Rice os crlk1 protein and its encoding nucleic acid for improving plant resistance to abiotic stress

Abstract

The application belongs to the technical field of molecular biology, and discloses the use of rice OsCRLK1 protein and its coding gene in improving the resistance of plants to abiotic stress. The application uses overexpression and CRISPR knockout technology to overexpress or knockout the rice OsCRLK1 gene in rice, and finds that overexpression of the OsCRLK1 gene in rice can significantly improve the ability of rice to resist low-temperature stress according to the phenotype changes of the transgenic rice plants. Therefore, the rice OsCRLK1 protein and its coding gene can be applied to improve the resistance of plants to abiotic stress, including overexpressing the rice OsCRLK1 gene in plants to obtain transgenic plants, and the resistance of the obtained transgenic plants to low-temperature stress is obviously enhanced.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the application of rice OsCRLK1 protein and its encoded nucleic acid in improving plant resistance to abiotic stress. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the major food crops, serving as the primary food source for more than half of the world's population. However, rice often encounters various abiotic stresses at different stages of growth, leading to severe yield reductions, such as drought, salt stress, heat stress, and low-temperature stress. Rice mainly grows in tropical or subtropical regions and is particularly sensitive to low temperatures at all stages of its growth. Especially during the seedling stage, low temperatures often inhibit seedling growth or even kill seedlings, ultimately affecting yield. Therefore, it is of great significance to elucidate the molecular genetic mechanisms of cold tolerance in rice seedlings and to breed cold-resistant rice varieties by utilizing existing germplasm resources. Cold tolerance in rice seedlings is a complex quantitative trait regulated by multiple quantitative trait loci (QTLs). Based on a parental cross, a mapping population was established, and linkage maps were constructed using molecular markers, resulting in the discovery of more than 25 major-effect QTLs related to cold tolerance in rice seedlings. However, among these QTLs, only a small number of key genes have been identified and their functional studies conducted.

[0003] Studies have shown that the phosphorylation state of calmodulin receptor kinase CRLK1 may affect its kinase activity and function in plant cells. CRLK1 has been reported to participate in plant responses to hormones such as gibberellin (GA) and abscisic acid (ABA). Furthermore, CRLK1 can be induced by drought and salt stress, suggesting its potential involvement in plant stress responses. Low temperature stress induces intracellular calcium... 2+ The concentration increased dramatically. Calmodulin receptor kinase CRLK1 positively regulates plant cold resistance in Arabidopsis by inhibiting the activity of protein kinases MPK3 / 6. However, to date, there have been no reports of CRLK1 protein or its encoding gene enhancing cold stress tolerance in rice. Summary of the Invention

[0004] The main objective of this invention is to provide the application of rice OsCRLK1 protein and its encoded nucleic acid in improving plant resistance to abiotic stress, especially in resistance to low temperature stress.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0006] This invention provides the application of rice OsCRLK1 protein and its encoded nucleic acid in improving plant resistance to abiotic stress.

[0007] Preferably, the OsCRLK1 protein comprises the amino acid sequence shown in SEQ ID NO: 2.

[0008] Preferably, the encoding nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 1 or its degenerate sequence, or comprises the nucleotide sequence shown in SEQ ID NO: 3 or its degenerate sequence.

[0009] Preferably, the application specifically involves overexpressing the nucleic acid encoding the rice OsCRLK1 protein in plants to obtain transgenic plants.

[0010] Preferably, the application specifically includes: (1) Construct a recombinant expression vector containing the encoding nucleic acid of rice OsCRLK1 protein overexpression; (2) Transform the constructed recombinant expression vector into plant tissues or plant cells; (3) Transgenic plants with improved resistance to abiotic stresses were obtained through cultivation and screening.

[0011] Preferably, the abiotic stress includes low temperature stress; more preferably, the low temperature stress is below 6°C; and more preferably, the low temperature is below 5°C, 4°C, 3°C, 2°C, 1°C, or 0°C.

[0012] Preferably, the plants include, but are not limited to, monocotyledonous or dicotyledonous plants; more preferably, the plants include crops, vegetables or ornamental plants, fruit trees, etc., such as rice, cotton, corn, sorghum, wheat, soybean, potato, barley, tomato, sugarcane or Arabidopsis thaliana, etc., with rice being the most preferred.

