Application of cotton GhZF-HD19 gene in regulation and control of plant cold tolerance

Abstract

The invention discloses application of a cotton GhZF-HD19 gene in regulation and control of plant cold tolerance, and belongs to the field of gene engineering. According to the invention, a GhZF-HD19 protein of a homeobox protein family is identified in upland cotton, the amino acid sequence of the GhZF-HD19 protein is shown as SEQ ID NO.2, and the nucleotide sequence of the coding gene GhZF-HD19 of the GhZF-HD19 protein is shown as SEQ ID NO.1. An overexpression vector and a silence vector of the gene are constructed, arabidopsis thaliana and cotton are transformed, and transgenic experiment results show that after cold stress treatment, the growth state of a GhZF-HD19 overexpression plant is obviously superior to that of a wild plant, and the cold damage phenotype of a silence plant is more serious, which indicates that the GhZF-HD19 gene positively regulates the cold stress tolerance of the plant. The invention lays a foundation for cold stress-resistant breeding of plants, and has great economic benefits and wide application prospects for plant production.

Description

Technical Field

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

[0002] Cotton is a major economic crop and a vital source of natural fiber worldwide. Cotton fiber is the primary 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 subject to various environmental stresses, such as drought, salinity, low temperatures, and high temperatures. Plants inevitably encounter various abiotic and biotic stresses during their growth and development. Cold stress is a key factor affecting plant growth and development and restricting its geographical distribution. When plants are exposed to continuous or brief cold stress, physiological and biochemical metabolic activities within their cells are affected, photosynthesis is inhibited, and ultimately growth and development are slowed, yield is reduced, and quality is compromised. Plants have developed a complex set of stress response mechanisms to enhance tolerance to various abiotic and biotic stresses. The 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. While the structure and function of ZF-HD proteins in animals are extensively and intensively studied, only a few ZF-HD proteins have been elucidated in plants. Whether ZF-HD proteins are involved in plant cold stress tolerance or adaptation remains unknown. Therefore, there is an urgent need for a ZF-HD protein that can regulate plant cold stress tolerance and the use of such a ZF-HD protein in improving plant cold stress resistance. Summary of the Invention

[0004] The purpose of the present invention is to provide an application of the cotton GhZF-HD19 gene in regulating plant cold tolerance to solve the problems existing in the above-mentioned prior art. The GhZF-HD19 gene provided by the present 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 object, the present invention provides the following solutions:

[0006] The present 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 cultivating transgenic plants with improved tolerance to cold stress;

[0009] (3) Application in the preparation of products for improving plant 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 a use 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 cultivating transgenic plants with improved tolerance to cold stress;

[0014] (3) Application in the preparation of products for improving plant tolerance to cold stress;

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

[0016] Preferably, the expression level of the encoding gene is up-regulated in the plant to improve the plant's tolerance to cold stress.

[0017] Preferably, the step of increasing the expression of the coding gene in the plant comprises the following steps: constructing an overexpression vector comprising the coding gene, transforming the overexpression vector into Agrobacterium, and then infecting the plant;

[0018] The plant is cotton or Arabidopsis thaliana.

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

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

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

[0022] (3) Application in the preparation of products for improving plant tolerance to cold stress.

[0023] The present invention also provides a use of an engineered bacterium comprising the above-mentioned overexpression vector in any of the following:

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

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

[0026] (3) Application in the preparation of products for improving plant tolerance to cold stress.

[0027] The present invention also provides a method for improving plant cold stress tolerance, comprising the steps of upregulating the expression of a gene encoding a cotton GhZF-HD19 protein in a plant to improve the plant's cold stress tolerance;

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

[0029] Preferably, the method of increasing the expression of the gene encoding the cotton GhZF-HD19 protein comprises the following steps:

[0030] An overexpression vector targeting the coding gene of the cotton GhZF-HD19 protein is constructed, and the overexpression vector is transformed into Agrobacterium, which then infects plant plants.

