Application of AhHTF1 gene in improving high temperature resistance of plant

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an AhHTF1 gene in improving high temperature resistance of plants. The nucleotide sequence of the AhHTF1 gene is shown as SEQ ID NO.1. According to the invention, a recombinant expression vector pCAMBIA1307-AhHTF1 is constructed, and the recombinant expression vector is introduced into a model plant tobacco, so that a transgenic tobacco plant over-expressing the AhHTF1 is obtained. A high-temperature stress treatment experiment shows that overexpression of the AhHTF1 gene can improve the tolerance of the plant to high-temperature stress, and the AhHTF1 gene can be used for carrying out genetic improvement on the heat resistance of the plant.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving AhHTF1 The application of genes in improving the heat resistance of plants. Background Technology

[0002] Suitable temperature is essential for the normal growth and development of plants. When the temperature exceeds the optimum temperature, causing irreversible damage to the normal growth, development, or physiological functions of plants, it is considered high-temperature stress or heat stress. High-temperature stress varies in its timing, intensity, and duration, and has a wide range of effects on plants. When the temperature rises above a threshold level, causing irreversible damage to the normal growth or physiological functions of plants, it is considered high-temperature stress. High temperatures affect the normal growth and development of plants in many ways, such as causing organ malformation, imbalance in physiological processes, and disordered cell metabolism, thereby hindering plant growth and significantly increasing the consumption of assimilates, accelerating plant senescence and death. During the reproductive growth stage, high temperatures can lead to loss of pollen activity, malformation of floral organs, abnormal fruit coloring, and a significant decline in fruit quality.

[0003] Peanuts have high nutritional value and a wide range of uses. Global warming-induced high-temperature stress poses a serious threat to peanut production. High-temperature stress is a significant abiotic stress factor in peanut growth and yield formation, especially during the flowering to pod-setting stages. Discovering and screening key candidate genes regulating peanut heat tolerance is crucial for breeding heat-resistant peanut varieties, studying the response mechanisms of peanuts to high-temperature stress, and enhancing the resilience of peanuts. Summary of the Invention

[0004] To address the above problems, the present invention provides AhHTF1 The application of genes in improving the heat tolerance of plants: This invention discovers an overexpression gene. AhHTF1 It can improve the plant's tolerance to high temperature stress, utilizing AhHTF1 Genes can be used to genetically modify the heat tolerance of plants.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides AhHTF1 The application of genes in improving the heat tolerance of plants, the AhHTF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is tobacco or peanut.

[0006] Furthermore, the method of application is as follows: improving the quality of the plant through transgenic technology or gene editing technology. AhHTF1 The expression level of genes is adjusted to improve the plant's tolerance to high temperatures.

[0007] A second aspect of this invention provides the application of AhHTF1 protein in improving the heat tolerance of plants, wherein the AhHTF1 protein is derived from the above-described... AhHTF1 Gene encoding.

[0008] Furthermore, the amino acid sequence of the AhHTF1 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the plant is tobacco or peanut.

[0010] A third aspect of the present invention provides the application of a recombinant expression vector in improving the heat tolerance of plants, wherein the recombinant expression vector contains the above-described... AhHTF1 Gene.

[0011] Furthermore, the recombinant expression vector is pCAMBIA1307- AhHTF1 .

[0012] Furthermore, the plant is tobacco or peanut.

[0013] A fourth aspect of the present invention provides the application of a recombinant bacterium in improving the heat tolerance of plants, wherein the recombinant bacterium contains the above-described... AhHTF1 Gene.

[0014] Furthermore, the plant is tobacco or peanut.

[0015] The fifth aspect of this invention provides a method for cultivating heat-resistant transgenic plants, comprising the following steps: The above-mentioned AhHTF1 Genes are linked to expression vectors to construct recombinant expression vectors; The recombinant expression vector was introduced into plants using Agrobacterium-mediated transformation. AhHTF1 Genes are overexpressed in plants to obtain heat-resistant transgenic plants.

[0016] Furthermore, the expression vector is pCAMBIA1307.

