A rice brown planthopper-resistant mutant gene, protein and application thereof

Abstract

The present application relates to the field of agricultural technology, in particular to a rice brown planthopper resistant mutant gene, protein and application thereof. The present application provides a rice brown planthopper resistant mutant gene, and the nucleotide sequence of the rice brown planthopper resistant mutant gene is shown as SEQ ID NO. 1. The rice brown planthopper resistant mutant gene provided by the present application is a wild type rice OsCOI1a gene which has a G to A mutation at the 1636th nucleotide, and can improve the insect resistance of rice at the seedling stage and the adult stage, and provides a new gene resource and molecular target for breeding insect-resistant rice.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a mutant gene and protein for rice resistance to brown planthoppers and its applications. Background Technology

[0002] Rice is one of the world's most important food crops, feeding half of the global population. The brown planthopper (BPH) is a monophagous piercing-sucking insect that feeds on rice and is currently the most serious pest affecting rice-growing areas in my country and Asia.

[0003] Brown planthoppers pierce rice plants with their stylets to suck sap, consuming plant nutrients and causing yellowing leaves, aging plants, and empty grains, severely impacting rice yield and quality. Furthermore, their honeydew excrement creates a breeding ground for bacteria on rice plants, and brown planthoppers are also vectors for rice toothed leaf dwarf virus and grassy dwarf virus. In addition, brown planthoppers reproduce rapidly and are difficult to detect as they inhabit the base of rice plants, leading to explosive outbreaks of damage and posing a significant threat to rice production.

[0004] Utilizing the inherent resistance of rice to breed and cultivate insect-resistant varieties is considered the most effective, economical, and environmentally friendly strategy for controlling brown planthoppers. Discovering and identifying brown planthopper-resistant genes is the prerequisite and foundation for breeding insect-resistant varieties.

[0005] Gene editing technology is a revolutionary tool in crop breeding. It can directly target genes and loci for precise modification, achieve accurate trait improvement, quickly respond to the agricultural production demand for new varieties, and significantly reduce time and labor costs. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a mutant gene, protein, and application of rice resistant to brown planthoppers. The mutant gene provided by this invention is a mutation from G to A at position 1636 of the wild-type rice OsCOI1a gene. This mutation enhances the insect resistance of rice during the seedling and mature stages, providing a new gene resource and molecular target for breeding insect-resistant rice.

[0007] Therefore, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides, in optional embodiments, a rice brown planthopper-resistant mutant gene, the nucleotide sequence of which is shown in SEQ ID NO.1. The nucleotide sequence of the wild-type rice OsCOI1a gene is shown in SEQ ID NO.3.

[0009] Secondly, in an optional embodiment, the present invention provides a rice mutant protein resistant to brown planthoppers, the amino acid sequence of which is shown in SEQ ID NO.2. The amino acid sequence of wild-type rice OsCOI1a protein is shown in SEQ ID NO.4.

[0010] Thirdly, in an optional embodiment, the present invention provides a plasmid containing the aforementioned rice brown planthopper-resistant mutant gene.

[0011] Fourthly, in an optional embodiment, the present invention provides a plant expression vector containing the above-mentioned rice brown planthopper-resistant mutant gene.

[0012] Fifthly, in optional embodiments, the present invention provides a recombinant cell or recombinant protein comprising the aforementioned rice brown planthopper-resistant mutant gene.

[0013] In a sixth aspect, the present invention provides an engineered bacterium in an optional embodiment, comprising the above-mentioned rice brown planthopper-resistant mutant gene.

[0014] In a seventh aspect, the present invention provides, in optional embodiments, the application of the above-mentioned rice brown planthopper-resistant mutant gene, the above-mentioned rice brown planthopper-resistant mutant protein, the above-mentioned plasmid, the above-mentioned plant expression vector, the above-mentioned recombinant cell or recombinant protein, or the above-mentioned engineered bacteria in improving rice brown planthopper resistance.

[0015] Eighthly, in an optional embodiment, the present invention provides a method for improving rice resistance to brown planthoppers, comprising the following steps:

[0016] By editing the OsCOI1a gene in wild-type rice into the aforementioned mutant gene for resistance to brown planthoppers, the resistance of rice to brown planthoppers was improved.

