Application of substances that regulate the expression of protein OsRNT in regulating heat tolerance in rice

Abstract

This invention discloses the application of substances regulating the expression of the protein OsRNT in regulating heat tolerance in rice. This invention belongs to the field of plant breeding, specifically relating to the application of substances regulating the expression of the protein OsRNT in regulating heat tolerance in rice. The protein OsRNT of this invention is as follows: A1) Amino acids as shown in sequence 3; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1), possessing more than 75% identity and the same function as the protein shown in A1); A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2). After CRISPR gene editing and knockout of the gene encoding the OsRNT protein, the OsRNT knockout mutant exhibits a heat-tolerant phenotype after high-temperature stress treatment, indicating that… OsRNT Genes play an important role in regulating the heat resistance of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding, specifically relating to the application of substances that regulate the expression of the protein OsRNT in regulating the high-temperature tolerance of rice. Background Technology

[0002] Extreme temperatures and global climate change threaten crop yields, with temperature being a major environmental factor influencing plant growth and development. With continuous population growth and industrialization, global temperatures are rising annually, and extreme heat severely restricts plant growth and crop yields, leading to significant reductions in the production of grains such as rice, wheat, and corn. High-temperature stress reduces the photosynthetic rate and accelerates transpiration in rice, inhibiting its growth and development, resulting in decreased grain filling rate and thousand-grain weight. Synergistic improvement of crop yield and stress resistance is an effective strategy to address the frequent occurrence of extreme weather events such as population growth and global warming. In-depth research into heat-resistant genes and their regulatory mechanisms is crucial for improving crop heat tolerance and is of great significance for the genetic improvement of heat-resistant traits in crops. Summary of the Invention

[0003] The main problem this invention aims to solve is how to improve the heat resistance of plants.

[0004] To address the above problems, the present invention provides a protein.

[0005] The protein provided by this invention may be any of the following proteins:

[0006] a1) A protein with the amino acid sequence SEQ ID No:3;

[0007] a2) A protein having the same function as the amino acid sequence shown in SEQ ID No:3, but with one or more amino acid residues substituted and / or deleted and / or added.

[0008] Proteins that have more than 75% identity with the amino acid sequence defined in (a3), (a1), or (a2) and have the same function;

[0009] The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), (a3).

[0010] The protein described in a1) above is named OsRNT.

[0011] 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.

[0012] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

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

[0014] Those skilled in the art can readily mutate the nucleotide sequence encoding the OsRNT protein 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 OsRNT protein isolated in this invention, provided they encode and function as the OsRNT protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0015] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0016] In this article, identity refers to the similarity between amino acid sequences 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 or nucleotide sequences, then the identity value (%) can be obtained.

[0017] In this document, the 80% or more of 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% of the identity.

[0018] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0019] In this article, the protein is derived from rice ( Oryza sativa L . ).

[0020] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following:

[0021] B1) Nucleic acid molecules that encode the proteins described above;

[0022] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0023] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] 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);

[0025] 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);

[0026] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0027] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0028] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0029] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0030] C3) contains an expression cassette containing the gene encoding described in C2);

[0031] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0032] 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);

[0033] 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);

[0034] 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);

[0035] 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).

[0036] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a gene as shown in E1) or E2) below:

[0037] E1) The coding sequence is a cDNA molecule or DNA molecule of SEQ ID No:2;

[0038] E2) The nucleotide sequence is the cDNA molecule or DNA molecule of SEQ ID No:1.

[0039] The DNA molecule shown in SEQ ID No:2 (which regulates plant heat tolerance) OsRNT The gene encodes the protein OsRNT, whose amino acid sequence is SEQ ID No:3.

[0040] The nucleotide sequence shown in SEQ ID No:2 is the nucleotide sequence of the protein OsRNT encoding gene (CDS).

[0041] The present invention OsRNT Genes can be any nucleotide sequence that encodes the protein OsRNT. Considering codon degeneracy and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0042] 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.

[0043] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No:2 and originate from the same species.

[0044] 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.

[0045] 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.

[0046] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0047] The microorganisms mentioned in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from the genus *Escherichia* (…). Escherichia Erwinia ( Erwinia Agrobacterium tumefaciens ( ), Agrobacterium tumefaciens Agrobacterium Flavobacterium ( Flavobacterium Alcaligenes ( ) Alcaligenes ), Pseudomonas ( Pseudomonas ), Bacillus spp. ( Bacillus (e.g., Agrobacterium tumefaciens EHA105).

[0048] The present invention also provides the use of the above-mentioned protein or gene expression substance or substance regulating the activity or content of said protein in any of the following:

[0049] 1) Application in regulating plant heat tolerance;

[0050] 2) Application in the preparation of products that regulate plant heat resistance;

[0051] 3) Applications in cultivating plants with altered heat tolerance;

[0052] 4) Applications in the preparation of products containing plants with altered heat resistance;

[0053] 5) Applications in plant breeding.

