Application of GhZAT11 gene in improving cold tolerance of cotton

Abstract

The application discloses application of a GhZAT11 gene in improving 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. Overexpression of the GhZAT11 gene in cotton can improve the cold resistance of the cotton. The application firstly explicitly defines the functional role of the gene GhZAT11 in the cold resistance of cotton, and provides a target gene for enhancing the cold resistance of cotton. The application finds through various tests that silencing of the GhZAT11 gene can reduce the cold resistance of a plant, and overexpression of the GhZAT11 gene can enhance the cold resistance of the plant. The results show that the GhZAT11 gene plays a positive regulation role in the cold resistance of cotton. The GhZAT11 gene can be applied to preventing and treating cold damage of cotton, and can be specifically applied to genetic improvement or molecular breeding of the cold resistance of cotton, such as cultivating a cold-resistant cotton variety.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to the application of the GhZAT11 gene in improving the cold resistance of cotton. Background Technology

[0002] Cotton is one of my country's important economic crops, a vital resource related to national welfare and people's livelihood, and occupies a very important position in the national economy. Cotton (Gossypium spp.) is a typical warm-season crop, with an optimal growth temperature typically between 20℃ and 30℃. Within this temperature range, cotton's photosynthesis, respiration, and growth and development reach their optimal levels. However, cotton's sensitivity to low temperatures presents numerous challenges during its growth. Especially during the seedling and flowering stages, low temperatures significantly impact cotton growth, leading to slow plant growth, poor root development, and even bud drop and poor fruit development. These low-temperature hazards not only affect cotton growth and development but may also lead to a significant decrease in yield, thereby impacting farmers' economic income. ZAT family genes (Zinc Finger of Arabidopsis Thaliana, ZAT) are important transcription factors that primarily function in plants. They are characterized by zinc finger domains and participate in regulating plant growth and development, stress responses, and physiological processes. In Osmanthus fragrans, the expression level of the OfZAT35 gene was found to continuously increase under low-temperature stress. Overexpression of OfZAT35 in tobacco significantly increased relative electrolyte leakage (REL) levels, along with increased activities of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX). Under low-temperature stress, overexpression of the NlZAT12 gene in Arabidopsis promoted the expression of several cold-response protein genes. Transgenic Arabidopsis showed lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA) but higher levels of soluble sugars, indicating that overexpression of the NlZAT12 gene enhances cold tolerance. Transgenic Arabidopsis lines overexpressing the GhZAT6 gene exhibited good salt tolerance on MS medium containing 100 mM NaCl and showed superior growth compared to the wild type. Furthermore, cotton plants with silenced GhZAT6 genes showed more pronounced leaf wilting compared to the control group after treatment with 400 mM NaCl.

[0003] Currently, cotton-growing areas are susceptible to cold damage stress, which severely impacts 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 the cold tolerance of cotton seedlings and promote the healthy and rapid development of cotton, it is imperative to improve cotton cold tolerance and explore genetic regulatory sites and key genes related to cotton cold tolerance. Summary of the Invention

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

[0005] The technical solution of the present invention is: the application of the GhZAT11 gene or an expression vector containing the GhZAT11 gene in regulating the cold resistance of cotton, wherein the GhZAT11 gene is a DNA fragment encoding a protein with the 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 resistance refers to overexpressing the GhZAT11 gene in cotton to improve its cold resistance.

[0008] A method for breeding cold-resistant cotton varieties involves detecting the expression level of the GhZAT11 gene in cotton. Varieties with higher GhZAT11 gene expression levels are considered to be more cold-resistant cotton varieties. 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) This invention is the first to clearly define the functional role of the gene GhZAT11 in the cold resistance of cotton, providing a target gene for enhancing the cold resistance of cotton.

[0011] (2) Through various experiments, this invention found that silencing the GhZAT11 gene reduces the cold tolerance of the plant; while overexpression of the GhZAT11 gene enhances the cold tolerance of the plant. The results show that GhZAT11 plays a positive regulatory role in the cold tolerance of cotton.

