Application of the cotton GhZF-HD19 gene in regulating plant cold tolerance

Abstract

This invention discloses the application of the cotton GhZF-HD19 gene in regulating plant cold tolerance, belonging to the field of genetic engineering. This invention identified a homeobox protein, GhZF-HD19, in upland cotton, with its amino acid sequence shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene, GhZF-HD19, shown in SEQ ID NO.1. This invention constructed overexpression and silencing vectors for this gene, transformed Arabidopsis thaliana and cotton, and the transgenic experiments showed that after cold stress treatment, the growth status of GhZF-HD19 overexpressing plants was significantly better than that of wild-type plants, while the silencing plants showed more severe cold-induced damage phenotypes, indicating that the GhZF-HD19 gene positively regulates plant cold stress tolerance. This invention lays the foundation for cold stress tolerance breeding in plants and has significant economic benefits and broad application prospects for plant production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to the application of the cotton GhZF-HD19 gene in regulating plant cold tolerance. Background Technology

[0002] Cotton is one of the world's most important economic crops and a significant source of natural fiber. Cotton fiber is a major source of natural fiber and a crucial raw material for the textile industry. With global climate change and environmental degradation, cotton growth and development are affected by various environmental pressures, such as drought, salinity, low temperature, and high temperature stresses. Plants inevitably encounter various abiotic and biotic stresses during their growth and development. Among these, cold stress is a significant factor affecting plant growth and development and limiting its geographical distribution. After continuous or brief exposure to cold stress, related physiological and biochemical metabolic activities within plant cells are affected, photosynthesis is inhibited, ultimately leading to slow growth, reduced yield, and decreased quality. Plants have developed a series of complex stress response mechanisms to enhance their tolerance to various abiotic and biotic stresses. Induction of various stress-related genes is considered one of the mechanisms plants employ to enhance their adaptability to stress factors.

[0003] ZF-HD (Zinc finger-homeodomain) proteins belong to the homeobox protein family. Currently, the structure and function of ZF-HD proteins in animals have been extensively and thoroughly studied, while the functions of only a few ZF-HD proteins in plants have been elucidated. Whether ZF-HD proteins are related to plant cold stress tolerance or adaptation has not yet been reported. Therefore, there is an urgent need to provide a ZF-HD 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 GhZF-HD19 gene in regulating plant cold tolerance, so as to solve the problems existing in the prior art. The GhZF-HD19 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 great 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 GhZF-HD19 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 GhZF-HD19 protein is shown in SEQ ID NO.2.

[0011] The present invention also provides an application of the above-mentioned cotton GhZF-HD19 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] Preferably, the expression level of the encoding gene is upregulated in the plant to improve the plant's tolerance to cold stress.

[0017] Preferably, 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, and then infecting 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, comprising the step of upregulating the expression level of the gene encoding the cotton GhZF-HD19 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] Preferably, the upregulation of the expression level of the gene encoding the cotton GhZF-HD19 protein includes the following steps:

[0030] An overexpression vector targeting the gene encoding the cotton GhZF-HD19 protein was constructed, and the overexpression vector was transformed into Agrobacterium, which was then used to infect plant plants.

[0031] Preferably, 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 GhZF-HD19 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 homeobox protein, GhZF-HD19, in upland cotton, with its amino acid sequence shown in SEQ ID NO.2 and the nucleotide sequence of its encoding gene, GhZF-HD19, shown in SEQ ID NO.1. Overexpression and silencing vectors of this gene were constructed and transformed into Arabidopsis thaliana and cotton. Transgenic experiments showed that after cold stress treatment, the growth status of GhZF-HD19-overexpressing plants was significantly better than that of wild-type plants, while the silencing plants exhibited more severe cold-induced damage phenotypes, indicating that the GhZF-HD19 gene positively regulates plant cold stress tolerance. This invention lays the foundation for cold stress tolerance breeding in plants and has significant economic benefits and broad application prospects for plant production. 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 Figure 1 shows the results of the experiment on upland cotton plants injected with the GhZF-HD19 gene via VIGS. A represents the albino phenotype 20 days after injection; TRV2::PDS represents TRV2::00 plants after PDS injection, and TRV2::Empty represents TRV2::00; B represents phenotypic changes after cold stress treatment; TRV2::Empty represents TRV2::0; C represents the RT-qPCR results of VIGS plants; W1 represents the wild type, T1 represents TRV2::00, and 1-4 represent TRV2::GhZF-HD19 silent lines.