[0017] Furthermore, the Agrobacterium is GV3101.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to propose and verify AhHTF1 Genes can regulate a plant's heat tolerance. By constructing genes containing... AhHTF1 The recombinant expression vector pCAMBIA1307- AhHTF1 The recombinant expression vector was introduced into the model plant tobacco using the Agrobacterium-mediated leaf disc method, and overexpression was obtained. AhHTF1 Transgenic tobacco plants. Obtained through high-temperature stress experiments. AhHTF1Gene overexpression can improve the heat tolerance of plants, utilizing AhHTF1 Genes can be used to genetically modify a plant's ability to withstand high temperatures. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0020] Figure 1 The spectrum of expression vector pCAMBIA1307 is shown. * indicates the expression vector pCAMBIA1307. BsaB The I restriction site is blocked by Dam methylation.

[0021] Figure 2 Image showing positive detection and screening results for genetically modified tobacco; Figure 2 Figure A shows the electrophoresis diagram for positive detection of transgenic tobacco. M represents the DNA Marker. Lane WT corresponds to wild-type tobacco plants, lane - is the template-free negative control, and lane + corresponds to pCAMBIA1307-. AhHTF1 The recombinant expression vector, and the samples corresponding to lanes 1 to 8 are all parallel samples of transgenic tobacco; Figure 2 Figure B Figure 2 The C diagram and Figure 2 Figure D shows a parallel experimental plate diagram of T1 generation transgenic tobacco hygromycin screening.

[0022] Figure 3 The figure shows the germination of wild-type tobacco plants and overexpressing tobacco plants after high-temperature treatment. WT represents wild-type tobacco plants, while OE-1 and OE-2 are overexpressing tobacco plants. The dashed lines in the figure are used to distinguish the distribution areas of seeds from different genotypes of tobacco plants.

[0023] Figure 4 The germination rate of wild-type tobacco plants and overexpression tobacco plants after high temperature treatment is shown in the figure. WT represents wild-type tobacco plants, and OE-1 and OE-2 are overexpression tobacco plants. Figure 4 Figure A shows the germination rate statistics for the room temperature control treatment; Figure 4 Figure B is a statistical chart of germination rate when treated at 42℃.

[0024] Figure 5 The root length phenotype and data statistics of wild-type tobacco plants and overexpressing tobacco plants after high temperature treatment are presented. WT represents wild-type tobacco plants, and OE-1 and OE-2 are overexpressing tobacco plants. Figure 5Figure A is a root length phenotype. The three wild-type tobacco plants corresponding to WT are parallel samples, the three OE-1 plants corresponding to OE-1 are parallel samples, and the three OE-2 plants corresponding to OE-2 are parallel samples. Scale bar: 1cm. Figure 5 Figure B is Figure 5 The average root length statistics for WT, OE-1, and OE-2 in Figure A, ** represent p <0.01, *** indicates p <0.001.

[0025] Figure 6 The phenotypes of wild-type tobacco plants and overexpressing tobacco plants after high temperature stress during the seedling stage are shown. WT represents wild-type tobacco plants, and OE-1 and OE-2 are overexpressing tobacco plants. Figure 6 Figure A shows the phenotypic diagrams of 4-week-old wild-type tobacco plants and overexpression tobacco lines after heat treatment. Figure 6 Figure B shows the chlorophyll content statistics of wild-type tobacco plants and overexpression tobacco plants after being subjected to high temperature stress. ** indicates p <0.01; Figure 6 Figure C shows the chlorophyll content of 4-week-old wild-type tobacco plants overexpressing the tobacco line after heat treatment. The 3 wild-type tobacco plants corresponding to WT are parallel samples, the 3 OE-1 plants corresponding to OE-1 are parallel samples, and the 3 OE-2 plants corresponding to OE-2 are parallel samples.

[0026] Figure 7 The physiological changes of 8-week-old wild-type tobacco plants and overexpressing tobacco plants after high temperature stress are shown. WT represents wild-type tobacco plants, and OE-1 and OE-2 are overexpressing tobacco plants. Figure 7 Figure A is a statistical chart of soluble sugar content. Figure 7 Figure B is a statistical chart of malondialdehyde (MDA) content. Figure 7 Graph C is a statistical graph of peroxidase activity. Figure 7 The D-chart is a statistical chart of soluble protein content. Figure 7 The E-chart is a statistical chart of proline content. Figure 7 The F-plot is a statistical graph of superoxide dismutase activity, where * indicates... p <0.05, ** indicates p <0.01.

