Application of GhZAT11 gene in improving cold resistance of cotton

Abstract

The invention discloses application of a GhZAT11 gene to improvement of cold resistance of cotton, and belongs to the technical field of plant molecular biology, an amino acid sequence coded by the GhZAT11 gene is shown as SEQ ID No.2, and the cold resistance of the cotton can be improved by overexpressing the GhZAT11 gene in the cotton. According to the invention, the function of the gene GhZAT11 in the cold resistance of the cotton is defined for the first time, and a target gene is provided for enhancing the cold resistance of the cotton. Various experiments show that silencing of the GhZAT11 gene can reduce the cold resistance of the plant; the overexpression of the GhZAT11 gene enhances the cold resistance of the plant, and the result shows that the GhZAT11 plays a positive regulation role in the cold resistance of the cotton. The GhZAT11 gene can be applied to prevention and treatment of cotton cold damage, and particularly can be applied to enhancement of cotton cold tolerance genetic improvement or molecular breeding, such as cultivation of cold tolerance cotton varieties.

Description

Technical Field

[0001] The invention relates to the technical field of plant molecular biology, and in particular to application of a GhZAT11 gene in improving cold tolerance of cotton. Background Art

[0002] Cotton is one of the important economic crops in my country. It is an important material related to the national economy and people's livelihood, and occupies a very important position in the entire national economy. Cotton (Gossypium spp.) is a typical thermophilic crop, and the suitable growth temperature is usually between 20℃ and 30℃. Within this temperature range, cotton's photosynthesis, respiration, and growth and development can all reach the best state. However, cotton's sensitivity to low temperatures makes it face many challenges during its growth. Especially in the seedling and flowering stages, low temperature environments will have a significant impact on the growth of cotton, resulting in slow plant growth, poor root development, and even bud shedding and poor fruit development. These low temperature hazards not only affect the growth and development of cotton, but may also lead to a significant decrease in yield, thereby affecting farmers' economic benefits. ZAT family genes (Zinc Finger ofArabidopsis Thaliana, ZAT) are an important class of transcription factors that mainly play a role in plants. They are characterized by zinc finger domains and are involved in regulating plant growth and development, stress response, and physiological processes. In Osmanthus fragrans, it was found that the expression level of the OfZAT35 gene continued to increase under low temperature stress. After overexpression of OfZAT35 in tobacco, it was observed that its relative electrolyte leakage (REL) level was significantly increased, and the activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) were also increased. Under low temperature stress conditions, overexpression of the NlZAT12 gene in Arabidopsis promoted the expression of some cold-responsive protein genes. The reactive oxygen species and malondialdehyde (MDA) content in transgenic Arabidopsis were lower, while the soluble sugar content was higher, indicating that overexpression of the NlZAT12 gene could enhance the cold tolerance of Arabidopsis. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed good salt tolerance on MS medium containing 100mM NaCl, and the growth condition was better than that of the wild type. In addition, cotton plants with silenced GhZAT6 gene showed more obvious leaf wilting compared with control plants under 400mM NaCl treatment.

[0003] At present, cotton-growing areas are susceptible to cold stress, which seriously affects cotton production. However, research on cold damage prevention and control is very limited, and there is a lack of cotton germplasm resources with high cold tolerance. Therefore, in order to enhance cotton cold tolerance in the seedling stage and promote the healthy and rapid development of cotton, it is urgent to improve cotton cold tolerance and explore cotton cold tolerance genetic regulatory sites and key genes. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a gene related to cotton cold tolerance and its application.

[0005] The technical solution of the present invention is: application of GhZAT11 gene or expression vector containing GhZAT11 gene in regulating cold tolerance of cotton, wherein the GhZAT11 gene is a DNA fragment encoding a protein with an amino acid sequence shown in SEQ ID No.2.

[0006] Furthermore, the nucleotide sequence of the GhZAT11 gene is shown in SEQ ID No.1.

