Use of osrnt protein or material regulating expression of osrnt protein in regulating heat tolerance of rice

Abstract

Disclosed in the present invention is a use of an OsRNT protein or a material regulating the expression of the OsRNT protein in regulating the heat tolerance of rice. The present invention relates to the field of plant breeding, and specifically to a use of an OsRNT protein or a material regulating the expression of the OsRNT protein in regulating the heat tolerance of rice. The OsRNT protein of the present invention is as follows: A1) an amino acid sequence as shown in SEQ ID NO:3; A2) a protein, which is obtained by subjecting the protein in A1) to substitution and / or deletion and / or addition of amino acid residues and has 75% or more identity to and the same function as the protein in A1); and A3) a fusion protein obtained by linking a protein tag to the N-terminus or / and C-terminus in A1) or A2). After a gene encoding the OsRNT protein is knocked out by CRISPR gene editing, an OsRNT-knockout mutant exhibits a heat-tolerant phenotype under heat stress, indicating that the OsRNT gene plays an important role in regulating the heat tolerance of rice.

Description

Application of substances that regulate the expression of protein OsRNT in regulating heat tolerance in rice 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 seed setting 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. Technical issues

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

[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 of SEQ ID No:3, by substitution and / or deletion and / or addition of one or more amino acid residues;

[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 of 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% 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] The protein described in this article 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) can be a gene as shown in E1) or E2):

[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 of SEQ ID No:2 (the OsRNT gene that regulates plant heat tolerance) encodes the protein OsRNT, which is the amino acid sequence of SEQ ID No:3.

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

[0041] The OsRNT gene described in this invention can be any nucleotide sequence capable of encoding 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 specific species as needed.

[0042] B1) The nucleic acid molecule may also include nucleic acid molecules obtained by codon preference modification based on the nucleotide sequence of 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 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 described in this article can be yeast, bacteria, algae, or fungi. Among them, bacteria can originate from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*. Specifically, *Agrobacterium tumefaciens* EHA105 is an example.

[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) The gene encoding the OsRNT protein was knocked out by replacing 5'-CCGCATTACGTGACCTTGGT-3' (SEQ ID No:6) in the gene encoding the protein in the rice genomic DNA with 5'-CCGCATATACGTGACCTTGGT-3' (SEQ ID No:5);

[0069] 2) Replace 5'-CCGCATTACGTGACCTTGGT-3' (SEQ ID No:6) in the gene encoding the protein in the rice genomic DNA with 5'-CGCATACCTTGGT-3' (SEQ ID No:7) to knock 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 ways: when treated at 45°C for 48 hours, the survival rates of the knockout mutant plants Osrnt1 and Osrnt2 increased by 30% and 50%, respectively, compared with the control plants. Beneficial effects

[0079] This invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out specific targets in the gene encoding the heat-resistant protein OsRNT in rice, providing new materials for the breeding of heat-resistant rice varieties and playing a positive role in accelerating the improvement of rice varieties. Attached Figure Description

[0080] Figure 1 is a schematic diagram of the structure of the recombinant plasmid SG2027-OsRNT.

[0081] Figure 2 shows the sequencing results of the mutation sites and their surrounding nucleotides. The sequence of the 93-11 mutation site and its surrounding nucleotides is SEQ ID No:6; the sequence of the Osrnt-1 mutation site and its surrounding nucleotides is SEQ ID No:5; and the sequence of the Osrnt-2 mutation site and its surrounding nucleotides is SEQ ID No:7. In the figure, "-" indicates a nucleotide deletion at this position relative to the Osrnt-1 nucleotide sequence (SEQ ID No:5).

[0082] Figure 3 shows the phenotypes and statistics of rice under high temperature stress. A is the phenotype of the 93-11 and OsRNT mutants under high temperature stress; B is the survival rate statistics of the 93-11 and OsRNT mutants. Embodiments of the present invention

[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 may 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 differences comparison of data.

