Rice cold tolerance gene COLD8.1 and application thereof in plant cold tolerance
By mining the cold-resistant gene COLD8.1 in rice and genetically regulating it, the problem of rice's sensitivity to cold stress was solved, significantly improving the cold resistance of rice seedlings and enhancing their growth and survival rate in low-temperature environments.
Patent Information
- Application Number
- CN202411251747.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2026-02-27
- Estimated Expiration
- 2044-09-09
AI Technical Summary
Rice is sensitive to cold stress, which can lead to seedling death and pollen abortion during the booting stage, severely affecting yield. There are few existing cold-resistant genes and no effective solutions have been found for rice growth in low-temperature environments.
The cold-resistant gene COLD8.1 in rice was discovered and isolated. By overexpressing or knocking out this gene, the cold resistance of the plant was regulated. The recombinant expression vector was used to carry out genetic transformation in rice to improve or reduce its cold resistance in the seedling stage.
It significantly improved the cold resistance of rice seedlings, enhanced the growth and survival rate of rice under low temperature conditions, and proved the role of the COLD8.1 gene in regulating plant cold resistance.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a rice cold tolerance gene COLD8.1 and application thereof in plant cold tolerance. BACKGROUND
[0002] Rice is one of the most important crops, providing 21% of the global per capita calorie intake. Rice originated in tropical and subtropical regions and is sensitive to cold stress. In high-latitude and high-altitude regions, cold stress seriously restricts the growth and production of rice. Seedling death and pollen abortion at the booting stage are two major factors causing yield loss under cold stress. Therefore, it is urgent to improve the cold tolerance of rice to maintain the safety of rice production and expand its planting area to lower temperature geographical regions.
[0003] Rice cold tolerance is a quantitative trait that is jointly regulated by multiple genetic factors and the environment. The genetic basis of rice cold tolerance is still in its infancy. Many quantitative trait loci (QTLs) have been located and cloned, such as qLTG3-1, bZIP73, OsTPP1, Ctb1, COLD1, qCT-9, CTB4a, qPSR10, and HAN1. Among them, natural variations in COLD1, CTB4a, qPSR10, and HAN1 genes have been confirmed to enhance the adaptability of rice to low temperature environments. However, few natural alleles with cold tolerance have been discovered. SUMMARY
[0004] The application provides a rice cold tolerance gene COLD8.1 and application thereof in plant cold tolerance, and the cold tolerance gene COLD8.1 can significantly regulate the cold tolerance performance of plants.
[0005] The application provides a rice cold tolerance gene COLD8.1, and the amino acid sequence of the protein encoded by the cold tolerance gene COLD8.1 is shown in SEQ ID No. 1.
[0006] In one specific embodiment of the application, the full-length nucleotide sequence of the cold tolerance gene COLD8.1 is shown in SEQ ID No. 2.
[0007] In one specific embodiment of the application, the CDS nucleotide sequence of the cold tolerance gene COLD8.1 is shown in SEQ ID No. 3.
[0008] The application also provides a pair of primers for cloning the above-mentioned cold tolerance gene COLD8.1, including an upstream primer with a nucleotide sequence shown in SEQ ID No. 4 and a downstream primer shown in SEQ ID No. 5.
[0009] The application also provides a recombinant expression vector containing and expressing the above-mentioned cold tolerance gene COLD8.1.
[0010] The application also provides application of the cold-resistant gene COLD8.1 or the recombinant expression vector in regulating cold resistance of a plant at a seedling stage.
[0011] In one specific embodiment of the application, regulating cold resistance of a plant at a seedling stage comprises improving the cold resistance of the plant at the seedling stage when the cold-resistant gene COLD8.1 is overexpressed, and reducing the cold resistance of the plant at the seedling stage when the cold-resistant gene COLD8.1 is knocked out.
[0012] The application also provides a method for improving the cold resistance of a plant at a seedling stage, comprising overexpressing the cold-resistant gene COLD8.1 in the genome of the plant.
[0013] The application also provides application of the cold-resistant gene COLD8.1 or the recombinant expression vector in creating a cold-resistant plant germplasm.
[0014] The application also provides a method for creating a cold-resistant plant germplasm, comprising overexpressing the cold-resistant gene COLD8.1 in the genome of a target plant.