[0007] Furthermore, the regulation of cotton cold tolerance refers to overexpressing the GhZAT11 gene in cotton, thereby improving the cold tolerance of cotton.

[0008] A method for breeding cold-resistant cotton varieties, comprising detecting the expression level of a GhZAT11 gene in cotton, wherein a variety with a higher expression level of the GhZAT11 gene is a relatively cold-resistant cotton variety, and the nucleotide sequence of the GhZAT11 gene is shown in SEQ ID No.1.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] (1) The present invention clarifies for the first time the functional role of the gene GhZAT11 in cotton cold tolerance, and provides a target gene for enhancing cotton cold tolerance.

[0011] (2) Through various experiments, the present invention found that silencing of the GhZAT11 gene would reduce the cold tolerance of the plant; while overexpression of the GhZAT11 gene would enhance the cold tolerance of the plant. The results showed that GhZAT11 plays a positive regulatory role in the cold tolerance of cotton.

[0012] (3) The gene GhZAT11 discovered in the present invention can be used to prevent and treat cotton chilling injury, and can be used to enhance cotton chilling tolerance in genetic improvement or molecular breeding, such as breeding chilling-tolerant cotton varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 , cold stress expression analysis of GhZAT11 gene;

[0014] Figure 2 , CUT&Tag-qPCR analysis of GhZAT11 gene;

[0015] Figure 3 , GhZAT11 gene silencing reduces cotton cold tolerance;

[0016] Figure 4 , GhZAT11 gene overexpression enhances cold tolerance in cotton. DETAILED DESCRIPTION

[0017] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0018] The present invention has found through a large number of studies that the GhZAT11 gene has a special function in the cold tolerance of cotton. The cold tolerance of cotton plants with silenced GhZAT11 gene expression is significantly reduced. The application provides a target gene and theoretical support for enhancing the cold tolerance of cotton. The GhZAT11 gene is a DNA fragment with a protein function encoding the amino acid sequence shown in SEQ ID No.2.

[0019] It should be noted that the “having” means that the nucleotide sequence of the GhZAT11 gene may only have the nucleotide sequence shown in SEQ ID No.1, or may be composed of the nucleotide sequence shown in SEQ ID No.1 and other nucleotide sequences, such as nucleotide sequences encoding functional units such as labels for protein purification, fluorescent protein markers and DNA binding sites, or encoding elements that have regulatory effects on gene transcription and expression, including but not limited to promoters, strong promoters, enhancers or transcription factor binding sites, etc.; the “having” may also mean that the nucleotide sequence shown in SEQ ID No.1 is discontinuous in the GhZAT11 gene, but the cDNA of the nucleotide sequence shown in SEQ ID No.1 can be produced.

[0020] Wherein "identity" refers to the similarity with the nucleotide sequence shown in SEQ ID No.1. The difference with the nucleotide sequence shown in SEQ ID No.1 may be caused by the change, deletion or insertion of one or more nucleotides, and the degeneracy of codons. These changes make the gene sequence not completely consistent with the nucleotide sequence shown in SEQ ID No.1, but at least have more than 90% identity, for example, it can be but not limited to 90%, 95%, 98%, 99% or higher identity. At the same time, these genes with more than 90% identity with the nucleotide sequence shown in SEQ ID No.1 have the same function as the gene with the nucleotide sequence shown in SEQ ID No.1. The identity can be evaluated by naked eyes or computer software, for example, using conventional blast software in the art for comparison. When the GhZAT11 gene has the nucleotide sequence shown in SEQ ID No.1, it is better to be applied to enhance the cold tolerance of cotton.

[0021] It is understood that, in addition to the GhZAT11 gene itself, biological materials containing the above-mentioned GhZAT11 or proteins encoding the GhZAT11 gene can also be used to enhance the cold tolerance of cotton. The biological material can be, for example, but not limited to, an expression cassette, a vector or a transgenic cell line.

[0022] The present invention will be further described below by way of examples.

