Application of cotton GhABF3 gene in regulating cold tolerance of plants

Abstract

The application discloses application of a cotton GhABF3 gene in regulation of cold tolerance of plants and belongs to the field of genetic engineering.A kind of ABF family GhABF3 protein is identified in Gossypium hirsutum in the application, and its amino acid sequence is shown as SEQ ID NO.2, and the nucleotide sequence of its encoding gene GhABF3 is shown as SEQ ID NO.1.The overexpression vector and the silencing vector of the gene are constructed, and Arabidopsis and cotton are transformed, and the transgenic experiment result shows that after cold stress treatment, compared with wild type plants, the GhABF3 overexpression plants exhibit better growth trend, and the damage phenotype of the silencing plants is significantly aggravated after cold treatment.The application provides key gene resources and breeding methods for plant cold stress tolerance breeding, and is expected to bring significant economic benefits in the field of plant production, and has extremely wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of the cotton GhABF3 gene in regulating plant cold tolerance. Background Technology

[0002] Cotton fiber, as a core source of natural fibers, occupies a pivotal position in the textile industry, widely used in clothing, home textiles, and other fields; cottonseed is an important raw material for extracting edible oils such as cottonseed oil. As a typical warm-climate crop, cotton is mainly distributed in tropical and subtropical regions, and its entire growth and development process is highly sensitive to temperature changes. In recent years, influenced by global climate change and the distribution of arable land resources, cotton planting areas have been continuously expanding to higher latitudes. Under this trend, the risk of cotton encountering abiotic stresses such as low temperatures during its growth process has significantly increased, seriously threatening cotton yield and quality. Therefore, in-depth analysis of the molecular mechanisms of cotton's response to cold stress and the cultivation of new cotton germplasm with both excellent fiber quality and stress resistance traits have become the core direction of current cotton genetic breeding research.

[0003] ABRE-binding factors (ABFs) are an important subfamily belonging to the basic leucine zipper (bZIP) family. Researchers have successfully cloned and functionally analyzed ABF family genes in Arabidopsis thaliana and various other plants. Studies have found that the expression levels of these family members are significantly upregulated under abiotic stresses such as drought, high salinity, and hypoxia, as well as under exogenous ABA treatment. This phenomenon indicates that ABF family genes play a crucial regulatory role in ABA-mediated stress signal transduction pathways. However, the low-temperature response mechanism of ABF family genes in cotton remains unclear. Therefore, there is an urgent need to provide an ABF protein that can regulate plant cold stress tolerance and its application in improving plant resistance to cold stress. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the cotton GhABF3 gene in regulating plant cold tolerance, so as to solve the problems existing in the prior art. The GhABF3 gene provided by this invention positively regulates the cold stress tolerance of plants, which is of great significance for the cultivation of cold-resistant plants and has outstanding economic benefits and broad application prospects for plant production.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides an application of cotton GhABF3 protein in any of the following:

[0007] (1) Application in regulating plant tolerance to cold stress;

[0008] (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress;

[0009] (3) Application in the preparation of products that improve the plant's tolerance to cold stress;

[0010] The amino acid sequence of the cotton GhABF3 protein is shown in SEQ ID NO.2.

[0011] The present invention also provides an application of the above-mentioned cotton GhABF3 protein encoding gene in any of the following:

[0012] (1) Application in regulating plant tolerance to cold stress;

[0013] (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress;

[0014] (3) Application in the preparation of products that improve the plant's tolerance to cold stress;

[0015] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0016] Furthermore, upregulating the expression of the encoding gene in plants enhances the plant's tolerance to cold stress.

[0017] Furthermore, the upregulation of the expression level of the coding gene in plants includes the following steps: constructing an overexpression vector containing the coding gene, transforming the overexpression vector into Agrobacterium, culturing the transformed Agrobacterium to obtain a bacterial solution, and using the bacterial solution to infect plant plants;

[0018] The plant in question is cotton or Arabidopsis thaliana.

[0019] The present invention also provides the use of an overexpression vector comprising the above-described encoding gene in any of the following:

[0020] (1) Application in regulating plant tolerance to cold stress;

[0021] (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress;

[0022] (3) Application in the preparation of products that improve the tolerance of plants to cold stress.

[0023] The present invention also provides the use of engineered bacteria comprising the above-described overexpression vector in any of the following:

[0024] (1) Application in regulating plant tolerance to cold stress;

[0025] (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress;

[0026] (3) Application in the preparation of products that improve the tolerance of plants to cold stress.