[0054] 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 OsRNT.

[0055] In this invention, the regulation can be up-regulation, enhancement, or increase. The regulation can also be suppression, reduction, or down-regulation.

[0056] In this article, the upregulation, enhancement, or increase of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase, or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0057] In this article, regulating the expression of the gene encoding the protein can also be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0058] 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 described above.

[0059] The present invention also provides a method for improving the heat tolerance of plants, the method comprising step P, wherein step P is to inhibit or reduce or silence the activity and / or content of the aforementioned proteins in the receptor plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned proteins, thereby improving the heat tolerance of plants.

[0060] In the above method, the inhibition, reduction, or silencing of the expression level and / or activity of the gene encoding the protein OsRNT in the recipient plant can be achieved by using gene mutation, gene knockout, gene editing, or gene knockdown techniques to reduce or inactivate the activity of the gene encoding the protein OsRNT in the recipient plant genome.

[0061] The present invention also provides a method for reducing the heat tolerance of plants, the method comprising step M, wherein step M is to enhance, increase or upregulate the activity and / or content of the aforementioned proteins in the recipient plant, or / and enhance, increase or upregulate the expression level of the encoding genes of the aforementioned proteins, thereby reducing the heat tolerance of plants.

[0062] The present invention provides a method for cultivating plants with enhanced heat resistance, comprising inhibiting or reducing or silencing the expression and / or content and / or activity of the coding gene of the aforementioned protein in the recipient plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the aforementioned protein, thereby obtaining a plant with enhanced heat resistance.

[0063] In this invention, the purpose of plant breeding may include cultivating plants with enhanced heat resistance.

[0064] In the above method, the inhibition, reduction, or silencing of the expression level and / or activity of the gene encoding the protein OsRNT in the recipient plant can be achieved by using gene mutation, gene knockout, gene editing, or gene knockdown techniques to reduce or inactivate the activity of the gene encoding the protein OsRNT in the recipient plant genome.

[0065] In the above method, the gene knockout is achieved using the CRISPR / Cas9 system.

[0066] The target site for gene editing in the CRISPR / Cas9 system is positions 3343-3362 of SEQ ID No:1 or positions 359-378 of SEQ ID No:2.

[0067] In the above method, knocking out the gene encoding the target rice protein can be achieved by mutating the protein of sequence 1 in the rice genome by at least one of the following methods:

[0068] 1) Replace 5'-CCGCATTACGTGACCTTGGT-3' in the gene encoding the protein in the rice genomic DNA with 5'-CCGCATATACGTGACCTTGGT-3', thereby knocking out the gene encoding the OsRNT protein;

[0069] 2) Replace “5'-CCGCATTACGTGACCTTGGT-3'” in the gene encoding the protein in the rice genomic DNA with 5'-CGCATACCTTGGT-3', thereby knocking out the gene encoding the OsRNT protein.

[0070] In this article, the high-temperature treatment conditions can be: treating three-week-old rice seedlings at 45℃ for 48 hours.

[0071] In the above applications or methods, the plant is any one of the following:

[0072] N1) Monocotyledonous or dicotyledonous plants;

[0073] N2) Plants of the order Poales;

[0074] N3) Gramineae plants;

[0075] N4) Rice plants;

[0076] N5) rice.

[0077] In this article, the plant heat resistance performance may include plant heat stress phenotype and improved survival rate.

[0078] In this invention, the enhanced heat resistance is specifically manifested in the following way: when treated at 45°C for 48 hours, compared with the control plant, the knockout mutant plant... Osrnt1 and Osrnt2 Survival rates increased by 30% and 50%, respectively.

[0079] This invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out specific targets in the gene encoding OsRNT, a protein that regulates heat tolerance in rice. 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

[0080] Figure 1 For recombinant plasmid SG2027- OsRNT A structural diagram.

[0081] Figure 2 The sequencing results are for the mutation site and its surrounding nucleotides.

[0082] Figure 3 This is a phenotypic and statistical graph of rice under high temperature stress. Where A represents 93-11 and... OsRNT Phenotypic diagram of mutants under high temperature stress; B represents 93-11 and OsRNT Mutant survival rate statistics chart. Detailed Implementation

[0083] 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.

[0084] 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.

[0085] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0086] The SG2027 vector in the following examples is 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.

[0087] The rice varieties 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 Indica rice 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 replicating the experiments of this invention and shall not be used for any other purpose.

[0088] The following examples use EXCEL software for statistical analysis of data and SPASS software for multiple variance comparisons of data.