[0013] Compared with the prior art, the present invention has the following technical advantages: This invention investigates phenotypic changes in rice plants encoding the OsCRLK1 protein under 6℃ low-temperature conditions using overexpression and CRISPR knockout, then performs gene cloning and functional analysis to analyze the relationship between candidate genes and abiotic stress responses in rice seedlings. The results show that overexpression of the OsCRLK1 protein in rice... OsCRLK1 Genes can significantly improve rice's ability to resist low-temperature stress. This invention has significant theoretical and practical implications for improving and enhancing rice's stress resistance, cultivating high-yielding and stress-tolerant varieties, and accelerating the process of stress-resistant molecular breeding. Attached Figure Description

[0014] Figure 1 PCR positivity identification of transgenic plants for overexpression and knockout materials. Lanes 1-6 were used for overexpression material identification, lanes 7-9 for knockout mutant primer identification, lane 10 for vector plasmid positive control, and lane 11 for negative control with added double-distilled water.

[0015] Figure 2 For rice OsCRLK1 Knockout details for each gene lineage. Mutation type 1 involves a 5-base deletion (bp) in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 42. Mutation type 2 involves an addition of 1 bp in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 46. Mutation type 3 involves a 1-base deletion in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 14.

[0016] Figure 3 For rice OsCRLK1 Expression levels of the gene overexpression material in various lines. A represents overexpression lines with expression levels 5-16 times higher than the wild type. B represents... OsCRLK1 Immunoblotting images of GFP protein expression in various lines of the gene overexpression material.

[0017] Figure 4 Analysis of OsCRLK1 expression patterns in rice. A represents subcellular localization of the OsCRLK1 protein, and B represents expression patterns at the rice seedling stage. OsCRLK1 Gene expression levels under low-temperature stress. C represents... OsCRLK1 Gene expression in different tissues (roots, stems, internodes, leaves, flowers).

[0018] Figure 5 For overexpression OsCRLK1 The cold tolerance phenotypes of CRISPR knockout transgenic plants and wild-type plants at 6℃ during the seedling stage. A shows the phenotypes of CRISPR knockout transgenic plants before and 7 days after 6℃ treatment; B shows the survival rate statistics of CRISPR knockout transgenic plants before and after 6℃ treatment; C shows the phenotypes of OsCRLK1 overexpressing plants before and 7 days after 6℃ treatment; D shows the survival rate statistics of OsCRLK1 overexpressing plants before and after 6℃ treatment. Detailed Implementation

[0019] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0020] Definitions of terms involved in this invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] The terms "polynucleotide" and "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, specific nucleic acid sequences implicitly encompass variants of their conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0022] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terminology applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terminology covers amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0023] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, an engineered selection marker for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0024] In this invention, the term "transformation" refers to the genetic transformation of polynucleotides or polypeptides into plants by introducing the gene encoding the rice PD1 protein into plant cells. Methods for introducing such polynucleotides or polypeptides into plants are well known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation. "Stable transformation" refers to the integration of the introduced polynucleotide construct into the genome of the plant cell and its inheritance through its progeny; "transient transformation" refers to the introduction of a polynucleotide into a plant but its temporary expression or presence in the plant.

[0025] In this invention, the term "germination rate" refers to the percentage of rice seeds that germinate normally within a specified period under optimal germination conditions. Rice seedling emergence rate refers to the percentage of rice seeds that successfully grow into mature, healthy seedlings from sowing to the appearance of a certain number of mature leaves in the paddy field within a certain timeframe, out of the total number of sown seeds. It reflects the viability of rice seeds and their emergence after sowing.

[0026] Example 1

[0027] Using two restriction endonucleases Kpn1 and BamH1 The pUN1301 vector was digested, linearized, and recovered.

[0028] Primers were designed based on the full-length Nipponbare sequence. Using Nipponbare cDNA as a template, the full-length coding region of the OsCRLK1 protein was amplified. The stop codon (TGA) was removed, and adapters linearized from the pUN1301 vector were added to the 5′ and 3′ ends. The amplification primers are as follows: F: 5'-cgggatccATGGGGAGGATGAAAGGGGTT-3' (SEQ ID NO:4, where the lowercase part is the connector sequence); R: 5'-ggggtaccGACATCTGCAAGATCAGAGAC-3' (SEQ ID NO: 5, where the lowercase part is the connector sequence).

[0029] The target DNA fragment (1306 bp) was amplified and recovered by PCR. Homologous recombination of the target fragment with the linearized vector was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, Code no: C115-01). Positive clone plasmids were verified by PCR and sequencing. Sequencing results showed that the OsCRLK1 gene fragment shown in SEQ ID NO:1 was inserted between the two restriction sites of the p1300S vector, resulting in the recombinant vector named pUN1301-. OsCRLK1 .

[0030] Example 2

[0031] according to OsCRLK1 The target sites for gene cDNA sequence knockout were designed, and the knockout target sites are shown in Table 1.