[0023] The experimental materials used in the embodiments of the present invention are described as follows:

[0024] The transgenic cotton recipient material used in this example is Upland cotton (Gossypium hirsutum) "ZM113".

[0025] The vectors used in this example are as follows: pTRV2: a silencing gene vector used for silencing material transformation; pCambia2300-GFP: an overexpression vector used for overexpression material transformation.

[0026] The strains used in this example are as follows: competent Escherichia coli DH5α, competent Agrobacterium GV3101.

[0027] As for other materials or reagents, unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0028] Example 1 Cotton Planting and Processing

[0029] Cotton planting: Select ZM13 seeds with full grains, wash them with sterile water, soak them in a container, and place them in a 28℃ artificial climate incubator overnight to promote germination. When the seeds turn white, transplant them into moist nutrient soil, cover them with mulch, and place them in a 28℃ artificial climate incubator with alternating natural light for 16h / darkness for 8h.

[0030] Cotton treatment: When the cotton seedlings grew to two leaves and one heart, the cotton in the treatment group was placed in an artificial climate incubator at 4°C, and the cotton in the control group was placed in an artificial climate incubator at 28°C, with alternating natural light of 16 h / darkness of 8 h.

[0031] Example 2 Expression analysis of GhZAT11 gene under cold stress

[0032] Root tissues of upland cotton ZM113 were sampled at 0h, 1h, 3h, 6h, 12h and 24h after 4℃ stress treatment, and the expression of GhZAT11 gene was analyzed by RT-qPCR, with GhUBQ7 gene as the internal reference gene.

[0033] The RT-qPCR reaction system is as follows:

[0034]

[0035] The RT-qPCR reaction procedure is as follows:

[0036]

[0037] The primers used for RT-qPCR are as follows:

[0038]

[0039] Data analysis and utilization 2 -ΔΔCT Methods, three replicates, t-test, ** indicates P < 0.01. The results showed that with the increase of cold stress time, the expression level of GhZAT11 gene increased significantly ( Figure 1 ). During 0-12h, the expression level of GhZAT11 gene continued to increase and reached a peak at 12h. At 24h, the expression level of GhZAT11 gene began to decrease. These results prove that GhZAT11 gene responds to cold stress.

[0040] Example 3 GhZAT11 gene CUT&Tag-qPCR analysis

[0041] Root tissue samples were taken at 28°C and 4°C for 72 h, ground with a mortar and pestle, and transferred into a 10 mL centrifuge tube. Cell nuclei were extracted using the CELLYTPN1 kit. The specific steps are as follows:

[0042] (1) Dissolve DTT in 1× NIB Buffer to a final concentration of 1 mM. Pipette 3 mL into a 10 mL centrifuge tube containing root powder and vortex for 1 min.

[0043] (2) Filter the liquid from the previous step into a 50 mL centrifuge tube and divide the filtered liquid evenly into two 1.5 mL centrifuge tubes.

[0044] (3) Centrifuge the 1.5 mL centrifuge tube at 4°C, 1200 × g for 10 min and remove the supernatant.

[0045] (4) Resuspend the tissue pellet at the bottom of the centrifuge tube with 1 mL of NIBA solution, and then add 10 μL of 10% TRITONTMX-100 solution to lyse the cell membrane.

[0046] (5) Take a 2 mL centrifuge tube, add 800 μL of 2.2 M sucrose solution, then add the lysate from the previous step, centrifuge at 4°C, 12,000 × g, and remove the supernatant after 10 min.

[0047] (6) Resuspend the tissue pellet at the bottom of the centrifuge tube with 1 mL of NIBA solution and centrifuge at 4°C, 12,000 × g, for 5 min. Remove the supernatant.

[0048] (7) Add a certain amount of Nuclei PURE Storage Buffer according to the amount of cell nuclei precipitate to resuspend the cell nuclei.

