Application of OsCTOR gene or OsCTOR protein in regulating cold tolerance of plants

Abstract

The present invention discloses the application of the OsCTOR gene or the OsCTOR protein in regulating the cold tolerance of plants, belonging to the technical field of plant genetic engineering. The rice OsCTOR gene involved in the present invention is derived from the cultivated rice Nipponbare. In the present invention, the OsCTOR gene is knocked out by the CRISPR gene editing technology to obtain a rice mutant with a loss-of-function of this gene, and the cold stress experiment proves that the reduction of the expression level of the OsCTOR gene can effectively improve the cold tolerance of rice. Therefore, the OsCTOR gene of the present invention and the lipid transfer protein encoded thereby have broad application potential in enhancing the cold tolerance of rice and cultivating cold-tolerant rice varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly to the application of the OsCTOR gene or OsCTOR protein in regulating the cold tolerance of plants. Background Art

[0002] During the growth of rice (Oryza sativa L.), low-temperature stress (cold stress) is one of the important abiotic stress factors affecting rice yield and quality, and it also limits the geographical distribution of plants. The low-temperature environment can lead to a decrease in the fluidity of the plant cell membrane, affect the normal functions of cells, and at the same time inhibit the activity of enzymes and metabolic processes, thus interfering with the normal physiological activities of plants. Under cold stress, the photosynthesis of plants is significantly weakened, the synthesis of photosynthetic products is blocked, and further affects the growth and development of plants. In addition, low temperature can also cause difficulties in water and nutrient absorption of crops, resulting in nutritional imbalance in plants, further weakening the disease resistance and production performance of crops. Cold stress is often accompanied by freezing damage, which destroys cell structure, causes irreversible damage, and even leads to the death of crops in severe cases. These adverse effects have become particularly prominent under the background of global climate change, and the cold climate conditions pose new challenges to crop cultivation in tropical and subtropical regions.

[0003] Due to the increasing frequency and intensity of cold stress, which pose a threat to the stability and safety of agricultural production, how to improve the cold tolerance of crops and reduce the negative impacts brought by cold stress has become an important topic in agricultural scientific research. During the long-term adaptation to cold stress, plants sense external stimuli, generate and transduce corresponding stress signals, and thus activate various defense mechanisms to cope with the harm of cold stress. Significant reprogramming of the transcriptome occurs during the low-temperature stress response in different species, and many proteins have thus become important factors in this adaptive response.

[0004] During the long-term adaptation to cold stress, plants sense external stimuli, generate and transduce corresponding stress signals, and thus activate various defense mechanisms to cope with the harm of cold stress. Major transcriptome reprogramming occurs during the low-temperature stress response in different species, and many proteins are considered to be important factors in this adaptive response.

[0005] Studies have found that lipid transfer proteins are responsible for the transport of lipid molecules in plants, regulate processes such as membrane structure and signal transduction, and participate in the low-temperature response mechanism. Since lipid transfer proteins can mediate the transmembrane transport and distribution of different lipids, they regulate related signal pathways by maintaining the fluidity and stability of the membrane when plants respond to low-temperature stress.

[0006] Lipid transfer proteins are responsible for the transport of lipid molecules in plants, regulating processes such as membrane structure and signal transduction, and participating in the cold response mechanism. Since lipid transfer proteins can mediate the transmembrane transport and distribution of different lipids, they regulate related signal pathways by maintaining the fluidity and stability of the membrane when plants respond to cold stress. However, how to regulate cold tolerance in rice through lipid transfer proteins has not been reported yet. Summary of the Invention

[0007] The object of the present invention is to provide the application of the OsCTOR gene or OsCTOR protein in regulating plant cold tolerance to solve the problems existing in the above-mentioned prior art.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] One of the technical solutions of the present invention is the application of the OsCTOR gene or OsCTOR protein in regulating plant cold tolerance.

[0010] Another technical solution of the present invention is the application of the OsCTOR gene or OsCTOR protein in breeding cold-tolerant plant varieties.

