Application of rice Oscam3 gene in regulation and control of high temperature resistance of rice

Abstract

The invention discloses application of a rice Oscam3 gene in regulation and control of high temperature resistance of rice. The invention belongs to the field of plant breeding, and particularly relates to application of a rice Oscam3 gene in regulation and control of high temperature resistance of rice. The method for cultivating the plant with the high temperature resistance changed comprises the following steps: 1) inhibiting or reducing or silencing the expression quantity of a coding gene of a protein in a receptor plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the protein to obtain a plant with enhanced high temperature resistance; and 2) increasing, enhancing and / or up-regulating the expression quantity of the coding gene of the protein in the receptor plant, or / and increasing, enhancing and / or up-regulating the activity and / or content of the coding gene of the protein to obtain the plant with weakened high temperature resistance. After high-temperature stress treatment, the Oscam3 knockout mutant has a high-temperature-resistant phenotype, which indicates that the Oscam3 gene plays an important role in regulating and controlling the high-temperature resistance of the rice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of rice Oscam3 gene in regulating high temperature resistance of rice. Background Art

[0002] In recent years, extreme temperatures and water shortages caused by climate change have become a global problem that needs to be solved urgently. With the continuous growth of population and the reduction of available fresh water, the imbalance between food supply and demand has been further aggravated. High temperature is one of the key stresses that significantly affects plant growth and development. With the rise of global temperatures, the harm of high temperature stress to plants has become more serious. The main harm of high temperature to plant growth is that it leads to a decrease in photosynthesis and a decrease in organic matter accumulation, thereby reducing energy supply; rising temperatures can also cause the accumulation of reactive oxygen species (ROS) in plants, which destroy the stability of various biomacromolecules such as proteins and nucleic acids in plants and interfere with the metabolic balance in cells; in addition, high temperature environments can destroy subcellular structures such as plasma membranes, leading to low seed germination efficiency, slow seedling growth, reproductive organ deformities, decreased pollen vitality, and reduced crop yield and quality. Therefore, it is of great significance to study the mechanism by which high temperature in the environment affects plant growth and development. Summary of the invention

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

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for cultivating plants with altered high temperature tolerance, comprising the following 1) or 2):

[0005] 1) inhibiting, reducing or silencing the expression of a protein encoding gene in a receptor plant, or / and inhibiting, reducing or silencing the activity and / or content of a protein encoding gene, to obtain a plant with enhanced high temperature tolerance;

[0006] 2) increasing, enhancing and / or up-regulating the expression of a protein encoding gene in a recipient plant, or / and increasing, enhancing and / or up-regulating the activity and / or content of a protein encoding gene, to obtain a plant with reduced high temperature tolerance;

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

[0008] A1) a protein with an amino acid sequence as shown in SEQ ID No: 3;

[0009] A2) a protein having more than 75% identity with the protein shown in A1) and having the ability to regulate the high temperature resistance of plants obtained by replacing and / or deleting and / or adding amino acid residues of the protein shown in A1); for example, a person skilled in the art can obtain a protein mutant having the same function as the amino acid sequence shown in SEQ ID No: 3 by replacing, deleting and / or adding one or more amino acids without affecting its activity according to the conventional technical means in the art such as the amino acid sequence shown in SEQ ID No: 3 and conservative substitution of amino acids;

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

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

[0012] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No: 3 in the sequence listing.

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

[0014] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) 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 (monomeric red fluorescent protein) or AviTag tag protein.

[0015] A person skilled in the art can easily mutate the nucleotide sequence encoding the protein Oscam3 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein Oscam3 isolated by the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein Oscam3 and have the function of the protein Oscam3.

[0016] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0017] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for a pair of amino acid sequences or nucleotide sequences to calculate the identity, then the value (%) of the identity can be obtained.

[0018] Herein, the 80% or greater identity may 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] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0020] In the above, the protein is derived from rice (Oryza sativa L.).

[0021] In a specific embodiment, a method for cultivating a plant with enhanced high temperature tolerance comprises the following steps: inhibiting the expression of a nucleic acid molecule encoding an Oscam3 protein in a target plant to obtain a transgenic plant with enhanced high temperature tolerance.

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

[0023] Herein, the knockout is achieved by the CRISPR / Cas9 system.

[0024] Herein, the knockout of the coding gene of the protein of the target plant may be to mutate the coding gene of the protein in the plant genome (the nucleotide sequence of the genome is SEQ ID No: 1) as follows:

[0025] 1) replacing 5'-GGGAACTGTCATGCGTTCGC-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'-GGGAACTGTCATGCGTTACGC-3', thereby knocking out the gene encoding the Oscam3 protein;

[0026] 2) replacing 5'-GGGAACTGTCATGCGTTCGC-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'-GGGAACTGTCATGCGTTGC—3', thereby knocking out the gene encoding the Oscam3 protein.