[0027] Figure 8 To overexpress the accumulation of reactive oxygen species in tobacco leaves under normal and high temperature conditions, WT represents wild-type tobacco plants, while OE-1 and OE-2 are overexpressing tobacco plants. The top row of images shows the leaf phenotype after treatment at normal temperature, the middle row shows the leaf phenotype after CAB staining under high temperature treatment, and the bottom row shows the leaf phenotype after NBT staining under high temperature treatment.

[0028] Figure 9 For subcellular localization analysis of AhHTF1, Figure 9 (a) diagram Figure 9 (b) diagram Figure 9 (c) diagram and Figure 9 Figure (d) is a comparison image of the unloaded system. Figure 9 (e) diagram, Figure 9 (f) diagram, Figure 9 (g) diagram and Figure 9 The (h) diagram is for EGFP- AhHTF1 image, Figure 9 Figure (a) and Figure 9 Figure (e) shows the image observed through a green fluorescent protein filter. Figure 9 Figure (b) and Figure 9 Figure (f) shows the image observed through a red fluorescent protein filter. Figure 9 (c) diagram and Figure 9 Image (g) is a bright-field image. Figure 9 Figure (d) is Figure 9 (a) diagram Figure 9 Figure (b) and Figure 9 The image after merging the (c) figures, Figure 9 The (h) diagram is Figure 9 (e) diagram, Figure 9 (f) diagram and Figure 9 The image after merging the (g) plots; scale bar: 20μm. Detailed Implementation

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0030] In this invention, Agrobacterium EHA105 was purchased from Qingke Biotechnology, and the pCAMBIA1307 expression vector and the vector pCAMBIA1300-35S-EGFP were kindly provided by Professor Xu Jie of Jiangxi Agricultural University.

[0031] Peanuts, as an important economic crop, are highly susceptible to environmental factors during their growth and development. With global warming and increasingly frequent high-temperature weather, peanut cultivation faces severe challenges. High temperatures adversely affect multiple growth stages of peanuts, including reducing seed germination rates, inhibiting plant growth, impacting flowering and pollination, and decreasing pod formation, ultimately leading to reduced yield and quality. Identifying and screening key candidate genes that regulate peanut heat tolerance is crucial for breeding heat-resistant peanut varieties, studying the response mechanisms of peanuts to high-temperature stress, and enhancing the resilience of peanuts.

[0032] This invention provides AhHTF1 Application of genes in improving the heat tolerance of plants. This invention first constructs a recombinant expression vector pCAMBIA1307- AhHTF1 This recombinant expression vector was introduced into the model plant tobacco to obtain overexpression. AhHTF1 Transgenic tobacco plants. Through high-temperature stress treatment experiments, it was found that the overexpressed gene... AhHTF1 It can improve the plant's tolerance to high temperature stress, utilizing AhHTF1 Genes can be used to genetically modify the heat tolerance of plants.

[0033] Example 1: AhHTF1 Gene cloning and construction of recombinant expression vectors In the early stages of this invention, a candidate gene associated with high-temperature stress-induced expression in peanuts was discovered during multi-omics studies. AhHTF1 , AhHTF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoding the gene is shown in SEQ ID NO.2.

[0034] SEQ ID NO.1: 。

[0035] SEQ ID NO.2: MEVKGSSNSNKSAVLISQSEEEEMMDLRRRGPWTVDEDLTLINYIATHGEGLKRTGKSCRLRWLNYLRPDVRRGNITLEEQLLILELHTRWGNRWSKIAQYLPGRTDNEIKNYWRTRVQKHAKQLKCDVNSKQFKDAMRYLWMPRLVERIQAAAAASTT TTTVVAPTNTSINNNNNAFTYNNNLNNNFEVHNGKMMLPTSTPPSVMANNDFVGSQITTQSYNTPENSSTAASSDSFGTPISDFNDYYNSNNINNLGDNNFYQASQTLFSHEQGMDLQSMMMVESNTPWMQSGDTSDNFWNVENMLFLEQQLMNDNM.