[0017] In a ninth aspect, the present invention provides, in an optional embodiment, a method for obtaining brown planthopper-resistant plants, comprising the following steps:

[0018] By using gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction, plants can be made to contain the aforementioned mutant gene for resistance to brown planthoppers in rice, thereby obtaining plants resistant to brown planthoppers.

[0019] Preferably, the plant is rice.

[0020] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0021]

[0022] The amino acid sequence of the said SEQ ID NO.2 is shown as follows:

[0023] MGGEVPEPRRLNRALSFDDWVPDEALHLVMGHVEDPRDREAASRVCRRWHRIDALTRKHVTVAFCYAARPARLRERFPRLESLSLKGKPRAAMYGLIPDDWGAYAAPWIDELAAPLECLKALHLRRMTVTDADIAALVRARGHMLQELKLDKCIGFSTDALRLVARSCRSLRTLFLEECHITDKGGEWLHELAVNNSVLVTLNFYMTELKVAPADLELLAKNCKSLISLKMSECDLSDLISFFQTANALQDFAGGAFYEVGELTKYEKVKFPPRLCFLGLTYMGTNEMPVIFPFSMKLKKLDLQYTFLTTEDHCQIIAKCPNLLILEVRNVIGDRGLEVVGDTCKKLRRLRIERGDDDPGLQEEQGGVSQLGLTAVAVGCRELEYIAAYVSDITNGALESIGTFCKNLYDFRLVLLDRERQVTDLPLDNGVCALLRNCTKLRRFALYLRPGGLSDDGLSYIGQYSGNIQYMLLGNVGESDHGLIRFAVGCTNLQKLELRSCCFSERALSLAVLQMPSLRYIWVQGYRASQTGLDLLLMARPFWNIKFTPPSPESFNHMTEDGEPCVDSHAQVLAYYSLAGRRSDCPQWVIPLHPA。

[0024] The nucleotide sequence of the said SEQ ID NO.3 is shown as follows:

[0025]

[0026] The amino acid sequence of SEQ ID NO.4 is shown below:

[0027] .

[0028] Compared with the prior art, the present invention has one of the following beneficial effects:

[0029] 1. The rice brown planthopper resistant mutant gene provided by this invention is a mutation from G to A at position 1636 of the wild-type rice OsCOI1a gene, which can improve the insect resistance of rice in the seedling and mature stages, and provides a new gene resource and molecular target for breeding insect-resistant rice. Attached Figure Description

[0030] Figure 1 This is a comparison chart of the seedling stage of wild-type and insect-resistant rice mutant rtb1 in Example 1 of this invention, a phenotypic chart of brown planthopper inoculation treatment, and a statistical analysis chart of seedling mortality rate.

[0031] Figure 2This is a comparison of the nucleotide and amino acid sequence mutation sites of the OsCOI1a gene in wild-type and insect-resistant rice mutant rtb1 in Example 1 of this invention.

[0032] Figure 3 This is a sequencing peak diagram of the OsCOI1a site-edited plant and the wild type in Example 3 of this invention.

[0033] Figure 4 This is a phenotypic diagram of resistance to brown planthoppers and a statistical analysis result of seedling mortality rate in OsCOI1a-edited plants and wild-type plants in Example 4 of this invention.

[0034] Figure 5 This is a phenotypic diagram of resistance to brown planthopper and a statistical analysis result of seedling mortality rate in mature plants of OsCOI1a-edited plants and wild-type plants in Example 4 of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1