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

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

[0033] overexpressing a gene encoding a cotton GhZF-HD19 protein in plant cells, then cultivating the plant cells, and obtaining regenerated plants using the plant cells, thereby obtaining the transgenic plant with improved cold stress tolerance;

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

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

[0036] The present invention identified a GhZF-HD19 protein from the homeobox protein family in upland cotton. The amino acid sequence of the protein is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding it, GhZF-HD19, is shown in SEQ ID NO. 1. The present invention constructed overexpression and silencing vectors for the gene and transformed 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 cold damage phenotype of silenced plants was more severe, indicating that the GhZF-HD19 gene positively regulates the plant's cold stress tolerance. The present invention lays a foundation for plant breeding for cold stress tolerance and has great economic benefits and broad application prospects for plant production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Figure 1 shows the results of the GhZF-HD19 gene VIGS injection experiment on upland cotton plants; A shows the albino phenotype 20 days after injection; TRV2::PDS shows the TRV2::00 plant after PDS injection, and TRV2::Empty shows TRV2::00; B shows the phenotypic changes after cold stress treatment; TRV2::Empty shows TRV2::00; C shows the RT-qPCR results of the VIGS plants; W1 is the wild type, T1 is TRV2::00, and 1-4 are TRV2::GhZF-HD19 silenced lines;

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

[0040] Figure 3 Graph showing the germination rate and root elongation of Arabidopsis thaliana under control and cold stress, including overexpression line (OE), complementation (AT-Comp), mutation (mutation), and wild type (WT);

[0041] Figure 4 This is a statistical chart of the results of the Arabidopsis overexpression experiment of the GhZF-HD19 gene; among them, a is the water loss of leaves; b is the relative water content of leaves. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The experimental supplies involved in the present invention are:

[0048] The pBI1300 cloning vector and LATaq enzyme were purchased from TaKaRa Biotechnology; the RNA extraction kit was purchased from Omega; the cDNA first-strand reverse transcription kit, T4 ligase, and rapid restriction endonucleases were purchased from Fermentas Biotechnology; the RNA reverse transcription kit, LATaq DNA Polylnerase, and other related reagents were purchased from Dalian Bao Biotechnology; Xba I, BamH I, Kpn I, and other related enzymes were purchased from Fermentas; the 2× Taq PCR Master Mix, DNA gel recovery kit, and plasmid extraction micro kit were purchased from TIANGEN. All chemical reagents used in the experiment, including rifampicin (Rif), kanamycin (Kan), gentamicin (GEN), MES, acetosyringone (AS), MgCl2, and culture medium preparation, were of domestic analytical grade and purchased from Shanghai Shenggong Biotechnology Co., Ltd. PCR primer synthesis and DNA sequencing were performed by Xinjiang Youkang Biological Co., Ltd.

[0049] The present invention's upland cotton TM-1 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 promises to release the above-mentioned biological material to the public within 20 years from the filing date of this invention.

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

[0051] Total RNA from the upland cotton TM-1 sample was extracted according to the instructions of the plant total RNA extraction kit of Omega Company. After its integrity was checked by 1.0% agarose gel electrophoresis, cDNA was synthesized using a reverse transcription kit.

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

[0053] Referring to 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] PCR amplification was performed using cotton cDNA as a template. The reaction system was as follows: cDNA (50 ng / μL) 1 μL, 2× TaqPCR Master Mix 10 μL, primer GhZF-HD19-F 0.5 μL, primer GhZF-HD19-R 0.5 μL, ddH2O 8 μL, a total of 20 μL.

[0057] The amplification program was as follows: 95°C for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and storage at 4°C. The amplified product was detected by electrophoresis on a 1% agarose gel, and the target band was recovered. The sample was sent to Xinjiang Youkang Biological 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 expressed thereby 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 GhZF-HD19 gene plant expression vector

[0064] The plant expression vector pBI1300, containing the 35S promoter, was digested with Kpn I / Xba 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. Upon identification, the correct recombinant plasmid was named 35S::GhZF-HD19. In this example, Arabidopsis thaliana was used as the transgenic plant material.

