Rice OsCOI1a mutant gene, protein and application thereof

Abstract

The invention relates to the technical field of agriculture, in particular to a rice OsCOI1a mutant gene, a protein and application of the rice OsCOI1a mutant gene and the protein. The invention provides a rice OsCOI1a mutant gene. The nucleotide sequence of the rice OsCOI1a mutant gene is that mutation from C to A occurs at the 308th site of the nucleotide sequence of a wild rice OsCOI1a gene. The rice OsCOI1a mutant gene provided by the invention is characterized in that the 308th site of a nucleotide sequence of a wild rice OsCOI1a gene is mutated from C to A, so that alanine at the 103th site of an amino acid sequence of the OsCOI1a protein is mutated into aspartic acid, and the rice mutant gene or mutant protein not only can improve the brown planthopper resistance of rice, but also can enhance the high-temperature tolerance of the rice, so that the rice mutant gene or mutant protein has a good application prospect. The growth and the yield of the rice are not obviously influenced, and the method has important production and application values.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a rice OsCOI1a mutant gene, protein, and its applications. Background Technology

[0002] Rice is one of the world's most important food crops. The brown planthopper (BPH) is a monophagous piercing-sucking insect that feeds on rice. It pierces the plant with its stylet to suck sap, consuming plant nutrients and causing yellowing leaves, aging plants, and empty grains, severely impacting rice yield and quality. Furthermore, brown planthoppers can transmit rice toothed leaf dwarf virus and rice grass dwarf virus, further exacerbating the damage. Utilizing rice's own resistance by breeding and planting 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.

[0003] High temperatures are one of the major abiotic stresses affecting rice growth and production. Throughout its entire growth cycle, rice is highly sensitive to high temperatures. High temperatures not only cause leaf damage, stunted growth, and reduced pollen viability and seed setting rate, leading to yield reduction, but also cause abnormal grain filling and increased chalkiness, affecting rice quality. Currently, heat-resistant genes in rice are relatively scarce. Therefore, identifying superior heat-resistant genes in rice is crucial and of great significance for ensuring food security.

[0004] Gene editing technology is a revolutionary tool in life science research and crop breeding. It can quickly and efficiently edit target genes and target sites to achieve precise improvement of target traits, greatly improve the efficiency of germplasm creation and variety selection, and significantly reduce time and labor costs. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a rice mutant gene and protein with resistance to brown planthoppers, and their applications. This rice OsCOI1a mutant gene is a mutation from C to A at position 308 of the wild-type rice OsCOI1a gene. This mutant gene not only enhances rice's resistance to brown planthoppers but also strengthens its high-temperature tolerance, without significantly affecting rice growth and yield, thus possessing significant value for production applications.

[0006] Therefore, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a rice OsCOI1a mutant gene in an optional embodiment, wherein the nucleotide sequence of the rice OsCOI1a mutant gene is a mutation from C to A at position 308 of the wild-type rice OsCOI1a gene.

[0008] Preferably, the nucleotide sequence of the rice OsCOI1a mutant gene 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 OsCOI1a mutant protein, wherein the amino acid sequence of the rice OsCOI1a mutant protein is a mutation in which alanine is changed to aspartic acid at position 103 of the amino acid sequence of the wild-type rice OsCOI1a protein.

[0010] Preferably, the amino acid sequence of the rice mutant protein is shown in SEQ ID NO.2. The amino acid sequence of the wild-type rice OsCOI1a protein is shown in SEQ ID NO.4.

[0011] Thirdly, in an optional embodiment, the present invention provides a recombinant plasmid containing the above-mentioned rice OsCOI1a mutant gene.

[0012] Fourthly, in an optional embodiment, the present invention provides a plant expression vector containing the above-mentioned rice OsCOI1a mutant gene.

[0013] Fifthly, in an optional embodiment, the present invention provides a recombinant cell comprising the above-mentioned rice OsCOI1a mutant gene.

[0014] In a sixth aspect, the present invention provides, in optional embodiments, a recombinant protein comprising the above-mentioned rice OsCOI1a mutant gene.

[0015] In a seventh aspect, the present invention provides, in an optional embodiment, a method for obtaining rice plants with improved resistance to brown planthoppers, comprising the following steps:

[0016] Plants can be made to contain the above-mentioned rice OsCOI1a mutant gene through gene editing, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] The plant in question is rice.