[0039] Figure 2 Phenotypic diagrams of Arabidopsis thaliana under control and cold stress: overexpression line (OE), complementation (AT-Comp), mutation, and wild type (WT);

[0040] Figure 3 The figures show the germination rate and root elongation measurements of Arabidopsis thaliana under control, overexpression line (OE), complementation line (AT-Comp), mutation line, and wild-type line (WT) under cold stress.

[0041] Figure 4 The results of the Arabidopsis thaliana overexpression experiment of the GhZF-HD19 gene are shown in the figure; where a is the leaf water loss and b is the relative water content of the leaf. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Experimental supplies involved in this invention:

[0048] pBI1300 cloning vector and LA Taq enzyme were purchased from TaKaRa Biotechnology Co., Ltd.; RNA extraction kit was purchased from Omega Biotechnology Co., Ltd.; cDNA first-strand reverse transcription kit, T4 ligase, and rapid restriction endonuclease were purchased from Fermentas Biotechnology Co., Ltd.; RNA reverse transcription kit, LA Taq DNA Polylnerase, and other related reagents were purchased from Dalian Takara Bio Inc.; XbaⅠ, BamHI, KpnⅠ, and other related enzymes were purchased from Fermentas; 2×Taq PCR Master Mix, DNA gel extraction kit, and plasmid extraction micro kit were purchased from TIANGEN Biotechnology Co., Ltd.; Rifampicin (Rif), kanamycin (Kan), gentamicin (GEN), MES, acetylsuccinone (AS), MgCl2, and culture medium preparation reagents used in the experiment were all domestically produced analytical grade and purchased from Shanghai Sangon Biotech Co., Ltd. The synthesis of primers and DNA sequencing for PCR were performed by Xinjiang Youkang Biotechnology Co., Ltd.

[0049] 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.

[0050] Example 1: Extraction of total RNA from cotton and synthesis of cDNA

[0051] Total RNA was extracted from the upland cotton TM-1 sample according to the instructions of Omega's 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.

[0052] Example 2 Cloning of the cotton GhZF-HD19 gene

[0053] Based on the transcriptome sequencing results of upland cotton, primers GhZF-HD19-F and GhZF-HD19-R were designed, and their nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0054] GhZF-HD19-F: ATGGAGGTAGTCAACACTAAAACC (SEQ ID NO.3);

[0055] GhZF-HD19-R: TCATTGTTGTGGGGTGTCAC (SEQ ID NO. 4).

[0056] Using cotton cDNA as a template, PCR amplification was performed. The reaction system was as follows: cDNA (50 ng / μL) 1 μL, 2×Taq PCR Master Mix 10 μL, primer GhZF-HD19-F 0.5 μL, primer GhZF-HD19-R 0.5 μL, ddH2O 8 μL, total 20 μL.

[0057] 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.

[0058] The nucleotide sequence of the GhZF-HD19 gene is shown in SEQ ID NO.1, and the amino acid sequence of the GhZF-HD19 protein it expresses is shown in SEQ ID NO.2.

[0059] SEQ ID NO.1:

[0060] ATGGAGGTAGTCAACACTAAAACCACCCCATACCCACAATCTGAGTCTTCATCAGAGACACACAACCCCAACGAGGCCACGACCAAGTCTTTAACCCTCCTCAAGAGCTGCACAATCCACCACCATATGGTGGTTTCTTACAAAGAGTGCCTCAAAAACCATGTTGCCAGCTTGGGTGGACATGCCTTGGATGGTTGTGGTGAGTTCATGCCTAGCCCTACTTCTACCCCGACTGACCCTGTCTCTCTCAAATGTGCTGCCTGTGGTTGCCACCGTAACTTCCACCGTCGTGACTCATATGATGCTCCTCCTGCTTTCATCCACCGCCTGCCACCCCCACCTACTCATCATAGCTCTAGTCCAAGCCCAACCCACACCCCTGGTCTAAGCCCTAGCCCTAGCCCTAGCCCAACCCATACCCCGCCTTCACCCGTCCCATACTCTTACTACTCTTCTGCACCCCACATGCTGCTTGCCTTGAGCACTGGCTACTCAGGGCCATTGGATGAGTATCACCACCATCCCAGAGTTGGAGTGATAGAGAAAAACAACAACAACCCAAGTGGGAGAAAGAGGTCAAGGACAAAGTTCAGCAAGGAACAGAAACAAAAGATGCATGATTTTGCTGTGAGGGTTGGTTGGAGGATGCCAAAGGGTGAAGAAAAGCTAGTCAAAGAGTTTTGCGATGAAGTTGGGGTCGATAGAGGGGTCCTCAAGGTCTGGATGCACAATAACAAGAACAATTTTGGCAAGAAATTAGAGGTACTAGCTGTTGGTAATCTCAACCCTGATAGCAACAACAACAACAACAGTGAAGACAATCCAAATGGCAATGCTACAATCAGCTTTGATTCCAATAGTGACACCCCACAACAATGA。