[0027] Herein, the purpose of plant breeding includes cultivating plants with enhanced / weakened high temperature tolerance.

[0028] The present invention also provides a method for regulating the high temperature resistance of plants, comprising regulating the activity and / or content of the above-mentioned protein in the target plant, or / and the expression level of the gene encoding the protein, to regulate the high temperature resistance of the plant.

[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, comprises introducing a substance that inhibits the expression of the gene encoding the protein Oscam3 into the recipient plant, thereby obtaining the target plant with altered high temperature resistance; the Oscam3 encoding gene encodes the protein Oscam3.

[0030] The above proteins also fall within the scope of protection claimed by the present invention.

[0031] The present invention also provides a biological material related to the above protein, and the biological material may be any one of the following:

[0032] B1) a nucleic acid molecule encoding the protein 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) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism 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 molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0038] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0039] C1) a nucleic acid molecule that inhibits, reduces or silences the expression of a gene encoding the protein described above;

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

[0041] C3) an expression cassette containing the coding gene described in C2);

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

[0043] C5) a recombinant microorganism containing the coding 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 coding 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 coding 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 coding 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 biological material, the nucleic acid molecule described in B1) may be the gene shown in E1) or E2) below:

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

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

[0050] The DNA molecule shown in SEQ ID No: 2 (Oscam3 gene regulating the high temperature tolerance of plants) encodes the protein Oscam3 with the amino acid sequence of SEQ ID No: 3.

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

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

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

[0054] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0055] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (ie, cosmid), Ti plasmid or viral vector.

[0056] Further, the vector in C4) may be a recombinant vector SG2027-Oscam3, the nucleotide sequence of the recombinant vector SG2027-Oscam3 is sequence 4 in the sequence table, the SG2027-Oscam3 expresses an sgRNA targeting the Oscam3 gene, the target sequence is: 5'-GGGAACTGTCATGCGTTCGC-3' (SEQ ID No: 5), the target of the sgRNA is located in the second exon of the Oscam3 gene, and the nucleotide sequence of the target of the sgRNA is 987-1006 of SEQ ID No: 1 (corresponding to 99-118 of SEQ ID No: 2). The microorganism described herein may be yeast, bacteria, algae or fungi. The bacteria may be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc. Specifically, it may be Agrobacterium tumefaciens EHA105.

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

[0058] U1) Application in regulating plant's ability to tolerate high temperatures;

[0059] U2) Application in the preparation of products for regulating the high temperature tolerance of plants;

[0060] U3) Application in cultivating plants with high temperature tolerance;

[0061] U4) Application in the preparation of products for cultivating plants with high temperature tolerance;

[0062] U5) Application in plant breeding.

[0063] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein Oscam3.

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

[0065] In the above applications and methods, the regulation may be increasing, enhancing or up-regulating; the regulation may also be inhibiting, decreasing or silencing.

[0066] In the present invention, the enhanced high temperature tolerance can be manifested in that when three-week-old seedlings are subjected to 45°C high temperature stress, the survival rate of Oscam3 gene knockout mutant plants is increased by 38% in the NIP background compared with the control plants. In the 93-11 background, the survival rate of Oscam3 gene knockout mutant plants is increased by 18% compared with the control plants.

[0067] In the above application or method, the plant is any one of the following:

[0068] N1) monocots or dicots;

[0069] N2) Gramineae;

[0070] N3) Gramineae;

[0071] N4) Oryza plants;

[0072] N5) Rice.

[0073] The present invention uses CRISPR / Cas9-mediated gene editing technology to perform site-directed mutation or knockout of specific targets in the gene encoding the Oscam3 protein that regulates rice heat resistance. The results show that after high-temperature treatment, the Oscam3 mutant strain has a lower curling rate of rice leaves and a significantly higher survival rate under high temperature than the control plants, which provides new materials for the breeding of heat-resistant rice varieties and plays a positive role in accelerating the improvement of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic diagram of the structure of the recombinant plasmid SG2027-Oscam3.

[0075] Figure 2 The nucleotide sequence of the mutation site and its surrounding nucleotides.

[0076] Figure 3 These are the high temperature stress phenotypes of rice NIP and Oscam3 mutants and the statistical graphs of survival rates after high temperature stress treatment.