[0032] Table 1. Knockout target sites (5'-3')

[0033] Primers CRLK1target1-BsF, CRLK1target1-F0, CRLK1target2-R0, and CRLK1target2-BsR were designed based on the target. Four-primer PCR amplification was performed using a 100-fold diluted pCBC-MT1T2 plasmid as a template. The PCR products were purified and recovered, and the final vector was constructed using an enzyme digestion-ligation system. The vector was named CRISPR- OsCRLK1 .

[0034] Table 2 Gene knockout primers (5'-3')

[0035] Example 3 The freeze-thaw method was used to express the expression vector pUN1301- OsCRLK1 and the knockout vector CRISPR- OsCRLK1 Transfected into Agrobacterium EHA105 competent cells, the specific experimental method is as described in Molecular Cloning Laboratory Guide.

[0036] Example 4 1) Sterilization: Remove the shells from healthy, plump Nipponbare seeds, soak them in 70% ethanol for 1-2 minutes, add 50% bleach and place on a shaker (200 rpm) for about 1.5 hours. Rinse five times with sterile water, place the seeds on sterile filter paper to absorb excess moisture, and then evenly place them in NBD medium and incubate in the dark at 26°C. All the above steps are performed in a laminar flow hood.

[0037] 2) Subculture: After about 15 days of dark culture, the rice seed buds are separated and transferred to NBD subculture medium for continued dark culture at 26°C; after 10 days, the seeds are separated from the callus and the callus is transferred to a new NBD subculture medium. After about 4-5 days of dark culture at 26°C, Agrobacterium transformation can be carried out. 3) During this period, Agrobacterium containing plasmids is streaked on the corresponding antibiotic culture medium. After 2 days, single clones are picked, streaked again, and cultured for 1 day. 4) Collect the bacterial cells from the culture medium and vortex them in NBC1 medium containing acetylsuccinone (AS), adjusting the OD to approximately 0.1-0.2; 5) Transformation: Select healthy callus tissue into a sterile Erlenmeyer flask, add the above-prepared suspension, gently shake at room temperature for about 10 minutes, discard the bacterial solution, place the callus tissue on sterile filter paper, absorb the excess bacterial solution, and place it in a laminar flow hood to blow air until the callus tissue turns slightly white. Then transfer the callus tissue to NBC2 medium with a layer of sterile filter paper, and co-culture at 22°C in the dark for 2 days. 6) Screening: Transfer the co-cultured callus to NBS1 medium containing the corresponding antibiotic, incubate in the dark at 26°C for 10-12 days, then transfer to NBS2 medium and continue incubation in the dark at 26°C for 10-12 days. 7) Differentiation: After transferring the callus to NBR1 medium, culture it in the dark at 26°C for 6 days, then transfer it to an artificial climate incubator with 15 h light / 9 h dark for 15-20 days at 26°C. During this period, callus tissue with green spots is transferred to NBR2 medium for culture until it differentiates into seedlings. 8) Cut the roots and leaves of the transgenic seedlings that are about 5 cm tall, transfer them to the rooting medium, and culture them at 26°C in an artificial climate incubator with 12 h light / 12 h darkness. 9) Hardening off seedlings: Once the root system of the transgenic seedlings is sufficiently developed, open the culture bottle for about 2 days, wash off the culture medium, place the seedlings in water for 1 week, and then transfer them to the soil for planting.

[0038] The culture medium formulations used in the above genetic transformation process are shown in Table 3; the preparation of hormone and antibiotic storage solutions in the culture medium is shown in Table 4.

[0039] Table 3 Genetic transformation culture medium

[0040] Table 4. Preparation of Hormone and Antibiotic Storage Solutions

[0041] Example 5 The generated T0 generation transgenic seedlings were validated by PCR and propagated through planting. T1 generation transgenic seeds were harvested, and positive results were verified and sequenced to obtain the corresponding homozygous mutant materials, such as... Figure 1 As shown. The obtained homozygous mutant materials were further propagated by planting, and the offspring were screened for seeds using hygromycin (50 mg / L) to obtain the corresponding Cas9-free homozygous mutant materials.

[0042] The overexpression and mutant line identification primers used to determine homozygosity are shown in Table 5.

[0043] Table 5 Primers for molecular identification of transgenic plants (5'-3')

[0044] Example 6 (1) T3 generation OsCRLK1 knockout mutant and seeds of Nipponbare were selected.

[0045] (2) Rice cultivation in soil: After the newly harvested seeds break dormancy, soak them in a 28℃ incubator for 3 days until they germinate, and then sow them. Select seeds with uniform germination and sow them evenly in a mixture of nutrient soil and vermiculite prepared in a ratio of 3:1. Cover the surface with a layer of vermiculite and then let them grow normally in a 28℃ incubator. Water them once every 2-3 days during this period.

