Rice gene Ocaml5 and application thereof in regulating and controlling high temperature resistance of rice

Abstract

The invention discloses a rice gene Ocaml5 and application of the rice gene Ocaml5 in regulation and control of high temperature resistance of rice. The invention belongs to the technical field of biology, and particularly relates to a rice gene Ocaml5 and application of the rice gene Ocaml5 to regulation and control of high temperature resistance of rice. The protein is any one of the following proteins: A1) a protein with an amino acid sequence as shown in SEQ ID No: 1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the protein of A1), has 80% or more of identity with the protein of A1) and has the character of regulating and controlling high temperature resistance of plants; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). Experiments prove that the protein Ocaml5 can negatively regulate and control the high-temperature resistance of plants and has important theoretical significance on rice breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a rice gene Oscaml5 and an application thereof in regulating the high temperature resistance of rice. Background Art

[0002] With the continuous growth of population, people have put forward higher requirements for rice yields, and are also facing more unfavorable climate challenges related to extreme climatic conditions. Rice is one of the most important food crops in my country and occupies an irreplaceable and important position in the development of the national economy. Extreme temperature affects the changes in global extreme climate, which makes crops face the threat of reduced yields. Data surveys show that high temperature stress can reduce the photosynthetic rate of rice, accelerate the transpiration rate, inhibit the growth and development of rice, and lead to a decrease in its fruit set rate and thousand-grain weight. The coordinated improvement of crop yield and stress resistance is an effective response strategy to solve the frequent occurrence of extreme weather such as population growth and global warming. Exploring the key genes that regulate rice's high temperature resistance is the key to accelerating rice improvement. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a protein.

[0004] The protein provided by the present invention may be any of the following proteins:

[0005] A1) the amino acid sequence is the protein shown in SEQ ID No: 1;

[0006] A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having more than 80% identity with the protein of A1) and having the function of regulating plant stress resistance;

[0007] A3) A fusion protein obtained by connecting a protein tag to one end of A1) or A2).

[0008] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0009] The label protein includes, but is not limited to, GST (glutathione sulfhydryl transferase) label protein, His6 label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA label protein, Myc label protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. A person skilled in the art can easily mutate the nucleotide sequence encoding the protein Oscaml5 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 Oscaml5 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 Oscaml5 and have the function of the protein Oscaml5.

[0010] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0011] 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.

[0012] 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.

[0013] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0014] Among the above proteins, the protein is derived from rice (Oryza sativa L.).

[0015] The present invention also provides a biological material related to the above protein, and the biological material may be any one of the following:

[0016] B1) a nucleic acid molecule encoding the protein described above;

[0017] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0018] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0019] 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);

[0020] 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);

[0021] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0022] 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);

[0023] C1) a nucleic acid molecule that inhibits, reduces or silences the expression of a gene encoding the protein described above;

[0024] C2) expressing the gene encoding the nucleic acid molecule described in C1);

[0025] C3) an expression cassette containing the coding gene described in C2);

[0026] C4) a recombinant vector containing the coding gene described in C2) or a recombinant vector containing the expression cassette described in C3);

[0027] 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);

[0028] 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);

[0029] 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);

[0030] 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).

[0031] In the above biological material, the nucleic acid molecule described in B1) may be the gene shown in E1) or E2) below:

[0032] E1) the coding sequence is a cDNA molecule or a DNA molecule of SEQ ID No: 2;

[0033] E2) The nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No: 3.

[0034] The DNA molecule shown in SEQ ID No:2 (Oscam15 gene regulating high temperature tolerance of plants) encodes the protein Oscam15 with the amino acid sequence of SEQ ID No:1.

[0035] The nucleotide sequence shown in SEQ ID No: 2 is the nucleotide sequence of the protein Oscam15 encoding gene (CDS).

[0036] The Oscam15 gene of the present invention can be any nucleotide sequence that can encode the protein Oscam15. Considering the degeneracy of codons and the preference of codons of different species, those skilled in the art can use codons suitable for expression of a specific species as needed.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, phages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors. Specifically, it can be vector SG2027.

[0041] In the above biological materials, the expression cassettes described in B2) and C3) refer to DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription, but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both of which can be induced by methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0042] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0043] The present invention also provides the use of the above-mentioned protein Oscam15 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:

[0044] U1) Application in regulating plant high temperature resistance;

[0045] U2) Application in the preparation of products for regulating the high temperature resistance of plants;

[0046] U3) Application in cultivating plants with high temperature resistance;

[0047] U4) Application in the preparation of products for cultivating plants with high temperature resistance;

[0048] U5) Application in plant breeding.