[0039] Obtaining the OsCOI1a insect-resistant haplotype

[0040] Using the rice variety Nanjing 9108 as the background material, chemical mutagenesis was induced using ethyl methanesulfonate (EMS), and the mutant library was phenotypically identified using the seedling group identification method. Seeds of the test materials were disinfected, soaked, and germinated before being sown in 6 cm square plastic pots. When the seedlings reached the three-leaf stage, brown planthoppers were inoculated at a density of 10 second- to third-instar nymphs per seedling. The brown planthopper resistance of the tested rice materials was evaluated by statistically analyzing the seedling mortality rate. When the seedling mortality rate of the background material Nanjing 9108 reached 90%, the number of dead seedlings in each test material was investigated and the average mortality rate was calculated. The results showed that when the seedling mortality rate of Nanjing 9108 reached 90%, one mutant had a mortality rate of approximately 30%, and this brown planthopper-resistant rice material was named rtb1. Subsequently, the rtb1 mutant was crossed with Nanjing 9108, and extreme resistance and extreme susceptibility F2 plants were selected for BSA analysis. Whole-genome sequencing revealed that the rtb1 mutant exhibits a G-to-A base mutation at position 1636 of the nucleotide sequence of the OsCOI1a gene (gene number Os01g0853400) on rice chromosome 1, resulting in a glutamic acid to lysine mutation at position 546 (see [link to original text]). Figure 2 ).

[0041] Example 2

[0042] Construction of the OsCOI1a gene-editing vector PE-OsCOI1a-E546K

[0043] Primers for a site-directed gene editing vector were designed to target the G-to-A mutation at position 1636 of the OsCOI1a gene. The relevant sequences are shown in Table 1. After primer synthesis, annealing was performed to obtain the target sgRNA fragment and RTPBS fragment corresponding to the OsCOI1a gene editing vector. The reaction mixture consisted of 10 μL each of forward and reverse primers, 5 μL of 10×T4 ligation buffer, 1 μL of T4 PNK, and 24 μL of ddH2O.

[0044] The Phuc422-PEmax-NC vector was digested with enzymes in the following reaction mixture: 15 μL plasmid, 4 μL CutSmart buffer, 1 μL BasI enzyme, and 20 μL ddH2O. After centrifugation, the mixture was digested overnight at 37°C. The digested product was then inactivated at 65°C for 20 min.

[0045] The inactivated PE vector and target fragment were ligated using Goldengate technology to form a complete recombinant vector. The reaction mixture consisted of 1 μL of inactivated vector, 1 μL each of the two target fragments, 1 μL of sg2.0 fragment, 1 μL of evopreQ1 fragment, 1 μL of T4 buffer, 1 μL of T4 ligase, 1 μL of CutSmart, 1 μL of BasI enzyme, and 1 μL of ddH2O. After mixing and centrifugation, the mixture was placed in a PCR instrument. The Goldengate program was as follows: 37℃ for 5 min, 25℃ for 5 min, 25 cycles; 80℃ for 10 min.

[0046] The recombinant PE vector was transformed into E. coli and cultured overnight on Kanamycin-resistant medium. After single-clon sequencing verification, the PE-OsCOI1a-E546K positive plasmid was obtained.

[0047] Table 1. Vector primer sequences

[0048]

[0049] Example 3

[0050] Obtaining the OsCOI1a gene-edited plant E546K

[0051] The constructed editing vector plasmid PE-OsCOI1a-E546K was transformed into Agrobacterium and infected the callus tissue of the rice variety Nanjing 9108. After co-culture, recovery, screening, differentiation, and rooting, transgenic rice seedlings were obtained.

[0052] Genotyping of the obtained rice seedlings was further performed. Genomic DNA was extracted from the rice plants, and primer pairs were designed to amplify the sequences flanking the target editing site using PCR. The primer sequences are shown in Table 2. The PCR reaction system consisted of: 1 μL DNA template, 1 μL each of forward and reverse primers, 10 μL 2×Phanta Mix Master Mix, and 7 μL ddH2O. The PCR amplification program was: 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for 35 cycles; 72 ℃ extension for 5 min.

[0053] The PCR products were subjected to electrophoresis, gel excision, and recovery. The purified PCR products were sent to a company for sequencing, yielding the homozygous edited positive plantlet E546K. Sequencing peak diagrams of wild-type and site-edited rice plants are shown below. Figure 3 As shown.

