Blwrky15 gene, protein and application thereof in regulating drought stress of betula utilis

Abstract

The application discloses a BlWRKY15 gene, a protein and application of the BlWRKY15 gene and the protein in regulating drought stress of Betula luminifera, relates to the technical field of plant genetic engineering, and the amino acid sequence of the BlWRKY15 protein of the Betula luminifera is any one of the following (1) or (2): (1) the amino acid sequence shown in SEQ ID NO. 2; (2) a protein having more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO. 2 and having a function of regulating drought stress response of plants. The application constructs a BlWRKY15 overexpression vector and transforms white birch; under normal conditions, each index of the transgenic white birch and the wild type is not significantly different; after 10 days of natural drought, the transgenic white birch is more serious than the wild type in wilting, and the electrolyte leakage rate and the MDA content are significantly increased, and the POD activity is significantly reduced, indicating that the drought sensitivity of the transgenic white birch is enhanced. The above results prove that the BlWRKY15 negatively regulates the drought resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a BlWRKY15 gene and protein and its application in regulating drought stress in *Betula sclerophylla*. Background Technology

[0002] The frequency, duration, and spatial extent of droughts are currently on the rise globally, and drought has become a major constraint on global plant growth. Plants respond to drought stress by regulating hormone levels, sucrose metabolism, secondary metabolic processes, and antioxidant systems, thereby improving their survival capabilities. *Betula luminifera* is a valuable and fast-growing timber species in southern my country and a representative species of the *Betula* genus in central subtropical regions. Due to its strong adaptability and wide range of uses, it is widely promoted in afforestation. For broad-leaved tree species like *Betula luminifera*, which have important ecological and economic value, their plantation cultivation and industrial development are heavily threatened by seasonal and extreme drought events. Therefore, in-depth analysis of the molecular mechanisms of drought response in forest trees and the exploration of key drought-resistant genetic resources are of urgent practical significance for cultivating highly stress-resistant new varieties through molecular breeding techniques.

[0003] The WRKY family is one of the important transcription factor families in plants. WRKY proteins, through their unique WRKY domains, specifically recognize and bind to W-box elements in the promoter regions of target genes, thereby activating or inhibiting the transcription of downstream genes and widely participating in the regulation of plant growth and development, secondary metabolism, and responses to biotic and abiotic stresses. In recent years, numerous studies have confirmed the crucial role of WRKY transcription factors in plant drought resistance. In Arabidopsis, AtWRKY43 has been shown to positively regulate the expression of the sugar transporter gene AtSWEET5, promoting sugar accumulation and distribution, thereby enhancing the plant's osmotic regulation and drought tolerance. In apple, MdWRKY71 interacts with MdARF3, synergistically binding to and activating the promoter of the iron superoxide dismutase gene MdFeSOD, promoting the scavenging of reactive oxygen species (ROS), thus significantly improving the drought tolerance of transgenic plants. Overexpression of PtrWRKY75 in poplar induces the accumulation of ROS in guard cells via the salicylic acid pathway, thereby reducing stomatal aperture, decreasing water transpiration, and ultimately improving the plant's drought tolerance.

[0004] While the drought resistance function of the WRKY family in herbaceous model plants (such as Arabidopsis thaliana and rice) has been extensively reported, research on its role in woody plants with long growth cycles and complex genetic backgrounds remains relatively lagging. As a fast-growing, high-quality timber and ecological restoration tree species, research on the drought resistance of *Betula sclerotium* is currently limited to phenotypic observations and assessments at the physiological and biochemical levels. To date, there are no reports of cloning the WRKY15 gene from *Betula sclerotium* and verifying its function in regulating drought stress.

[0005] Therefore, studying the drought-resistant genes of *Betula glabra* and analyzing their application in drought stress regulation is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a BlWRKY15 gene, protein, and its application in regulating drought stress in *Betula sclerophylla*, in order to address the aforementioned shortcomings in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a *Betula sclerotiorum* BlWRKY15 protein, wherein the amino acid sequence of the *Betula sclerotiorum* BlWRKY15 protein is any one of the following (1) or (2):

[0008] (1) The amino acid sequence as shown in SEQ ID NO. 2;

[0009] (2) A protein that has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.2 and has the function of regulating plant drought stress response.