[0036] 1. AhHTF1 Cloning of genes In this invention, the flood-tolerant peanut variety 'Changhua 18' is labeled as CH18. Total RNA is extracted from the leaves of CH18 plants, and the extracted qualified total RNA is used to synthesize the first strand of cDNA using a reverse transcription kit from Takara.

[0037] use AhHTF1 A pair of specific primers was designed based on the coding region-specific cDNA sequence of the gene. Using the cDNA as a template, amplification was performed according to the PCR reaction system in Table 1 and the reaction procedure in Table 2. AhMFT1 Genes were extracted, and PCR products were obtained.

[0038] Specific primer set contains AhHTF1 -F and AhHTF1 -R, AhHTF1 The nucleotide sequence of -F is shown in SEQ ID NO.3. AhHTF1 The nucleotide sequence of -R is shown in SEQ ID NO.4: SEQ ID NO.3: 5'-ATGGAAGTAAAAGGAAGCAG-3'; SEQ ID NO. 4: 5'-CATGTTGTCATTCATGAGTT-3'.

[0039] Table 1 PCR reaction system

[0040] Table 2 PCR amplification program

[0041] After detecting the PCR products by agarose gel electrophoresis and obtaining a single band of the expected size, the target gene fragment was recovered using a DNA gel recovery kit from Takara and stored at -20°C for later use.

[0042] 2. Construction of recombinant expression vectors 2.1 Enzyme digestion of expression vector pCAMBIA1307 Using rapid digestion enzymes Xba I and BamH I digested the expression vector pCAMBIA1307 with enzymes. The map of the expression vector pCAMBIA1307 is as follows: Figure 1 As shown in Table 3, the linearized expression vector pCAMBIA1307 was obtained by digestion at 37°C for 0.5 h according to the enzyme digestion system shown in Table 3.

[0043] Table 3. Enzyme digestion system of expression vector pCAMBIA1307

[0044] 2.2 Enzyme digestion of the target fragment The target gene fragment with the corresponding restriction enzyme sites was amplified using the specific upstream primer AhHTF1-1307F and downstream primer AhHTF1-1307FR.

[0045] The nucleotide sequence of AhHTF1-1307F is shown in SEQ ID NO.5, and the nucleotide sequence of AhHTF1-1307F is shown in SEQ ID NO.6. SEQ ID NO.5: 5'-CCGGAATTCATGGAAGTAAAAGGAAGCAG-3'; SEQ ID NO. 6: 5'-CGCGGATCCCATGTTGTCATTCATGAGTT-3'.

[0046] The target fragment was digested with a restriction fast digester, and the reaction was carried out at 37°C for 0.5 h according to the reaction system in Table 4.

[0047] Table 4. Enzyme digestion system for the target fragment

[0048] 2.3 Connection The linearized expression vector pCAMBIA1307 obtained in step 2.1 and the enzyme-digested target fragment obtained in step 2.2 were ligated according to the ligation system in Table 5. The ligation reaction conditions were: 50℃ for 10 min. After ligation, pCAMBIA1307- was obtained. AhHTF1 Recombinant expression vectors.

[0049] Table 5 Connection System

[0050] Example 2: Obtaining recombinant bacteria 1. Transformation and plasmid extraction of Escherichia coli The recombinant expression vector pCAMBIA1307- obtained in Example 1 was used. AhHTF1 The heat shock transformation into E. coli DH5α competent cells is carried out through the following steps: (1) Take the Escherichia coli DH5α competent cells stored at -80℃ out of the refrigerator and place them in ice to thaw.

[0051] (2) Take 6 μL of ligation product and add it to 100 μL of Escherichia coli DH5α competent cells. Mix well by pipetting and then incubate on ice for 5 min, heat shock in water bath at 42℃ for 60 s, and immediately incubate on ice for 3 min. Then transfer to 700 μL of LB liquid medium and revive at 37℃ for 1 h.

[0052] (3) Centrifuge the culture medium that has been revived for 1 hour at 5000 rpm for 5 min, collect the bacterial precipitate, and resuspend the bacterial precipitate in 200 μL of supernatant.