[0011] Still another technical solution of the present invention is the application of the OsCTOR gene or OsCTOR protein in cold-tolerant plant breeding.

[0012] A fourth technical solution of the present invention is the application of a recombinant expression vector, overexpression vector, interference vector, recombinant virus, recombinant bacterium or recombinant gene expression cassette containing the OsCTOR gene in regulating plant cold tolerance.

[0013] A fifth technical solution of the present invention is a method for regulating plant cold tolerance by regulating the expression of the OsCTOR gene in the plant to regulate the cold tolerance of the plant.

[0014] Based on the above technical solutions, the present invention has the following technical effects:

[0015] The rice OsCTOR gene involved in the present invention is derived from the cultivated rice Nipponbare. In the present invention, the OsCTOR gene was knocked out by CRISPR gene editing technology to obtain a rice mutant with a functional deletion of this gene, and cold stress experiments confirmed that the reduction of the expression level of the OsCTOR gene can effectively improve the cold tolerance of rice. Therefore, the OsCTOR gene of the present invention and the lipid transfer protein encoded by it have broad application potential in enhancing the cold tolerance of rice and cultivating cold-tolerant rice varieties.

[0016] 1. The present invention first cloned and analyzed the lipid transfer protein gene OsCTOR in rice, and published its nucleotide sequence, amino acid sequence and promoter sequence, which is of great significance for elucidating the molecular mechanism of cold tolerance regulation in rice and the cultivation of cold-tolerant materials.

[0017] 2. The present invention first confirmed the participation of the OsCTOR gene in rice cold tolerance by transgenic means. After knocking out the OsCTOR gene, the cold tolerance of rice can be significantly enhanced, indicating that OsCTOR negatively regulates rice cold tolerance.

[0018] 3. The method for cultivating cold-tolerant plants according to the present invention has the advantages of simple operation and short cycle, and is suitable for wide application. This method has important theoretical and practical values in the research of the molecular mechanism of cold tolerance, the breeding of cold-resistant varieties and cold-tolerant molecular breeding, and provides an economical, rapid and efficient way to improve plant cold tolerance. The present invention has broad application prospects and market potential in the agricultural field. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is a schematic diagram of the protein structure and the analysis of the expression pattern of OsCTOR. Among them: (A) is the protein structure of OsCTOR, and OsCTOR encodes a lipid transfer protein; (B) is the prediction of the protein structure of OsCTOR on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). It can be seen from the figure that OsCTOR contains an AAI domain.

[0021] Figure 2 It is the gene structure diagram of OsCTOR and the gene editing situation of the OsCTOR transgenic knockout mutant. Among them, Figures (A) and (B) are the gene structure diagrams and the sequencing results of the homozygous gene editing lines, and (C) is the change in the amino acid sequence caused by gene editing.

[0022] Figure 3Phenotypic differences of OsCTOR transgenic materials under cold stress and plant height comparison of OsCTOR transgenic lines after 6 days of treatment at 4°C and 6 days of recovery at 28°C. Among them: (A) is a photo of plant phenotypes, showing the cold tolerance differences between transgenic materials and wild type after 6 days of cold treatment at 4°C and 6 days of recovery at 28°C when the seedling length is 5 mm. WT: wild type, CTOR-1, CTOR-2: gene-edited lines of OsCTOR. (B) is the plant height comparison of OsCTOR gene-edited materials and wild type after 6 days of recovery.

[0023] Figure 4 Data from the Ricedata website (https: / / www.ricedata.cn / ) shows the expression levels of the OsCTOR gene at different growth stages and after different treatments. Among them: (A) is the expression level of this gene in different parts of the plant at different growth and development stages. (B) is the expression level of this gene under different stress conditions during the seedling stage of the plant.