[0089] Example 1: Creation of OSRNT gene knockout rice

[0090] 1. Obtaining the rice OSRNT gene

[0091] Leaf DNA was extracted from rice variety 93-11. Using this DNA as a template, primers OSRNT-F: 5'-ATGAAAATATGCACCT-3' (SEQ ID No:8); OSRNT-R: 5'-CTATGCGGCCTTTGG-3' (SEQ ID No:9) were used to perform the Phanta ®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 OSRNT gene (nucleotide sequence is SEQ ID No:1). Positions 2058-2210 of SEQ ID No:1 represent exon 1, positions 3137-3467 represent exon 2, positions 3618-4027 represent exon 3, positions 4030-4181 represent exon 4, positions 5141-5213 represent exon 5, and positions 5562-5730 represent exon 6.

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

[0093] 2. Construction of the recombinant plasmid SG2027-OsRNT with OsRNT gene knocked out

[0094] The nucleotide sequence of the recombinant plasmid SG2027-OsRNT is SEQ ID No:4 in the sequence listing. SEQ ID No:4 is the gene sequence obtained by Sanger sequencing, which includes the sgRNA sequence, the OsRNT coding region sequence, and the SG2027 vector sequences flanking the insert sequence. The SG2027-OsRNT vector map is shown in Figure 1.

[0095] SG2027-OsRNT expresses an sgRNA targeting the OsRNT gene, with the target sequence being 5'-CCGCATTACGTGACCTTGGT-3' (SEQ ID No:6). The target site of this sgRNA is located in the second exon of the OsRNT gene, and the nucleotide sequence of the target site is positions 3343-3362 of SEQ ID No:1 (corresponding to positions 359-378 of SEQ ID No:2).

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

[0097] The recombinant plasmid SG2027-OsRNT obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 (Shanghai Weidi Company) to obtain recombinant Agrobacterium. Using the Agrobacterium infection method, the recombinant Agrobacterium was used to genetically transform embryogenic callus tissue of rice 93-11. Then, resistant callus tissue was 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 husks, and select 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-OsRNT constructed in step 2 above and introduce it into Agrobacterium tumefaciens EHA105 to obtain recombinant bacteria EHA105 / SG2027-OsRNT.

[0102] (4) Take the recombinant bacteria EHA105 / SG2027-OsRNT 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-OsRNT bacterial suspension.

[0103] (5) Immerse the 93-11 callus tissue from step (2) in the EHA105 / SG2027-OsRNT 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.

[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 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 OsRNT gene knockout rice plants.

[0107] The transgenic plants obtained using the recombinant vector SG2027-OsRNT are designated as OsRNT transgenic plants.

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

[0109] 4. Identification of OsRNT gene knockout rice

[0110] The plants to be tested were 93-11 (control group) and the candidate OsRNT gene knockout plants obtained in step 3.

[0111] Genomic DNA was extracted from the leaves of rice plants with the OsRNT gene knockout to be tested. Using the genomic DNA as a template, PCR amplification was performed using primer pairs consisting of primers OsRNT-F1 and OsRNT-R1.

[0112] OsRNT-F1: 5'-GGGCTGATTTCCCCTAGCAG-3' (SEQ ID No: 10);

[0113] OsRNT-R1:5'-GGTTGTTGACATGTGGTTCCT-3' (SEQ ID No: 11).

[0114] The SG2027-OsRNT 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] Sequencing analysis (Figure 2) revealed that, compared to the genomic DNA of 93-11, the genes encoding the OsRNT protein in the two homologous chromosomes of mutation type RNT-1 underwent the following mutation: "5'-CCGCATTACGTGACCTTGGT-3' (SEQ ID No:6, corresponding to positions 3343 to 3362 of SEQ ID No:1 and positions 359 to 378 of SEQ ID No:2)" was mutated to "5'-CCGCATATACGTGACCTTGGT-3' (SEQ ID No:5), i.e., the insertion of one base "A", causing a frameshift mutation in OsRNT, resulting in its loss of function and thus knocking out the gene encoding the OsRNT protein. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 2.

[0116] Compared with the genomic DNA of 93-11, the gene encoding the OsRNT protein in the two homologous chromosomes of mutation type RNT-2 underwent the following mutation: "5'-CCGCATTACGTGACCTTGGT-3' (SEQ ID No:6, corresponding to positions 3343 to 3362 of SEQ ID No:1 and positions 359 to 378 of SEQ ID No:2)" was mutated to "5'-CGCATACCTTGGT-3' (SEQ ID No:7), that is, the deletion of 6 bases "TACGTG", which caused a frameshift mutation in OsRNT, resulting in its loss of function. The sequencing results of the gene encoding the OsRNT protein and the surrounding nucleotides are shown in Figure 2.