[0053] (4) Spread the resuspended bacterial solution onto LB solid medium containing 50 μg / mL kanamycin antibiotic and incubate upside down at 37°C.

[0054] (5) Pick and label the single colonies that grow on the plate, perform colony PCR identification, select positive clones and transfer them to LB liquid medium containing 50 μg / mL kanamycin antibiotic and culture overnight. Then, take 400 μL of the cultured bacterial solution and send it to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0055] (6) A culture medium containing a positive clone with correct sequencing results is considered to contain [the bacteria]. AhHTF1 The recombinant Escherichia coli was prepared by mixing 40 v / v glycerol solution with the positive clone culture at a volume ratio of 1:1 and then storing at -20°C.

[0056] 2. Transformation of Agrobacterium The recombinant expression vector pCAMBIA1307- was prepared using the freeze-thaw method. AhHTF1 The specific steps for transforming Agrobacterium-competent cells GV3101 are as follows: (1) The plasmids containing the above-constructed plasmids were extracted using a high-purity plasmid miniprep kit from Takara. AhHTF1 The recombinant expression vector pCAMBIA1307- in recombinant Escherichia coli AhHTF1 .

[0057] (2) Take 3 μL of recombinant expression vector and add it to 100 μL of Agrobacterium competent cells GV3101. After mixing, incubate on ice for 10 min, freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and incubate on ice for 5 min. Then transfer it to 700 μL of LB liquid medium and revive at 28℃ for 3 h.

[0058] (3) Take 200 μL of culture medium that has been cultured for 3 hours and spread it on an LB medium solid plate containing 50 μg / mL kanamycin and 25 μg / mL rifampin. Incubate at 28°C with the plate inverted.

[0059] (4) Select single colonies obtained from the culture and culture them in LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin for 36 h. Perform bacterial PCR identification. Positive clones are those containing kanamycin. AhHTF1 Agrobacterium recombinant strain, containing AhHTF1 The recombinant Agrobacterium was stored in a refrigerator at -80°C.

[0060] Example 3: Cultivation of Transgenic Tobacco 1. Agrobacterium-mediated genetic transformation of native tobacco (1) Preparation of Agrobacterium infection solution: The Agrobacterium infection solution prepared in Example 2 containing... AhHTF1 Recombinant Agrobacterium was inoculated onto LB solid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin and streak-strike activated. Activated positive colonies were picked and incubated in LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin at 28°C until the bacterial culture reached OD500. 600 The value was 0.6; the bacterial culture was centrifuged at 600g for 10 min at room temperature, the bacterial cells were collected, and the bacterial cells were resuspended in 30mL of 1 / 2MS medium to obtain a resuspended bacterial culture; the resuspended bacterial culture was mixed evenly and poured into a plastic plate so that the bacterial culture completely covered the surface of the plastic plate.

[0061] (2) Preparation and co-culture of explants: Healthy and intact leaves from 8-week-old wild-type tobacco plants cultured in a greenhouse were selected. After rinsing the leaf surface with pure water to remove dust, the leaves were disinfected in a clean bench with 70% ethanol for 1 min, 0.1% sodium hypochlorite for 10 min, and then rinsed with sterile water 5 times. The moisture on the leaf surface was absorbed with filter paper, avoiding the veins, and the leaf margins were cut off. The leaves were then cut into 25 mm pieces. 2 The leaves were placed in the above Agrobacterium infection solution for 10 min. The infected leaves were then spread flat on MS medium and co-cultured at 28°C in the dark for 3 days.

[0062] (3) Subculture and bud regeneration: After co-culture, the leaves were rinsed 5 times with sterile water and transferred to MS selective medium containing 125 mg / mL cephalosporin. They were cultured at 25°C. When the leaf callus tissue began to sprout, the explants with buds were transferred to a new MS medium containing 125 mg / mL cephalosporin.

[0063] (4) Rooting culture and transplanting: When the young shoots elongate to 3cm~4cm, cut off the shoots and transfer them to a rooting medium containing 125mg / mL cephalosporin and 25mg / mL hygromycin for screening and culture; after the seedling roots have developed well, gently remove the tissue culture seedlings from the medium, rinse them with sterile water to remove the mucilage, and carefully transfer them into sterile nutrient soil for adaptive culture.