[0024] Figure 5 Subcellular localization of the OsCTOR gene, specifically the subcellular localization of OsCTOR in tobacco epidermal cells (the second row). From left to right, they are: GFP channel, bright field, and merged channel. Detailed implementation manners

[0025] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present application are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art. The reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0031] The embodiments of the present invention provide the application of the OsCTOR gene or OsCTOR protein in regulating plant cold tolerance.

[0032] In some specific embodiments, the nucleotide sequence of the OsCTOR gene is as shown in SEQ ID NO.1 or has 95% or more identity or homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein;

[0033] The amino acid sequence of the OsCTOR protein is as shown in SEQ ID NO.3 or a fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.3.

[0034] In some specific embodiments, the nucleotide sequence of the cDNA of the transcript of the OsCTOR gene is as shown in SEQ ID NO.2.

[0035] In some specific embodiments, silencing or knocking out the OsCTOR gene improves the cold tolerance of plants.

[0036] In some specific embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0037] In some specific embodiments, the plant includes rice.

[0038] The embodiments of the present invention also provide the application of the OsCTOR gene or OsCTOR protein in breeding cold-tolerant plant varieties.

[0039] The embodiments of the present invention also provide the application of the OsCTOR gene or OsCTOR protein in cold-tolerant plant breeding.

[0040] The methods of the cold-tolerant plant breeding specifically may all include the step of using plants with a lower expression level of the OsCTOR protein as parents for hybridization.

[0041] The cold tolerance characteristics are mainly reflected in showing strong cold resistance at the bud stage (for example, when the bud grows to about 5 cm) and / or at the seedling stage (for example, the two-leaf and one-heart stage).

[0042] The embodiments of the present invention also provide the application of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette containing the OsCTOR gene in regulating the cold tolerance of plants.

[0043] The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

[0044] The construction of the recombinant expression vector can be completed with the help of a variety of existing plant expression vectors. Commonly used vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment, such as pGreen0029, pCAMBIA series (such as 3301, 1300, 2301), pBI121, pBin19, etc. Other derivative plant expression vectors can also be used. These vectors may contain the 3' untranslated region of the foreign gene, including polyadenylation signals and other functional fragments that promote mRNA processing or gene expression, to direct the polyadenylation of the mRNA precursor at the 3' end.

[0045] When constructing the vector, different types of promoters can be added before the transcription start site to enhance the gene expression effect, such as enhanced, constitutive, tissue-specific or inducible promoters, such as CaMV 35S, Ubiquitin promoter (pUbi), stress-inducible promoter Rd29A, etc. The promoter can be used alone or in combination with other plant promoters. In addition, enhancer elements can be added during vector construction, including enhancers for translation or transcription, to ensure the consistency of the reading frame, thereby achieving correct protein translation. The enhancer region can select the ATG start codon or the start codon in its adjacent region.

[0046] The translation control signal and the start codon can be natural or synthetic, and the translation initiation region may be derived from the transcription initiation region or a specific structural gene. To facilitate the screening and identification of transgenic plants or plant cells, the recombinant vector can be optimized, such as inserting a gene expressing color change or luminescence marker, or a gene with an antibiotic or chemical resistance marker. In addition, marker genes can also not be used, and transgenic plants can be directly obtained through stress screening.

[0047] The embodiments of the present invention also provide a method for regulating the cold tolerance of plants, by regulating the expression of the OsCTOR gene of the plant to regulate the cold tolerance of the plant.

[0048] The regulation of the cold tolerance of plants specifically refers to improving the cold tolerance of plants.

[0049] In the present invention, the OsCTOR gene was successfully knocked out in Nipponbare rice by the CRISPR / Cas9 gene editing method, and the decrease in the expression level of the OsCTOR gene under cold stress was confirmed using this material, that is, this gene can effectively enhance the cold tolerance of rice.

[0050] In the quantitative tests in the following examples, three repeated experiments were set, and the results were averaged. The primer synthesis and sequencing work were both completed by Shanghai Sangon Biotech Co., Ltd.