[0089] Example 1 OSRNT The creation of gene knockout rice

[0090] 1. Rice OSRNT Acquisition of genes

[0091] DNA was extracted from leaves of rice variety 93-11. Using this DNA as a template, primers were then used... OSRNT- F: 5'-ATGAAATATGCACCT -3'; OSRNT- R: 5'-CTATGCGGCCTTTGG -3', PCR amplification was performed using Phanta® Max Super-Fidelity DNA Polymerase (catalog number: P505-d1, Vazyme) to obtain the amplification product, i.e. OSRNT The genome of the gene (nucleotide sequence is SEQ ID No: 1). Positions 2058-2210 of SEQ ID No: 1 are exon 1, positions 3137-3467 are exon 2, positions 3618-4027 are exon 3, positions 4030-4181 are exon 4, positions 5141-5213 are exon 5, and positions 5562-5730 are exon 6.

[0092] OsRNT The coding sequence of the gene in rice variety 93-11 is SEQ ID No:2, encoding the OsRNT protein with the amino acid sequence SEQ ID No:3. The genomic DNA of rice 93-11 encodes... OsRNTThe genomic nucleotide sequence of the protein is SEQ ID No:1.

[0093] 2. Knockout OsRNT Recombinant plasmid SG2027 of the gene -OsRNT Construction

[0094] Recombinant plasmid SG2027 -OsRNT The nucleotide sequence is SEQ ID No:4 in the sequence listing. SEQ ID No:4 is the gene sequence obtained by Sanger sequencing, containing the sgRNA sequence. OsRNT The coding region sequence and the SG2027 vector sequences flanking the insertion sequence. SG2027 -OsRNT See the vector map Figure 1 .

[0095] SG2027 -OsRNT Expression targeting OsRNT The gene's sgRNA has a target sequence of 5'-CCGCATTACGTGACCTTGGT-3', and the target site of this sgRNA is located at... OsRNT The second exon of the gene, the nucleotide sequence of the target site of this sgRNA is positions 3343-3362 of SEQ ID No:1 (corresponding to positions 359-378 of SEQ ID No:2).

[0096] 3. OsRNT Obtaining and identifying gene knockout rice

[0097] The recombinant plasmid SG2027 obtained in step 2 -OsRNT Recombinant Agrobacterium tumefaciens EHA105 (Shanghai Weidi Company) was introduced to obtain Agrobacterium tumefaciens. Using the Agrobacterium tumefaciens inoculation method, the recombinant Agrobacterium tumefaciens 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.

[0098] The specific steps are as follows:

[0099] (1) Take out the mature seeds of rice 93-11, remove the shells, and select the plump, clean seeds without sterile spots for disinfection.

[0100] (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.

[0101] (3) Take the recombinant vector SG2027 constructed in step 2 above. -OsRNT By introducing Agrobacterium tumefaciens EHA105, recombinant strain EHA105 / SG2027- was obtained. OsRNT .

[0102] (4) Take the recombinant bacteria EHA105 / SG2027- obtained in step (3). OsRNT The bacterial cells were resuspended in infection medium (MS liquid medium + 50 g / L sucrose + 50 μL / L Silwet L-77) to obtain EHA105 / SG2027- OsRNT bacterial suspension.

[0103] (5) Immerse the 93-11 callus tissue from step (2) in the EHA105 / SG2027 prepared in step (4). -OsRNT Infect the bacterial suspension for 20 minutes. 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 hours.

[0104] (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.

[0105] (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.

[0106] (8) After completing step (7), take fresh hygromycin-resistant callus tissue, inoculate it into pre-regeneration medium (MS basal medium), and culture it in the dark at 28°C for 7 days. Then, place it in a light culture room (12h light / 12h dark) and continue to culture for 7 days. After that, transfer it to regeneration medium and continue to culture under light until regenerated plants grow, thus obtaining candidate plants. OsRNT Gene knockout rice plants.

[0107] The recombinant vector SG2027 will be used. -OsRNT The resulting transgenic plants are denoted as OsRNT Genetically modified plants.

[0108] Both the induction medium and the differentiation medium were formulated as MS medium (Solepro, M8521).

[0109] 4. OsRNT Identification of gene knockout rice

[0110] Plants to be tested: 93-11 (control group) and candidate plants obtained in step 3 OsRNT Gene knockout plants.

[0111] Extract the test OsRNTGenomic DNA from rice plant leaves was knocked out using gene knockout as a template, and primers were used. OsRNT- F1 and primers OsRNT- PCR amplification was performed using primers consisting of R1.

[0112] OsRNT- F1: 5'-GGGCTGATTTCCCCTAGCAG -3';

[0113] OsRNT- R1:5'-GGTTGTTGACATGTGGTTCCT-3'.

[0114] Using SG2027- OsRNT The 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.