[0065] Example 4 Transformation of Agrobacterium

[0066] Operate in a clean bench and transform the identified plant expression vector into Agrobacterium GV1301. 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 minutes;

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

[0069] (3) Heat shock at 37°C 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 culture at 28°C, 200 rpm, with shaking for 5 h;

[0071] (5) 5000 rpm, centrifugation for 5 min;

[0072] (6) Discard the supernatant and spread 100 μL of the remaining bacterial solution on LB solid medium containing Rif (100 mg / L), Gen (50 mg / L), and Kan (50 mg / L). Incubate in a 28°C incubator for 2 days. A single colony was selected from the plate and confirmed by PCR, indicating successful transfer of the target gene into Agrobacterium.

[0073] Example 5: Infection of Arabidopsis thaliana by the Flower Drop Method

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

[0075] (2) Preparation of infection solution: Activate the identified Agrobacterium containing the target gene in LB liquid containing three antibodies (50 mg / L Kan, 50 mg / L Gen, 20 mg / L Rif), culture overnight at 28°C with shaking, inoculate the overnight cultured bacteria into 100 mL LB liquid containing three antibodies at a ratio of 1:100 and expand the culture for about 8 hours. 600 When the OD value reached 2.5, the bacterial solution was taken out and centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded and the precipitate was suspended in liquid 1 / 2MS (5-6% sucrose + 0.02% Silwet L-77) to make the OD 600 Reach 0.8;

[0076] (3) Use a 200 μL pipette to draw up the infection solution and carefully drop it onto the flower buds. After incubating the infected Arabidopsis in the dark for 24 hours, culture normally and inoculate approximately twice a week for a total of 6 times. After infection, incubate the Arabidopsis under long-term light conditions. When the pods turn yellow, collect the seeds for later experiments.

[0077] Example 6 Identification of transgenic Arabidopsis

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

[0079] Example 7 Germination rate and root elongation determination

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

[0081] Example 8 Verification of the role of the GhZF-HD19 gene in cotton 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 that had been double-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 tumefaciens LBA4404, using Agrobacterium TRV2::00 as a negative control and Agrobacterium TRV2::PDS as a positive control. The OD value of the Agrobacterium culture was raised to 1.5 before VIGS injection. Upland cotton TM-1 was used as the injection medium. After soaking, the seeds were transferred to small black pots filled with nutrient soil. The greenhouse was maintained at 28°C during the day and 25°C at night, with a 16-hour light / 8-hour dark cycle. VIGS injection was performed after both cotyledons of the seedlings were flattened. The underside of the cotyledons was scratched with a syringe, and the bacterial solution was injected throughout the cotyledons. After injection, the cotton plants were grown in the dark for 24 hours before being transferred to normal culture. An albinism phenotype appeared approximately 10 days after PDS injection and was still evident 20 days later, 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. Figure 1Center C shows that GhZF-HD19 gene expression remained unchanged in WT and TRV2::00 plants, but expression in TRV2::GhZF-HD19 plants was significantly reduced and lower than that in WT and TRV2::00 plants, indicating successful gene silencing. After the plants developed three true leaves, they were transferred to a 4°C growth chamber for cold stress treatment. Leaf samples were collected at 0 and 12 hours of treatment. After sampling, leaf samples were immediately collected in liquid nitrogen and stored in a -80°C freezer until further use.

[0087] Example 9 Identification of gene-silenced cotton phenotypes to resist cold stress

[0088] like Figure 1 As shown in Figures A and B, before cold stress treatment, the growth of WT, TRV2::00, and TRV2::GhZF-HD19 plants was consistent under control conditions. After 12 hours of cold stress, no significant differences were observed between the WT and TRV2::00 empty vector plants, but GhZF-HD19 gene-silenced plants suffered more severe damage than the WT and empty vector plants. After silencing, the stems of the plants softened and showed slight signs of reversal, indicating that gene silencing reduced the plants' cold tolerance.