[0051] To obtain the OsCTOR gene and the OsCTOR gene knockout mutant lines of the present invention, you can contact Yan Tao of the College of Agronomy, Hunan Agricultural University, Address: Room 3-346, Building 3, Yuelushan Laboratory, Furong District, Changsha City, Hunan Province; Zip Code: 410128.

[0052] Example 1

[0053] Protein structure of OsCTOR

[0054] Prediction through the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) found that the OsCTOR gene encodes a lipid transfer protein ( Figure 1 A).

[0055] The full length of the OsCTOR gene is shown in SEQ ID NO.1.

[0056] SEQ ID NO.1: AGCAGCCAGCAGCCAACTGCATCGATATCGATCGATCACTCG ACCGACCATCTCACAAGCAAAGCAAAAGCTCGTGGCAACAATGGCGAAGTGGGCGGCGATCATGGCGGTGCTGCTGCTGGCGGCGGCGTGGGCGCCGGCGCCGGCGACGGCGCAGTGCAACGCCGGGCAGCTGGCGATCTGCGCGGGCGCGATCATCGGCGGGTCGACGCCGTCGGCGTCGTGCTGCTCCAACCTGCGCGCGCAGAGGGGGTGCTTCTGCCAGTACGCGCGCAACCCGGCGTACGCCTCCTACATCAACAGCGCCAACGCCCGCAAGACCCTCACCTCCTGCGGCATCGCCATCCCCCGCTGCTAGGCACGCTCGATCTCCCGCCGCCGCGCGCCGCCGCTCGCCGGCGCCGGCGCCGGCCATGGTGCGTGGCAAATATATATATATATATATATATATATATATATATATATATATATACTGTGTGTACGTGCGCTTGAATAAAGGACGTGAGTTAATTTGATCGGTGTCGATGGTGTGTTTTTTTGGTATTCAAAGTTTGAATTCCGAATTTGGTCATCAGTTTATATATCTTGAGCTCA。

[0057] To further verify the structure of the OsCTOR protein, the protein structure of OsCTOR was predicted through the NCBI website. The results are as Figure 1 shown. OsCTOR mainly contains an AAI domain ( Figure 1 B), and the specific starting position of the structure is 26aa - 91aa of the whole protein.

[0058] Example 2

[0059] Identification of Nipponbare and mutant (CTOR) plant mutants and cloning of genes

[0060] To clarify the function of the OsCTOR gene in rice cold tolerance, the inventors constructed a CRISPR / Cas9-based loss-of-function mutant of the OsCTOR gene using the rice variety Nipponbare as the background. After surface sterilizing the obtained mutant seeds with 3% (v / v) hydrogen peroxide for 30 minutes, they were washed 5 times with distilled water and sown on a germination box, and cultured in an incubator for 10 days. The young and tender leaves of rice were cut, and rice DNA was extracted by the CTAB method. Using the extracted DNA as a template, PCR amplification was performed with the primer OsCTOR-CDS-F (CGACCATCTCACAAGCAAAGCA) and the primer OsCTOR-CDS-R (AGATCGAGCGTGCCTAGCAG). The target fragment amplified using the wild-type Nipponbare DNA as a template was the coding region of the OsCTOR gene. After sequencing, it was shown that the obtained fragment was the nucleotide sequence shown in SEQ ID NO.2.

[0061] SEQ ID NO.2: ATGGCGAAGTGGGCGGCGATCATGGCGGTGCTGCTGCTGGC GGCGGCGTGGGCGCCGGCGCCGGCGACGGCGCAGTGCAACGCCGGGCAGCTGGCGATCTGCGCGGGCGCGATCATCGGCGGGTCGACGCCGTCGGCGTCGTGCTGCTCCAACCTGCGCGCGCAGAGGGGGTGCTTCTGCCAGTACGCGCGCAACCCGGCGTACGCCTCCTACATCAACAGCGCCAACGCCCGCAAGACCCTCACCTCCTGCGGCATCGCCATCCCCCGCTGCTAG.