[0015] Beneficial effects: The application provides a new cold-resistant gene mined and separated from Jiangxi Dongxiang wild rice (referred to as "Dongye", DY) with extremely strong cold resistance. The amino acid sequence coded by the cold-resistant gene COLD8.1 is shown as SEQ ID No. 1.
[0016] In the embodiments of the application, COLD8.1 gene overexpression transgenic rice is constructed. After low-temperature stress, the growth condition and survival rate of the COLD8.1 gene overexpression transgenic rice are significantly better than those of rice GZX49. Meanwhile, COLD8.1 gene Crispr knockout plants are also constructed. After low-temperature stress, the growth condition and survival rate of the COLD8.1 gene Crispr knockout plants are significantly worse than those of rice Zhonghua 11. It is proved that the COLD8.1 gene has the function of regulating the cold resistance of a plant. After overexpression, the cold resistance at a seedling stage can be significantly improved. After knockout mutation, the cold resistance at a seedling stage can be significantly reduced. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 Fig. 3 is a result graph of expression quantity identification of COLD8.1 overexpression materials;
[0018] Figure 2 Fig. 5 is a phenotype graph of COLD8.1 overexpression materials after cold treatment;
[0019] Figure 3 Fig. 7 is a survival rate result graph of COLD8.1 overexpression materials after cold treatment;
[0020] Figure 4 Figure for sequencing results of mutation of target site of COLD8.1 knockout material;
[0021] Figure 5 Figure for survival rate of COLD8.1 knockout material after cold treatment. DETAILED DESCRIPTION
[0022] The present application provides a cold tolerance gene COLD8.1 of rice, wherein the amino acid sequence of the protein encoded by the cold tolerance gene COLD8.1 is shown as SEQ ID No. 1.
[0023] The amino acid sequence is obtained based on translation of coding sequence (CDS) by Primer 3 software (http: / / frodo.wi.mit.edu / ), and totally encodes 101 amino acids, and the specific sequence is shown as SEQ ID No. 1: MTRDGALPASGGGGAAEGPRRCSQCGHHGHNARTCTARGPVKLFGVRIGDKPPTAAAGGGGGMRKSASMGSLAQLAEGRRRRREGGGVRLRWERRQTAEAR
[0024]
[0025] In one specific embodiment of the present application, the CDS nucleotide sequence of the cold-resistant gene COLD8.1 is 306 bp, and its nucleotide sequence is shown in SEQ ID No. 3: ATGACGCGTGACGGCGCGCTGCCGGCGAGCGGCGGCGGCGGGGCGGCGGAGGGGCCGAGGCGGTGCTCGCAGTGCGGGCACCACGGGCACAACGCGCGGACGTGCACGGCGAGGGGCCCCGTGAAGCTGTTCGGCGTGCGGATCGGCGACAAGCCGCCGACCGCGGCCGCGGGCGGAGGAGGAGGGATGAGGAAGAGCGCCAGTATGGGGAGCCTCGCGCAGCTGGCGGAGGGGCGGCGGCGGCGGCGGGAGGGAGGAGGGGTACGGCTCCGATGGGAACGACGACAAACGGCGGAAGCGAGGTGA.
[0026] The present application also provides a pair of primers for cloning the above-mentioned cold-resistant gene COLD8.1, which comprises an upstream primer with a nucleotide sequence shown in SEQ ID No. 4 and a downstream primer shown in SEQ ID No. 5.
[0027] SEQ ID No. 4: 5'-ATGGTTATAGATGATGTGAGTAT-3';
[0028] SEQ ID No. 5: 5'-TCACTTTGCAGGAATCAGTCCATTT-3'.
[0029] In one specific embodiment of the present application, the full-length sequence of the cold-resistant gene COLD8.1 is obtained by using the genomic DNA of Dongye rice as a template, using the above-mentioned primer pair synthesized by a biological company to configure a PCR amplification system, and performing PCR amplification. The PCR amplification procedure for the full-length amplification of the present application comprises: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 6 minutes, 35 cycles; 72℃ extension for 10 minutes.
[0030] In one embodiment of the present application, the CDS sequence of the cold tolerance gene COLD8.1 is amplified by using the primers and the cDNA of Dongye as template.