[0046] (3) Rice hydroponics: Select seeds with consistent germination and sow them in a 96-well PCR plate with the bottom removed. Place the plate in a 28 ℃ incubator and grow. Change the water every 2-3 days. Add an appropriate amount of nutrient solution when the third leaf just emerges. Change the water to clean water after the third leaf has fully unfolded.

[0047] (4) After the disinfected rice seeds germinate at room temperature, they are sown. Each experimental group has at least 3 replicates. After two weeks of cultivation under light at 28℃, the seeds are treated at 12℃ for 2-4 days (depending on the actual situation), and then transferred to 28℃ to resume growth for one week.

[0048] (5) For the identification of mutants, the genome near the target site was amplified as shown in SEQ ID NO.3 and sent to Sangon Biotech Co., Ltd. for sequencing and comparison of sequencing results.

[0049] like Figure 2 As shown, mutation type 1 involves a 5-base deletion (bp) in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 42. Mutation type 2 involves an addition of 1 bp in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 46. Mutation type 3 involves a 1-base deletion in the Target 1 sequence at exon 1, resulting in a missense mutation at amino acid 10, leading to premature translation termination at amino acid 14.

[0050] Example 7 Rice RNA extraction and quantitative real-time PCR Rice leaves were ground into powder using liquid nitrogen. For every 50-100 mg of rice leaves, 1 mL of TRIPure (Tsingke Biotech, Beijing) was added and incubated at room temperature for 5 min. Then, 200 μL of chloroform was added, and the mixture was centrifuged at 12000 rpm at 4℃ for 15 min. After centrifugation, the mixture separated into three layers. 400 μL of the top aqueous layer was transferred to a new RNA-free centrifuge tube, and the same volume of isopropanol was added. The mixture was inverted and incubated for 20 min at room temperature, then centrifuged at 12000 rpm at 4℃ for 10 min. The supernatant was discarded, and the resulting flaky precipitate was RNA. The precipitate was washed twice with 70% ethanol solution (prepared with enzyme-free sterile water), dried, and then dissolved in 40-60 μL of enzyme-free sterile water. The quality of the RNA was evaluated using a UV spectrophotometer; an A260 / 280 value between 1.8 and 2.0 indicated good RNA quality.

[0051] For qualified RNA, the first strand of cDNA was synthesized using the Novozymes HiScript II RT SuperMix reverse transcription kit.

[0052] Real-time quantitative PCR (qRT-PCR) was performed using AceQ qPCR SYBR Green Master Mix enzyme, following the CFX96 (Bio-Rad) operating instructions. This study used rice... Actin As an internal reference gene, the experimental results were obtained using 2 -ΔΔCT The method is used for analysis.

[0053] Rice protein extraction and immunoblotting experiments Rice leaves were ground into powder using liquid nitrogen. Extraction solution was added at a ratio of 500 μL per 0.3 g of powder, vortexed to mix, and incubated on ice for 20 min. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was transferred to a new centrifuge tube, centrifuged again, and 5X loading buffer was added. After denaturation at 95℃ for 10 min, SDS-polyacrylamide gel electrophoresis was performed.

[0054] For electrophoresis, cover the electrophoresis tank with the black and red electrodes in place. Adjust the voltage (80 V for stacking gel, 120 V for separating gel). Stop electrophoresis when the bromophenol blue migrates to the bottom of the gel and prepare for transfer. Open the transfer clamps, keeping the black side horizontal. Peel the gel from the glass plate and, following the order of sponge-filter paper-gel-NC membrane-filter paper-sponge, close the clamps and secure. Place the "sandwich" into the transfer tank, with the clamps facing each other. Transfer at 100 V for 90 min in ice water. After transfer, remove the apparatus and block the NC membrane in 5% skim milk powder at room temperature for 1 h or overnight at 4°C. After blocking, wash with 1X PBST buffer for 30 min. Incubate the washed NC membrane in 3% skim milk containing the appropriate primary antibody at room temperature for 2 h or overnight at 4°C. Wash with 1X PBST buffer for 30 min. Incubate the washed NC membrane in 1X PBST containing the appropriate secondary antibody at room temperature for 1 h. Wash with 1X PBST buffer for 30 min. Image using a gel imaging system (Bio-Rad, USA).

[0055] The results are as follows Figure 3 As shown in Figure A, the expression levels of all overexpression lines were 5-16 times higher than those of the wild type. Figure B shows... OsCRLK1 Immunoblotting images of GFP protein expression in various lines of the gene overexpression material.