[0064] 2. Positive identification of transgenic tobacco plants 2.1 Extraction of DNA from tobacco plants using CTAB and organic solvent methods: (1) When the T0 generation transgenic tobacco seedlings infected by the above leaf disc method grow new leaves, cut some leaves into a mortar, add 600 μL of preheated CTAB, add quartz sand, grind the leaves into a slurry, and transfer the slurry into a 1.5 mL centrifuge tube.

[0065] (2) Place the centrifuge tube in a water bath and heat it at 65°C for 60 minutes, shaking the centrifuge tube during the process; then add an extraction solvent of equal volume to the slurry. The extraction solvent is obtained by mixing phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 and mixing thoroughly by inverting for 5 minutes.

[0066] (3) Centrifuge the above solution at 12000 rpm for 10 min, transfer the supernatant, and add an equal volume of mixed solvent 1 to the supernatant. Mixed solvent 1 is obtained by mixing chloroform and phenol in a 1:1 volume ratio. Then centrifuge for 10 min and transfer the supernatant.

[0067] (4) Add an equal volume of mixed solvent 2 to the supernatant obtained in step (3). Mixed solvent 2 is obtained by mixing chloroform and isoamyl alcohol in a volume ratio of 24:1. Mix well and centrifuge for 10 min, and take the supernatant again.

[0068] (5) Add an equal volume of -20℃ pre-cooled isopropanol to the supernatant obtained in step (4), let it stand at -20℃ for 30 min, then centrifuge for 10 min, retain the precipitate, add 75 v / v% ethanol to the precipitate, shake gently to suspend the precipitate, and let it stand for 5 min.

[0069] (6) Discard the supernatant and retain the precipitate. After drying at room temperature, dissolve it in sterile water and store at -20℃ for later use to obtain... AhHTF1 DNA from a genetically modified tobacco plant sample.

[0070] To obtain AhHTF1 Using DNA from transgenic tobacco plant samples as templates, AhHTF1 -F and AhHTF1 -R performed PCR amplification according to the PCR system in Table 1 and the reaction procedure in Table 2. The PCR products obtained from the PCR amplification were then subjected to gel electrophoresis, and the results are as follows: Figure 2 As shown, the striped areas indicate successfully cultivated transgenic tobacco seedlings that were selected through screening. AhHTF1 The transgenic tobacco plants overexpressing the gene were selected in this invention. AhHTF1 Parallel samples OE-1 and OE-2 of tobacco plants overexpressing the expression were used for subsequent experiments.

[0071] 3. Phenotypic identification (1) Germination test Wild-type tobacco plants of the same generation harvested at the same time under completely identical growth conditions and AhHTF1 The seeds of the overexpressed transgenic tobacco lines were tested. Wild-type tobacco plants and T2 generation were compared. AhHTF1 Transgenic tobacco seeds were disinfected sequentially with 75% v / v ethanol for 1 min, then with 10% v / v H2O2 for 15 min, rinsed five times with sterile ultrapure water, and then spread evenly on MS medium. The control group was cultured normally, while the treatment group seeds were cultured under normal conditions after being incubated at 42℃ for 12 h. Germination status was recorded daily starting the next day, and germination potential and germination rate were calculated. Germination was defined as the seed showing more than half its own length emerging from the germination process.

[0072] Germination potential = (Number of seeds germinated within 10 days / Total number of seeds tested) × 100%; Germination rate = (Number of all normally germinated seeds within 10 days / Total number of seeds tested) × 100%.

[0073] like Figure 3 and Figure 4 As shown, after high-temperature treatment, wild-type tobacco plants and AhHTF1 The germination initiation time of seeds in overexpressed transgenic tobacco lines was delayed and the germination rate was slowed down, but the seed germination of wild-type tobacco plants was more severely affected by heat stress.

[0074] (2) Root length test Seeds of wild-type plants and transgenic tobacco lines of the same generation, harvested under the same growth conditions, were sterilized and germinated. After normal culture for 2 days, they were transferred to MS solid medium and cultured vertically for 7 days. The treatment group was immersed in a 42℃ water bath for 3 hours and then cultured normally. The length of primary roots of different lines was counted on the 14th day after sowing.