[0010] A *Betula glabra* BlWRKY15 gene, wherein the *Betula glabra* BlWRKY15 gene encodes the *Betula glabra* BlWRKY15 protein, and the nucleotide sequence thereof is any one of the following (3) or (4):

[0011] (3) The nucleotide sequence as shown in SEQ ID NO. 1;

[0012] (4) A DNA molecule that has more than 90% sequence identity with the nucleotide sequence shown in SEQ ID NO.1 and encodes the amino acid sequence shown in SEQ ID NO.2.

[0013] As a preferred option, a recombinant expression vector containing the *Betula glabra* BlWRKY15 gene is used.

[0014] Preferably, the host cell contains the *Betula lucida* BlWRKY15 gene or the recombinant expression vector of claim 3.

[0015] Preferably, the application of the *Betula glabra* BlWRKY15 protein or the *Betula glabra* BlWRKY15 gene in regulating plant drought stress response.

[0016] A method for regulating plant drought tolerance includes the steps described in the above scheme: introducing the BlWRKY15 gene of *Betula sclerophylla* into a plant and overexpressing it to obtain a transgenic plant with altered drought tolerance.

[0017] Preferably, the recombinant expression vector is an overexpression vector obtained by inserting the nucleotide sequence shown in SEQ ID NO. 1 between the CaMV35S promoter and the OCS terminator of the plant expression vector pXHKFGuv.

[0018] Preferably, the host cell is an Agrobacterium cell, and more preferably Agrobacterium rhizogenes Arqual.

[0019] Preferably, the plant is a birch species, and more preferably, *Betula sclerophylla* or *Betula sylvestris*.

[0020] Preferably, the method includes the following steps:

[0021] S01. Construct an overexpression vector containing the nucleotide sequence shown in SEQ ID NO. 1;

[0022] S02. Transform the overexpression vector into Agrobacterium to obtain a transgenic engineered bacterium;

[0023] S03. Transform plant recipients using the aforementioned transgenic engineered bacteria and screen for transgenic positive plants.

[0024] In the above technical solution, the BlWRKY15 gene, protein and its application in regulating drought stress in birch have the following beneficial effects: The present invention provides the full-length genome and cDNA sequence, protein sequence, etc. of the BlWRKY15 gene, and confirms that BlWRKY15 can negatively regulate its drought resistance by transforming birch. When wild-type and transgenic birch were subjected to natural drought stress treatment, there was no difference between the two without stress treatment. After 10 days of stress treatment, the wilting degree of the transgenic line was significantly more severe than that of the wild type.

[0025] Further physiological parameters were measured. Electrolyte leakage rate and MDA content results showed that under normal conditions, all plants exhibited similar electrolyte leakage rates and MDA contents. Under drought stress, the electrolyte leakage rate and MDA content of the transgenic lines were significantly higher than those of the wild type, indicating that the transgenic lines were more sensitive to drought. Meanwhile, POD results showed that under normal conditions, there was no significant difference in POD activity among all plants; however, under drought stress, the POD activity of the transgenic lines was significantly lower than that of the wild type. These results indicate that BlWRKY15 negatively regulates the drought tolerance of birch. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0027] Figure 1 The electrophoresis diagram provided for this embodiment of the invention is an amplification of the full-length cDNA of the BlWRKY15 gene from *Betula sclerophylla*.

[0028] Figure 2 This is a schematic diagram of the domain distribution of BlWRKY15 provided for an embodiment of the present invention;

[0029] Figure 3 A schematic diagram of the phylogenetic tree of BlWRKY15 and proteins such as Arabidopsis thaliana is provided for embodiments of the present invention;

[0030] Figure 4 This is a schematic diagram of multiple sequence alignment between BlWRKY15 and proteins such as Arabidopsis thaliana, provided for an embodiment of the present invention.