[0049] DNA was extracted using the Hyperactive pG-MNase CUT&RUN Assay Kit for PCR / qPCR from Novagen. The specific steps are as follows:

[0050] (1) Magnetic bead treatment: Take a 1.5 mL centrifuge tube, aspirate 10 μL of magnetic beads, add 100 μL of prepared Binding Buffer solution, and place it on a magnetic bead separator. After 3 minutes, remove the supernatant. Repeat the operation twice, and then add 10 μL of Binding Buffer solution.

[0051] (2) Add the cell nucleus to a 2 mL centrifuge tube and centrifuge at room temperature at 800 × g for 5 min. Keep the cell nucleus and discard the liquid. Add 500 μL Wash Buffer solution and gently pipette. Centrifuge at room temperature at 800 × g for 5 min. Keep the cell nucleus and discard the liquid. Finally, add 50 μL Wash Buffer solution to the cell nucleus.

[0052] (3) Pipette 50 μL of cell nuclei and transfer them into the magnetic beads treated in (1). Mix well and react for 10 min at room temperature. Place the mixture on a magnetic bead separator and let it stand for 3 min before discarding the supernatant.

[0053] (4) Pipette 100 μL of pre-cooled Antibody Buffer solution into the eight-well plate, add 1 μL of antibody, mix well, and place at 4°C for 12 h.

[0054] (5) Place the liquid from the previous step on a magnetic bead separator and let it sit for 3 minutes before removing the supernatant.

[0055] (6) Add 800 μL of Dig-wash Buffer, invert, centrifuge briefly, and place the eight-row array on a magnetic bead separator. Let it sit for 3 min and then remove the supernatant. Repeat twice.

[0056] (7) Add 100 μL of pG-MNase Enzyme mixture, mix well, react at 4°C for 1 h, place on a magnetic bead separator, let stand for 3 min, and then remove the supernatant.

[0057] (8) Add 800 μL of Dig-wash Buffer, invert, centrifuge briefly, and place the eight-row array on a magnetic bead separator. Let it sit for 3 min and remove the supernatant. Repeat twice.

[0058] (9) Add 100 μL CaCl2 Mix the mixture, stir well, and react on ice for 1 h.

[0059] (10) Add 100 μL Stop Buffer solution, mix well, react at 37°C for 20 min, centrifuge at 4°C, 12,000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0060] (11) Add 1 mL of Buffer GDP solution, mix well, react at room temperature for 10 min, centrifuge, transfer 650 μL of the solution to the FastPure gDNA Mini Columns adsorption column, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid.

[0061] (12) Pipette 700 μL of Buffer GW solution into the adsorption column and centrifuge at 10,000 rpm for 2 min to remove the waste liquid.

[0062] (13) Open the lid and let dry.

[0063] (14) Place the adsorption column in a clean 1.5 mL tube and add 20 μL ddHO. 2 O was added to the center of the adsorption column, allowed to stand for 2 min, and centrifuged at 10,000 rpm for 2 min.

[0064] (15) The qPCR reaction system is as follows:

[0065]

[0066] The RT-qPCR reaction procedure is as follows:

[0067]

[0068] The primers used for RT-qPCR are as follows:

[0069]

[0070] Data analysis used the 2-ΔΔCT method, three replicates, t-test, ** indicates P < 0.01. The results showed that after cold stress, the H3K4me3 modification of the GhZAT11 gene increased ( Figure 1 ), which increased by about 2.1 times, demonstrating that the GhZAT11 gene responded to cold stress.