[0012] (3) The gene GhZAT11 discovered in this invention can be applied to prevent and control cold damage in cotton. Specifically, it can be used to enhance the genetic improvement or molecular breeding of cotton cold resistance, such as to cultivate cold-resistant cotton varieties. Attached Figure Description

[0013] Figure 1 Analysis of GhZAT11 gene expression under cold stress;

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

[0015] Figure 3 Silencing the GhZAT11 gene reduces the cold resistance of cotton.

[0016] Figure 4 Overexpression of the GhZAT11 gene enhances the cold resistance of cotton. Detailed Implementation

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0018] This invention, through extensive research, has discovered that the GhZAT11 gene plays a specific role in cotton's cold tolerance. Cotton plants with silenced GhZAT11 gene expression exhibit significantly reduced cold tolerance. This application provides a target gene and theoretical support for enhancing cotton's cold tolerance. The GhZAT11 gene is a DNA fragment that encodes a protein with the amino acid sequence shown in SEQ ID No. 2.

[0019] It should be noted that "having" means that the GhZAT11 gene nucleotide sequence may only have the nucleotide sequence shown in SEQ ID No. 1, or it may be composed of the nucleotide sequence shown in SEQ ID No. 1 and other nucleotide sequences, such as nucleotide sequences encoding functional units for protein purification tags, fluorescent protein markers, and DNA binding sites, or encoding elements that regulate gene transcription and expression, including but not limited to promoters, strong promoters, enhancers, or transcription factor binding sites; "having" may also mean that the nucleotide sequence shown in SEQ ID No. 1 is not continuous in the GhZAT11 gene, but can produce cDNA with the nucleotide sequence shown in SEQ ID No. 1.

[0020] "Identity" here refers to the similarity to the nucleotide sequence shown in SEQ ID No. 1. Differences from the nucleotide sequence shown in SEQ ID No. 1 may be caused by alterations, deletions, or insertions of one or more nucleotides, as well as codon degeneracy. These alterations make the gene sequence not completely identical to the nucleotide sequence shown in SEQ ID No. 1, but it has at least 90% identity, for example, but not limited to 90%, 95%, 98%, 99%, or higher. Furthermore, genes with more than 90% identity to the nucleotide sequence shown in SEQ ID No. 1 have the same function as genes with the nucleotide sequence shown in SEQ ID No. 1. Identity can be evaluated visually or using computer software, such as comparison using conventional BLAST software in the art. When the GhZAT11 gene has the nucleotide sequence shown in SEQ ID No. 1, it is more effective in enhancing the cold resistance of cotton.

[0021] It is understood that, in addition to the GhZAT11 gene itself, biological materials containing the aforementioned GhZAT11 or proteins encoding the GhZAT11 gene can also be used to enhance the cold resistance of cotton. Among these, biological materials may include, but are not limited to, expression cassettes, vectors, or transgenic cell lines.

[0022] The present invention will be further illustrated by the following examples.

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

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

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

[0026] The strains used in this embodiment are as follows: Escherichia coli competent cells DH5α and Agrobacterium competent cells GV3101.

[0027] Unless otherwise specified, the materials or reagents used in the examples were prepared according to existing methods or were purchased directly from the market.

[0028] Example 1: Cotton Planting and Treatment

[0029] Cotton cultivation: Select plump ZM13 seeds, wash them with sterile water, soak them in a container, and place them in a 28℃ artificial climate incubator overnight to promote germination. After the seeds show signs of sprouting, transplant them into moist nutrient soil, cover them with mulch, and place them in a 28℃ artificial climate incubator with alternating natural light for 16 hours and darkness for 8 hours.

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

[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 the GhZAT11 gene was analyzed by RT-qPCR. The internal reference gene was the GhUBQ7 gene.

[0033] The RT-qPCR reaction system is shown below:

[0034]

[0035] The RT-qPCR reaction procedure is shown below:

[0036]

[0037] The primers used for RT-qPCR are shown below:

[0038]

[0039] Data analysis utilizes 2 -ΔΔCT Methods: Triple replicates, t-test, ** indicates P < 0.01. Results showed that the expression level of the GhZAT11 gene significantly increased with increasing cold stress time. Figure 1 During the 0-12h period, the expression level of the GhZAT11 gene continuously increased, peaking at 12h. At 24h, the expression level of the GhZAT11 gene began to decline. These results demonstrate that the GhZAT11 gene responds to cold stress.

