Application of rice Oscam3 gene in regulating high temperature tolerance of rice

Abstract

The application discloses application of a rice Oscam3 gene in regulating high-temperature tolerance of rice and belongs to the field of plant breeding, and particularly relates to application of the rice Oscam3 gene in regulating high-temperature tolerance of rice. The application cultivates a plant with changed high-temperature tolerance by the following method: 1) inhibiting or reducing or silencing the expression amount of a gene coding a protein in a receptor plant, and / or inhibiting or reducing or silencing the activity and / or content of the gene coding the protein, to obtain a plant with enhanced high-temperature tolerance; and 2) increasing, enhancing and / or up-regulating the expression amount of a gene coding a protein in a receptor plant, and / or increasing, enhancing and / or up-regulating the activity and / or content of the gene coding the protein, to obtain a plant with weakened high-temperature tolerance. The Oscam3 knockout mutant shows a high-temperature tolerance phenotype after high-temperature stress treatment, indicating that the Oscam3 gene plays an important role in regulating high-temperature tolerance of rice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the rice Oscam3 gene in regulating rice heat tolerance. Background Technology

[0002] In recent years, extreme temperatures and water scarcity caused by climate change have become urgent global problems. The continuous population growth and decreasing availability of freshwater have further exacerbated the imbalance between food supply and demand. High temperature is one of the key abiotic stresses that significantly affects plant growth and development, and with rising global temperatures, the harm caused by high temperature stress to plants is becoming increasingly severe. The most significant harm of high temperature to plant growth is the reduction in photosynthesis and organic matter accumulation, thereby reducing energy supply. Elevated temperatures can also cause the accumulation of reactive oxygen species (ROS) in plants, which disrupt the stability of various biomolecules such as proteins and nucleic acids, interfering with intracellular metabolic balance. Furthermore, high temperatures damage subcellular structures such as the plasma membrane, leading to low seed germination efficiency, slow seedling growth, reproductive organ malformations, decreased pollen viability, and reduced crop yield and quality. Therefore, studying the mechanisms by which high temperatures affect plant growth and development is of great significance. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to regulate the heat resistance of plants to obtain rice varieties with strong heat resistance.

[0004] To address the problems existing in the prior art, the present invention provides a method for cultivating plants with altered heat tolerance, comprising the following 1) or 2):

[0005] 1) Inhibit or reduce or silence the expression level of protein-coding genes in recipient plants, and / or inhibit or reduce or silence the activity and / or content of protein-coding genes to obtain plants with enhanced heat resistance.

[0006] 2) Increase, enhance and / or upregulate the expression level of protein-coding genes in recipient plants, or / and increase, enhance and / or upregulate the activity and / or content of protein-coding genes to obtain plants with weakened heat resistance.

[0007] The protein may be any of the following:

[0008] A1) A protein with the amino acid sequence shown in SEQ ID No:3;

[0009] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 75% identity with the protein shown in A1) and has the ability to regulate the heat resistance of plants; for example, those skilled in the art can, based on the amino acid sequence shown in SEQ ID No:3 and conventional techniques such as the conserved substitution of amino acids, obtain a protein mutant with the same function as the amino acid sequence shown in SEQ ID No:3 by substituting, deleting and / or adding one or more amino acids without affecting its activity.

[0010] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] The protein described in A1) above is named Oscam3. The Oscam3 protein consists of 149 amino acids.

[0012] To facilitate the purification or detection of the protein in A1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:3 in the sequence listing.

[0013] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0014] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0015] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein Oscam3 of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein Oscam3 isolated in this invention, provided they encode and function as protein Oscam3, are derived from and equivalent to the nucleotide sequence of this invention.

[0016] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0017] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences or nucleotide sequences, then the identity value (%) can be obtained.

[0018] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0020] The protein mentioned above is derived from rice (Oryza sativa L.).

[0021] In one specific embodiment, a method for cultivating plants with enhanced heat resistance includes the following steps: inhibiting the expression of nucleic acid molecules encoding the Oscam3 protein in the target plant to obtain transgenic plants with enhanced heat resistance.

[0022] The inhibition of the expression of nucleic acid molecules encoding the Oscam3 protein in the target plant can be achieved by introducing a knockout vector or interference vector that targets the nucleic acid molecules encoding the Oscam3 protein into the target plant.