[0018] Eighthly, in an optional embodiment, the present invention provides an application of the above-mentioned rice OsCOI1a mutant gene in improving the high-temperature tolerance of rice.

[0019] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0020]

[0021] The amino acid sequence of SEQ ID NO.2 is shown below:

[0022] MGGEVPEPRRLNRALSFDDWVPDEALHLVMGHVEDPRDREAASRVCRRWHRIDALTRKHVTVAFCYAARPARLRERFPRLESLSLKGKPRAAMYGLIPDDWGDYAAPWIDELAAPLECLKALHLRRMTVTDADIAALVRARGHMLQELKLDKCIGFSTDALRLVARSCRSLRTLFLEECHITDKGGEWLHELAVNNSVLVTLNFYMTELKVAPADLELLAKNCKSLISLKMSECDLSDLISFFQTANALQDFAGGAFYEVGELTKYEKVKFPPRLCFLGLTYMGTNEMPVIFPFSMKLKKLDLQYTFLTTEDHCQIIAKCPNLLILEVRNVIGDRGLEVVGDTCKKLRRLRIERGDDDPGLQEEQGGVSQLGLTAVAVGCRELEYIAAYVSDITNGALESIGTFCKNLYDFRLVLLDRERQVTDLPLDNGVCALLRNCTKLRRFALYLRPGGLSDDGLSYIGQYSGNIQYMLLGNVGESDHGLIRFAVGCTNLQKLELRSCCFSERALSLAVLQMPSLRYIWVQGYRASQTGLDLLLMARPFWNIEFTPPSPESFNHMTEDGEPCVDSHAQVLAYYSLAGRRSDCPQWVIPLHPA。

[0023] The nucleotide sequence of SEQ ID NO.3 is shown below:

[0024]

[0025] The amino acid sequence of SEQ ID NO.4 is shown below:

[0026] .

[0027] Compared with the prior art, the present invention has one of the following beneficial effects:

[0028] 1. The rice OsCOI1a mutant gene provided by this invention is a mutation from C to A at position 308 of the wild-type rice OsCOI1a gene, resulting in a mutation of alanine to aspartic acid at position 103 of the OsCOI1a protein amino acid sequence. This rice mutant gene or mutant protein can not only improve the resistance of rice to brown planthoppers, but also enhance the high temperature tolerance of rice, and the growth and yield of rice are not significantly affected, which has significant production application value. Attached Figure Description

[0029] Figure 1This is a phenotypic diagram and statistical analysis result of the brown planthopper-resistant rice mutant rbph1 and wild-type rice plants after brown planthopper inoculation treatment in Example 1 of the present invention.

[0030] Figure 2 This is a schematic diagram comparing the nucleotide and amino acid sequence mutation sites of the OsCOI1a gene in the brown planthopper-resistant rice mutant rbph1 and wild-type rice in Example 1 of this invention.

[0031] Figure 3 These are sequencing peak diagrams of the OsCOI1a site-edited plant A103D and the wild-type plant in Example 3 of this invention.

[0032] Figure 4 This is a graph showing the resistance phenotype and seedling mortality statistics of OsCOI1a-edited plant A103D and wild-type brown planthopper in Example 4 of this invention.

[0033] Figure 5 This is a phenotypic diagram of the high temperature tolerance of OsCOI1a-edited plant A103D and wild-type plants in Example 5 of the present invention, and a statistical analysis result of their survival rate.

[0034] Figure 6 This is a schematic diagram comparing the agronomic traits of OsCOI1a-targeted material A103D and wild-type rice plants in Example 6 of the present 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 superior haplotype of OsCOI1a against brown planthopper

[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. After disinfection, soaking, and germination, the seeds of the test materials were sown in plastic pots with 6 cm sides. 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 counted, 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%. This brown planthopper-resistant rice material was named rbph1 (see [link to relevant documentation]). Figure 1 The rtb1 mutant was then crossed with Nanjing 9108, and F2 plants exhibiting extreme insect resistance and extreme insect susceptibility were selected for BSA analysis. Whole-genome sequencing revealed that the rtb1 mutant had a C-to-A base mutation at position 308 of the nucleotide sequence of the OsCOI1a gene (gene number Os01g0853400) on rice chromosome 1, resulting in a mutation of alanine to aspartic acid at position 103 (see [link to original text]). Figure 2 ).