[0075] The results are as follows Figure 5As shown, under normal conditions, the root lengths of transgenic tobacco lines and wild-type tobacco plants are identical, with no significant difference. After high-temperature treatment, the primary roots of the transgenic lines elongate faster than those of the wild-type, indicating that overexpression... AhHTF1 This improved the tobacco's tolerance to high-temperature stress during seed germination.

[0076] (3) High-temperature phenotypic identification of seedlings Seeds from wild-type plants and transgenic tobacco lines of the same generation, harvested under identical growth conditions, were sterilized and germinated on MS solid medium, then cultured in a greenhouse at 25°C. At the four-leaf stage, uniformly growing seedlings were selected and transplanted into mixed soil, covered with plastic film to retain moisture, and the film was removed after 3 days. Wild-type tobacco plants and transgenic tobacco lines cultured in petri dishes for 4 weeks in the greenhouse were then compared. AhHTF1 Overexpressing tobacco lines were treated at 42℃ for 12 hours, and the plant phenotype was observed and identified.

[0077] like Figure 6 As shown, after 12 hours of high-temperature treatment, all 4-week-old tobacco seedlings showed wilting and leaf chlorosis, with chlorophyll content decreasing compared to before treatment. Compared to wild-type plants, the transgenic lines showed less wilting, and most plants remained green.

[0078] (4) Determination of physiological changes in transgenic tobacco plants under high temperature stress Wild-type tobacco plants were used as the control group. AhHTF1 The overexpression-expressing tobacco strains were used as the experimental group. After being soil-cultured to 8 weeks of age and treated at 42℃ for 24 hours, relevant physiological indicators were measured in both the control and experimental groups to investigate the effects. AhHTF1 Internal factors that improve plant heat resistance.

[0079] like Figure 7 As shown, compared to wild-type tobacco plants, AhHTF1 The overexpressed transgenic tobacco lines showed an average increase of 17.91%, 62.98%, and 33.55% in soluble sugar, soluble protein, and proline, respectively, while the average malondialdehyde (MDA) content decreased by 25.69%. The average enzyme activities of peroxidase and superoxide dismutase increased by 28.27% and 16.39%, respectively. Significant differences were observed in the activities of soluble protein, proline, MDA, and antioxidant enzymes, indicating that... AhHTF1 The high-temperature tolerance of tobacco may be improved by accumulating osmotic regulators, especially soluble proteins and proline, as well as by increasing the content of antioxidants.

[0080] like Figure 8 As shown, compared to wild-type tobacco plants, AhHTF1 The leaves of the overexpressed transgenic tobacco lines showed less reactive oxygen species accumulation and fewer spots under high-temperature conditions, indicating that... AhHTF1The transgenic tobacco lines overexpressing the gene have stronger antioxidant defense mechanisms, thus better resisting oxidative damage caused by high temperatures.

[0081] Example 4: Subcellular localization of AhHTF1 S1. PCR Amplification: Based on the nucleotide sequence shown in SEQ ID NO.1, using the cDNA from Example 2 as a template, and AhHTF1-EGFP-F and AhHTF1-EGFP-R as amplification primers, amplification was performed according to the amplification system in Table 6 and the amplification procedure in Table 2. AhHTF1 Genes were extracted, and PCR products were obtained. The nucleotide sequence of AhHTF1-EGFP-F is shown in SEQ ID NO.7, and the nucleotide sequence of AhHTF1-EGFP-R is shown in SEQ ID NO.8.

[0082] SEQ ID NO.7: 5'-CGCGGATCCATGGAAGTAAAAGGAAGCAG-3'; SEQ ID NO. 8: 5'-GAATTCCATGTTGTCATTCATGAGTT-3'.

[0083] The PCR amplification system is shown in Table 6.

[0084] Table 6 Amplification System

[0085] S2, Electrophoresis and Gel Recovery: The PCR products were subjected to agarose gel electrophoresis, and the successfully amplified gene fragments were cut and recovered from the gel.