[0062] The protein sequence encoded by the nucleotide sequence shown in SEQ ID NO.2 is as shown in SEQ ID NO.3.

[0063] SEQ ID NO.3: MAKWAAIMAVLLLAAAWAPAPATAQCNAGQLAICAGAIIGGST PSASCCSNLRAQRGCFCQYARNPAYASYINSANARKTLTSCGIAIPRC.

[0064] Using wild-type Nipponbare DNA as a template, PCR amplification was performed with primer OsCTOR-promoter-F (CAGGTACGTGTCATTCAGTAC) and primer OsCTOR-promoter-R (TAAATGACGATGTTAGTGGTG). A fragment of about 2 kb was amplified. After sequencing, it was shown that the upstream promoter of OsCTOR has the nucleotide sequence shown in SEQ ID NO.4. This promoter is about 2 kb and is used to direct the high expression of this gene in plant cells to meet the requirements for rapid synthesis of lipid transfer proteins and response to various stimuli or stresses.

[0065]

[0066] On this basis, the present invention selected two homozygous mutant lines (CTOR-1, CTOR-2). Compared with Nipponbare, CTOR-1 inserted a base A, and CTOR-2 deleted 5 bp, ( Figure 2 of A), and both types of mutations occurred in the exon region of the gene. Both types of mutations led to amino acid frameshift ( Figure 2 of B), indicating that the gene knockout lines were all homozygous mutants and could be used for subsequent research.

[0067] The PCR reaction system is shown in Table 1.

[0068] Table 1

[0069]

[0070]

[0071] The above PCR reaction procedure was as follows: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 20 s, 72°C for 5 min, 35 cycles.

[0072] Example 3

[0073] Cold tolerance identification of Nipponbare and mutant (CTOR) plants

[0074] Seeds of Nipponbare, CTOR-1, and CTOR-2 were disinfected with 3% H2O2 for 30 minutes and soaked in deionized water at 37°C for about 2 days to germinate. The germinated seeds were transferred to a greenhouse (28°C day / 25°C night, 12 h / 12 h light / dark cycle, 85% relative humidity) and placed in a petri dish with two layers of filter paper. Early seedlings (bud length about 5 mm) were treated at low temperature (4°C) in an incubator for 6 days and then recovered at 28°C for 6 days. Finally, the plant height of each type was measured to evaluate its cold tolerance.

[0075] The results showed that after cold treatment, the plant height of the OsCTOR mutant rice was significantly higher than that of Nipponbare, ( Figure 3 in A). These results indicate that knocking out the OsCTOR gene based on the CRISPR / Cas9 technology improved the cold tolerance of rice.

[0076] Example 4

[0077] Analysis of the expression pattern of the OsCTOR gene

[0078] According to the data on the Rice data website (https: / / www.ricedata.cn / ), the expression pattern of the OsCTOR gene can be known. From Figure 4As shown in (A), the expression level of the OsCTOR gene varies significantly during different growth stages of rice. Among them, the expression level of the OsCTOR gene is significantly higher in the pre-flowering and flowering stages of rice than in other growth stages; from Figure 4 As shown in (B), under different treatment conditions, the expression level of the OsCTOR gene in rice is different. Among them, the expression level of the OsCTOR gene under cold treatment conditions in rice is significantly lower than that under normal conditions and other treatment conditions.

[0079] Example 5

[0080] Subcellular localization experiment of OsCTOR

[0081] To explore the subcellular localization of OsCTOR, the inventor constructed a subcellular localization vector of OsCTOR, transformed it into the competent Agrobacterium cell EHA105, and expressed it in Nicotiana benthamiana.