[0041] Example 2

[0042] Construction of the OsCOI1a gene-editing vector PE-OsCOI1a-A103D

[0043] Primers for a site-directed gene editing vector were designed to target the C-to-A mutation at position 308 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-A103D positive plasmid was obtained.

[0047] Table 1. Vector primer sequences

[0048]

[0049] Example 3

[0050] Obtaining the OsCOI1a gene-edited plant A103D

[0051] The constructed editing vector plasmid PE-OsCOI1a-A103D 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 flanking the target editing site for PCR amplification. 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 site-edited positive plantlet A103D. 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 resistance in brown planthoppers using OsCOI1a gene-edited material A103D.

[0058] The brown planthopper resistance phenotype of OsCOI1a-edited plant A103D was identified using the seedling group identification method. Edited plant A103D 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 under the following conditions: 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 mortality rate of wild-type rice plants reached 90%, the total number of seedlings and the number of dead seedlings were statistically analyzed, 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 A103D wilted, with most seedlings growing well; its 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 A103D was only about 30%, indicating a significant increase in brown planthopper resistance (see [link to relevant documentation]). Figure 4 ).

[0059] Example 5

[0060] High-temperature tolerance identification of OsCOI1a gene-editing material A103D

[0061] After disinfection, soaking, and germination treatment, seeds of OsCOI1a gene-edited material A103D and wild-type rice were selected based on consistent germination rates and sown. Wild-type rice seeds were sown in the left six rows of a 96-well hydroponic container, while the OsCOI1a gene-edited material A103D was sown in the right six rows. The seedlings were cultured in pure water until they developed one leaf and one bud, then the nutrient solution was changed to Yashida solution every two days. Two weeks after seedling emergence, the seedlings were subjected to high-temperature stress treatment at 45℃ for 72 hours, followed by a 7-day recovery period at 28℃. Phenotypic observation and data analysis were then performed. The results showed that almost all wild-type plants wilted and died under high temperature, while the OsCOI1a gene-edited material A103D plants grew well, with only a few seedlings dying. Statistical analysis showed that the survival rate of wild-type seedlings under high temperature stress was only 10%, while the survival rate of edited material A103D was as high as 70%, indicating a significant improvement in high temperature tolerance.

[0062] Example 6

[0063] Agronomic traits determination of OsCOI1a gene-edited material A103D

[0064] Field observations revealed that the OsCOI1a gene-edited material A103D and wild-type rice plants exhibited similar growth patterns at the mature stage, with no significant adverse effects observed (see [link to relevant documentation]). Figure 6 Further analysis of agronomic traits revealed that the OsCOI1a gene-edited material A103D showed no significant differences from the wild type in plant height, tillering, and thousand-grain weight. This indicates that the mutant OsCOI1a protein or gene of this invention improves rice resistance to brown planthoppers and high-temperature tolerance without significantly affecting rice growth and yield, demonstrating significant application value.

[0065] 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 rice OsCOI1a mutant gene, characterized in that, The nucleotide sequence of the rice OsCOI1a mutant gene is that the wild-type rice OsCOI1a gene has a mutation from C to A at position 308.

2. The rice OsCOI1a mutant gene according to claim 1, characterized in that, The nucleotide sequence of the rice OsCOI1a mutant gene is shown in SEQ ID NO.

1.

3. A mutant protein of rice OsCOI1a, characterized in that, The amino acid sequence of the rice OsCOI1a mutant protein is that of the wild-type rice OsCOI1a protein, where alanine is replaced by aspartic acid at position 103.

4. The rice OsCOI1a mutant protein according to claim 3, characterized in that, The amino acid sequence of the rice mutant protein is shown in SEQ ID NO.

2.

5. A recombinant plasmid comprising the rice OsCOI1a mutant gene as described in claim 1 or 2.

6. A plant expression vector comprising the rice OsCOI1a mutant gene as described in claim 1 or 2.

7. A recombinant cell comprising the rice OsCOI1a mutant gene as described in claim 1 or 2.

8. A recombinant protein comprising the rice OsCOI1a mutant gene as described in claim 1 or 2.

9. A method for obtaining rice plants with enhanced resistance to brown planthoppers, characterized in that, Includes the following steps: Plants can be made to contain the rice OsCOI1a mutant gene as described in claim 1 or 2 by means of gene editing, hybridization, backcrossing, self-pollination or asexual reproduction. The plant in question is rice.

10. The application of the rice OsCOI1a mutant gene as described in claim 1 or 2 in improving the high-temperature tolerance of rice.

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