[0031] Figure 5 This is a schematic diagram of the subcellular localization of BlWRKY15 provided for an embodiment of the present invention;

[0032] Figure 6 This is a schematic diagram illustrating the molecular-level identification of transgenic plants, provided as an embodiment of the present invention.

[0033] Figure 7 This is a schematic diagram of fluorescence detection in transgenic plants provided as an embodiment of the present invention;

[0034] Figure 8 The diagram provided for embodiments of the present invention illustrates the phenotypic and physiological indicators of transgenic plants under drought stress. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Example 1: Cloning of BlWRKY15 full-length DNA and cDNA

[0037] I. Extraction of total DNA and total RNA from *Betula sclerophylla*

[0038] Young leaves of *Birchia glabra* NP-10 were collected, and total genomic DNA was extracted using a modified CTAB method. The concentration and quality of the DNA samples were evaluated using 1% agarose gel electrophoresis and UV spectrophotometry. DNA samples were stored at -20°C. Leaves of wild-type *Birchia glabra* and materials overexpressing BlWRKY15 were collected during the seedling stage. Total RNA was extracted using a plant total RNA extraction kit from TIANGEN and dissolved in pure water. The quality and purity of RNA were assessed using 1% agarose gel electrophoresis and UV spectrophotometry. Electrophoresis results showed intact three RNA bands (28S, 18S, 5S), with no significant degradation or DNA contamination, meeting the requirements for subsequent experiments. RNA samples were stored at -80°C.

[0039] II. Synthesis of the first strand of cDNA from *Betula sclerophylla*

[0040] After mixing equal amounts of the RNA extracted in step 1 above, reverse transcription was performed using the Takara PrimeScript™ 1st Strand cDNA Synthesis Kit according to its instructions. The reverse transcription product was stored at -20°C for later use.

[0041] III. Cloning of the full-length cDNA of *Betula glabra* BlWRKY15

[0042] Using cDNA reverse transcribed from *Betula sclerophylla* leaf RNA as a template, the full-length cDNA sequence of BlWRKY15 was amplified using upstream primer P1 (SEQ ID NO. 3) and downstream primer P2 (SEQ ID NO. 4). PCR amplification was performed using PhantaFlashMasterMix (Vazyme, Nanjing). The PCR reaction system was prepared according to the enzyme's instructions, with the following reaction program: 98℃ pre-denaturation for 3 min; then 35 cycles of 98℃ for 15 s, 54℃ for 10 s, and 72℃ for 15 s; and a final extension at 72℃ for 5 min. Agarose gel electrophoresis showed the PCR product to be the expected size. After gel recovery, ligation into a cloning vector, transformation into *E. coli*, PCR, and sequencing, the full-length cDNA of BlWRKY15 was confirmed to be 822 bp in length. Figure 1 It was named BlWRKY15mRNA, with the sequence shown in SEQ ID NO.1 and the encoded amino acid sequence shown in SEQ ID NO.2.

[0043] Example 2: Bioinformatics Analysis of the BlWRKY15 Gene

[0044] The full-length cDNA of BlWRKY15 is 822 bp in length, encoding 273 amino acid residues. The domains of BlWRKY15 were analyzed using an online website. Figure 2The results showed that BlWRKY15 possesses a typical WRKY domain. A phylogenetic tree was constructed using MEGA software with a neighbor-joining method. Phylogenetic analysis revealed that BlWRKY15 clustered with Arabidopsis thaliana AtWRKY7 / 11 / 15 / 17 proteins in one group. Figure 3 Comparison of the amino acid sequences of BlWRKY15 protein with those of homologous proteins in Arabidopsis thaliana revealed that BlWRKY15 possesses a typical WRKY domain. Figure 4 This domain contains a highly conserved WRKYGQK heptapeptide sequence and a zinc finger structure.