[0049] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0050] Herein, the regulation may be up-regulation, enhancement or increase, or down-regulation, inhibition or reduction.

[0051] Herein, the regulation of the high temperature resistance of the plant may be up-regulation, enhancement or improvement of the high temperature resistance of the plant, or may be down-regulation, inhibition or reduction of the high temperature resistance of the plant.

[0052] Herein, the high temperature tolerance of the plant may include the plant having a high temperature stress tolerance phenotype and an improved survival rate.

[0053] The high temperature stress phenotype of the plant can be manifested as: when the three-week-old seedlings are treated at 45°C for 36 hours, the survival rate of OsCaml5-1 and OsCaml5-2 is significantly reduced compared with 93-11; when the treatment is continued in the field high shed, compared with the control plants, the plant height of the knockout mutant plants is reduced, the number of tillers is significantly reduced, and the fruit setting rate is reduced.

[0054] Herein, the regulation of the expression of the coding gene of the protein may be inhibition, reduction or down-regulation of the coding gene expression. Inhibition, reduction or down-regulation of the coding gene expression may be achieved by gene knockout or gene silencing.

[0055] The gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0056] The present invention also provides a method for regulating the high temperature resistance of rice, comprising the following steps:

[0057] 1) Up-regulating, enhancing or increasing the expression of the gene encoding the protein mentioned above in the recipient rice, so as to improve the high temperature resistance of the rice;

[0058] 2) Down-regulating, inhibiting or reducing the expression of the gene encoding the protein mentioned above in the recipient rice, so as to reduce the high temperature resistance of the rice.

[0059] In a specific embodiment, the method for downregulating, inhibiting or reducing the high temperature resistance of rice may include the following steps:

[0060] A) using the CRISPR / Cas9 system to perform gene editing on the Oscaml5 gene encoding the Oscaml5 protein in the recipient rice, and causing the Oscaml5 gene to mutate, resulting in premature termination of the translation of the protein, to obtain the target rice;

[0061] B) self-pollinating the target rice to obtain homozygous rice, namely the target rice, wherein the high temperature resistance of the target rice is weaker than that of the recipient rice.

[0062] The Oscam15 gene is a DNA molecule as follows (a1) or (a2) or (a3):

[0063] (a1) the coding region of Oscam15 gene is a DNA molecule as shown in SEQ ID No: 2;

[0064] (a2) a DNA molecule that hybridizes with a defined DNA molecule under stringent conditions and encodes the protein Oscaml5; (a3) ​​a DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with a defined DNA molecule and encodes the protein.

[0065] The present invention also provides a method for cultivating rice with changed high temperature resistance, comprising the following steps:

[0066] 1) up-regulating or enhancing or increasing the expression of the gene encoding the protein mentioned above in the recipient rice to obtain the target rice, wherein the target rice has a stronger high temperature resistance than the recipient rice;

[0067] 2) down-regulating or inhibiting or reducing the expression of the gene encoding the protein mentioned above in the recipient rice to obtain the target rice, wherein the high temperature resistance of the target rice is weaker than that of the recipient rice.

[0068] The method for cultivating high temperature resistant rice provided by the present invention comprises the following steps: up-regulating or enhancing or improving the expression of the coding gene of the protein of the receptor rice to obtain the target rice, wherein the high temperature resistance of the target rice is stronger than that of the receptor rice.

[0069] Herein, the improvement of the high temperature resistance of rice is specifically embodied in increasing the survival rate of rice under high temperature stress conditions.

[0070] Herein, the down-regulation, inhibition or reduction of the expression of the gene encoding the protein in rice may be the knockout of the gene encoding the protein Oscam15 in rice.

[0071] In the above method, the knockout is achieved through the CRISPR / Cas9 system.

[0072] In the above method, the target of gene editing by the CRISPR / Cas9 system is positions 199-218 of SEQ ID No: 3 or positions 78-97 of corresponding SEQ ID No: 2.