[0027] The present invention also provides a method for improving the cold stress tolerance of plants, including the step of upregulating the expression level of the gene encoding the cotton GhABF3 protein in plants to improve the cold stress tolerance of said plants;

[0028] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0029] Furthermore, the upregulation of the expression level of the gene encoding the cotton GhABF3 protein includes the following steps:

[0030] An overexpression vector targeting the encoded gene was constructed, the overexpression vector was transformed into Agrobacterium, the transformed Agrobacterium was cultured to obtain a bacterial solution, and the bacterial solution was used to infect plant plants.

[0031] Furthermore, the plant is cotton or Arabidopsis thaliana.

[0032] This invention also provides a breeding method for transgenic plants with improved tolerance to cold stress, comprising the following steps:

[0033] The gene encoding the cotton GhABF3 protein was overexpressed in plant cells, the plant cells were then cultured, and regenerated plants were obtained using the plant cells, thus obtaining the transgenic plant with improved tolerance to cold stress.

[0034] The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0035] The present invention discloses the following technical effects:

[0036] This invention identified a GhABF3 protein of the ABF family in upland cotton, the amino acid sequence of which is shown in SEQ ID NO. 2, and the nucleotide sequence of its encoding gene GhABF3 is shown in SEQ ID NO. 1. This invention constructed overexpression and silencing vectors for this gene, and transformed Arabidopsis thaliana and cotton. Transgenic experiments showed that, after cold stress treatment, GhABF3-overexpressing plants exhibited superior growth compared to wild-type plants; while silent plants showed significantly aggravated damage phenotypes after cold treatment. This invention provides key gene resources and breeding methods for cold stress tolerance breeding in plants, and is expected to bring significant economic benefits in the field of plant production, with extremely broad application prospects. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The figure shows the results of the Arabidopsis thaliana overexpression experiment of the GhABF3 gene; where A represents the growth of Arabidopsis thaliana seedlings; B represents the root system statistics; and C represents the growth of Arabidopsis thaliana plants. Compared with WT, * indicates P<0.05, and ** indicates P<0.01.

[0039] Figure 2 The graph shows the albino phenotype of the positive control TRV2:GhPDS plant (A) and the relative expression level of the GhABF3 gene in the TRV2:GhABF3 silent plant (B); where, compared with TRV2:00, *** indicates P<0.001;

[0040] Figure 3 Figure 1 shows the results of an experiment injecting the GhABF3 gene into upland cotton plants using VIGS; where A represents the growth of upland cotton plants; B represents the statistical results of antioxidant performance; compared with WT, *** indicates P<0.001. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Experimental supplies involved in this invention:

[0047] The total RNA extraction kit, cDNA first-strand reverse transcription kit, 2×TaqPCRMaster Mix II, DNA gel recovery kit, and plasmid extraction kit used in the embodiments of this invention were all purchased from TIANGEN Corporation; Real-time PCR reagents were purchased from TaKaRa Biotechnology Co., Ltd.; Restriction endonucleases Kpn I, Sal I, and Xba I were purchased from TaKaRa Biotechnology Co., Ltd.; Ampicillin, kanamycin, gentamicin, MES, rifampin, acetylsuccione, MgCl2, and culture medium preparation reagents were all domestically produced analytical grade and purchased from Shanghai Sangon Biotech Co., Ltd.; Escherichia coli competent strain DH5α was purchased from Beijing TransGen Biotech Co., Ltd.; PCR primer synthesis and DNA sequencing were performed by Xinjiang Youkang Biotechnology Co., Ltd. and Shanghai Sangon Biotech Co., Ltd.

[0048] The upland cotton TM-1 of this invention has been disclosed in Li, Fuguang et al. “Genome sequence of cultivated upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution.” Nature biotechnology vol.33, 5(2015):524-30. doi:10.1038 / nbt.3208. The applicant undertakes to release the above-mentioned biological material to the public within 20 years from the date of this application.

[0049] Example 1: Extraction of total RNA from cotton and synthesis of cDNA

[0050] Total RNA was extracted from the upland cotton TM-1 sample according to the instructions of the TIANGEN plant total RNA extraction kit. After the integrity of the RNA was checked by 1.0% agarose gel electrophoresis, cDNA was synthesized using a reverse transcription kit.