[0040] Example 3: GhZAT11 gene cut & tag-qPCR analysis

[0041] Root tissue samples were collected at 28℃ and 4℃ for 72 hours, ground in a mortar and pestle, and transferred to 10mL centrifuge tubes. Cell nuclei were extracted using the CELLYTPN1 kit. The specific steps are as follows:

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

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

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

[0045] (4) Resuspend the clump of tissue 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 lysis buffer from the previous step, incubate at 4 °C, 12000 × g for 10 min, and then remove the supernatant.

[0047] (6) Resuspend the tissue mass at the bottom of the centrifuge tube in 1 mL of NIBA solution, 4 °C, 12000 × g, for 5 min, then remove the supernatant.

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

[0049] DNA was extracted using the Novizan Hyperactive pG-MNase CUT&RUN Assay Kit for PCR / qPCR. 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, place it on a magnetic bead separator, remove the supernatant after 3 min, repeat the operation twice, and then add 10 μL of Binding Buffer solution.

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

[0052] (3) Transfer 50 μL of cell nuclei into the magnetic beads prepared in (1), mix well, react at room temperature for 10 min, place on a magnetic bead separator, let stand for 3 min and then remove the supernatant.

[0053] (4) Take 100 μL of pre-cooled Antibody Buffer solution and add it to the eight-segment, 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 the magnetic bead separator and let it stand for 3 minutes before removing the supernatant.

[0055] (6) Add 800 μL of Dig-wash Buffer, invert the container, centrifuge briefly, place the eight-pack on the magnetic bead separator, let stand for 3 minutes, then remove the supernatant. Repeat twice.

[0056] (7) Add 100 μL of pG-MNase Enzyme mixture, mix well, react at 4℃ 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 the container, centrifuge briefly, place the eight-pack on the magnetic bead separator, let stand for 3 minutes, remove the supernatant, and repeat twice.

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

[0059] (10) Add 100 μL of Stop Buffer solution, mix well, react at 37°C for 20 min, then centrifuge at 4°C and 12000 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, and then transfer 650 μL into FastPure gDNAMini Columns adsorption column. Centrifuge at 10000 rpm for 1 min and then remove the waste liquid.

[0061] (12) Pipette 700 μL of Buffer GW solution into the adsorption column, centrifuge at 10000 rpm for 2 min, and then remove the waste liquid.

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

[0063] (14) Place the adsorption column into a clean 1.5 mL tube, then add 20 μL ddH2O to the center of the adsorption column, let stand for 2 min, and centrifuge at 10000 rpm for 2 min.

[0064] (15) The qPCR reaction system is shown below:

[0065]

[0066] The RT-qPCR reaction procedure is shown below:

[0067]

[0068] The primers used for RT-qPCR are shown below:

[0069]

[0070] Data analysis was performed using the 2-ΔΔCT method with three replicates and t-tests. ** indicates P < 0.01. Results showed that H3K4me3 modification of the GhZAT11 gene increased with cold stress. Figure 1 The number of cells increased by approximately 2.1 times, demonstrating that the GhZAT11 gene responds to cold stress.

[0071] Example 4: Construction of GhZAT11 gene expression vector

[0072] 1. Recovery of target fragments and linearized vectors

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

[0074]

[0075] (2) Obtaining the target fragment: Using cotton cDNA as a template, the sequence was amplified using Novizan's high-fidelity enzyme 2×PhantaMax Master Mix (Dye Plus). The PCR reaction system is 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 vector were purified using the EasyPure Quick Gel Extraction Kit from Beijing TransGen Biotech Co., Ltd. The specific steps are as follows:

[0082] (a) Quickly cut the agarose gel containing the target band under UV light to avoid prolonged exposure to UV light that could damage the DNA. Cut the gel into small pieces and place them into a clean 1.5 mL centrifuge tube for subsequent gel dissolution.

