Application of OsCTOR gene or OsCTOR protein in regulation of plant cold tolerance

Abstract

The invention discloses application of an OsCTOR gene or an OsCTOR protein in regulation of plant cold tolerance, and belongs to the technical field of plant genetic engineering. The rice OsCTOR gene involved in the invention is derived from cultivated rice Nipponbare. According to the invention, the OsCTOR gene is knocked out through a CRISPR gene editing technology, a rice mutant with the gene function deleted is obtained, and a cold stress experiment proves that the reduction of the expression quantity of the OsCTOR gene can effectively improve the cold resistance of rice. Therefore, the OsCTOR gene and the lipid transfer protein coded by the OsCTOR gene have wide application potential in the aspects of enhancing the cold resistance of the rice and cultivating cold-resistant rice varieties.

Description

Technical Field

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

[0002] During the growth of rice (Oryza sativa L.), low temperature stress (cold stress) is one of the important abiotic stress factors that affect rice yield and quality, and also limits the geographical distribution of plants. Low temperature environment will cause the fluidity of plant cell membranes to decrease, affecting the normal function of cells, while inhibiting enzyme activity and metabolic processes, thereby interfering with the normal physiological activities of plants. Under cold stress, plant photosynthesis is significantly weakened, and the synthesis of photosynthetic products is blocked, which in turn affects the growth and development of plants. In addition, low temperature will cause difficulties in the absorption of water and nutrients by crops, leading to 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 and causes irreversible damage, and even leads to crop death in severe cases. These adverse effects have become particularly prominent in the context of global climate change, and cold climatic conditions have posed new challenges to crop cultivation in tropical and subtropical regions.

[0003] As the frequency and intensity of cold stress increase, it threatens the stability and safety of agricultural production. How to improve the cold tolerance of crops and reduce the negative impact of cold stress has become an important topic in agricultural science research. In the long-term process of adapting to cold stress, plants sense external stimuli, generate and transduce corresponding adversity signals, and thus initiate a variety of defense mechanisms to cope with the harm of cold stress. Different species have undergone significant reprogramming of the transcriptome during the low temperature stress response, and many proteins have become important factors in this adaptive response.

[0004] In the long-term adaptation to cold stress, plants sense external stimuli, generate and transduce corresponding stress signals, and thus initiate a variety of defense mechanisms to cope with the harm of cold stress. Different species undergo significant transcriptome reprogramming during the low temperature stress response, and many proteins are considered to be important factors in this adaptive response.

[0005] Studies have found that lipid transport 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 transport proteins can mediate the transmembrane transport and distribution of different lipids, they regulate related signal pathways by maintaining membrane fluidity and stability when plants respond to cold stress.

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

[0007] The purpose of the present invention is to provide an application of an OsCTOR gene or an OsCTOR protein in regulating plant cold tolerance, so as 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 use of OsCTOR gene or OsCTOR protein in regulating plant cold tolerance.

[0010] The second technical solution of the present invention is the use of OsCTOR gene or OsCTOR protein in breeding cold-resistant plant varieties.

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

[0012] A fourth technical solution of the present invention is the use 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 plant cold tolerance.

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

[0014] Based on the above technical solution, 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. The present invention knocks out the OsCTOR gene through CRISPR gene editing technology to obtain a rice mutant with a loss of gene function, and confirms through cold stress experiments that the reduction in the expression 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 cloned and analyzed for the first time the lipid transport protein gene OsCTOR that responds to cold stress in rice, and published the nucleotide sequence, amino acid sequence and promoter sequence of the gene, which is of great significance for clarifying the molecular mechanism of rice cold tolerance regulation and the cultivation of cold-resistant materials.

[0017] 2. The present invention, for the first time, uses transgenic means to confirm that the OsCTOR gene is involved in the cold tolerance of rice. Knocking out the OsCTOR gene can significantly enhance the cold tolerance of rice, indicating that OsCTOR negatively regulates the cold tolerance of rice.

[0018] 3. The method of the present invention is used to cultivate cold-resistant plants, which has the advantages of simple operation and short cycle, and is suitable for wide application. The method has important theoretical and practical value in the study of molecular mechanisms of cold resistance, breeding of cold-resistant varieties and cold-resistant molecular breeding, and provides an economical, fast and efficient way to improve plant cold resistance. 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 embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0021] Figure 2 Figure 2 is the gene structure diagram of OsCTOR and the gene editing status of OsCTOR transgenic knockout mutants. Figures (A) and (B) are the gene structure diagram and the sequencing results of the homozygous gene-edited strain, and (C) is the amino acid sequence changes caused by gene editing.