[0023] In this paper, the knockout is implemented using the CRISPR / Cas9 system.

[0024] In this article, knocking out the gene encoding the protein in the target plant can be achieved by mutating the gene encoding the protein in the plant genome (the nucleotide sequence of the genome is SEQ ID No:1) as follows:

[0025] 1) Replace 5'-GGGAACTGTCATGCGTTACGC-3' in the gene encoding the protein in the plant genomic DNA (corresponding to positions 987 to 1006 of SEQ ID No:1 and positions 99 to 118 of SEQ ID No:2) with 5'-GGGAACTGTCATGCGTTACGC-3' to knock out the gene encoding the Oscam3 protein;

[0026] 2) Replace 5'-GGGAACTGTCATGCGTTGC-3' (corresponding to positions 987 to 1006 of SEQ ID No:1 and positions 99 to 118 of SEQ ID No:2) in the gene encoding the protein in the plant genomic DNA with 5'-GGGAACTGTCATGCGTTCGC-3', thereby knocking out the gene encoding the Oscam3 protein.

[0027] The purpose of plant breeding described in this article includes cultivating plants with enhanced / weak heat resistance.

[0028] The present invention also provides a method for regulating the heat tolerance of plants, including regulating the activity and / or content of the proteins described above in the target plant, and / or the expression level of the genes encoding the proteins, to regulate the heat tolerance of plants.

[0029] In the above method, regulating the activity and / or content of the protein Oscam3 in the target plant, or / and the expression level of the gene encoding the protein, includes introducing a substance into the recipient plant that inhibits the expression of the gene encoding the protein Oscam3, thereby obtaining a target plant with altered heat tolerance; the gene encoding Oscam3 encodes the protein Oscam3.

[0030] The aforementioned proteins also fall within the scope of protection claimed in this invention.

[0031] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following:

[0032] B1) Nucleic acid molecules that encode the proteins described above;

[0033] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0034] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0035] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0036] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0037] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0038] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0039] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0040] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0041] C3) contains an expression cassette encoding the gene described in C2);

[0042] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0043] C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0044] C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0045] C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4);

[0046] C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0047] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a gene as shown in E1) or E2) below:

[0048] E1) The coding sequence is a cDNA molecule or DNA molecule of SEQ ID No:2;

[0049] E2) The nucleotide sequence is the cDNA molecule or DNA molecule of SEQ ID No:1.

[0050] The DNA molecule shown in SEQ ID No:2 (the Oscam3 gene that regulates the heat tolerance of plants) encodes the protein Oscam3, whose amino acid sequence is the same as that in SEQ ID No:3.

[0051] The nucleotide sequence shown in SEQ ID No:2 is the nucleotide sequence of the gene encoding the protein Oscam3 (CDS).

[0052] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No:2.

[0053] B1) The nucleic acid molecule may also include nucleic acid molecules that have more than 95% identity with the nucleotide sequence shown in SEQ ID No:2 and originate from the same species.

[0054] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0055] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0056] Further, the vector described in C4) can be the recombinant vector SG2027-Oscam3, the nucleotide sequence of which is sequence 4 in the sequence listing. SG2027-Oscam3 expresses an sgRNA targeting the Oscam3 gene, with the target sequence being 5'-GGGAACTGTCATGCGTTCGC-3' (SEQ ID No: 5). The target site of this sgRNA is located in exon 2 of the Oscam3 gene, and the nucleotide sequence of the target site is positions 987-1006 of SEQ ID No: 1 (corresponding to positions 99-118 of SEQ ID No: 2). The microorganisms described herein can be yeast, bacteria, algae, or fungi. The bacteria can originate from genera such as Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, and Bacillus. Specifically, it could be Agrobacterium tumefaciens EHA105.

[0057] The present invention also provides the use of the protein Oscam3 described above, or a substance regulating gene expression, or a substance regulating the activity or content of said protein, in any of the following:

[0058] Application of U1 in regulating plant heat tolerance;

[0059] Application of U2 in the preparation of products that regulate the heat resistance of plants;

[0060] U3) Applications in cultivating plants with high-temperature resistance;

[0061] U4) Applications in the preparation of products that cultivate heat-resistant plants;

[0062] U5) Applications in plant breeding.

[0063] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein Oscam3.

[0064] In the above applications, the substance that regulates gene expression or the substance that regulates the activity or content of the protein can be a biological material related to the protein, and the biological material can be the biological material described above.