[0045] Example 3: Subcellular localization analysis of BlWRKY15

[0046] I. Construction of the BlWRKY15-GFP fusion expression vector

[0047] Using the cloned full-length cDNA plasmid BlWRKY15 as a template, primers with restriction enzyme sites were designed to amplify the target fragment. The upstream primer was P5: SEQ ID NO. 7, and the downstream primer was P6: SEQ ID NO. 8. Simultaneously, the plasmid pCAMBIA1305-GFP was double-digested with restriction endonucleases KpnI and BamHI (Takara). The gel-recovered target gene fragment and the digested plasmid fragment were ligated using the homologous recombination ClonExpressII OneStep Cloning Kit (Vazyme, Nanjing). The reaction system followed the enzyme's instructions. After transformation into E. coli, the recombinant plasmid was verified by bacterial culture and sequencing. The correctly sequenced recombinant plasmid was named 35S::BlWRKY15-GFP.

[0048] II. Agrobacterium-mediated transformation

[0049] Take one tube (100 μl) of Agrobacterium tumefaciens GV3101 (Weidi Biotechnology) competent cells and thaw them on ice. Add 4 µl of 35S::BlWRKY15-GFP recombinant plasmid, gently mix, and place on ice for 5 min. Then, freeze in liquid nitrogen for 5 min, and immediately place in a 37°C water bath for 5 min, followed by an ice bath for 2 min. Add 700 μl of antibiotic-free YEB liquid medium and incubate at 28°C with shaking at 200 rpm for 3-4 h. Spread the bacterial culture evenly on YEB solid medium containing 50 µg / ml kanamycin and 50 µg / ml rifampin, and incubate upside down at 28°C for 48 h. Pick single clones for PCR detection. Store positive Agrobacterium cells for later use.

[0050] III. Observation of Tobacco Transient Transformation and Subcellular Localization

[0051] Add 200 μl of the above-mentioned positive Agrobacterium-positive bacterial suspension to 20 mL of YEB liquid medium containing 50 µg / mL kanamycin and 50 µg / mL rifampicin, and incubate at 28 °C and 200 rpm with shaking for 12-16 h. Add 1 mL of the bacterial suspension to 100 mL of fresh YEB liquid medium containing 50 µg / mL kanamycin and 50 µg / mL rifampicin, and incubate under the same conditions with shaking for 8-12 h until the OD600 is approximately 0.8-1.2. Centrifuge at 4000 rpm for 5 min, discard the supernatant, and resuspend twice in a suspension prepared with 10 mL MMES, 10 mL MgCl2, and 100 µM MAS until the OD600 = 0.5. Incubate in the dark for 1-3 h for later use. One day in advance, the tobacco leaves were thoroughly watered. The bacterial solution was injected into the lower epidermis of the tobacco leaves using a syringe. After 24 hours of incubation in the dark at 23°C, the leaves were removed and cultured normally. In this experiment, the empty vector pCAMBIA1305-GFP Agrobacterium was used as a control. 48-72 hours after injection, the location of fluorescence in the epidermal cells of the tobacco leaves in the control and experimental groups was observed and photographed using a laser confocal microscope. Figure 5 The results showed that the control group GFP empty vector was fluorescent in both the cell membrane and nucleus of tobacco mesophyll cells, while the fluorescence signal of the 35S:BlWRKY15-GFP fusion protein was localized only in the nucleus of tobacco mesophyll cells, indicating that BlWRKY15 is localized in the nucleus, which is consistent with its functional characteristics as a transcription factor.

[0052] Example 4: Construction and genetic transformation of the BlWRKY15 gene overexpression vector

[0053] I. Construction of the BlWRKY15 gene overexpression vector

[0054] The plant overexpression vector pXHKFGuv was digested with restriction endonucleases SpeI and PmeI (Takara). The linearized vector fragment was recovered by 1% agarose gel electrophoresis. Homologous sequences of SpeI and PmeI were introduced at both ends of the upstream and downstream primers of the gene cloning to construct new primers: upstream primer P3: SEQ ID NO. 5, downstream primer P4: SEQ ID NO. 6. PCR amplification yielded the target gene fragment containing homologous sequences to both ends of the linearized vector. The target gene fragment was ligated to the linearized vector using the ClonExpress II One-Step Cloning Kit (Vazyme, Nanjing), following the manufacturer's instructions. After incubation at 37°C for 30 min, the ligation product was transformed into *E. coli* DH5α competent cells. Single clones were picked for colony PCR detection, and positive clones were sent for sequencing for further validation. The sequencing results were consistent with the gene cloning results, indicating that the BlWRKY15 gene was successfully inserted between the CaMV35S promoter and the OCS terminator in the overexpression vector pXHKFGuv. The recombinant vector was named 35S::BlWRKY15.

