the present invention, preferably, the Bph54 gene comprises any one of the following nucleotide sequences: (1) A nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4; (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 as 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.

[0020] According to the present invention, a method for improving plant resistance to brown planthopper is provided, wherein the plant is preferably a grass.

[0021] More preferably, the plant is a member of the genus *Oryza*.

[0022] More preferably, the plant is rice.

[0023] According to the present invention, a method for improving plant resistance to brown planthopper is provided. Preferably, the method for improving the expression level of rice Bph54 protein and / or Bph54 gene in the plant includes, but is not limited to, transgenic, hybridization, backcrossing, self-pollination or asexual reproduction.

[0024] More preferably, the method for increasing the expression level of rice Bph54 protein and / or Bph54 gene in the plant includes: crossing the plant carrying rice Bph54 protein and / or Bph54 gene with other plants, and retaining the offspring carrying rice Bph54 protein and / or Bph54 gene.

[0025] In a sixth aspect, the present invention also provides a method for creating plants resistant to brown planthoppers, comprising: introducing rice Bph54 protein and / or Bph54 gene into the plant; The rice Bph54 protein includes any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.3.

[0026] According to a method for creating plants resistant to brown planthoppers provided by the present invention, preferably, the Bph54 gene comprises any one of the following nucleotide sequences: (1) A nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4; (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 as 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.

[0027] According to the present invention, a method for creating plants resistant to brown planthoppers is provided, wherein the plant is preferably a grass.

[0028] More preferably, the plant is a member of the genus *Oryza*.

[0029] More preferably, the plant is rice.

[0030] According to the present invention, a method for creating plants resistant to brown planthoppers preferably includes: hybridizing a plant carrying the rice Bph54 protein and / or the Bph54 gene with other plants, and retaining the offspring carrying the rice Bph54 protein and / or the Bph54 gene.

[0031] In a seventh aspect, the present invention also provides plants obtained according to a method for improving the resistance of plants to brown planthoppers or a method for creating brown planthopper-resistant plants.

[0032] Those skilled in the art will understand that molecular markers designed or generated based on the rice Bph54 protein and / or Bph54 gene sequence disclosed in this invention can be used for the breeding of rice resistant to brown planthoppers.

[0033] The present invention has the following beneficial effects: This invention successfully cloned the rice Bph54 gene using map-based cloning, and performed functional annotation and identification. The gene exhibits complete dominance, and both homozygous and heterozygous genotypes confer equal resistance to brown planthoppers in rice. This characteristic is beneficial for the application of this gene in breeding. Based on this, the Bph54 gene can be introduced into brown planthopper-susceptible plant varieties through traditional breeding techniques such as hybridization and backcrossing, or through genetic engineering, to cultivate new brown planthopper-resistant varieties with stable genetic traits. This invention provides important germplasm resources for effectively reducing insect pests and achieving increased and stable crop yields. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 This is provided in Embodiment 2 of the present invention. Bph54 The results of resistance identification of transgenic plants to brown planthopper using genome complementation vectors of the phenological gene, among which 15344 is... Bph54 Parental materials: ZS97 is the susceptible receptor Zhenshan 97; G54-1, G54-12, G54-13, and G54-14 are... Bph54 Transgenic plants with genomic complementation vectors for candidate genes.

[0036] Figure 2 This is provided in Embodiment 3 of the present invention. Bph54 The results of resistance identification of transgenic plants with overexpressed gene to brown planthoppers, among which 15344 is... Bph54 Parental materials: ZS97 is the susceptible receptor Zhenshan 97; OE54-21 and OE54-26 are...Bph54 Gene overexpression transgenic plants. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0039] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0040] The rice material used in this invention is as follows: Bph54 The parental line IRGC 15344 was obtained from the International Rice Research Institute, and the susceptible brown planthopper cultivar Yangdao 6 (9311) and the susceptible recipient material Zhenshan 97 were obtained from the National Germplasm Bank of the Institute of Crop Science, Chinese Academy of Agricultural Sciences. These biological materials are available to the public from the applicant.

[0041] Example 1 Bph54 Determining the location interval sequence To shrink Bph54 Gene location regions, using Bph54 Markers at both ends of the gene mapping region screened 3552 BC2F2 monoclonal samples, resulting in 38 recombinant monoclonal samples. Genotypic analysis of these recombinant monoclonal samples was performed using the developed molecular markers, combined with phenotypic analysis of their resistance to brown planthoppers. Bph54 Fine mapping of genes.