[0031] The present application also provides a recombinant expression vector containing and expressing the cold tolerance gene COLD8.1.
[0032] In one embodiment of the present application, the recombinant expression vector is an overexpression vector, and the basic backbone vector of the overexpression vector can be a common vector such as pC1300S vector. In one embodiment of the present application, the full-length sequence of the cold tolerance gene COLD8.1 is inserted into the KpnI and BamHI double enzyme digestion sites of the pC1300S vector, thereby constructing the overexpression vector.
[0033] In one embodiment of the present application, KpnI and BamHI double enzyme digestion sites are introduced on both sides of the full-length sequence of the cold tolerance gene COLD8.1, and the design primer can be:
[0034] OX-F (SEQ ID No. 6): 5'-gagctttcgcgagctcggtaccATGGTTATAGATGATGTGAGTAT-3';
[0035] OX-R (SEQ ID No. 7): 5'-caggtcgactctagaggatccTCACTTTGCAGGAATCAGTCCATTT-3'.
[0036] The present application also provides the application of the cold tolerance gene COLD8.1 or the recombinant expression vector in regulating the cold tolerance of plant seedlings.
[0037] In one embodiment of the present application, the regulation of the cold tolerance of plant seedlings includes improving the cold tolerance of plant seedlings when overexpressing the cold tolerance gene COLD8.1, and reducing the cold tolerance of plant seedlings when knocking out the cold tolerance gene COLD8.1.
[0038] The present application also provides a method for improving the cold tolerance of plant seedlings, which comprises overexpressing the cold tolerance gene COLD8.1 in the genome of the plant.
[0039] In one specific embodiment of the present application, the overexpression can be achieved by genetic transformation, i.e. transforming the overexpression vector into Agrobacterium, and then transforming the overexpression vector into the target plant by Agrobacterium-mediated genetic transformation, thereby obtaining the transgenic plant with overexpression. In one specific embodiment of the present application, the target plant is rice.
[0040] The present application also provides the application of the cold-resistant gene COLD8.1 or the recombinant expression vector in creating cold-resistant plant germplasm.
[0041] The cold-resistant gene COLD8.1 or the recombinant expression vector can be used to create germplasm with different cold resistance by knocking out or overexpressing the cold-resistant gene COLD8.1.
[0042] The present application also provides a method for creating cold-resistant plant germplasm, which comprises overexpressing the cold-resistant gene COLD8.1 in the genome of the target plant.
[0043] The method for overexpression is not particularly limited in the present application, and in one embodiment, it can be Agrobacterium-mediated genetic transformation.
[0044] In order to further illustrate the present application, the rice cold-resistant gene COLD8.1 and its application in plant cold resistance provided by the present application are described in detail below with reference to the examples, but they should not be understood as limiting the scope of protection of the present application.
[0045] In the examples of the present application, the primers are synthesized by Shanghai Shengong, and the sequence determination is determined by Shanghai Shengong. The DNA, RNA extraction, PCR and reagent formula are carried out according to J. Sambrook et al., Molecular Cloning Laboratory Manual (3rd Edition, Jin Dongyan et al. (translated), Science Press, 2002).
[0046] Example 1 Cloning of gene COLD8.1
[0047] 1. Genomic DNA of rice variety Dongye was extracted, and primers shown as SEQ ID No. 4 and SEQ ID No. 5 were used for PCR. The obtained PCR product was sequenced to obtain the full-length sequence of rice gene COLD8.1, which consists of 1545 bases, and the nucleotide sequence is shown as SEQ ID No. 2.
[0048] PCR system: DNA (3 ng); 10 μmol / L of upper and lower primers, 0.3 μl each; 2x PCR buffer for KOD FX 10 μl; KOD FX (1.0 U / μl) 0.4 μl; 2 mM dNTPs 4 μl; add water to a total volume of 20 μl.
[0049] PCR program: 94℃ pre-denaturation 5 minutes; 94℃ denaturation 30 seconds, 55℃ annealing 30 seconds, 72℃ extension 6 minutes, 35 cycles; 72℃ extension 10 minutes.