[0065] In the above applications and methods, the regulation can be to increase, enhance, or upregulate; the regulation can also be to inhibit, reduce, or silence.

[0066] In this invention, the enhanced heat resistance is manifested in the following ways: under 45°C high-temperature stress, three-week-old seedlings, on a NIP background, show a 38% increase in survival rate of Oscam3 gene knockout mutant plants compared to control plants. On a 93-11 background, the survival rate of Oscam3 gene knockout mutant plants is 18% higher than that of control plants.

[0067] In the above applications or methods, the plant is any one of the following:

[0068] N1) Monocotyledonous or dicotyledonous plants;

[0069] N2) Plants of the order Poales;

[0070] N3) Gramineae plants;

[0071] N4) Rice plants;

[0072] N5) rice.

[0073] This invention utilizes CRISPR / Cas9-mediated gene editing technology to perform site-directed mutations or knockouts on specific targets of the Oscam3 gene, which regulates the heat tolerance of rice. The results show that after high-temperature treatment, the Oscam3 mutant lines exhibited less leaf curling and a significantly higher survival rate under high temperatures compared to the control plants. This provides new materials for the breeding of heat-resistant rice varieties and plays a positive role in accelerating the improvement of rice varieties. Attached Figure Description

[0074] Figure 1 This is a schematic diagram of the structure of the recombinant plasmid SG2027-Oscam3.

[0075] Figure 2 The sequencing results are for the mutation site and its surrounding nucleotides.

[0076] Figure 3 Phenotypic diagrams of rice NIP and Oscam3 mutants under high temperature stress and statistical graphs of survival rates after high temperature stress treatment.

[0077] Figure 4 Phenotypic diagrams of rice 93-11 and Oscam3 mutants under high temperature stress and statistical graphs of survival rates after high temperature stress treatment. Detailed Implementation

[0078] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0079] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0080] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0081] The SG2027 vector in the following examples has been described in Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses. Molecular plant, 2018, 11(12):1492-1508. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating the relevant experiments of this invention and shall not be used for any other purpose.

[0082] The rice varieties Nipponbare (NIP) and 93-11 described in the following examples are described in: Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indicarice genomes and its association with gene expression, plant development, and stress responses. Molecular plant, 2018, 11(12): 1492-1508. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating the relevant experiments of this invention and shall not be used for any other purpose.

[0083] The following examples use EXCEL software for statistical analysis of data and SPASS software for multiple differences comparison of data.

[0084] Example 1: Creation of Oscam3 gene knockout rice

[0085] 1. Obtaining the Oscam3 gene in rice

[0086] Leaf DNA was extracted from rice varieties NIP and 93-11. Using this DNA as a template, primers Oscam3-F (5'-ATGGCGGACCAGCTC-3') and Oscam3-R (5'-TTACTTGGCCATCAT-3') were used to... PCR amplification was performed using Max Super-Fidelity DNA Polymerase (catalog number: P505-d1, Vazyme) to obtain the amplified product, which is the genomic sequence of the Oscam3 gene. The nucleotide sequence of the genomic gene encoding the Oscam3 protein in the genomic DNA of rice 93-11 is SEQ ID No:1. Positions 107-177 of SEQ ID No:1 are exon 1, and positions 964-1338 are exon 2.

[0087] The coding sequence of the Oscam3 gene in rice varieties NIP and 93-11 is SEQ ID No:2, and the coding amino acid sequence of the Oscam3 protein is SEQ ID No:3.

[0088] 2. Construction of the recombinant plasmid SG2027-Oscam3 with the Oscam3 gene knocked out

[0089] The nucleotide sequence of recombinant plasmid SG2027-Oscam3 is SEQ ID No:4. The image of plasmid SG2027-Oscam3 can be found here. Figure 1 .

[0090] Sequence 4 is the gene sequence obtained by Sanger sequencing, containing the sgRNA sequence, the Oscam3 coding region sequence, and the SG2027 vector sequence flanking the insertion sequence. SG2027-Oscam3 expresses an sgRNA targeting the Oscam3 gene, with the target sequence being: 5'-GGGAACTGTCATGCGTTCGC-3' (SEQ ID No:1). The target site of this sgRNA is located in exon 2 of the Oscam3 gene, and the nucleotide sequence of the target site is positions 987-1006 of SEQ ID No:1 (corresponding to positions 98-118 of sequence 2).