[0042] In order to determine Bph54 Locating the genome sequence of the region and using the PacBio HiFi third-generation genome sequencing strategy. Bph54The parental IRGC 15344 genome was sequenced and assembled. The sequencing results are summarized below: DNBseq data volume was 49.4 Gb, HiFi sequencing data volume was 24.6 Gb, and Hi-C sequencing data volume was 44.9 Gb. After HiFi sequencing, the BAM data was converted to FASTQ format using Smartlink 12.0.0.1, and the hifiasm (v0.19.8-r603) assembly method was used to obtain a preliminary assembled genome with a size of 427.5 Mb and a contig N50 of 31.7 Mb. After redundancy removal using purge_dup (v1.2.5), a genome size of 388 Mb was obtained, with a contig N50 of 31.7 Mb and a BUSCO value of 98.8%. By combining Hi-C crosslinking signals, contigs were oriented and clustered, and then attached to 12 chromosomes. The scaffold genome size was 388 Mb, the scaffold N50 was 31.7 Mb, the contig N50 was 31.7 Mb, and the BUSCO value was 98.8%. Finally, coverage was achieved. Bph54 Genomic sequence of the localized region.

[0043] Through gene prediction and annotation, in Bph54 There is a candidate gene encoding an LRR-like protein within the localization region, serving as... Bph54 Candidate genes.

[0044] Example 2 Bph54 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 pCAMBIA1300-Ubi1-LTP2-DsRed, constructed in the inventor's laboratory (i.e., the transgenic backbone vector pCAMBIA1300-Ubi1-LTP2-DsRed in prior art CN117701589A). The aleurone-specific promoter LTP2 drives the expression of the DsRed gene in the mature rice seed coat, making the seed coat appear red and facilitating 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.

[0045] (2) Full-length sequence amplification PCR amplification was successfully performed using KOD Plus Neo (Toyobo) high-fidelity polymerase to obtain... Bph54The full-length genome sequence (SEQ ID NO.1) of the candidate gene, including the corresponding promoter, expression cassette and terminator of the candidate gene.

[0046] (3) Connection Using the ClonExpress II One Step Cloning Kit (catalog number: C112-01) from Novizan, homologous recombination was performed to... Bph54 The full-length genome sequence of the candidate gene (SEQ ID NO.1) 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 genome complementation vector for the candidate gene (denoted as pCAMBIA1300-Gene-LTP2-DsRed).

[0047] 2. Construction of transgenic plants Using Agrobacterium-mediated genetic transformation, the genome complementation vector (pCAMBIA1300-Gene-LTP2-DsRed) of the above candidate gene was transferred into the insect-susceptible recipient material Zhenshan 97. The corresponding transgenic plants with genome complementation vectors of the candidate gene (represented as G54) were obtained. After harvesting the seeds, the corresponding T1 generation transgenic plants with genome complementation vectors were obtained.

[0048] 3. Identification of resistance to brown planthopper in transgenic plants The seedling group method described by Huang et al. (Huang et al. 2001 Identification and mapping of two brown planthopper resistance genes in rice. Theor Appl Genet 102, 929-934) was used to identify the resistance of the above-mentioned T1 generation transgenic plants (G54 represented by the genome-complementary transgenic plants) to brown planthoppers, with the susceptible receptor Zhenshan 97 as a control.

[0049] like Figure 1 As shown, four days after inoculation with brown planthoppers, the entire Zhenshan 97 recipient plant died, while transgenic plants with genome complementation vectors representing candidate genes G54-1, G54-12, G54-13, and G54-14 all grew healthily without leaf damage, exhibiting strong resistance to brown planthoppers.

[0050] The above insect resistance identification results fully demonstrate that this candidate gene is indeed the one being tested. Bph54Gene. Bph54 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0051] Example 3 Bph54 Verification of gene overexpression function 1. Construction of overexpression vectors (1) Amplification of full-length cDNA sequence by Bph54 Using the reverse transcription product of total RNA from the leaf sheath of parental IRGC 15344 as a template, according to Bph54 Primers were designed based on the predicted gene sequence, and the results were obtained by PCR. Bph54 A fragment of the cDNA sequence of a gene.