[0050] 2, Extract the RNA of Dongye leaves of rice varieties, reverse transcribe into cDNA, and use the primers shown in SEQ ID No. 4 and SEQ ID No. 5 to perform PCR, the size of the amplified product is 306 bp, the coding sequence (CDS) of the gene COLD8.1 is obtained by sequencing analysis of the obtained PCR product, which consists of 306 bases, and the nucleotide sequence is shown in SEQ ID No. 3.
[0051] PCR system: 4 μl of reverse transcribed cDNA; 0.3 μl of 10 μmol / L upper and lower primers respectively; 10 μl of 2x PCR buffer for KOD FX; 0.4 μl of KOD FX (1.0 U / μl); 4 μl of 2 mM dNTPs; add water to a total volume of 20 μl.
[0052] PCR program: 94℃ pre-denaturation 5 minutes; 94℃ denaturation 30 seconds, 55℃ annealing 30 seconds, 72℃ extension 1 minute, 30 cycles; 72℃ extension 7 minutes.
[0053] Example 2 Overexpression of COLD8.1 gene improves the cold tolerance of rice seedlings
[0054] 1, Construction of overexpression vector
[0055] According to the full-length sequence of the rice gene COLD8.1 amplified in Example 1, a pC1300S vector KpnI, BamHI double enzyme digestion site homologous recombination adapter was introduced on the upstream and downstream primers, and the primer pair shown in SEQ ID No. 6 and SEQ ID No. 7 was designed as OX-F and OX-R.
[0056] Using the Dongye genome obtained in Example 1 as a template, primers OX-F and OX-R were used to perform PCR, and the product size was 1545 bp.
[0057] PCR system: DNA (3 ng); 0.3 μl of 10 μmol / L upper and lower primers respectively; 10 μl of 2x PCR buffer for KOD FX; 0.4 μl of KOD FX (1.0 U / μl); 4 μl of 2 mM dNTPs; add water to a total volume of 20 μl.
[0058] PCR program: 94℃ pre-denaturation 5 minutes; 94℃ denaturation 30 seconds, 55℃ annealing 30 seconds, 72℃ extension 1 minute, 30 cycles; 72℃ extension 7 minutes.
[0059] The PCR-amplified isolated COLD8.1 gene fragment was ligated to the pC1300S vector digested with Kpnl and BamHI by homologous recombination. After sequencing alignment, the COLD8.1 gene overexpression vector was obtained by transforming into Agrobacterium EHA105.
[0060] 2. Obtaining of overexpression transgenic plants
[0061] The COLD8.1 gene overexpression vector was transformed into indica rice variety Ganxiao indica 49 (GZX49) by Agrobacterium-mediated genetic transformation. After selection culture, differentiation, rooting, and seedling, the T0 generation transgenic plants were obtained (referring to Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal, 1994, 6: 271-282). The seeds were collected after planting all the transgenic materials, which completed the expansion and obtained the stably inherited T1 generation materials. For a certain T1 generation transgenic line, if all the T2 generation plants obtained by sampling selfing are transgenic plants, the T1 generation transgenic line is a homozygous transgenic line. Three homozygous transgenic lines OX-1, OX-2, and OX-3 were selected for subsequent analysis.
[0062] 3. Identification of overexpression transgenic plants
[0063] (1) Expression level identification
[0064] The 2-leaf stage seedlings were taken, total RNA was extracted, cDNA was obtained by reverse transcription, and fluorescent real-time quantitative PCR was performed. The primers for detecting the expression level of COLD8.1 gene were as follows:
[0065] RT-F (SEQ ID No. 8): 5'-GTCAGAAGAGGAGCACAAAAAG-3';
[0066] RT-R (SEQ ID No. 9): 5'-GCGTCCTTGAACCAACATAATT-3'.
[0067] The reagent for quantitative analysis was FastStart Universal SYBR Green Master (ROX). The instrument used was the real-time fluorescent quantitative PCR instrument ViiA7 of the American Applied Biosystems company. The Ubqtin gene was used as an internal reference.
[0068] UBQ-F (SEQ ID No. 17): 5'-AACCAGCTGAGGCCCAAGA-3';
[0069] UBQ-R (SEQ ID No. 18): 5'-ACGATTGATTTAACCAGTCCATGA-3'.