[0086] S3, subcellular localization vector EGFP- AhHTF1 Build: Following the enzyme digestion system shown in Table 7, use restriction endonucleases. EcoR I and restriction endonucleases BamH I performed double digestion of the vector pCAMBIA1300-35S-EGFP and the recovered gene fragment to obtain the linearized pCAMBIA1300-35S-EGFP vector and the digested gene fragment, respectively.

[0087] Table 7 EGFP empty vector digestion system

[0088] Then, using T4 ligase, following the ligation reaction system in Table 8, the linearized pCAMBIA1300-35S-EGFP vector and the enzyme-digested gene fragment were ligated to obtain the subcellular localization vector EGFP- AhHTF1 .

[0089] Table 8 Connection System

[0090] S4. Transformation: Referring to the steps in Example 2, the subcellular localization vector EGFP- constructed in step S3 of this example is transformed. AhHTF1 The recombinant bacteria were sequentially introduced into *E. coli* DH5α competent cells and *Agrobacterium* EHA105 competent cells, and cultured to obtain recombinant bacteria for subcellular localization. *Agrobacterium* strains transformed only with the pCAMBIA1300-35S-EGFP vector were used as control strains and labeled as empty vector *Agrobacterium*.

[0091] S5. Preparation of Infection Solution: The recombinant bacteria and empty-vectored Agrobacterium for subcellular localization were cultured separately in a shaker at 28°C until the logarithmic growth phase. The bacterial cells were then collected by centrifugation at 5000 rpm for 10 min at room temperature. The bacterial cells were resuspended in the infection solution until the OD of the bacterial suspension was reached. 600 The pH value was 1.0, and the solution was allowed to stand at room temperature for 2 hours. The inoculum was a liquid containing 10 mM 2-morpholine ethanesulfonic acid, 150 μM acetylsyl syringone, and 10 mM MgCl2, with a pH value of 5.6.

[0092] S6, Agrobacterium-infected tobacco Young leaves of wild-type native tobacco cultivated in a greenhouse were selected. The underside of the leaves was carefully punctured with a needle, and cultured empty-vectored Agrobacterium and recombinant bacteria for localization were slowly injected into the leaf mesophyll along the wound using a syringe. Residual liquid on the leaf surface was then aspirated. After the injected tobacco plants were cultured at 21°C for 2 days, the infected area was torn off and prepared into slides for observation under a laser confocal microscope.

[0093] like Figure 9 As shown, the subcellular localization vector EGFP- AhHTF1 The green fluorescent signal appears only in the cell nucleus, and overlaps with the red nuclear localization fluorescent signal in the cell nucleus to appear yellow, indicating that AhHTF1 is localized in the cell nucleus.

[0094] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, preferred embodiments are described in this invention to avoid redundancy.

[0095] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0096] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. AhHTF1 The application of genes in improving the heat tolerance of plants is characterized by, The AhHTF1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is tobacco or peanut.

2. As described in claim 1 AhHTF1 The application of genes in improving the heat tolerance of plants is characterized by, The application method is as follows: Improving the [specific] content in plants through transgenic technology or gene editing technology. AhHTF1 The expression level of genes is adjusted to improve the plant's tolerance to high temperatures.

3. An application of AhHTF1 protein in improving the heat tolerance of plants, characterized in that, The AhHTF1 protein is as described in claim 1. AhHTF1 Genetic encoding; the plant is tobacco or peanut.

4. The application of a recombinant expression vector in improving the heat tolerance of plants, characterized in that, The recombinant expression vector contains the features described in claim 1. AhHTF1 Genes; the plant in question is either tobacco or peanut.

5. The application of a recombinant bacterium in improving the heat resistance of plants, characterized in that, The recombinant bacteria contain the components described in claim 1. AhHTF1 Genes; the plant in question is either tobacco or peanut.

6. A method for cultivating heat-resistant transgenic plants, characterized in that, Includes the following steps: The claim 1 AhHTF1 Genes are linked to expression vectors to construct recombinant expression vectors; The recombinant expression vector was introduced into plants to enable... AhHTF1 Genes are overexpressed in plants to obtain heat-resistant transgenic plants.

7. The method for cultivating heat-resistant transgenic plants according to claim 6, characterized in that, The expression vector is pCAMBIA1307.

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