[0071] Example 4 Construction of GhZAT11 gene expression vector

[0072] 1. Recovery of target fragment and linearized vector

[0073] (1) Linearization of expression vector: The gene silencing vector pTRV2 and the overexpression vector pCambia2300-GFP were incubated at 37°C for 1 h and double-digested to obtain linearized vectors according to the following reaction system:

[0074]

[0075] (2) Obtaining the target fragment: Using cotton cDNA as a template, the sequence was amplified using the high-fidelity enzyme 2×PhantaMax Master Mix (Dye Plus) produced by Novozymes. The PCR reaction system was as follows:

[0076]

[0077] The PCR amplification system is as follows:

[0078]

[0079] Primer information

[0080]

[0081] (3) The above PCR products and linearized expression vectors were recovered and purified using the EasyPure Quick Gel Extraction Kit from Beijing Quanshijin Biotechnology Co., Ltd. The specific steps are as follows:

[0082] (a) Quickly cut the agarose gel containing the target band under ultraviolet light to avoid damage to the DNA caused by prolonged exposure to ultraviolet light. Cut the gel into small pieces as much as possible and place them in a 1.5 mL clean centrifuge tube for subsequent sol preparation.

[0083] (b) Weigh the gel using an electronic balance, assuming that 100 mg equals 100 μL. Add 3 times the volume of Buffer GDP and incubate at 55°C in a water bath for 15 min. Invert and mix 2-3 times to completely dissolve the gel.

[0084] (c) Place the FastPure DNA Mini Columns-G adsorption column in a 2 mL collection tube, carefully transfer the sol liquid cooled to room temperature into the adsorption column, and centrifuge at 12000 rpm for 1 min.

[0085] (d) Discard the filtrate and return the adsorption column to the collection tube. Add 300 μL of Buffer GDP to the adsorption column, let it stand at room temperature for 3 minutes, and then centrifuge at 12,000 rpm for 1 minute.

[0086] (e) Discard the filtrate and return the adsorption column to the collection tube. Add 700 μL Buffer GW and centrifuge at 12,000 rpm for 1 min.

[0087] (f) Repeat step (5).

[0088] (g) After discarding the filtrate, put the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 min.

[0089] (h) Place the adsorption column in a sterilized 1.5 ml centrifuge tube, add 40 μL Elution Buffer (preheated to 55°C in a water bath) to the center of the adsorption column, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 1 min to elute the DNA, and store at -20°C for later use.

[0090] 2. Construction of vector and transformation of E. coli: The vector was constructed by using the Ultra OneStep Cloning Kit performs homologous recombination between the linearized vector and the target fragment to construct the vector. Prepare the following liquid system on ice:

[0091]

[0092] React at 50°C for 10 min, cool on ice immediately, and then transform the recombinant product into competent E. coli.

[0093] (1) Add all 10 μL of the recombinant ligation product to 100 μL of freshly melted DH5α competent medium and let it stand on ice for 30 min.

[0094] (2) Heat shock at 42°C in a water bath for 45 seconds and immediately place on ice for 2 minutes.

[0095] (3) Add 500 μL of LB liquid culture medium without antibiotics and shake at 37°C for 1 h.

[0096] (4) Centrifuge at 5,000 r / min for 2 min, discard 400 μL of supernatant in a clean bench, and spread 100 μL of bacterial solution on LB solid medium containing the corresponding antibiotics. After drying, invert and culture in a 37°C incubator overnight.

[0097] (5) On the next day, single clones were picked from the plates and shaken in 500 μL of LB liquid culture medium containing the corresponding antibiotics until the bacterial solution became turbid. PCR was performed on the bacterial solution and the positive clones were sent to Shanghai Biotechnology Co., Ltd. for sequencing. The sequencing results returned by the company were compared with the target sequence. The clones with completely correct results were preserved.

[0098] 3. Plasmid extraction: using Beijing Quanshijin Plasmid MiniPrep Kit was used to extract E. coli plasmids. The specific steps are as follows:

[0099] (1) Take 5 mL of E. coli cultured overnight at 37°C in a shaking incubator and place it in a 10 mL centrifuge tube. Centrifuge at 12,000 rpm for 5 min and discard the supernatant.

[0100] (2) Add 300 μL of colorless solution RB (containing RNase A) and vortex vigorously to suspend the bacterial precipitate. No small bacterial clumps should remain.

[0101] (3) Add 300 μL of blue solution LB and gently invert the tube for 6-8 times to fully lyse the bacteria until the solution turns clear blue, indicating complete lysis. The operation should be completed within 5 min.