[0083] (b) Weigh the gel using an electronic balance. Add 3 times the volume of BufferGDP according to the formula 100mg equals 100μL. Incubate in a 55°C water bath for 15 minutes, inverting and mixing 2-3 times during the process 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 then 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 min, and then centrifuge at 12000 rpm for 1 min.

[0086] (e) Discard the filtrate and then return the adsorption column to the collection tube. Add 700 μL of 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 sterile 1.5 ml centrifuge tube, add 40 μL of Elution Buffer (preheated to 55 °C in a water bath) to the center of the adsorption column, and incubate at room temperature for 5 min. Elute the DNA by centrifugation at 12000 rpm for 1 min, and store at -20 °C for later use.

[0090] 2. Vector construction and transformation of E. coli: using vectors from Nanjing Novizan Biotechnology Co., Ltd. The Ultra OneStep Cloning Kit performs homologous recombination of linearized vectors and target fragments to construct vectors, which are then prepared in the following liquid systems on ice:

[0091]

[0092] The reaction was carried out at 50°C for 10 min, and then immediately cooled on ice. The recombinant product was then transformed into competent E. coli cells.

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

[0094] (2) Heat shock in a 42℃ water bath for 45 seconds, then immediately place on ice for 2 minutes.

[0095] (3) Add 500 μL of LB liquid culture medium without antibiotics and place it on a shaker 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, leave 100 μL of bacterial culture and spread it on LB solid medium containing the corresponding antibiotic, blow dry and invert it in a 37℃ incubator overnight.

[0097] (5) On the second day, pick single clones from the plate and shake them in 500 μL of LB liquid medium containing the corresponding antibiotic until the bacterial culture is turbid. Perform bacterial culture PCR and send positive clones to Shanghai Sangon Biotech Co., Ltd. for sequencing. Compare the sequencing results returned by the company with the target sequence. If the comparison results are completely correct, preserve the bacteria.

[0098] 3. Plasmid extraction: Using Beijing Total Gold plasmid extraction equipment... The Plasmid MiniPrep Kit is used for the extraction of E. coli plasmids. The specific steps are as follows:

[0099] (1) Take 5 mL of Escherichia coli culture that has been cultured overnight at 37℃ in a shaker and put it into a 10 mL centrifuge tube. Centrifuge at 12,000 r / min for 5 min and discard the supernatant.

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

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

[0102] (4) Add 450 μL of yellow solution NB, gently mix by turning it upside down 6-8 times until the solution color changes from blue to yellow and forms a firm yellow aggregate, indicating that the neutralization is complete. Let it stand at room temperature for 2 minutes.

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

[0104] (6) Add 700 μL of rinsing solution WB, 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 dry for 5 min to remove residual rinsing solution.

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

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

[0107] 4. Agrobacterium-mediated transformation: Agrobacterium-mediated transformation was performed using GV3101 from Shanghai Weidi Biotechnology Co., Ltd. The specific steps are as follows:

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

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

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

[0111] (4) Centrifuge at 3000 rpm for 3 min, collect the bacterial cells, discard part of the supernatant in a clean bench, and resuspend 150 μL of bacterial cells by pipetting. Spread the bacterial solution evenly on LB solid medium containing Kan and Rif resistance. Incubate upside down in an incubator at 28℃ for 2 days.

[0112] (5) Positive clone detection: Select single clone strains and culture them in 600 μL of LB liquid medium containing Kan and Rif resistance for 12 h at 28℃ and 200 rpm.

[0113] (6) Use bacterial culture as a template for PCR verification.

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

[0115] (1) Select self-pollinated ZM113 seeds preserved in the laboratory, soak the seeds in sterile water overnight at 37°C, select the seeds with white sprouts, plant them with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite, cover with a transparent lid, and inject Agrobacterium after the cotyledons have fully unfolded.

[0116] (2) Take 40 mL of Agrobacterium tumefaciens culture containing gene silencing vector that has been cultured overnight into a 50 mL clean centrifuge tube, centrifuge at 4000 rpm for 10 min to enrich the bacterial cells, and discard the supernatant.