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

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

[0024] Figure 5 It is the subcellular localization of OsCTOR gene, specifically the subcellular localization of OsCTOR in tobacco epidermal cells (second row), from left to right: GFP channel, bright field and fusion channel. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are 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, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0031] The embodiments of the present invention provide the use of 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 transcript cDNA of the OsCTOR gene is shown as SEQ ID NO.2.

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

[0036] In some specific embodiments, the plant is a monocot or a dicot.

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

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

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

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

[0041] The cold-resistant property is mainly manifested in that the plant exhibits strong cold-resistant ability in the bud stage (for example, when the bud grows to about 5 cm) and / or the seedling stage (for example, the two-leaf and one-heart stage).

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

[0043] The recombinant vector may 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 various 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 can contain the 3' untranslated region of the foreign gene, including polyadenylation signals and other functional fragments that promote mRNA processing or gene expression to guide the polyadenylation of the mRNA precursor at the 3' end.

[0045] When constructing a vector, different types of promoters can be added before the transcription start site to enhance the expression of the gene, such as enhanced, constitutive, tissue-specific or inducible promoters, such as CaMV 35S, Ubiquitin promoter (pUbi), stress-induced promoter Rd29A, etc. Promoters can be used alone or in combination with other plant promoters. In addition, enhancing elements can be added to the 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 use the ATG start codon or the start codon of its adjacent region.

[0046] Translation control signals and start codons can be natural or synthetic, while the translation initiation region may be derived from the transcription initiation region or a specific structural gene. In order to facilitate the screening and identification of transgenic plants or plant cells, the recombinant vector can be optimized, such as inserting a gene that expresses a color change or luminescent marker, or a gene with an antibiotic or chemical resistance marker. In addition, it is also possible to obtain transgenic plants directly through adversity screening without using marker genes.

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

[0048] The regulating plant cold tolerance is specifically to improve the plant cold tolerance.

[0049] The present invention successfully knocked out the OsCTOR gene in the rice Nipponbare by using the CRISPR / Cas9 gene editing method, and used the material to confirm the reduction in the expression level of the OsCTOR gene under cold stress, that is, the gene can effectively enhance the cold tolerance of rice.

[0050] The quantitative tests in the following examples were repeated three times, and the results were averaged. Primer synthesis and sequencing were completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0051] To obtain the OsCTOR gene and the OsCTOR gene knockout mutant strain of the present invention, please contact Yan Tao of the College of Agriculture of Hunan Agricultural University, address: 3-346, Building 3, Yuelushan Laboratory, Furong District, Changsha City, Hunan Province; Postal Code: 410128.

[0052] Example 1

[0053] Protein structure of OsCTOR

[0054] The prediction from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) revealed that the OsCTOR gene encodes a lipid transport 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.

[0057] In order to further verify the structure of OsCTOR protein, the protein structure of OsCTOR was predicted through the NCBI website. Figure 1 As shown, OsCTOR mainly contains an AAI domain ( Figure 1 B), the specific structure starting position is 26aa-91aa of the entire protein.

[0058] Example 2

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

[0060] In order to clarify the function of OsCTOR gene in rice cold tolerance, the inventors constructed a CRISPR / Cas9-based OsCTOR gene functional loss mutant with the rice variety Nipponbare as the background. The obtained mutant seeds were surface sterilized with 3% (v / v) hydrogen peroxide for 30 minutes, washed 5 times with distilled water, sown on a germination box, and cultured in an incubator for 10 days. The young leaves of rice were cut, and rice DNA was extracted by CTAB method. The extracted DNA was used as a template, and PCR amplification was performed with primers OsCTOR-CDS-F (CGACCATCTCACAAGCAAAGCA) and primers OsCTOR-CDS-R (AGATCGAGCGTGCCTAGCAG). The target fragment amplified using wild-type Nipponbare DNA as a template was the OsCTOR gene coding region. 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 GGCGGCGTGGGCCGGCCGGCGACGGCGCAGTGCAACGCCGGGCAGCTGGCGATCTGCGCGGGCGCGATCATCGGCGGTCGACGCCGTCGGCGTCGCTGCTCCAACCTGCGCGCGCAGAGGGGGTGCTCTGCCAGTACGCGCGCAACCCGGCGTACGCCTCCTACATCAACAGCGCCAACGCCCGCAAGACCCTCACCTCCTGCGGCATCGCCATCCCCCGCTGCTAG.

[0062] The protein sequence encoded by the nucleotide sequence shown in SEQ ID NO.2 is 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 primers OsCTOR-promoter-F (CAGGTACGTGTCATTCAGTAC) and primers OsCTOR-promoter-R (TAAATGACGATGTTAGTGGTG), and a fragment of about 2 kb was obtained. After sequencing, it was shown that the upstream promoter of OsCTOR had a nucleotide sequence as shown in SEQ ID NO. 4. The promoter is about 2 kb and is used to guide the gene to be expressed in large quantities in plant cells to meet the needs of rapid synthesis of lipid transport proteins and response to various stimuli or stresses.