[0051] Example 2 Cloning of the cotton GhABF3 gene

[0052] Based on the transcriptome sequencing results of upland cotton, primers GhABF3-F and GhABF3-R were designed, and their nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0053] GhABF3-F: ATGGGGACTAACATGAACTTTGG (SEQ ID NO.3);

[0054] GhABF3-R: CCAAGGACCGGTCTGGGTT (SEQ ID NO. 4).

[0055] Using cotton cDNA as a template, PCR amplification was performed. The reaction system was as follows: 1 μL cDNA (50 ng / μL), 10 μL 2×TaqPCR Master Mix, 0.5 μL primer GhABF3-F, 0.5 μL primer GhABF3-R, 8 μL ddH2O, for a total of 20 μL.

[0056] The amplification program was as follows: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min; stored at 4℃. The amplified products were detected by 1% agarose gel electrophoresis, and the target band was recovered. The samples were then sent to Xinjiang Youkang Biotechnology Co., Ltd. for sequencing.

[0057] The nucleotide sequence of the GhABF3 gene is shown in SEQ ID NO.1, and the amino acid sequence of the GhABF3 protein it expresses is shown in SEQ ID NO.2.

[0058] SEQ ID NO.1:

[0059]

[0060] SEQ ID NO.2:

[0061] MGTNMNFGSNPPPSGDCGGNKPPGNNLLTRQPSIYSLTFDEFQSTMGGIGKDFGSMNMDELLRNIWSAEEIQTMASSGGVLEGNGGLQRQGSLTLPRTLSQKTV DEVWKDISKEYSLGKDGIGGGGGTNNMPQRQQTLGEMTLEEFLVRAGVVREDTQLAGKVNNGGFFGGNNTGFEIGFQQGGKGPNLMGTRIPDGGNQIGIQASNLH PNVNGVRSNQHQLAQQHQHQQPIFPKQTGVGYGAQIPLQSGGQLGSPGIRSGMHGIGDQGISNGLIQAGALQGGGMGMVGLGTGSPANQLSSDGIGKSSGDTSS VSPVPYVFNGSMRGRKYSAVEKVAERRQRRMIKNRESAARSRARKQAYTMELEAEVAKLKEEENQELRKKHAEIMEMQKNQVIEMVDMQQGAKKRCLRRTQTGPW.

[0062] Example 3: Construction of a plant expression vector for the GhABF3 gene

[0063] The plant expression vector pCAMBIA1300-eGFP containing the 35S promoter was digested with Kpn I and Sal I to obtain the vector fragment. The target gene fragment and the vector fragment were recovered. The target gene fragment and the vector fragment were ligated in vitro, and the correctly identified recombinant plasmid was named 35S:GhABF3. Arabidopsis thaliana was selected as the transgenic plant material in this embodiment of the invention.

[0064] Example 4 Transformation of Agrobacterium

[0065] The identified plant expression vector was transformed into Agrobacterium GV1301 within a clean bench. The transformation steps are as follows:

[0066] (1) Take 5 μL of the identified plasmid and add it to 100 μL of Agrobacterium competent cells. Mix well and incubate on ice for 20 min.

[0067] (2) Place in liquid nitrogen for 5 minutes;

[0068] (3) Heat shock at 37℃ for 5 minutes, followed by ice bath for 2 minutes;

[0069] (4) Add 600 μL of fresh LB medium without any antibiotics, mix well and incubate at 28℃, 200 rpm for 5 h with shaking.

[0070] (5) Centrifuge at 5000 rpm for 5 min;

[0071] (6) Discard the supernatant, leaving 100 μL of bacterial culture, and spread it on LB solid medium containing Rif (100 mg / L), Gen (50 mg / L), and Kan (50 mg / L). Incubate at 28°C for 2 days. Pick single clones from the plate and confirm them by PCR. If the PCR results are correct, it means that the Agrobacterium has been successfully transformed with the target gene.

[0072] Example 5: Inoculation of Arabidopsis thaliana using the flower-dipping method

[0073] (1) When the transplanted Arabidopsis thaliana reaches its peak flowering period one week after its first flowering, the previous siliques are cut off and then the plants are infected.

[0074] (2) Preparation of inoculum: Agrobacterium tumefaciens identified as containing the target gene was activated in LB broth containing triple antibodies (50 mg / L Kan, 50 mg / L Gen, 20 mg / L Rif) and cultured overnight at 28°C with shaking. The overnight cultured bacteria were then inoculated into 100 mL of LB broth containing triple antibodies at a volume ratio of 1:100 for expansion culture for about 8 hours.