[0052] Primers were then designed based on the obtained cDNA sequence, and the TaKaRa 5' and 3' Full RACE kits were used to obtain... Bph54 The 5' and 3' end sequences of the gene.

[0053] Based on the above amplification products, and through comparison, it was determined that... Bph54 The transcription start and termination sites of genes ultimately yield... Bph54 The full-length cDNA sequence of the gene (SEQ ID NO.2), the amino acid sequence of the protein it encodes is shown in SEQ ID NO.3, and the ORF sequence is shown in SEQ ID NO.4.

[0054] (2) ORF sequence amplification by Bph54 Using the full-length cDNA of the gene as a template, the amplification was performed using KOD Plus Neo (Toyobo) high-fidelity polymerase. Bph54 The ORF sequence of the gene (SEQ ID NO.4).

[0055] (3) Vector single enzyme digestion The pCAMBIA1300-Ubi1-LTP2-DsRed vector was digested with BamHI to obtain the linearized pCAMBIA1300-Ubi1-LTP2-DsRed vector.

[0056] (4) Connection Using the ClonExpress II One Step Cloning Kit (catalog number: C112-01) from Novizan, homologous recombination was performed to separately... Bph54The ORF fragment of the gene (SEQ ID NO.4) was ligated into the linearized pCAMBIA1300-Ubi1-LTP2-DsRed vector after single enzyme digestion. Transformation and sequencing were performed according to the method described in this embodiment. Plasmids with correct sequencing were retained, yielding the corresponding results. Bph54 The gene overexpression vector (denoted as pCAMBIA1300-Ubi1-Gene-LTP2-DsRed). Bph54 The gene is controlled by the maize Ubi1 strong promoter.

[0057] 2. Construction of overexpression transgenic plants Using Agrobacterium-mediated genetic transformation, the above-mentioned... Bph54 The gene overexpression vector (pCAMBIA1300-Ubi1-Gene-LTP2-DsRed) was transformed into the insect-susceptible recipient material Zhenshan 97, and the corresponding results were obtained. Bph54 Transgenic plants overexpressing the gene (denoted as OE54). After harvesting the seeds, the corresponding T1 generation overexpressing transgenic plants are obtained.

[0058] 3. Identification of resistance to brown planthopper in transgenic plants overexpressing the gene The seedling group method described by Huang et al. (Huang et al. 2001 Identification and mapping of two brown planthopper resistance genes in rice. Theor Appl Genet 102, 929-934) was used to identify the resistance of the above-mentioned T1 generation overexpressing transgenic plants (overexpressing transgenic plants represented by OE54) to brown planthopper, with the susceptible receptor Zhenshan 97 as a control.

[0059] like Figure 2 As shown, four days after inoculation with brown planthoppers, the entire Zhenshan 97 recipient plant died, as did plants represented by OE54-21 and OE54-26. Bph54 All positive lines of the gene overexpression transgene grew healthily, with no leaf damage, and showed strong resistance to brown planthopper.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rice Bph54 protein, characterized in that, The rice Bph54 protein includes any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that, It encodes the rice Bph54 protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule includes any of the following nucleotide sequences: (1) A nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4; (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 as 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.

4. A biomaterial, characterized in that, The biological material contains the nucleic acid molecule as described in claim 2 or 3, wherein the biological material is recombinant DNA, expression cassette, transposon, vector, microorganism or cell.

5. The use of the rice Bph54 protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the biomaterial of claim 4 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).

6. The application according to claim 5, characterized in that, The plant is a grass family plant, preferably a rice plant, and more preferably rice.

7. A method for improving plant resistance to brown planthoppers, characterized in that, include: Increase the expression level of rice Bph54 protein and / or Bph54 gene in the plant; The rice Bph54 protein includes any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.

3.

8. A method for creating plants resistant to brown planthoppers, characterized in that, include: Introduce rice Bph54 protein and / or Bph54 gene into the plant; The rice Bph54 protein includes any of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.3; (2) the amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.

3.

9. The method according to claim 7 or 8, characterized in that, The Bph54 gene includes any of the following nucleotide sequences: (1) A nucleotide sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.4; (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 as 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.

10. The plant obtained by the method according to any one of claims 7 to 9.

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