[0073] In the above method, the knockout of the coding gene of the protein in the target rice may be to perform at least one of the following mutations on the coding gene of the protein having the amino acid sequence of SEQ ID No: 1 in the rice genome:

[0074] 1) replacing 5'-CGGGGACGGGTGCATCACGC-3' in the gene encoding the protein in rice genomic DNA with 5'-CGGGGACGGGTGCATCCGC-3', thereby knocking out the gene encoding the Oscaml5 protein;

[0075] 2) replacing 5'-CGGGGACGGGTGCATCACGC-3' in the gene encoding the protein in rice genomic DNA with 5'-CGGGGACGGGTGCAATCCGC-3', thereby knocking out the gene encoding the Oscaml5 protein;

[0076] Herein, the high temperature treatment condition is: treating three-week-old rice seedlings at 45°C for 36 hours.

[0077] The present invention utilizes CRISPR / Cas9-mediated gene editing technology to perform site-directed mutation or knockout of a specific target in the gene encoding the Oscaml5 protein related to regulating rice heat resistance, providing new materials for the breeding of heat-resistant rice varieties, and playing a positive guiding role in accelerating the improvement of rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of the structure of the recombinant plasmid SG2027-Oscaml5.

[0079] Figure 2 The nucleotide sequence of the mutation site and its surrounding nucleotides.

[0080] Figure 3 The phenotypes and statistical diagrams of rice under normal conditions (CK) and high temperature stress (HS) treatment. A is the phenotype diagram of 93-11 and oscaml5 mutants Oscaml5-1 and Oscaml5-2 under high temperature stress; B is the statistical diagram of the survival rate of 93-11 and oscaml5 mutants Oscaml5-1 and Oscaml5-2.

[0081] Figure 4 Statistical data of plant height (cm) and tillering (number) of Datian 93-11 and oscaml5-1 under normal conditions and after high temperature stress (HS) treatment.

[0082] Figure 5 Statistics of ear phenotype and fruit setting rate (%) of Datian 93-11 and oscaml5-1 under normal conditions and high temperature stress (HS) treatment. DETAILED DESCRIPTION

[0083] 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.

[0084] 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.

[0085] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0086] The SG2027 vector in the following embodiment 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.

[0087] The rice variety 93-11 in the following embodiment 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 experiment of the present invention and cannot be used for other purposes.

[0088] The following examples use EXCEL software to perform statistical analysis of data, and SPASS software to perform multiple difference comparisons of data.

[0089] Example 1: Creation of Oscam15 gene knockout rice

[0090] 1. Obtaining the Oscaml5 gene from rice

[0091] The DNA of the leaves of rice variety 93-11 was extracted and used as a template. The primers oscaml5-F: 5'-ATGGCGGAGGTGGAG-3'; Oscaml5-R: 5'-TTATTGGTCGGAGAG-3' were used. Max Super-Fidelity DNA Polymerase (Cat. No.: P505-d1, Vazyme) was used for PCR amplification to obtain the amplified product, namely the genome sequence of the Oscaml5 gene (nucleotide sequence is SEQ ID No: 3). Positions 121-622 of SEQ ID No: 3 are the first exon.

[0092] The coding sequence of OScaml5 gene in water 93-11 is SEQ ID No: 2, and the encoded amino acid sequence is SEQ ID No: 1 of OScaml5 protein.

[0093] 2. Construction of recombinant plasmid SG2027-Oscaml5 with knockout of Oscaml5 gene

[0094] The nucleotide sequence of the recombinant plasmid SG2027-Oscam15 is SEQ ID No: 4, which is a gene sequence obtained by Sanger sequencing, including the sgRNA sequence, the Oscam15 gene coding region sequence and the SG2027 vector backbone sequence. The map of the recombinant plasmid SG2027-Oscam15 is shown in Figure 1 .

[0095] SG2027-Oscaml5 expresses sgRNA targeting the Oscaml5 gene, and the target sequence is: 5'-CGGGGACGGGTGCATCACGCT-3'. The target site of the sgRNA is located in the first exon of the Oscaml5 gene, and the nucleotide sequence of the target site of the sgRNA is positions 199-218 of SEQ ID No: 3 (corresponding to positions 78-97 of SEQ ID No: 2).

[0096] 3. Obtaining and identifying Oscaml5 gene knockout rice

[0097] The recombinant plasmid SG2027-Oscam15 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 (Shanghai Weidi Company) 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.

[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, smooth and sterile seeds for disinfection.

[0100] (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.