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

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

[0119] (5) Carefully scratch the back of the cotton cotyledon with the needle of a 1mL sterile syringe (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 left to stand at room temperature in darkness for 24 hours, the plants were transferred to a greenhouse for cultivation. Once they reached the stage of two leaves and one bud, they were treated at 4℃. RNA was extracted from the leaves of TRV:00 and TRV:GhZAT11 plants, reverse transcribed, and then quantitatively analyzed using fluorescence. The results showed that the expression of the GhZAT11 gene was significantly reduced. Figure 3 (A). After treatment at 4℃ for 3 days, the results showed that the leaves of TRV:GhZAT11 plants were severely wilted. Figure 3(B). These results indicate that silencing the GhZAT11 gene reduces cotton's cold tolerance. Further analysis of MDA and POD contents revealed that TRV:GhZAT11 plants had higher MDA contents than TRV:00 plants, while TRV:GhZAT11 plants had lower POD contents than TRV:00 plants. Figure 4 (C and D) This result also proves that silencing GhZAT11 can reduce cold resistance.

[0121] Example 6: Overexpression of the GhZAT11 gene enhances cold resistance in cotton.

[0122] (1) In a clean bench, cut the sterile seedlings into small segments and put them into an Erlenmeyer flask containing Agrobacterium infection solution containing the GhZAT11 gene overexpression vector for 10 min. After infection, transfer the hypocotyl to a sterile large petri dish and wait for the surface bacterial solution to dry. Then transfer the hypocotyl to a co-culture medium and culture in the dark for 40-46 h. After co-culture, transfer it to sterile water containing CEF and soak for 20 min. Then wash it with sterile water 5-6 times until the residue is washed away and the surface moisture is dried. Then spread the hypocotyl evenly in hypocotyl induction medium containing termethin and kanamycin. Finally, place the hypocotyl in a greenhouse at 28±1℃ for culture. The induced callus tissue was transferred to a callus proliferation medium containing termethin and kanamycin and cultured. The culture was subcultured every 4 weeks. An appropriate amount of callus tissue was then transferred to an embryogenic callus induction medium containing termethin and kanamycin and cultured. The culture was subcultured every 3 weeks until embryogenic callus tissue appeared.

[0123] (2) The obtained embryogenic callus was transferred into an embryogenic callus proliferation medium containing termethin and kanamycin for expansion culture. Subculture was carried out every two weeks. When the embryogenic callus proliferated to a certain amount, it could be used for subsequent differentiation into seedlings.

[0124] Leaves of overexpressing plants were collected, DNA was extracted, and RT-qPCR amplification was performed using primers designed to target specific fragments. The results showed that the expression level of the GhZAT11 gene in the overexpressing lines was significantly higher than that in the Mock plants. Figure 4 (A) This study demonstrates the successful construction of the GhZAT11 gene overexpression line. Mock and T4 generation cotton seeds were selected and soaked in sterile water overnight at 37°C. Seeds with emerging white leaves were selected and planted with the radicle facing down in a 1:1 mixture of nutrient soil and vermiculite. A transparent lid was placed on the seed coat, and removed after the cotyledons unfolded. The plants were cultured in a greenhouse until the 6-leaf stage, then transferred to a light incubator with a temperature of 6°C, a 16-hour light-8-hour dark cycle, and a relative humidity of 60%. Phenotypic observation was performed after 3 days. The results showed that the leaves of the GhZAT11 gene overexpression line exhibited slight wilting, while the Mock plants showed more severe wilting, demonstrating that the GhZAT11 gene overexpression line had higher cold tolerance than the Mock plants. Figure 4 The presence of B indicates that the GhZAT11 gene can improve the cold resistance of cotton. Further analysis of MDA and POD content revealed that the MDA content of the OE strain was lower than that of the Mock strain, while the POD content of the OE strain was higher than that of the Mock strain. Figure 4 (C and D) This result also proves that the GhZAT11 gene can enhance cold resistance.

Claims

1. GhZAT11 Genes or containing GhZAT11 The application of gene expression vectors in regulating the cold resistance of cotton, the aforementioned GhZAT11 The amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

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

1.

3. The application according to claim 1 or 2, characterized in that, The regulation of cotton cold resistance refers to the overexpression of [a specific substance] in cotton. GhZAT11 Genes that improve the cold resistance of cotton.

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