[0055] II. Agrobacterium-mediated transformation of birch

[0056] The constructed 35S::BlWRKY15 vector was transformed into Agrobacterium rhizogenes Arqual competent cells using the freeze-thaw method: 100 μl of Agrobacterium rhizogenes Arqual competent cells were thawed on ice, 4 µl of the 35S::BlWRKY15 recombinant plasmid was added, and the mixture was gently mixed. The cells were then placed on ice for 5 min, frozen in liquid nitrogen for 5 min, and then quickly placed in a 37°C water bath for 5 min, followed by an ice bath for 2 min. 700 μl of YEB liquid medium was added, and the cells were cultured at 28°C with shaking at 200 rpm for 3 h. The cells were collected by short centrifugation and spread on solid YEB plates containing spectinomycin (Spec) and rifampicin (Rif). The plates were incubated upside down at 28°C for about 48 h. Agrobacterium single clones were picked for PCR detection and sequencing verification.

[0057] Transfer the correctly sequenced Agrobacterium single clones to 400 mL of YEB liquid medium containing 50 μg / mL rifampin and 50 μg / mL spectinomycin, and culture at 28 °C with shaking at 200 rpm until the OD600 is between 0.6 and 0.8. Centrifuge at 5000 rpm for 15 min at 4 °C, collect the precipitate and resuspend the cells in resuspending buffer (1 / 2 MS + 20 g / L sucrose + 150 µMAS + 0.02% Tween-20, pH 5.8-6.0) until the OD600 is 0.4 to 0.6. Select birch tissue culture seedlings that are about 1-2 months old. Take the first and second leaves from the top, and make a cut perpendicular to the vein with a blade. Place the leaves in a suspension for 10-15 minutes. Remove the infected leaves, blot off excess bacterial solution with sterile filter paper, and invert the leaves in co-culture medium (1 / 2 MS + 20 g / L sucrose + 6 g / L agar, pH 5.8-6.0) for 3 days in the dark. After the dark culture, transfer the leaves to hairy root induction and selection medium (1 / 2 MS + Tim 200 mg / L + Kan 50 mg / L + 20 g / L sucrose + 6 g / L agar, pH 5.8-6.0) for hairy root induction under light conditions. After about 21 days, transfer the induced positive hairy roots to callus induction and selection medium (WP). Callus induction was performed using WPM (Wheat Powder) + 0.8 mg / L 6-BA + 0.02 mg / L NAA + 0.5 mg / L GA3 + 200 mg / L Tim + 50 mg / L Kan + 20 g / L sucrose + 6 g / L agar, pH 5.8-6.0. The callus tissue was then transferred to differentiation medium (WPM + 1 mg / L 6-BA + 200 mg / L Tim + 50 mg / L Kan + 20 g / L sucrose + 6 g / L agar, pH 5.8-6.0) for differentiation and selection. The differentiated seedlings were then transferred to rooting medium (1 / 2 MS + 0.2 mg / L NAA + 200 mg / L Tim + 50 mg / L Kan + 20 g / L sucrose + 6 g / L agar, pH 5.8-6.0) for root induction and selection.