[0115] Identified by sequencing ( Figure 2 (Compared to the genomic DNA of 93-11, mutation types) RNT-1 The gene encoding the OsRNT protein in two homologous chromosomes underwent the following mutation: "5'-CCGCATTACGTGACCTTGGT-3' (corresponding to positions 3343 to 3362 in SEQ ID No:1 and positions 359 to 378 in SEQ ID No:2)" was mutated to "5'-CCGCATATACGTGACCTTGGT-3', i.e., the insertion of one base "A", causing a frameshift mutation in OsRNT, resulting in loss of function and thus knocking out the gene encoding the OsRNT protein. Sequencing results of this mutation site and its surrounding nucleotides are shown in [Figure number missing]. Figure 2 .

[0116] Compared with the genomic DNA of 93-11, the mutation types RNT-2 The gene encoding the OsRNT protein in two homologous chromosomes underwent the following mutation: "5'-CCGCATTACGTGACCTTGGT-3' (corresponding to positions 3343 to 3362 in SEQ ID No:1 and positions 359 to 378 in SEQ ID No:2)" was mutated to "5'-CGCATACCTTGGT-3', i.e., the deletion of 6 bases "TACGTG", causing a frameshift mutation in OsRNT, resulting in its loss of function, thus knocking out the gene encoding the OsRNT protein. Sequencing results of the mutation site and surrounding nucleotides are shown in [link to sequencing data]. Figure 2 .

[0117] Based on the above identification, two homozygous edited plants were obtained (i.e., the mutations on the two homologous chromosomes were identical), and were named respectively. Osrnt-1 and Osrnt-2 .

[0118] The above OsRNT homozygous mutation type OsRrnt-1 and Osrnt-2 The T1 generation rice mutant plants were further cultured and screened to obtain the T2 generation without transgenic elements. Osrnt The plants were examined and their phenotypic characteristics were identified.

[0119] Osrnt-1 Plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are called T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are called T2 generation seeds. Osrnt-1 The plant and its self-pollinated offspring are called Osrnt-1 strains.

[0120] Osrnt-2 Plants are self-pollinated and seeds are harvested. These seeds are then cultivated into plants, which are called T1 generation plants. T1 generation plants are self-pollinated and seeds are harvested, which are called T2 generation seeds. Osrnt-2 The plant and its self-pollinated offspring are called Osrnt-2 strains.

[0121] Example 2: Comparison of Rice Production Traits

[0122] The plants to be tested were: rice 93-11 and mutant strains. Osrnt-1 , Osrnt-2 The T3 generation homozygous strain of the line.

[0123] Seeds of all tested plants were germinated and seedlings were cultivated in a greenhouse (starting from the emergence of white hairs, for a total of 3 weeks). Three-week-old seedlings were obtained and subjected to high-temperature stress treatment (45℃ for 48 hours, abbreviated as HS). The survival rate was counted, and at least 60 individual plant data were collected for each material. Normal growth conditions without high-temperature stress treatment were used as the control CK.

[0124] The growth status of the plants to be tested can be seen in the following figures. Figure 3 Seven days after rehydration following high-temperature stress (HS), the 93-11 plants were mostly withered and yellow. Osrnt-1 and Osrnt-2 It is in a normal growth state ， The leaves are partially curled. Figure 3 (A); 93-11 survival rate was 0%. Osrnt-1 and Osrnt-2 The survival rates were 30% and 50%, respectively, significantly higher than the survival rate of 93-11. Figure 3 (B). This indicates that OsRNT is a negative regulatory gene for high-temperature response.

[0125] 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. The application of substances that knock out protein-coding genes in any of the following: U1) Applications in improving plant heat resistance; U2) Applications in the preparation of products that improve the heat resistance of plants; U3) Applications in the preparation of products that enhance the heat resistance of plants; U4) Applications in plant breeding; The protein is a protein with an amino acid sequence as shown in SEQ ID No:3; The plant in question is rice; The purpose of the breeding is to develop rice varieties with enhanced heat resistance; The substance that knocks out the protein-coding gene is any one of the following: C1) Knock out the nucleic acid molecules that express the gene encoding the protein; 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).

2. Use according to claim 1, characterized in that: C1) The nucleic acid molecule described below is a gene as shown in E1) or E2): E1) The coding sequence is a cDNA molecule or DNA molecule of SEQ ID No:2; E2) The nucleotide is a cDNA molecule or DNA molecule of SEQ ID No:

1.

3. A method for increasing the heat tolerance of a plant, characterized by: The method includes knocking out the protein described in claim 1 in the target plant to improve the plant's heat resistance; the plant is rice.

4. A method for cultivating plants with enhanced heat tolerance, comprising knocking out the gene encoding the protein described in claim 1 in a recipient plant to obtain a plant with enhanced heat tolerance; The plant in question is rice.

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