[0065]

[0066] On this basis, the present invention selected two homozygous mutant strains (CTOR-1, CTOR-2). Compared with Nipponbare, CTOR-1 has an insertion of a base A and CTOR-2 has a deletion of 5 bp. Figure 2 A), both mutation types occur in the exon region of the gene. Both mutation types lead to amino acid frameshift ( Figure 2 B), indicating that the gene knockout strains are all homozygous mutations and can be used for subsequent research.

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

[0068] Table 1

[0069]

[0070]

[0071] The PCR reaction program 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] Identification of cold tolerance of Nipponbare mutant (CTOR) plants

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

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

[0076] Example 4

[0077] Analysis of the expression pattern of OsCTOR gene

[0078] The expression pattern of OsCTOR gene can be known from the data of Ricedata website (https: / / www.ricedata.cn / ). Figure 4As shown in (A), there are significant differences in the expression levels of OsCTOR gene in different growth stages of rice, among which the expression levels of OsCTOR gene in the early flowering stage and flowering stage of rice are significantly higher than those in other growth stages; Figure 4 As shown in (B), the expression level of OsCTOR gene in rice is different under different treatment conditions, among which the expression level of OsCTOR gene under cold treatment of rice is significantly lower than that under normal conditions and other treatment conditions.

[0079] Example 5

[0080] Subcellular localization experiment of OsCTOR

[0081] In order to explore the subcellular localization of OsCTOR, the inventors constructed a subcellular localization vector of OsCTOR, transformed it into Agrobacterium competent cells EHA105, and expressed it in Nicotiana benthamiana.

[0082] Specific implementation method: Using the cloned OsCTOR coding region as a template, amplify with primers OsCTOR-ZL035-F (CTATTTACAATTACAGTCGACATGGCGAAGTGGGCGGCG) and primers OsCTOR-ZL035-R (CATGGATCCTCTAGAGTCGACGCAGCGGGGGATGGCGAT), and recover the obtained PCR product. 5 μg of subcellular localization vector ZL035 was digested with restriction endonuclease SalI, and the digestion reaction was 37°C for 30 min), and the obtained digestion product was recovered. OsCTOR was cloned into the ZL035 vector using the homologous recombination method (NoviZan C112), and after successful sequencing, it was transferred into the Agrobacterium strain EHA105. Take the successfully transformed Agrobacterium monoclone and use 5ml LB broth medium, culture overnight at 28℃ until OD600 is 1.0, centrifuge at 5000 rpm, collect the obtained bacteria, resuspend them in tobacco injection solution to OD600 of 0.8, let them stand at room temperature for 3 hours, mix them with tobacco cell membrane localization marker (AtPIP2A-RFP) Agrobacterium strain in equal volumes, and inject them into tobacco leaves. After culturing in the incubator for 48 hours, use laser confocal microscope (Zeiss LSM 980) to take pictures.

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

[0084] Table 2

[0085] Tobacco transformation buffer formula (pH = 5.6) concentration <![CDATA[MgCl2 (Magnesium Chloride)]]> 10mM MES (sodium methyl ester sulfonate) 10mM AS(Acetosyringone) 150μM <![CDATA[ddH2O (double distilled water)]]> -

[0086] The results showed that OsCTOR was localized in the extracellular region of the cell.

[0087] In summary, OsCTOR shows negative regulation on rice cold tolerance, and reducing the expression of this gene can significantly enhance the cold tolerance of rice.

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

[0089] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of OsCTOR gene or OsCTOR protein in regulating plant cold tolerance.

2. The use according to claim 1, characterized in that: 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; 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.

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

2.

4. The use according to claim 1, characterized in that: Silencing or knocking out the OsCTOR gene improves the cold tolerance of plants.

5. The use according to claim 1, characterized in that: The plant is a monocotyledon or a dicotyledon.

6. The use according to claim 5, characterized in that: The plants include rice.

7. Application of OsCTOR gene or OsCTOR protein in breeding cold-tolerant plant varieties.

8. Application of OsCTOR gene or OsCTOR protein in cold-tolerant plant breeding.

9. Use of a recombinant expression vector, an overexpression vector, an interference vector, a recombinant virus, a recombinant bacterium or a recombinant gene expression cassette comprising an OsCTOR gene in regulating plant cold tolerance.

10. A method for regulating plant cold tolerance, characterized in that: By regulating the expression of the OsCTOR gene of the plant, the cold tolerance of the plant is regulated.

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