[0070] The relative expression amount of COLD8.1 gene in the overexpression material is shown in Figure 1 Table 1. WT represents rice variety GZX49, and OX-1, OX-2 and OX-3 represent COLD8.1 gene overexpression transgenic lines. In the COLD8.1 gene overexpression transgenic lines, the relative expression amount of COLD8.1 gene is significantly higher than that of rice GZX49.
[0071] (2) Cold tolerance phenotype identification
[0072] Seedling culture: 100 healthy and full T2 generation seeds were selected and sealed in a parchment paper bag, soaked at 30°C for 2 days, and germinated for 1 day. Nutrient soil, perlite and vermiculite were mixed uniformly at a ratio of 8:1:1 with water. The mixed soil was loaded into a 6cm x 6cm x 7cm black small box. 30 healthy seeds were selected and sowed in the small box, and then covered with soil (3 replicates were set). The small box was placed in a bread tray and placed in a 30°C precision artificial climate box (KBWF240, parameters 0°C-70°C), with 16 hours of light and 8 hours of darkness per day. The seedlings were cultured to the one-leaf-one-core stage (about 8-10 days).
[0073] The one-leaf-one-core stage seedlings were placed in a 4°C low temperature incubator for 84h, and then placed in a 30°C precision artificial climate box, with 16 hours of light and 8 hours of darkness per day, and recovered for 7 days. The survival number of single plants and the total number of seedlings of the test material were counted, and the average value of the survival rate of seedlings of 3 replicates was used as the identification index of cold tolerance at the seedling stage. Seedling survival rate (%) = (survival seedling number / total seedling number) x 100%.
[0074] The results after treatment are shown in Figure 2 Table 2. WT represents rice variety GZX49, and OX1, OX2 and OX3 represent COLD8.1 gene overexpression transgenic plants, and the scale is 5 centimeters. After low temperature stress, the growth status of COLD8.1 gene overexpression transgenic plants was significantly better than that of rice GZX49.
[0075] The survival rate results are shown in Figure 3As shown, WT represents rice variety GZX49, and OX1, OX2 and OX3 represent COLD8.1 gene overexpression transgenic plants. After low temperature stress, the survival rate of COLD8.1 gene overexpression transgenic plants was significantly higher than that of rice GZX49.
[0076] Example 3 Crispr knockout of COLD8.1 gene reduces cold tolerance of rice seedlings
[0077] 1. Construction of Crispr knockout vector
[0078] According to the full-length gene sequence of rice gene COLD8.1 amplified in Example 1, the target site for COLD8.1 gene knockout was designed through the website http: / / skl.scau.edu.cn / targetdesign / , and the designed target site was SEQ ID No. 10: 5'-GCTCGCAGTGCGGGCACCAC-3'. The pCXUN_CAS9 vector KpnI enzyme cutting site flanking homologous recombination adapters were introduced on the upstream and downstream primers, respectively, and the primers were designed as follows:
[0079] U3-F (SEQ ID No. 11): 5'-CCCCTTTCGCCAGGGGTACCgtaattcatccaggtctccaag-3';
[0080] U3-R (SEQ ID No. 12): 5'-TACGAATTCGAGCTCGGTACCgctgtgccgtacgacggtacg-3';
[0081] Crispr-F (SEQ ID No. 13): 5'-GCTCGCAGTGCGGGCACCACgttttagagctagaaatagcaagtta-3';
[0082] Crispr-R (SEQ ID No. 14): 5'-GTGGTGCCCGCACTGCGAGCgccacggatcatctgcacaactc-3'.
[0083] OsU3 as a template, first use primer U3-F and Crispr-R for PCR, get PCR product 1, product size is 476bp. PCR program as follows: 94℃ pre-denaturation 5 minutes; 94℃ denaturation 30 seconds, 55℃ annealing 30 seconds, 72℃ extension 30 seconds, 30 cycles; 72℃ extension 7 minutes.
[0084] PCR was performed with the primer U3-R and Crispr-F using OsU3 as template to obtain PCR product 2, which was 368 bp in size. The PCR procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 30 cycles; extension at 72℃ for 7 min.
[0085] Finally, PCR was performed with the primer U3-F and U3-R using the mixture of PCR product 1 and PCR product 2 as template to obtain the final PCR product, which was 824 bp in size. The PCR procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, 30 cycles; extension at 72℃ for 7 min.