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

[0118] The T1 generation rice mutant plants of the above-mentioned homozygous OsRrnt-1 and Osrnt-2 OsRNT gene lines were further cultured and screened to obtain T2 generation Osrnt plants without transgenic elements, and then phenotypic identification was performed.

[0119] Osrnt-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. Osrnt-1 plants and their self-pollinated offspring are called the Osrnt-1 line.

[0120] Osrnt-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. Osrnt-2 plants and their self-pollinated offspring are called the Osrnt-2 line.

[0121] Example 2: Comparison of Rice Production Traits

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

[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 of the tested plants is shown in Figure 3. Seven days after rehydration following high-temperature stress (HS), the 93-11 plants were mostly withered and yellowed, while Osrnt-1 and Osrnt-2 showed normal growth, with some leaves curling (Figure 3A). The survival rate of 93-11 was 0%, while the survival rates of Osrnt-1 and Osrnt-2 were 30% and 50%, respectively, significantly higher than that of 93-11 (Figure 3B). 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.

[0126] Cross-references to related applications

[0127] This application claims priority to Chinese Patent Application No. 202510083172.8, filed on January 20, 2025, entitled "Application of a substance regulating the expression of protein OsRNT in regulating heat tolerance in rice", the entire contents of which are incorporated herein by reference. Industrial applicability

[0128] This invention utilizes CRISPR / Cas9-mediated gene editing technology to knock out specific targets in the gene encoding the heat-resistant protein OsRNT in rice, providing new materials for the breeding of heat-resistant rice varieties and playing a positive role in accelerating the improvement of rice varieties.

Claims

1. A protein, wherein the protein is any of the following: A1) A protein with the amino acid sequence SEQ ID No:3; A2) A protein that has more than 75% identity with and has the same function as the protein in A1) obtained by substituting and / or deleting and / or adding amino acid residues. A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The protein of claim 1, wherein: The protein is derived from rice (Oryza sativa L.).

3. A biomaterial relating to the protein of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); 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); 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); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); C1) A nucleic acid molecule that inhibits, reduces, or silences the expression of the gene encoding the protein described in claim 1 or 2; 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 biomaterial of claim 3, wherein: B1) The nucleic acid molecule 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.

5. The use of the protein or gene expression regulator or substance regulating the activity or content of said protein as described in claim 1 or 2 in any of the following: U1) Application in regulating plant heat tolerance; U2) Application in the preparation of products that regulate plant heat resistance; U3) Application in cultivating plants with enhanced heat resistance; U4) Applications in the preparation of products that enhance the heat resistance of plants; U5) Applications in plant breeding.

6. Use according to claim 5, characterized in that: The substance that regulates the expression of the gene or the substance that regulates the activity or content of the protein is a biological material related to the protein, and the biological material is the biological material according to claim 3 or 4.

7. A method of modulating the heat tolerance of a plant, comprising: This includes regulating the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and the expression level of the gene encoding the protein described in claim 1 or 2, to regulate the plant's heat tolerance.

8. The method according to claim 7, characterized in that: The regulation of the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and the expression level of the gene encoding the protein described in claim 1 or 2 includes the introduction of a substance that inhibits the expression of the gene encoding the protein into the recipient plant, resulting in a target plant with stronger heat resistance than the recipient plant; the gene encoding the protein described in claim 1 or 2.

9. Methods for cultivating plants with altered heat tolerance, including: 1) Inhibit or reduce or silence the expression level of the gene encoding the protein of claim 1 in the receptor plant, and / or inhibit or reduce or silence the activity and / or content of the gene encoding the protein of claim 1, to obtain a plant with enhanced heat resistance. 2) Increase, enhance and / or upregulate the expression level of the gene encoding the protein described in claim 1 in the recipient plant, or / and increase, enhance and / or upregulate the activity and / or content of the gene encoding the protein described in claim 1, to obtain a plant with reduced heat resistance.

10. The application according to claim 5 or 6, or the method according to any one of claims 7-9, characterized in that: The plant is any one of the following: N1) Monocotyledonous or dicotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Rice plants; N5) rice.

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