[0077] Figure 4 These are the high temperature stress phenotypes of rice 93-11 and Oscam3 mutants and the survival rate statistics after high temperature stress treatment. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0079] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0080] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0081] The SG2027 vector in the following examples has been recorded 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 public can obtain the biological material from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0082] The rice variety Nipponbare (NIP) and rice variety 93-11 in the following examples have been recorded 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 public can obtain the biological material from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0083] The following examples use EXCEL software to perform statistical analysis of data, and SPASS software to perform multiple difference comparisons of data.

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

[0085] 1. Obtaining the Oscam3 gene from rice

[0086] DNA from leaves of rice varieties NIP and 93-11 was extracted and used as template to generate the primers Oscam3-F: 5'-ATGGCGGACCAGCTC-3'; Oscam3-R: 5'-TTACTTGGCCATCAT-3'. Max Super-Fidelity DNA Polymerase (Cat. No.: P505-d1, Vazyme) was used for PCR amplification to obtain the amplified product, i.e., the genomic sequence of the Oscam3 gene. In the genomic DNA of rice 93-11, the genomic gene nucleotide sequence encoding the Oscam3 protein is SEQ ID No: 1. The 107th to 177th positions of SEQ ID No: 1 are the first exon, and the 964th to 1338th positions are the second exon.

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

[0088] 2. Construction of recombinant plasmid SG2027-Oscam3 with knockout of Oscam3 gene

[0089] The nucleotide sequence of the recombinant plasmid SG2027-Oscam3 is SEQ ID No: 4. The map of the plasmid SG2027-Oscam3 is shown in Figure 1 .

[0090] Sequence 4 is a gene sequence obtained by Sanger sequencing, including sgRNA sequence, Oscam3 coding region sequence and SG2027 vector sequence on both sides of the insertion sequence. SG2027-Oscam3 expresses sgRNA targeting the Oscam3 gene, and the target sequence is: 5'-GGGAACTGTCATGCGTTCGC-3' (SEQ ID No: 1). The target of the sgRNA is located in the second exon of the Oscam3 gene, and the nucleotide sequence of the target of the sgRNA is 987-1006 of SEQ ID No: 1 (corresponding to 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. The recombinant Agrobacterium was used to genetically transform the embryonic callus of rice 93-11 by Agrobacterium infection method, and then the resistant callus was screened (100 mg / L hygromycin was used for resistance screening), and then differentiation regeneration culture was carried out, and then rooting culture was carried out to obtain regenerated plants.

[0093] The specific steps are as follows:

[0094] (1) Take out the mature seeds of rice varieties Nipponbare (NIP) and 93-11, remove the shells, and select the plump, smooth and sterile seeds for disinfection.

[0095] (2) The sterilized rice 93-11 seeds were inoculated onto the induction medium and cultured at 28°C in the dark for about 14 days, and callus tissues with good appearance and good growth were selected.

[0096] (3) The recombinant vector SG2027-Oscam3 constructed in step 2 above was introduced into Agrobacterium tumefaciens EHA105 to obtain the recombinant bacterium EHA105 / SG2027-Oscam3.

[0097] (4) Take the recombinant bacteria EHA105 / SG2027-Oscam3 obtained in step (3), 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) Soak the 93-11 callus tissue from step (2) in the EHA105 / SG2027-Oscam3 bacterial suspension prepared in step (4) for infection for 20 minutes. After infection, pour out the bacterial suspension, take the callus tissue, dry it with sterile filter paper, and then place it on a co-culture medium (MS basic medium) with acetosyringone and glucose, and culture it in the dark at 28° C. for 50-55 hours.

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

[0100] (7) The callus tissue after the above culture was transferred to a screening medium (MS basic medium) supplemented with hygromycin and cephalosporin and cultured in the dark at 28°C for 30 days, with subculturing every 10 days.

[0101] (8) After completing step (7), fresh hygromycin-resistant callus tissue was taken and inoculated into a pre-regeneration medium (MS basic medium), cultured in the dark at 28° C. for 7 days, and then placed in a light culture room (12 h light / 12 h dark) for another 7 days, and then transferred to a regeneration medium and continued to be cultured in the light until regenerated plants grew out, thereby obtaining candidate Oscam3 gene knockout rice plants.

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

[0103] The formulas of induction medium and differentiation medium are both MS medium (Solabo, M8521).

[0104] 4. Identification of Oscam3 gene knockout rice

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

[0106] The genomic DNA of the leaves of the Oscam3 gene knockout rice plants to be tested was extracted, and PCR amplification was performed using the genomic DNA as a template and a primer pair consisting of primer Oscam3-F1 and primer 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 acceptor strain 93-11 was used as a negative control (CK). The resulting products were then sequenced.