[0056] Example 8 (1) Protoplast extraction: Take about 200 rice plants cultured for about 14 days, select the stem part from near the soil surface to the middle of the second leaf, cut it into small segments of 0.5 mm, put it into a conical flask wrapped with tin foil, add 10 mL of enzyme solution, shake gently, and vacuum for 1 h. Then put it in a shaker at 28 ℃, 40 rpm, for 4 h to fully release the protoplasts. Filter the enzyme solution with 4 layers of gauze, wash the protoplasts with W5 and transfer them to a 50 mL centrifuge tube, 350 g, 3 min, quickly discard the supernatant, resuspend the protoplasts (precipitate) with 2 mL of W5 solution, and suspend the protoplasts at a ratio of 1 mL protoplasts: 5 mL sucrose solution, 200 g, 7 min, the protoplasts separate into layers, use a cut pipette tip to pick up the protoplasts in the middle layer, then gently suspend the protoplasts with 20 mL of W5 solution, wash away the sucrose, 350 g, 3 min, discard the supernatant, suspend the precipitate with 2 mL of W5 solution and examine under a microscope. If the protoplasts are in good condition, incubate at 4 °C for more than 30 min. Then, add 350 g for 3 min, remove the supernatant, and add MMG solution as needed (100 μL MMG solution per tube of protoplasts), and wait for transformation.

[0057] (2) PEG-mediated transformation: Take a 2 mL centrifuge tube (enzyme-free and sterile), add 10 μg of plasmid, slowly add 100 μL of protoplasts, mix gently, add 110 μL of PEG solution, mix gently, place flat on the table, let stand for 7 min, add 780 μL of W5 solution, mix well to terminate the transformation, centrifuge at 100 g for 2 min at room temperature, remove the supernatant as much as possible, add 1 mL of W5 solution to resuspend the protoplasts, and place flat in a light-proof container.

[0058] (3) Subcellular localization experiment: OsCRLK1 The protein fused with the GFP tag was transferred into wild-type protoplasts, incubated overnight at 28°C, and the GFP fluorescence signal was observed using a laser confocal microscope.

[0059] like Figure 4 As shown, A represents the subcellular localization of the OsCRLK1 protein, and B represents the rice seedling stage. OsCRLK1 Gene expression levels under low-temperature stress. C represents... OsCRLK1 Gene expression in different tissues (roots, stems, internodes, leaves, flowers).

[0060] Example 9 Select rice seeds with uniform germination and sow them in 96-well PCR plates (with the bottom removed). Incubate at 28°C, changing the water every 2-3 days. Add an appropriate amount of nutrient solution when the third leaf just emerges, and replace with clean water after the third leaf is fully expanded. Place the rice seedlings from Example 5 that have reached the three-leaf stage in an artificial climate incubator at 6°C (10 h light / 14 h dark) for 2-3 days. crlk1 The mutant plants were treated for 2 days. CRLK1 After three days of overexpression treatment, the plants were returned to a 28°C artificial climate incubator (10 hours light / 14 hours darkness) for 7 days to recover. Survival rate was determined by the presence or absence of new leaves; plants with new leaves were considered alive, while those without were considered dead.

[0061] The results are as follows Figure 5 As shown in the figures, A represents the phenotypic images of CRISPR knockout transgenic plants before and after 6℃ treatment and 7 days after recovery; B represents the statistical results of the survival rate of CRISPR knockout transgenic plants before and after 6℃ treatment; C represents the phenotypic images of OsCRLK1 overexpressing plants before and after 6℃ treatment and 7 days after recovery; and D represents the statistical results of the survival rate of OsCRLK1 overexpressing plants before and after 6℃ treatment. The results indicate that overexpression of OsCRLK1 in rice... CRLK1 Genes can significantly improve the ability of rice to resist low temperature stress. Figure 5 CD), to rice CRLK1 Mutations or knockouts of genes significantly reduce the ability of rice to resist low-temperature stress. Figure 5Therefore, the rice OsCRLK1 protein, its encoding gene, and recombinant vector can be used to enhance the crop's resistance to abiotic stresses, especially low temperatures.

[0062] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. The application of nucleic acid encoding rice OsCRLK1 protein overexpression in improving rice resistance to low-temperature stress, characterized in that, The rice OsCRLK1 protein contains the amino acid sequence shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The applications specifically include, (1) Construct a recombinant expression vector containing the encoding nucleic acid of rice OsCRLK1 protein overexpression; (2) Transform the constructed recombinant expression vector into rice tissues or rice cells; (3) Transgenic rice with improved resistance to low-temperature stress was obtained through breeding and screening; The encoded nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a degenerate sequence thereof.

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