[0075] When the bacterial solution OD 600 When the OD reaches 2.5, remove the bacterial culture, centrifuge at 5000 rpm for 5 min at 4°C, discard the supernatant, and resuspend the precipitate in 1 / 2 MS liquid (6% sucrose + 0.02% Silwet L-77) to adjust the OD. 600 Reached 0.8;

[0076] (3) Soak the inflorescence in the infection solution for 1 minute, then shake off the remaining infection solution. Incubate the infected Arabidopsis thaliana in the dark for 24 hours, then culture normally. Repeat the soaking process 3 times every 5-7 days. After the infection is completed, culture the Arabidopsis thaliana under long light conditions. After the pods mature, collect the seeds for subsequent screening experiments.

[0077] Example 6: Identification of transgenic Arabidopsis thaliana

[0078] The collected infected Arabidopsis seeds were sown on 1 / 2 MS (containing 50 mg / L Kan) solid medium on a sterile operating table according to standard planting methods. After two to three weeks of growth in an artificial climate chamber, the untransformed Arabidopsis seedlings gradually turned white and died, while the successfully transformed Arabidopsis seedlings grew normally. The normally growing Arabidopsis were transferred to culture soil for further cultivation, and then DNA was extracted from the initially screened Arabidopsis for PCR identification.

[0079] Example 7: Observation of transgenic Arabidopsis thaliana phenotype

[0080] To examine the tolerance of transgenic and wild-type Arabidopsis seeds to cold stress, sterilized T3 generation Arabidopsis seeds (WT, GhABF3-OE, atabf3, and GhABF3-atabf3) were sown on 1 / 2 MS medium. Once seedlings had four leaves, they were transplanted into pots containing a 1:1 mixture of potting soil and vermiculite. After approximately three weeks of growth, plants with similar growth patterns were selected and placed in a 4°C incubator for three days. They were then transferred to normal culture conditions (22°C) and phenotypic changes were observed for 3-5 days. Alternatively, T3 generation Arabidopsis seeds were sown on 1 / 2 MS medium and grown for six days, then transferred to fresh 1 / 2 MS medium and placed in a 4°C incubator for vertical growth for six days. Root length was measured on day 7, and root elongation was calculated.

[0081] like Figure 1 As shown in Figure A, under normal conditions, there were no significant differences in growth status among wild-type (WT), overexpression line (GhABF3-OE), mutant (atabf3), and replacement line (GhABF3-atabf3). After low-temperature stress treatment, the root length of all lines decreased, but GhABF3-OE plants maintained a longer root length, while the root length of atabf3 mutant plants was inhibited, indicating that overexpression of GhABF3 can maintain root development and enhance stress adaptability. Figure 1 As shown in Figure B, after treatment at 4℃, the growth of WT and the replenished lines was significantly inhibited, and the leaves showed browning and curling. The atabf3 mutant was the most severely inhibited, with severely wrinkled and browned leaves. The growth inhibition of GhABF3-OE plants was significantly alleviated, and the degree of leaf damage was less than that of WT, indicating that overexpression of the GhABF3 gene improved the plant's ability to survive at low temperatures.

[0082] Example 8: Cotton VIGS Gene Silencing Experiment

[0083] RNA was extracted from upland cotton TM-1 and reverse transcribed into cDNA as a template. PCR amplification was performed using specific primers to construct the VIGS genetic transformation vector TRV2:GhABF3. The GhABF3 gene was amplified using forward primer F (SEQ ID NO.5) and reverse primer R (SEQ ID NO.6) and ligated into the TRV2:00 vector plasmid digested with Kpn I and Xba I.

[0084] F: aaggttaccgaattctctagaTCAAACCAGCACCAGTTGGC (SEQ ID NO. 5);

[0085] R: gagacgcgtgagctcggtaccTGCACTGTATTTCCTACCCCTCA (SEQ ID NO. 6).

[0086] The constructed TRV2:GhABF3 strain was transformed into Agrobacterium GV1301 using the VIGS genetic transformation vector. TRV2:00 Agrobacterium served as a negative control, and TRV2:GhPDS Agrobacterium served as a positive control. Before VIGS injection, the OD value of the Agrobacterium culture solution needed to be increased to 1.5. Upland cotton TM-1 was used as the VIGS injection material. After soaking, the seeds were transferred to small black pots filled with nutrient soil. Greenhouse conditions were 28℃ during the day and 25℃ at night, with a 16h light / 8h dark cycle. After the seedlings had fully expanded their two cotyledons, VIGS injection was performed. The back of the cotyledons was incised with a syringe, and the entire cotyledon was injected with the bacterial solution. After injection, the cotton was placed in darkness for 24 hours before being transferred to normal conditions for cultivation.