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

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

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

[0113] The T1 generation rice mutant plants of the above-mentioned Oscam3 gene homozygous mutant types Oscam3-1 and Oscam3-2 lines were further cultivated to screen out T2 generation Oscam3 plants without transgenic elements, and the phenotypes were identified.

[0114] The Oscam3-1 plant is self-pollinated and seeds are harvested, and the seeds are cultivated into plants, which are T1 generation plants. The T1 generation plants are self-pollinated and seeds are harvested, which are T2 generation seeds. The Oscam3-1 plant and its self-pollinated progeny are called Oscam3-1 strains.

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

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

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

[0118] The seeds of each plant to be tested were germinated and raised in a greenhouse (starting from the time of white appearance, for a total of 3 weeks) to obtain 3-week seedlings. The 3-week seedlings were subjected to high temperature stress treatment (treatment temperature was 45°C), and the survival rate was counted. At least 60 individual plant data were counted for each material, and the survival rate was calculated as follows: Survival rate (%) = (total number of rice individual plants - number of dead rice individual plants) / total number of rice individual plants × 100%.

[0119] The growth of the plants to be tested is shown in Figure 3 and Figure 4 When the three-week-old seedlings were subjected to high temperature stress at 45℃ for 36h, the survival rate of the NIP control was 18%, and that of the Oscam3-1 gene knockout mutant plants was 56%. When the three-week-old seedlings were subjected to high temperature stress at 45℃ for 36h, the survival rates of the NIP and Oscam3-2 plants 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 the three-week-old seedlings were subjected to high temperature stress at 45℃ for 48h, compared with the 93-11 control plants, the survival rate of the Oscam3 gene knockout mutant plants was significantly increased.

[0120] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for cultivating a plant with altered high temperature tolerance, comprising the following 1) or 2): 1) inhibiting, reducing or silencing the expression of a protein encoding gene in a receptor plant, or / and inhibiting, reducing or silencing the activity and / or content of a protein encoding gene, to obtain a plant with enhanced high temperature tolerance; 2) increasing, enhancing and / or up-regulating the expression of a protein encoding gene in a recipient plant, or / and increasing, enhancing and / or up-regulating the activity and / or content of a protein encoding gene, to obtain a plant with reduced high temperature tolerance; The protein is any of the following: A1) a protein with an amino acid sequence as shown in SEQ ID No: 3; A2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1) and having 80% or more identity with the protein of A1) and having the same function; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. The method according to claim 1, characterized in that: The protein is derived from rice (Oryza sativa L.).

3. A method for regulating the high temperature tolerance of plants, characterized in that: The method comprises regulating the activity and / or content of the protein described in claim 1 in a recipient plant, or / and the expression level of the gene encoding the protein described in claim 1, to regulate the high temperature resistance of the plant.

4. The method according to claim 3, characterized in that: The regulation includes introducing a substance that inhibits the expression of the gene encoding the protein into the recipient plant, thereby obtaining a target plant with a higher heat resistance than the recipient plant; the encoding gene encodes the protein described in claim 1.

5. The protein according to any one of claims 1 to 4.

6. Use of the protein according to claim 5 or a substance that regulates the activity or content of the protein or regulates the expression of a gene encoding the protein in any of the following: 1) Application in regulating plant's ability to tolerate high temperatures; 2) Application in the preparation of products for regulating the high temperature tolerance of plants; 3) Application in breeding plants with altered high temperature tolerance; 4) Application in the preparation of products for cultivating plants with altered high temperature tolerance; 5) Application in plant breeding.

7. The use according to claim 6, characterized in that: The substance that regulates the activity or content of the protein or regulates the expression of the gene encoding the protein is a biological material related to the protein of claim 5, and the biological material is any one of the following: B1) a nucleic acid molecule encoding the protein according to claim 5; 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) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism 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 molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ 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 according to claim 5; C2) expressing the gene encoding the nucleic acid molecule described in C1); C3) an expression cassette containing the coding gene described in C2); C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3); C5) a recombinant microorganism containing the coding 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 coding 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 coding 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 coding 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).

8. The use according to claim 7, characterized in that: C1) The nucleic acid molecule is a cDNA molecule or a DNA molecule of SEQ ID No:

5.

9. The biomaterial as claimed in claim 7 or 8.

10. The method according to any one of claims 1 to 4, or the use according to any one of claims 6 to 8, characterized in that: The plant is any one of the following: N1) monocots or dicots; N2) Gramineae; N3) Gramineae; N4) Oryza plants; N5) Rice.

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