[0101] (3) The recombinant vector SG2027-Oscam15 constructed in step (2) above is introduced into Agrobacterium tumefaciens EHA105 to obtain the recombinant bacterium EHA105 / SG2027-Oscam15.

[0102] (4) Take the recombinant bacteria EHA105 / SG2027-Oscam15 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-Oscam15 bacterial suspension.

[0103] (5) Soak the 93-11 callus tissue from step (2) in the EHA105 / SG2027-Oscam15 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.

[0104] (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.

[0105] (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.

[0106] (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 Oscaml5 gene knockout rice plants.

[0107] The transgenic plants obtained using the recombinant vector SG2027-Oscam15 were recorded as Oscam15 transgenic plants.

[0108] The formulas of induction medium and differentiation medium are both MS medium (Solabo, M8521).

[0109] 4. Identification of Oscaml5 gene knockout rice

[0110] Plants to be tested: 93-11 (control group) and candidate Oscaml5 gene knockout plants obtained in step 3.

[0111] The genomic DNA of the leaves of the Oscaml5 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 Oscaml5-F1 and primer Oscaml5-R1.

[0112] Oscaml5-F1:5'-GCCACTACACTAGCACACACT-3';

[0113] Oscaml5-R1:5'-CGGATCATCTGCTCCACCTC-3'.

[0114] The SG2027-Oscam15 plasmid was used as a positive control (V), and the recipient rice variety 93-11 was used as a negative control (CK). The resulting products were then sequenced.

[0115] Identified by sequencing ( Figure 2 ), compared with the genomic DNA of wild-type 93-11, the genes encoding Oscaml5 protein in the two homologous chromosomes of mutation type caml5-1 underwent the following mutation: "5'-CGGGGACGGGTGCATCCGC-3' (corresponding to positions 199 to 218 of SEQ ID No: 3, positions 78 to 97 of SEQ ID No: 2)" mutated to "5'-CGGGGACGGGTGCATCCGC-3'", resulting in amino acid mutation after the editing site. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2 .

[0116] Compared with the genomic DNA of wild-type 93-11, the genes encoding Oscaml5 protein in the two homologous chromosomes of mutation type caml5-2 underwent the following mutation: "5'-CGGGGACGGGTGCAATCCGC-3' (corresponding to positions 199 to 218 of SEQ ID No: 3 and positions 78 to 97 of SEQ ID No: 2)" mutated to 5'-GGGAACTGTCATGCGTTAGC-3', resulting in amino acid mutation after the editing site. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 2 .

[0117] Through the above identification, two homozygous edited plants (i.e., the mutations in the two homologous chromosomes were consistent) were obtained and named Oscaml5-1 and Oscaml5-2 respectively.

[0118] The T1 generation rice mutant plants of the above-mentioned Oscaml5 gene homozygous mutant types oscaml5-1 and oscaml5-2 lines were further cultivated to screen out T2 generation Oscaml5 plants without transgenic elements, and the phenotypic identification was performed.

[0119] The Oscam15-1 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 Oscam15-1 plant and its self-pollinated progeny are called Oscam15-1 strains.

[0120] The Oscam15-2 plant is self-pollinated and seeds are harvested, and the seeds are cultivated into plants, which are the T1 generation plants. The T1 generation plants are self-pollinated and seeds are harvested, which are the T2 generation seeds. The Oscam15-2 plant and its self-pollinated progeny are called the Oscam15-2 strain.

[0121] Example 2: Comparison of rice production traits

[0122] The plants to be tested are: T3 generation homozygous strains of rice 93-11, mutant strain Oscam15-1, and Oscam15-2.

[0123] The seeds of each plant to be tested were germinated and raised in a greenhouse (starting from the time of whitening, and cultivated for a total of 3 weeks) to obtain 3-week seedlings. The 3-week seedlings were subjected to high temperature stress treatment (45°C for 36 hours) and the survival rate (%) was calculated. At least 60 individual plant data were calculated for each material. The survival rate calculation formula is: survival rate (%) = (total number of rice individual plants - number of dead rice individual plants) / total number of rice individual plants × 100%.

[0124] The growth and survival rate of the tested plants are shown in Figure 3-Figure 5 .

[0125] Figure 3 In the experiment, after high temperature stress at 45℃, the survival rate of 93-11 was 42%, while that of Oscaml5-1 and Oscaml5-2 was 4.3% and 4%, respectively. The survival rates of Oscaml5-1 and Oscaml5-2 were significantly lower than those of 93-11, indicating that Oscaml5 is essential for high temperature response.