[0089] Example 10 Arabidopsis transgenic experiment and phenotypic identification

[0090] Sterilized T3 generation Arabidopsis seeds (WT, GhABF3-OE, atabf3, and GhABF3-atabf3) were sown on 1 / 2 MS culture medium. When the seedlings grew to 4 leaves, they could be transplanted into nutrient pots filled with a 1:1 mixture of nutrient soil and vermiculite. After growing for about 3 weeks, plants with basically the same growth in the nutrient pots were selected and placed in an incubator at 4°C for 3 days. They were then transferred to normal culture conditions (22°C) and observed for phenotypic changes after 3-5 days. Figure 2 As shown. T3 generation Arabidopsis seeds were sown on 1 / 2MS medium and grown for 6 days, then transferred to new 1 / 2MS medium and placed in an incubator at 4°C for 6 days. The root length was measured on the 7th day and the root elongation was calculated as follows: Figure 3 shown.

[0091] Under normal conditions, different treatments had no significant effect on leaf water loss. Under low temperature stress, GhZF-HD19-related treatments (including single GhZF-HD19 treatment and combined treatment with AtZF-HD19) could effectively reduce leaf water loss and improve the water retention capacity of plants under low temperature conditions, indicating that the GhZF-HD19 gene may play an important role in plants' response to low temperature stress and maintenance of water balance (e.g., Figure 4Under normal conditions, different treatments had no significant effect on leaf relative water content; under low temperature stress, GhZF-HD19-related treatments (including single GhZF-HD19 treatment and combined treatment with AtZF-HD19) increased leaf relative water content and enhanced 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., Figure 4 (as shown in b).

[0092] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a cotton GhZF-HD19 protein in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in cultivating transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products for improving plant tolerance to cold stress; The amino acid sequence of the cotton GhZF-HD19 protein is shown in SEQ ID NO.

2.

2. Use of the gene encoding the cotton GhZF-HD19 protein as claimed in claim 1 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in cultivating transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products for improving plant tolerance to cold stress; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that The expression level of the coding gene is up-regulated in the plant, thereby improving the plant's tolerance to cold stress.

4. The use according to claim 3, characterized in that The method of increasing the expression of the coding gene in plants comprises the following steps: constructing an overexpression vector comprising the coding gene, transforming the overexpression vector into Agrobacterium, and then infecting the plant; The plant is cotton or Arabidopsis thaliana.

5. Use of an overexpression vector comprising the coding gene according to any one of claims 2 to 4 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in cultivating transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products for improving plant tolerance to cold stress.

6. Use of an engineered bacterium comprising the overexpression vector according to claim 5 in any of the following: (1) Application in regulating plant tolerance to cold stress; (2) Application in cultivating transgenic plants with improved tolerance to cold stress; (3) Application in the preparation of products for improving plant tolerance to cold stress.

7. A method for improving plant cold stress tolerance, characterized in that: The method comprises the steps of up-regulating the expression of a gene encoding a cotton GhZF-HD19 protein in a plant to improve the plant's tolerance to cold stress; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, wherein The method of increasing the expression of the gene encoding the cotton GhZF-HD19 protein comprises the following steps: An overexpression vector targeting the coding gene is constructed, and the overexpression vector is transformed into Agrobacterium, which then infects plant plants.

9. The method according to claim 7 or 8, wherein The plant is cotton or Arabidopsis thaliana.

10. A method for breeding transgenic plants with improved tolerance to cold stress, characterized in that: The following steps are involved: overexpressing a gene encoding a cotton GhZF-HD19 protein in plant cells, then cultivating the plant cells, and obtaining regenerated plants using the plant cells, thereby obtaining the transgenic plant with improved cold stress tolerance; The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

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