[0102] (4) Add 450 μL of yellow solution NB and gently mix by inverting up and down 6-8 times until the solution changes color from blue to yellow and forms a compact yellow agglomerate, indicating complete neutralization. Let stand at room temperature for 2 min.

[0103] (5) After centrifugation at 12,000 r / min for 10 min, aspirate the supernatant into a centrifuge column, 800 μL at a time, and centrifuge at 12,000 r / min for 1 min. Discard the liquid in the collection tube.

[0104] (6) Add 700 μL of WB and centrifuge at 12,000 r / min for 1 min. Discard the liquid in the collection tube, centrifuge the empty tube at 12,000 r / min for 2 min, and place it on a clean bench to blow dry for 5 min to remove the residual rinsing solution.

[0105] (7) Place the centrifuge column in a clean 1.5 mL centrifuge tube and add 40 μL of 65°C preheated Elution Buffer to the center of the centrifuge column. After standing at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min to elute the plasmid DNA.

[0106] (8) The concentration of plasmid DNA was measured using NanoDrop 2000 and the obtained plasmid was stored at -20°C.

[0107] 4. Agrobacterium transformation: GV3101 from Shanghai Weidi Biotechnology Co., Ltd. was used for Agrobacterium transformation. The specific steps are as follows:

[0108] (1) Take 2 μL of plasmid and add it to 100 μL of freeze-thawed Agrobacterium competent cells (5 min of thawing is best), and gently shake the tube wall with your fingers to mix.

[0109] (2) Perform the following steps in sequence: place on ice for 5 min, place in liquid nitrogen for 5 min, place in a 37°C water bath for 5 min, and place on ice for 5 min.

[0110] (3) Add 600 μL of LB liquid culture medium to the clean bench and place in a shaking incubator at 200 rpm at 28°C for 3 h.

[0111] (4) Centrifuge at 3000 rpm for 3 min, collect the cells, discard part of the supernatant in a clean bench, and keep 150 μL of the cells. Use a pipette to resuspend them, and evenly apply the bacterial solution on LB solid medium containing Kan and Rif resistance. Incubate inverted in a 28°C incubator for 2 days.

[0112] (5) Positive clone detection: Single clone strains were selected and cultured in 600 μL LB liquid culture medium containing Kan and Rif resistance at 28°C and 200 rpm for 12 h.

[0113] (6) Perform PCR verification using bacterial culture as template.

[0114] Example 5 Silencing the expression of GhZAT11 gene reduces the cold tolerance of cotton

[0115] (1) Select the seeds of ZM113 of self-fertilization age stored in the laboratory, soak the seeds in sterile water at 37°C overnight, select the white seeds, plant them with the radicle facing downward in a 1:1 mixture of nutrient soil and vermiculite, cover them with a transparent lid, and inject Agrobacterium after the cotyledons are fully expanded.

[0116] (2) Take 40 mL of overnight cultured Agrobacterium containing the gene silencing vector into a 50 mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min to enrich the bacteria, and remove the supernatant.

[0117] (3) Resuspend the cells in the prepared resuspension solution and adjust the OD 600 =1.0.

[0118] (4) Incubate the cells at room temperature in the dark for 3 h.

[0119] (5) Use the needle of a 1 mL sterile syringe to carefully scratch the back of the cotton cotyledon (do not penetrate the leaf), and inject the bacterial solution into the leaf through the wound until the entire leaf is soaked.

[0120] (6) After being placed at room temperature for 24 h in the dark, the plants were moved to the greenhouse for cultivation. After growing to two leaves and one heart, the plants were treated at 4°C. RNA was extracted from the leaves of TRV:00 and TRV:GhZAT11 plants, and fluorescence quantitative identification was performed after reverse transcription. The results showed that the expression of the GhZAT11 gene was significantly reduced ( Figure 3 After 3 days of treatment at 4°C, the leaves of TRV:GhZAT11 plants were severely wilted ( Figure 3These results indicate that cotton cold tolerance decreased after silencing the GhZAT11 gene. Further testing of MDA and POD content showed that the MDA content of TRV:GhZAT11 plants was higher than that of TRV:00 plants, and the POD content of TRV:GhZAT11 plants was lower than that of TRV:00 plants ( Figure 4 This result also proves that silencing GhZAT11 can reduce cold tolerance.