[0082] Specific implementation method: Using the cloned OsCTOR coding region as a template, amplify with primer OsCTOR-ZL035-F (CTATTTACAATTACAGTCGACATGGCGAAGTGGGCGGCG) and primer OsCTOR-ZL035-R (CATGGATCCTCTAGAGTCGACGCAGCGGGGGATGGCGAT), and recover the obtained PCR product. Digest 5 μg of the subcellular localization vector ZL035 with the restriction enzyme SalI (the digestion reaction is at 37 °C for 30 min), and recover the obtained digested product. Clone OsCTOR into the ZL035 vector by homologous recombination method (Novizan C112), and transfer it into the Agrobacterium strain EHA105 after successful sequencing. Take the successfully transformed Agrobacterium monoclonal and culture it overnight in 5 ml of LB broth medium at 28 °C until the OD600 is 1.0. Centrifuge at 5000 revolutions of the centrifuge, collect the obtained bacterial cells, resuspend them with tobacco injection solution to an OD600 of 0.8, let it stand at room temperature for 3 h, and then mix it with the tobacco cell membrane localization Marker (AtPIP2A-RFP) Agrobacterium strain in equal volume and inject it into the tobacco leaves. After culturing in the incubator for 48 h, take pictures using a laser confocal microscope (Zeiss LSM 980).

[0083] The tobacco injection solution system is shown in Table 2.

[0084] Table 2

[0085] Tobacco transformation buffer formulation (pH = 5.6) Concentration <![CDATA[MgCl2 (Magnesium Chloride)]]> 10 mM MES (Methyl Ester Sulfonate) 10 mM AS (Acetosyringone) 150 μM <![CDATA[ddH2O (double distilled water)]]> -

[0086] The results show that OsCTOR is localized in the extracellular region of the cytoplasm.

[0087] In summary, OsCTOR negatively regulates cold tolerance in rice, and reducing the expression of this gene can significantly enhance the cold tolerance of rice.

[0088] OsCTOR in rice encodes a lipid transfer protein, and its gene nucleotide sequence is shown in SEQ ID NO.1. The constructed gene editing vector of OsCTOR was expressed in wild-type rice Nipponbare to obtain the OsCTOR gene editing lines. It was found that the corresponding indicators of cold tolerance of the genetically edited transgenic plants were significantly enhanced compared with those of the wild-type plants. Therefore, the lipid transfer protein encoded by OsCTOR in rice of the present invention is a potential target for improving rice cold tolerance by genetic engineering and has important application value.

[0089] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. OsCTOR The use of a gene or an OsCTOR protein in regulating plant cold tolerance is characterized in that: Silence or knock out the OsCTOR Genes that improve cold tolerance in plants OsCTOR The gene encodes the OsCTOR protein; OsCTOR The nucleotide sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence of the OsCTOR protein is shown as SEQ ID NO.3, and the plant includes rice.

2. The use according to claim 1, characterized in that: Said OsCTOR The nucleotide sequence of the gene transcript cDNA is shown in SEQ ID NO.

2.

3. OsCTOR The application of the gene or OsCTOR protein in breeding cold-tolerant plant varieties is characterized in that: Silence or knock out the OsCTOR Genes that improve cold tolerance in plants OsCTOR The gene encodes the OsCTOR protein; OsCTOR The nucleotide sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence of the OsCTOR protein is shown as SEQ ID NO.3, and the plant includes rice.

4. OsCTOR The application of the gene or OsCTOR protein in cold-tolerant plant breeding is characterized in that: Silence or knock out the OsCTOR Genes that improve cold tolerance in plants OsCTOR The gene encodes the OsCTOR protein; OsCTOR The nucleotide sequence of the gene is shown as SEQ ID NO.1, the amino acid sequence of the OsCTOR protein is shown as SEQ ID NO.3, and the plant includes rice.

5. Silence or Knockout OsCTOR The use of a recombinant vector of a gene in regulating plant cold tolerance is characterized in that: Said OsCTOR The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the plant includes rice.

6. A method for regulating plant cold tolerance, characterized in that: By silencing or knocking out the plant OsCTOR The expression of genes regulating the cold tolerance of plants, the OsCTOR The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the plant includes rice.

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