[0091] 3. Obtaining and identifying Oscam3 gene knockout rice

[0092] The recombinant plasmid SG2027-Oscam3 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 (Shanghai Weidi Company, EHA105) to obtain recombinant Agrobacterium. Using the Agrobacterium infection method, the recombinant Agrobacterium was used to genetically transform embryogenic callus tissue of rice 93-11. Resistant callus tissue was then screened (resistance screening used 100 mg / L hygromycin), followed by differentiation and regeneration culture, and then rooting culture to obtain regenerated plants.

[0093] The specific steps are as follows:

[0094] (1) Take out the mature seeds of Nipponbare (NIP) and 93-11 rice, remove the husks, and select plump, clean seeds without sterile spots for disinfection.

[0095] (2) Inoculate the disinfected rice 93-11 seeds onto the induction medium and culture them in the dark at 28°C for about 14 days. Select callus tissue with good appearance and good growth.

[0096] (3) Take the recombinant vector SG2027-Oscam3 constructed in step 2 above and introduce it into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria EHA105 / SG2027-Oscam3.

[0097] (4) Take the recombinant bacteria EHA105 / SG2027-Oscam3 obtained in step (3) and resuspend the bacteria in infection medium (MS liquid medium + 50 g / L sucrose + 50 μL / L Silwet L-77) to obtain EHA105 / SG2027-Oscam3 bacterial suspension.

[0098] (5) Immerse the 93-11 callus tissue from step (2) in the EHA105 / SG2027-Oscam3 bacterial suspension prepared in step (4) for 20 min. After infection, discard the bacterial suspension, take the callus tissue, blot dry with sterile filter paper, and then place it on a co-culture medium (MS basal medium) containing acetylsuccinone and glucose, and incubate in the dark at 28°C for 50-55 h.

[0099] (6) After completing step (5), select callus tissues without obvious Agrobacterium on the surface and transfer them to antibacterial medium (MS basic medium) with added cephalosporin, and incubate in the dark at 28°C for 3-4 days.

[0100] (7) The above-cultured callus tissue was transferred to the selection medium (MS basic medium) containing hygromycin and cephalosporin and cultured in the dark at 28°C for 30 days, and subcultured every 10 days.

[0101] (8) After completing step (7), take fresh hygromycin-resistant callus tissue, inoculate it in pre-regeneration medium (MS basic medium), culture it in the dark at 28°C for 7 days, and then place it in a light culture room (12h light / 12h dark) for 7 days. After that, transfer it to regeneration medium and continue to culture it in the light until regenerated plants grow, and obtain candidate Oscam3 gene knockout rice plants.

[0102] The transgenic plants obtained using the recombinant vector SG2027-Oscam3 are designated as Oscam3 transgenic plants.

[0103] Both the induction medium and the differentiation medium were formulated as MS medium (Solepro, M8521).

[0104] 4. Identification of Oscam3 gene knockout rice

[0105] The plants to be tested were 93-11 (control group) and the candidate Oscam3 gene knockout plants obtained in step 3.

[0106] Genomic DNA was extracted from the leaves of rice plants with the Oscam3 gene knockout to be tested. Using the genomic DNA as a template, PCR amplification was performed using primer pairs consisting of primers Oscam3-F1 and Oscam3-R1.

[0107] Oscam3-F1: 5'-ACCTGTAAATCCAGACAAGTCG-3';

[0108] Oscam3-R1:5'-CTTGAGAACACTTCCGGCCT-3'.

[0109] The SG2027-Oscam3 plasmid was used as a positive control (V), and the receptor variety 93-11 was used as a negative control (CK). The resulting products were then sequenced.

[0110] Identified by sequencing ( Figure 2 Compared to the genomic DNA of 93-11, the gene encoding the Oscam3 protein in the two homologous chromosomes of mutation type cam3-1 underwent the following mutation: "5'-GGGAACTGTCATGCGTTCGC-3' (corresponding to positions 987 to 1006 of SEQ ID No:1 and positions 99 to 118 of SEQ ID No:2)" was mutated to "5'-GGGAACTGTCATGCGTTACGC-3', i.e., the insertion of one base "A", causing a frameshift mutation in Oscam3, resulting in its loss of function and thus knocking out the gene encoding the Oscam3 protein. The sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 .