[0121] Example 6 Overexpression of GhZAT11 gene enhances cotton cold tolerance

[0122] (1) In a clean bench, cut the sterile seedlings into small pieces and put them into a conical flask containing Agrobacterium infection solution containing the GhZAT11 gene overexpression vector for infection for 10 minutes. After the infection, transfer the hypocotyls to a sterilized large culture dish and wait for the surface bacterial solution to dry. Then, transfer the hypocotyls to the co-cultivation medium and culture them in the dark for 40-46 hours. After the co-cultivation, transfer them to sterile water containing CEF and soak them for 20 minutes. Then, wash them with sterile water only for 5-6 times until the residue is washed off. Dry the surface water, and then evenly spread the hypocotyls in the hypocotyl induction medium containing timentin and kanamycin. Finally, place the hypocotyls in a greenhouse at 28±1℃ for culture. The induced callus tissue was transferred to a callus proliferation medium containing timentin and kanamycin and cultured, subcultured once every 4 weeks, and an appropriate amount of callus tissue was transferred to an embryonic callus induction medium containing timentin and kanamycin and cultured, subcultured every 3 weeks until embryonic callus tissue appeared.

[0123] (2) The obtained embryonic callus is transferred to an embryonic callus proliferation medium containing timentin and kanamycin for expansion culture, and subcultured every two weeks. When the embryonic callus proliferates to a certain amount, it can be used for subsequent differentiation into seedlings.

[0124] The leaves of the overexpressed plants were taken, DNA was extracted, and specific fragment primers were designed for RT-qPCR amplification. The results showed that the expression level of the GhZAT11 gene overexpression strain was much higher than that of the Mock plant ( Figure 4 A), proving that the GhZAT11 gene overexpression strain was successfully constructed. Mock and T4 cotton seeds were selected, soaked in sterile water, and kept at 37°C overnight. White seeds were selected and planted with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite. A transparent lid was covered and the transparent lid was removed after the cotyledons unfolded. After culturing in the greenhouse to the 6-leaf stage, the seeds were moved to a light incubator and set to a temperature of 6°C, a 16-hour light and 8-hour dark cycle, and a relative humidity of 60%. The phenotype was observed after 3 days of treatment. The results showed that the leaves of the GhZAT11 gene overexpression strain showed slight wilt, while the Mock plants showed more severe wilt, proving that the GhZAT11 gene overexpression strain has higher cold tolerance than the Mock plants ( Figure 4 B), indicating that the GhZAT11 gene can improve the cold tolerance of cotton. Further testing of MDA and POD content showed that the MDA content of the OE strain was lower than that of the Mock strain, and the POD content of the OE strain was higher than that of the Mock strain ( Figure 4 This result also proves that the GhZAT11 gene can enhance cold tolerance.

Claims

1. Application of the GhZAT11 gene or an expression vector containing the GhZAT11 gene in regulating the cold tolerance of cotton, wherein the amino acid sequence encoded by the GhZAT11 gene is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the GhZAT11 gene is shown in SEQ ID No.

1.

3. The use according to claim 1 or 2, characterized in that: The regulation of cotton cold tolerance refers to overexpressing the GhZAT11 gene in cotton, thereby improving the cold tolerance of cotton.

4. A method for breeding cold-resistant cotton varieties, characterized in that: The expression level of the GhZAT11 gene in cotton was detected. Varieties with higher expression levels of the GhZAT11 gene were relatively cold-resistant cotton varieties. The nucleotide sequence of the GhZAT11 gene was shown in SEQ ID No.1.

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