[0061] SEQ ID NO.2:

[0062] MEVVNTKTTPYPQSESSSETHNPNEATTKSLTLLKSCTIHHHMVVSYKECLKNHVASLGGHALDGCGEFMPSPTSTPTDPVSLKCAACGCHNFHRRDSYDAPPAFIHRLPPPPTHHSSSPSPTHTPGLSPSPSPSPTHTPPSPVPY SYYSSAPHMLLALSTGYSGPLDEYHHHPRVGVIEKNNNNPSGRKRSRTKFSKEQKQKMHDFAVRVGWRMPKGEEKLVKEFCDEVGVDRGVLKVWMHNNKNNFGKKLEVLAVGNLNPDSNNNNNSEDNPNGNATISFDSNSDTPQQ*.

[0063] Example 3: Construction of the GhZF-HD19 gene plant expression vector

[0064] The plant expression vector pBI1300 containing the 35S promoter was digested with KpnⅠ / XbaⅠ 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::GhZF-HD19. Arabidopsis thaliana was selected as the transgenic plant material in this embodiment of the invention.

[0065] Example 4 Transformation of Agrobacterium

[0066] The identified plant expression vector was transformed into Agrobacterium GV1301 within a clean bench. The transformation steps are as follows:

[0067] (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.

[0068] (2) Place in liquid nitrogen for 5 min;

[0069] (3) Heat shock at 37℃ for 5 min, followed by ice bath for 2 min;

[0070] (4) Add 600 µL of fresh LB medium without any antibiotics, mix well, and incubate at 28℃ and 200 rpm for 5 h with shaking.

[0071] (5) Centrifuge at 5000 rpm for 5 min;

[0072] (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. Select a single clone from the plate and confirm it by PCR. If the clone is correct, it means that the target gene has been successfully transferred into Agrobacterium.

[0073] Example 5: Inoculation of Arabidopsis thaliana using the drip method

[0074] (1) When the wild-type Arabidopsis thaliana to be planted reaches a height of 4 cm, cut off the terminal inflorescence to stimulate the growth of axillary inflorescences. Care should be taken to avoid damaging the axillary inflorescences. Once the axillary inflorescences have grown, the conversion can be carried out. Before the first conversion, pollinated flowers and pods should be removed to reduce the workload during screening.

[0075] (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 at a ratio of 1:100 into 100 mL of LB broth containing triple antibodies for expansion culture for about 8 h. When the bacterial culture 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 liquid 1 / 2 MS (5-6% sucrose + 0.02% Silwet L-77) to adjust the OD. 600 Reached 0.8;

[0076] (3) Using a 200 μL pipette, carefully drop the infection solution onto the flower buds. After incubating the infected Arabidopsis thaliana in the dark for 24 hours, it was cultured normally. The infection was repeated approximately twice a week for a total of 6 times. After the infection was completed, the Arabidopsis thaliana was cultured under long light conditions. After the pods turned yellow, the seeds were collected for subsequent 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 Germination rate and root elongation measurement

[0080] To examine the tolerance of transgenic and wild-type Arabidopsis seeds to cold stress, T3 generation seeds were sterilized, vernalized at 4°C, and sown on 1 / 2 MS (containing 50 mg / L Kan) solid medium. Separately, 1 / 2 MS solid medium containing seeds of homozygous transgenic lines and wild-type Arabidopsis seeds was subjected to cold stress treatment at 4°C. Germination rates of wild-type and transgenic lines were statistically analyzed after 10 days. The results showed that the germination rate and root elongation of the cold-stressed transgenic lines were significantly higher than those of the wild type.

[0081] Example 8: Validation of the role of the GhZF-HD19 gene in cotton's resistance to cold stress using VIGS

[0082] 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 TRV::GhZF-HD19. The GhZF-HD19 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 XbaⅠ and BamHⅠ.