[0126] Figure 4In the field, after high temperature stress, the plant height of the 93-11 control was 120 cm, and the plant height of the Oscaml5 gene knockout mutant plant Oscaml5-1 was 92 cm. The tiller number of the 93-11 control was 6, and the tiller number of the Oscaml5 gene knockout mutant plant Oscaml5-1 was 4. Compared with 93-11, the plant height and tiller number of Oscaml5-1 were significantly reduced, indicating that the knockout of Oscaml5-1 affected the tiller number under high temperature stress conditions.

[0127] The fruit setting rate of single ear under high temperature stress in the field was counted, and the number of counts was 10 single plants. The results showed that ( Figure 5 ): After high temperature stress, the fruit setting rate of the 93-11 control was 40%, and the fruit setting rate of the Oscaml5 gene knockout mutant plant Oscaml5-1 was 8%. Compared with 93-11, the fruit setting rate of Oscaml5-1 was reduced by 32%, which proved that the knockout of the Oscaml5 gene had a great effect on the rice fruit setting rate. Oscaml5 is a key regulatory protein for regulating yield in response to high temperature.

[0128] 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 protein, wherein the protein is any of the following: A1) a protein with an amino acid sequence as shown in SEQ ID No: 1; 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 protein according to claim 1, characterized in that: The protein is derived from rice (Oryza sativa L.).

3. A biological material related to the protein according to claim 1 or 2, wherein the biological material is any one of the following: B1) A nucleic acid molecule encoding the protein according to 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) 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 a gene encoding a protein according to claim 1 or 2; 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).

4. The biomaterial according to claim 3, characterized in that: B1) The nucleic acid molecule is the gene shown in E1) or E2) below: E1) the coding sequence is a cDNA molecule or a DNA molecule of SEQ ID No: 2; E2) The nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID No:

3.

5. Use of the protein or gene expression regulating substance according to claim 1 or 2 or the substance regulating the activity or content of the protein in any of the following: U1) Application in regulating plant high temperature resistance; U2) Application in the preparation of products for regulating the high temperature resistance of plants; U3) Application in cultivating plants with high temperature resistance; U4) Application in the preparation of products for cultivating plants with high temperature resistance; U5) Application in plant breeding.

6. A method for regulating the high temperature resistance of rice, comprising: 1) Up-regulating, enhancing or increasing the expression of the gene encoding the protein according to claim 1 or 2 in the recipient rice, so as to improve the high temperature resistance of the rice; 2) Down-regulating, inhibiting or reducing the expression of the gene encoding the protein according to claim 1 or 2 in the recipient rice, so that the high temperature resistance of the rice is reduced.

7. A method for cultivating rice with altered high temperature resistance, comprising: 1) up-regulating or enhancing or increasing the expression of the gene encoding the protein according to claim 1 or 2 in a recipient rice to obtain a target rice, wherein the target rice has a higher heat resistance than the recipient rice; 2) down-regulating, inhibiting or reducing the expression of the gene encoding the protein according to claim 1 or 2 in the recipient rice to obtain the target rice, wherein the high temperature resistance of the target rice is weaker than that of the recipient rice.

8. The method according to claim 6 or 7, characterized in that: The down-regulation, inhibition or reduction of the expression of the gene encoding the protein according to claim 1 or 2 in rice is to knock out the gene encoding the protein according to claim 1 or 2 in the target rice.

9. The method according to claim 8, characterized in that The knockout is achieved through the CRISPR / Cas9 system.

10. The method according to claim 8 or 9, characterized in that: The method of knocking out the gene encoding the protein of claim 1 or 2 in the target rice is to perform at least one of the following mutations on the protein whose amino acid sequence is shown in SEQ ID No: 1 in the rice genome: 1) replacing 5'-CGGGGACGGGTGCATCACGC-3' in the gene encoding the protein in rice genomic DNA with 5'-CGGGGACGGGTGCATCCGC-3', thereby knocking out the gene encoding the Oscaml5 protein; 2) Replacing 5'-CGGGGACGGGTGCATCACGC-3' in the gene encoding the protein in rice genomic DNA with 5'-CGGGGACGGGTGCAATCCGC-3', thereby knocking out the gene encoding Oscaml5 protein.

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