[0087] The results are as follows Figure 2 As shown in Figure A, the albinism phenotype appeared approximately 10 days after injection, and remained pronounced after 25 days, indicating successful gene silencing and a stable effect. Figure 2 As shown in Figure B, RT-qPCR was used to detect the expression of GhABF3 in TRV2:00 and TRV2:GhABF3 plants. The expression level of GhABF3 gene in TRV2:GhABF3 plants was significantly lower than that in TRV2:00, indicating successful gene silencing. After the plants developed three true leaves, they were transferred to a 4℃ growth chamber for cold stress treatment, and leaf samples were collected after 48 hours of treatment. Immediately after sampling, the leaf samples were collected in liquid nitrogen and stored in a -80℃ freezer for later use.

[0088] Example 9: Identification of gene-silenced cotton phenotypes to resist cold stress

[0089] like Figure 3 As shown in Figure A, before cold stress treatment, the growth of WT, TRV2:00, and TRV2:GhABF3 was consistent under the control conditions. After 12 hours of low-temperature stress treatment, there was no significant difference between WT and the empty vector TRV2:00, but the GhABF3 gene-silenced plants suffered more severe damage than WT and TRV2:00. After silencing, the leaves withered and the stems softened, indicating that the cold tolerance of plants decreased after GhABF3 gene silencing. The results of physiological and biochemical index analysis are as follows: Figure 3As shown in Figure B, compared with WT and TRV2:00 plants, the antioxidant system of TRV2:GhABF3 plants collapsed, with reduced SOD and POD enzyme activities, leading to weakened ROS scavenging capacity, osmotic imbalance, significantly reduced proline and soluble sugar content, aggravated membrane system damage, increased malondialdehyde (MDA) accumulation, and decreased chlorophyll content. These results indicate that GhABF3 gene silencing weakens antioxidant capacity, exacerbates cell membrane lipid peroxidation, and thus reduces the cold tolerance of cotton.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a cotton GhABF3 protein in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; The amino acid sequence of the cotton GhABF3 protein is shown in SEQ ID NO.2; Upregulating the expression level of the gene encoding the cotton GhABF3 protein in plants enhances the plant's tolerance to cold stress. The plant in question is cotton or Arabidopsis thaliana.

2. The use of the gene encoding the cotton GhABF3 protein as described in claim 1 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; Upregulating the expression of the encoded gene in plants enhances the plant's tolerance to cold stress; The plant in question is cotton or Arabidopsis thaliana.

3. The application as described in claim 2, characterized in that, The method of upregulating the expression level of the coding gene in plants includes the following steps: constructing an overexpression vector containing the coding gene, transforming the overexpression vector into Agrobacterium, culturing the transformed Agrobacterium to obtain a bacterial solution, and using the bacterial solution to infect plant plants.

4. The use of an overexpression vector comprising the encoding gene as described in claim 2 or 3 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; Upregulating the expression of the encoded gene in plants enhances the plant's tolerance to cold stress; The plant in question is cotton or Arabidopsis thaliana.

5. The use of an engineered bacterium comprising the overexpression vector of claim 4 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; Upregulating the expression of the encoded gene in plants enhances the plant's tolerance to cold stress; The plant in question is cotton or Arabidopsis thaliana.

6. A method for improving plant tolerance to cold stress, characterized in that, This includes the step of upregulating the expression of the gene encoding the cotton GhABF3 protein in plants to improve the plant's tolerance to cold stress; The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The plant in question is cotton or Arabidopsis thaliana.

7. The method as described in claim 6, characterized in that, The upregulation of the expression level of the gene encoding cotton GhABF3 protein includes the following steps: An overexpression vector for the encoded gene was constructed, the overexpression vector was transformed into Agrobacterium, the transformed Agrobacterium was cultured to obtain a bacterial solution, and the bacterial solution was used to infect plant plants.

8. A breeding method for transgenic plants with improved tolerance to cold stress, characterized in that, Includes the following steps: The gene encoding the cotton GhABF3 protein was overexpressed in plant cells, the plant cells were then cultured, and regenerated plants were obtained using the plant cells, thus obtaining the transgenic plant with improved tolerance to cold stress. The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; The plant in question is cotton or Arabidopsis thaliana.

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