[0083] F: aaggttaccgaattctctagaTGGACATGCCTTGGATGGTT (SEQ ID NO. 5);

[0084] R: gagacgcgtgagctcggtaccGCAAGCAGCATGTGGGGTG (SEQ ID NO. 6).

[0085] The constructed TRV2::GhZF-HD19 VIGS genetic transformation vector was transformed into Agrobacterium LBA4404, with TRV2::00 Agrobacterium as a negative control and TRV2::PDS Agrobacterium 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 maintained at 28℃ during the day and 25℃ at night, with a 16-hour light / 8-hour 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 plants were placed in darkness for 24 hours, after which they were transferred to normal conditions for cultivation. An albino phenotype appeared approximately 10 days after PDS injection, and the albino phenotype remained obvious after 20 days, indicating successful and stable gene silencing.

[0086] The expression of GhZF-HD19 in WT, TRV2::00, and TRV2::GhZF-HD19 was detected by RT-qPCR, and the results are as follows: Figure 1The results showed that the expression of the GhZF-HD19 gene did not change significantly in WT and TRV2::00, but its expression was significantly reduced in TRV2::GhZF-HD19 plants, and lower than that in WT and 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 at 0 and 12 h treatment times. Immediately after sampling, leaf samples were collected in liquid nitrogen and stored at -80℃ for later use.

[0087] Example 9: Phenotypic Identification of Cold Stress Resistance in Gene-Silenced Cotton

[0088] like Figure 1 As shown in Figures A and B, before cold stress treatment, WT, TRV2::00, and TRV2::GhZF-HD19 plants exhibited consistent growth under the control conditions. After 12 hours of cold stress treatment, there was no significant difference between WT and the empty TRV2::00 vector, but the GhZF-HD19 gene-silenced plants suffered more severe damage than WT and the empty vector. After silencing, the plant stems softened, and the plants began to show slight signs of inversion, indicating a decrease in cold resistance after gene silencing.

[0089] Example 10: Arabidopsis thaliana transgenic experiment and phenotypic identification

[0090] Sterilized T3 generation Arabidopsis seeds (WT, GhZF-HD19-OE, AtZF-HD19, and GhZF-HD19-AtZF-HD19) 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℃ incubator for three days. They were then transferred to normal culture conditions (22℃) and observed for 3-5 days for phenotypic changes. Figure 2 As shown. T3 generation Arabidopsis seeds were sown on 1 / 2 MS medium and grown for 6 days, then transferred to fresh 1 / 2 MS medium and placed in an incubator at 4°C for vertical growth for 6 days. Root length was measured on day 7, and root elongation was calculated. Figure 3 As shown.

[0091] Under normal conditions, different treatments had no significant effect on leaf water loss; however, under low-temperature stress, GhZF-HD19-related treatments (including GhZF-HD19 treatment alone and treatments combined with AtZF-HD19) effectively reduced leaf water loss and improved the plant's water retention capacity under low-temperature conditions, indicating that the GhZF-HD19 gene may play an important role in plant response to low-temperature stress and maintenance of water balance (e.g., Figure 4(As shown in Figure a). Under normal conditions, different treatments had no significant effect on the relative water content of leaves; under low-temperature stress, GhZF-HD19-related treatments (including GhZF-HD19 treatment alone and treatments combined with AtZF-HD19) could increase the relative water content of leaves and enhance the plant's ability to retain water under low-temperature conditions, indicating that the GhZF-HD19 gene plays an important role in plants' response to low-temperature stress and maintenance of water balance (e.g., as shown in Figure a). Figure 4 (As shown in b).

[0092] 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 GhZF-HD19 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; (3) Application in the preparation of products that improve the plant's tolerance to cold stress; The amino acid sequence of the cotton GhZF-HD19 protein is shown in SEQ ID NO.2; Upregulating the expression level of the gene encoding the cotton GhZF-HD19 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 GhZF-HD19 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; (3) Application in the preparation of products that improve the plant's 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, and then infecting 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 improving plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products that improve 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 improving plant tolerance to cold stress; (2) Application in the cultivation of transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products that improve 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 GhZF-HD19 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 GhZF-HD19 protein includes the following steps: An overexpression vector targeting the encoded gene was constructed, and the overexpression vector was transformed into Agrobacterium, which was then 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 GhZF-HD19 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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