[0058] III. Molecular Identification of Genetically Modified Birch

[0059] Gene expression levels in transgenic materials were analyzed. Quantitative primers were designed using Oligo software: upstream primer P7 (SEQ ID NO. 9) and downstream primer P8 (SEQ ID NO. 10), and real-time quantitative PCR was performed to analyze the expression level of the BlWRKY15 gene in wild-type and transgenic birch materials. Before performing real-time quantitative PCR, the designed quantitative primers and the internal reference gene Tubulin primers (upstream primer P9: SEQ ID NO. 11 and downstream primer P10: SEQ ID NO. 12) were tested, and the optimal annealing temperature (60℃) of the primers was analyzed. qRT-PCR was performed on a Bio-Rad CFX96 real-time quantitative PCR instrument using the SuperRealPreMixPlus (TIANGEN) kit, following the two-step method described in the instructions. The reaction program was: 95℃ pre-denaturation for 15 min; then 40 cycles of 95℃ for 10 s and 60℃ for 30 s. The expression level of the BlWRKY15 gene in transgenic materials and wild-type birch materials was analyzed. The results of qRT-PCR showed that the expression level of the BlWRKY15 gene in the transgenic materials was significantly higher than that in the control group. Figure 6 ).

[0060] Example 5: Phenotypic Analysis and Physiological Index Measurement of Transgenic Birch under Drought Stress

[0061] I. Fluorescence detection of transgenic plants

[0062] Wild-type birch and BlWRKY15-overexpressing transgenic birch were cultured under the same conditions (temperature 25±2℃, photoperiod 16h light / 8h dark, light intensity 2000Lux), and the fluorescence signal of the plants was observed under 365nm ultraviolet light. The results are as follows: Figure 7 As shown, no fluorescence was observed in the wild-type material, while obvious green fluorescence was observed in the BlWRKY15 overexpression material, indicating normal expression of the reporter gene and further confirming the successful acquisition of transgenic plants.

[0063] II. Treatment of Natural Drought Stress

[0064] Thirty wild-type birch and 30 transgenic birch plants (T2 generation positive lines) of uniform growth status were selected and divided into a control group and a drought treatment group, with 15 plants in each group. The control group was managed with normal watering, while the drought treatment group was subjected to natural drought stress treatment with watering stopped. Phenotypic changes of the plants were observed and recorded before treatment (day 0) and on day 10 after treatment, and leaf samples were collected for physiological index determination.

[0065] III. Phenotypic Observation

[0066] Without stress treatment (0 days), there were no significant differences between wild-type and transgenic birch in terms of plant height, leaf morphology, and color. After 10 days of natural drought treatment, the transgenic lines showed significantly more severe wilting than the wild-type, with obvious water loss, wrinkling, and yellowing of the leaves, while the wild-type only showed slight leaf drooping. Figure 8 ).

[0067] IV. Electrolyte Leakage Rate Measurement

[0068] Mature leaves from wild-type and transgenic birch were collected before drought treatment (day 0) and 10 days after drought treatment. After washing with deionized water, leaf discs of the same size were taken using a perforator and placed in centrifuge tubes containing 10 mL of deionized water. The samples were incubated at 25°C with shaking for 2 hours, and the initial conductivity (EC1) was measured using a conductivity meter. The samples were then heated in a boiling water bath for 15 minutes, cooled to room temperature, and the total conductivity (EC2) was measured. Electrolyte leakage rate (%) = (EC1 / EC2) × 100%. Results are as follows: Figure 8 As shown, under normal conditions, all plants exhibited similar electrolyte leakage rates; under drought stress, the electrolyte leakage rate of transgenic lines was significantly higher than that of wild-type, indicating that transgenic lines suffered more severe cell membrane damage and were more sensitive to drought.

[0069] V. Determination of Malondialdehyde (MDA) Content

[0070] Leaf samples of wild-type and transgenic birch were collected 0.2 g before drought treatment (day 0) and 10 days after drought treatment. 2 mL of 10% trichloroacetic acid (TCA) solution was added, and the mixture was homogenized in an ice bath. The homogenate was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. An equal volume of 0.67% thiobarbituric acid (TBA) solution was added, and the mixture was boiled in a boiling water bath for 15 min. After rapid cooling, the mixture was centrifuged at 4000 rpm for 10 min, and the absorbance of the supernatant was measured at 450 nm, 532 nm, and 600 nm. The MDA content was calculated using the formula. The results are as follows: Figure 8 As shown, under normal conditions, there was no significant difference in MDA content among all plants; under drought stress, the MDA content of transgenic lines was significantly higher than that of wild-type, indicating that transgenic lines experienced higher levels of membrane lipid peroxidation and more severe oxidative damage under drought stress.