[0086] The final PCR product amplified by separation was ligated to the pCXUN_CAS9 vector digested by KpnI through homologous recombination. After sequencing alignment, the COLD8.1 gene Crispr knockout vector was obtained by transformation into Agrobacterium EHA105.
[0087] 2. Obtaining of Crispr knockout plants
[0088] The COLD8.1 gene Crispr knockout vector was transformed into japonica rice Zhonghua 11 (ZH11) through Agrobacterium-mediated genetic transformation. The same method as in Example 2 was used to obtain T0 generation transgenic plants through selection culture, differentiation, rooting, and seedling. All the transgenic materials were propagated to obtain T1 generation materials with stable inheritance of homozygous mutation and no cas9 protein residue. Three homozygous transgenic lines CR1, CR2, and CR3 were selected for subsequent analysis.
[0089] 3. Identification of Crispr knockout plants
[0090] 1) Genotype identification
[0091] DNA was extracted from 2-leaf seedlings. PCR was performed using the primer sq-F and sq-R to obtain an amplification product of 419 bp in size. The obtained PCR product was sequenced to analyze the editing of the COLD8.1 gene target site. The PCR procedure was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, 32 cycles; extension at 72℃ for 7 min. The primer sequences used were as follows:
[0092] sq-F (SEQ ID No. 15): 5'-GGAGAAATCGCCTAACCCTA-3';
[0093] sq-R (SEQ ID No. 16): 5'-GGAACCACACCAAGCCAAGA-3'.
[0094] The sequencing results of the target sites of the Crispr knockout materials are shown in Figure 4 Figure 2. WT represents rice variety Zhonghua 11, and CR1, CR2 and CR3 represent COLD8.1 gene Crispr knockout lines. In the COLD8.1 gene Crispr knockout lines, the COLD8.1 gene is homozygously mutated at the target site.
[0095] 2) Cold tolerance phenotype identification
[0096] Seeds were soaked at 30°C for 2 days, and then germinated for 1 day. The germinated seeds were sowed in black square boxes filled with nutrient soil, and then cultured in a light incubator (light time: 16h / d, temperature: 30°C) to the 2-leaf stage. The 2-leaf seedlings were placed in a 4°C low-temperature incubator for 96h, and then returned to the light incubator for recovery for 1 week. Photographs were taken and survival rates were calculated. Each material was tested in triplicate.
[0097] The survival rate results are shown in Figure 5 Figure 3. WT represents rice variety Zhonghua 11, and CR1, CR2 and CR3 represent COLD8.1 gene Crispr knockout plants. After low-temperature stress, the survival rate of the COLD8.1 gene Crispr knockout plants was significantly lower than that of rice Zhonghua 11.
[0098] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiment without creativity, and these embodiments all belong to the protection scope of the present application.
Claims
1. Application of rice cold tolerance gene COLD8.1 in regulating cold resistance of rice seedlings, characterized in that, The cold tolerance of rice seedlings is improved by overexpressing the rice cold tolerance gene COLD8.1; The full-length nucleotide sequence of the rice cold tolerance gene COLD8.1 is shown as SEQ ID No. 2, and the CDS sequence is shown as SEQ ID No.
3.
2. A method for improving cold tolerance at the seedling stage of rice, characterized by, The cold tolerance of rice seedlings is improved by overexpressing the rice cold tolerance gene COLD8.1; The full-length nucleotide sequence of the rice cold tolerance gene COLD8.1 is shown as SEQ ID No. 2, and the CDS sequence is shown as SEQ ID No.
3.
3. Application of rice cold tolerance gene COLD8.1 in creating cold-tolerant rice germplasm, characterized in that, The cold tolerance of rice seedlings is improved by overexpressing the rice cold tolerance gene COLD8.1; The full-length nucleotide sequence of the rice cold tolerance gene COLD8.1 is shown as SEQ ID No. 2, and the CDS sequence is shown as SEQ ID No.
3.
4. A method of creating a cold water-tolerant rice germplasm, characterized by, The cold tolerance of rice seedlings is improved by overexpressing the rice cold tolerance gene COLD8.1; The full-length nucleotide sequence of the rice cold tolerance gene COLD8.1 is shown as SEQ ID No. 2, and the CDS sequence is shown as SEQ ID No. 3.