[0054] Table 2 Primer sequences for genotype detection

[0055]

[0056] Example 4

[0057] Identification of Brown Planthopper Resistance in OsCOI1a Gene-Edited Plant E546K

[0058] The brown planthopper resistance phenotype of OsCOI1a-edited plant E546K was identified using the seedling group identification method. E546K plants and wild-type seeds were simultaneously disinfected with 10% hydrogen peroxide solution for 1 hour. After rinsing the seeds with distilled water, they were soaked for two days. The rice seeds were then dried and germinated at 37℃. Uniformly germinated seeds were selected and sown in 6 cm square plastic pots for soil cultivation. The cultivation conditions were 28℃ for 14 hours of light, 25℃ for 10 hours of darkness, and 70% relative humidity. One week later, thinning was performed, removing small, weak, and mixed seedlings, leaving 20 uniformly growing rice plants per pot. When the rice seedlings reached the three-leaf stage, brown planthoppers were inoculated at a density of 10 second- to third-instar nymphs per seedling. Phenotypic changes were observed daily. When the wild-type rice plants reached 90%, the total number of seedlings and the number of dead seedlings were counted, and the mortality rate was calculated. Results showed that when almost all wild-type rice seedlings died, only a few seedlings of the OsCOI1a-edited plant E546K wilted, with most seedlings growing well; their growth and survival rate were significantly better than the wild type. Statistical analysis showed that under brown planthopper infestation, when the mortality rate of wild-type plants reached 90%, the mortality rate of the OsCOI1a-edited plant E546K was only about 30%, indicating a significant increase in brown planthopper resistance (see [link to relevant documentation]). Figure 4 ).

[0059] The resistance of the OsCOI1a gene-edited plant E546K to the brown planthopper at the adult stage was identified. Edited materials and wild-type rice seeds were disinfected, soaked, and germinated before being sown in seedbeds for seedling cultivation. Four weeks later, they were transplanted to the field, where normal water and fertilizer management was implemented, along with attention to pest and disease control. When the rice reached the jointing stage, the OsCOI1a edited materials and wild-type rice plants were dug up and transplanted into pots with a diameter of 12 cm and a height of 15 cm, continuing normal water and fertilizer management. After the rice plants had recovered from transplant shock, 20 egg-bearing short-winged female brown planthoppers were introduced into each plant, covered with a mesh screen, allowing them to lay eggs and reproduce on the rice plants. Phenotypic observations were conducted twice a week. When the wild-type rice plants died, the degree of damage to the identified rice plants was investigated, and the readings were graded. The grading standards for rice resistance to the brown planthopper at the adult stage are shown in Table 3. The results showed that when all wild-type rice plants died, the OsCOI1a-edited rice plants grew well, with only a small number of leaves turning yellow. Statistical analysis showed that when the resistance level of wild-type rice reached 8, the resistance level of the OsCOI1a-edited rice material E546K was 2, significantly improving its resistance to brown planthopper (see [link to study]). Figure 5 ).

[0060] Table 3 Grading standards for rice resistance to brown planthopper at adult stage

[0061]

[0062] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A mutant gene for rice resistance to brown planthopper, characterized in that, The nucleotide sequence of the rice brown planthopper-resistant mutant gene is shown in SEQ ID NO.

1.

2. A plasmid, characterized in that, It contains the rice brown planthopper resistant mutant gene as described in claim 1.

3. A plant expression vector, characterized in that, It contains the rice brown planthopper resistant mutant gene as described in claim 1.

4. A recombinant cell, characterized in that, It contains the rice brown planthopper resistant mutant gene as described in claim 1.

5. An engineered bacterium, characterized in that, It contains the rice brown planthopper resistant mutant gene as described in claim 1.

6. The application of the rice brown planthopper resistance mutant gene of claim 1, the plasmid of claim 2, the plant expression vector of claim 3, the recombinant cell of claim 4, or the engineered bacteria of claim 5 in improving rice brown planthopper resistance.

7. A method for improving rice resistance to brown planthopper, characterized in that, Includes the following steps: By editing the OsCOI1a gene in wild-type rice, the rice brown planthopper-resistant mutant gene as described in claim 1 is edited, thereby improving the rice's resistance to brown planthoppers.

8. A method for obtaining plants resistant to brown planthoppers, characterized in that, Includes the following steps: By using gene editing, hybridization, backcrossing, self-pollination or asexual reproduction, plants can be made to contain the rice brown planthopper resistant mutant gene as described in claim 1, thereby obtaining brown planthopper resistant plants; The plant in question is rice.

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