[0071] VI. Peroxidase (POD) Activity Assay

[0072] Leaf samples of wild-type and transgenic birch were collected before drought treatment (day 0) and 10 days after drought treatment, respectively. 2 mL of 0.1 mol / L phosphate buffer (pH 7.0) was added, and the samples were homogenized in an ice bath. The homogenate was centrifuged at 4000 rpm for 10 min, and the supernatant was used as the crude enzyme extract. POD activity was determined using the guaiacol method: the reaction system contained 2.9 mL of 0.1 mol / L phosphate buffer (pH 7.0), 1.0 mL of 0.2% guaiacol, 1.0 mL of 0.3% H₂O₂, and 0.1 mL of the crude enzyme extract. After mixing, the absorbance was immediately measured at 470 nm. One unit of enzyme activity (U) was defined as a change in absorbance of 0.01 μL per minute. Results are as follows: Figure 8 As shown, under normal conditions, there was no significant difference in POD activity among all plants; under drought stress, the POD activity of the transgenic lines was significantly lower than that of the wild type, indicating that the transgenic lines had a weaker ability to scavenge reactive oxygen species under drought stress and their antioxidant defense system was more severely damaged.

[0073] The results of the above measurements of electrolyte leakage rate, MDA content, and POD activity consistently indicate that BlWRKY15 overexpression enhances the sensitivity of birch to drought stress, meaning that BlWRKY15 negatively regulates the drought tolerance of plants. Figure 8 ).

[0074] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A BlWRKY15 protein from *Betula sclerophylla*, characterized in that, The amino acid sequence of the *Betula sclerophylla* BlWRKY15 protein is either (1) or (2) below: (1) The amino acid sequence as shown in SEQ ID NO. 2; (2) A protein that has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.2 and has the function of regulating plant drought stress response.

2. A BlWRKY15 gene from *Betula sclerophylla*, characterized in that, The *Betula glabra* BlWRKY15 gene encodes the *Betula glabra* BlWRKY15 protein of claim 1, and its nucleotide sequence is any one of the following (3) or (4): (3) The nucleotide sequence as shown in SEQ ID NO. 1; (4) A DNA molecule that has more than 90% sequence identity with the nucleotide sequence shown in SEQ ID NO.1 and encodes the amino acid sequence shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the BlWRKY15 gene of *Betula pulcherrima* as described in claim 2.

4. Host cells containing the *Betula lucida* BlWRKY15 gene as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The application of the BlWRKY15 protein of *Betula glabra* as described in claim 1 or the BlWRKY15 gene of *Betula glabra* as described in claim 2 in regulating plant drought stress response.

6. A method for regulating plant drought tolerance, characterized in that, The method includes the step of introducing the BlWRKY15 gene of *Betula glabra* as described in claim 2 into a plant to overexpress it, thereby obtaining a transgenic plant with altered drought resistance.

7. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is an overexpression vector obtained by inserting the nucleotide sequence shown in SEQ ID NO. 1 between the CaMV35S promoter and the OCS terminator of the plant expression vector pXHKFGuv.

8. The host cell according to claim 4, characterized in that, The host cell is an Agrobacterium cell, preferably Agrobacterium rhizogenes Arqual.

9. The application according to claim 5, characterized in that, The plant in question is a birch species, preferably Betula platyphylla or Betula platyphylla var. glabra.

10. The method according to claim 6, characterized in that, Includes the following steps: S01. Construct an overexpression vector containing the nucleotide sequence shown in SEQ ID NO. 1; S02. Transform the overexpression vector into Agrobacterium to obtain a transgenic engineered bacterium; S03. Transform plant recipients using the aforementioned transgenic engineered bacteria and screen for transgenic positive plants.

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