[0111] Identified by sequencing ( Figure 2 Compared to the genomic DNA of 93-11, the gene encoding the Oscam3 protein in the two homologous chromosomes of mutation type cam3-2 underwent the following mutation: "5'-GGGAACTGTCATGCGTTCGC-3' (corresponding to positions 987 to 1006 of SEQ ID No:1 and positions 99 to 118 of SEQ ID No:2)" was mutated to "5'-GGGAACTGTCATGCGTTAGC-3'", that is, the deletion of one base "C", causing Oscam3 to mutate and lose its function, thereby knocking out the gene encoding the Oscam3 protein. The sequencing results of this mutation site and its surrounding nucleotides are shown in [link to sequencing data]. Figure 2 .

[0112] Based on the above identification, two homozygous edited plants (i.e., the mutations on the two homologous chromosomes are consistent) were obtained and named Oscam3-1 and Oscam3-2, respectively.

[0113] The T1 generation rice mutant plants of the above-mentioned homozygous Oscam3 gene mutant types Oscam3-1 and Oscam3-2 were further cultured and screened to obtain T2 generation Oscam3 plants without transgenic elements, and then phenotypic identification was performed.

[0114] Oscam3-1 plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. Oscam3-1 plants and their self-pollinated offspring are called Oscam3-1 lines.

[0115] Oscam3-2 plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are the T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. Oscam3-2 plants and their self-pollinated offspring are called Oscam3-2 lines.

[0116] Example 2: Comparison of productive traits in gene-edited rice

[0117] The plants to be tested were: T3 generation homozygous lines of rice NIP and 93-11, mutant Oscam3-1, and Oscam3-2.

[0118] Seeds of all tested plants were germinated and seedlings were raised in a greenhouse (starting from the emergence of white shoots, for a total of 3 weeks). Three-week-old seedlings were obtained and subjected to high-temperature stress treatment (treatment temperature of 45℃). The survival rate was counted, with at least 60 individual plant data collected for each material. The survival rate was calculated using the following formula: Survival rate (%) = (Total number of rice plants - Number of dead rice plants) / Total number of rice plants × 100%.

[0119] The growth status of the plants to be tested can be seen in the following figures. Figure 3 and Figure 4 When three-week-old seedlings were subjected to high-temperature stress of 45℃ for 36 hours, the survival rate of the NIP control was 18%, and that of the Oscam3-1 gene knockout mutant plants was 56%. When subjected to high-temperature stress of 45℃ for 36 hours, the survival rates of NIP and Oscam3-2 were 0% and 21%, respectively. Compared with the NIP control plants, the survival rate of the Oscam3 gene knockout mutant plants was significantly increased. The survival rate of the 93-11 control was 0%, and the survival rates of the Oscam3 gene knockout mutant plants Oscam3-1 and Oscam3-2 were 19% and 21%, respectively. When three-week-old seedlings were subjected to high-temperature stress of 45℃ for 48 hours, the survival rate of the Oscam3 gene knockout mutant plants was significantly increased compared with the 93-11 control plants.

[0120] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Methods for cultivating plants with altered heat tolerance, including knocking out protein-coding genes in recipient plants to obtain plants with enhanced heat tolerance; The protein is a protein with an amino acid sequence as shown in SEQ ID No:3; The plant in question is rice.

2. A method for enhancing the heat resistance of plants, characterized in that: The invention includes knocking out the gene encoding the protein of claim 1 in a recipient plant to enhance the plant's heat tolerance; the gene encoding the protein of claim 1; and the plant being rice.

3. The use of the substance that knocks out the gene encoding the protein of claim 1 in any of the following: 1) Applications in enhancing the heat resistance of plants; 2) Applications in the preparation of products that enhance the heat resistance of plants; 3) Applications in cultivating plants with enhanced heat resistance; 4) Applications in the preparation of products that cultivate plants with enhanced heat resistance; 5) Applications in plant breeding; The plant in question is rice; The purpose of the breeding is to develop rice varieties with enhanced heat resistance; The substance is a biomaterial related to the protein of claim 1, and the biomaterial is any one of the following: C1) Knock out the nucleic acid molecule encoding the protein described in claim 1; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette containing the gene encoding described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

4. The application according to claim 3, characterized in that: C1) The target sequence of the nucleic acid molecule is the DNA molecule shown in SEQ ID No:5.

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