WRKY transcription factor gene StWRKY60 and application thereof

Abstract

This invention discloses the WRKY transcription factor gene StWRKY60 and its applications, belonging to the field of biotechnology. The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2. The StWRKY60 gene provided by this invention can not only be used to improve the drought resistance of potato varieties, but also, through genetic engineering technology, be applied to drought-resistant breeding of other crops (such as tomatoes, tobacco, and rice), providing important genetic resources and effective technical means for addressing global drought and ensuring food security.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the WRKY transcription factor gene StWRKY60 and its applications. Background Technology

[0002] With the intensification of global climate change, drought has become one of the major abiotic stresses restricting agricultural production and affecting food security. Exploring the stress-resistance gene resources within plants and cultivating drought-resistant new varieties through genetic engineering is an effective strategy to address this challenge.

[0003] WRKY transcription factors are a family of plant-specific transcriptional regulatory proteins that are widely involved in plant responses to biotic stresses (such as pathogen infection) and abiotic stresses (such as drought, high salinity, and low temperature). They play a core regulatory role in stress response networks by specifically binding to W-box cis-elements in the promoter regions of their conserved WRKY domains. Currently, studies have reported the functions of WRKY genes in multiple species. For example, ZmWRKY106 in maize has been shown to positively regulate drought resistance; inactivation of OsWRKY5 in rice may contribute to improved drought resistance. However, the functions of WRKY transcription factors exhibit high species specificity and functional diversity; the same WRKY gene may play drastically different roles in different species.

[0004] As a vital global food crop, potatoes are highly susceptible to drought stress in both growth and yield. Therefore, investigating the potato WRKY transcription factor gene StWRKY60 and clarifying its role in plant stress resistance physiology is of great significance for revealing the molecular mechanisms of drought resistance in potatoes and for improving crop drought resistance through genetic engineering. Summary of the Invention

[0005] The purpose of this invention is to provide the WRKY transcription factor gene StWRKY60 and its applications to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention is the application of the STWRKY60 gene or its encoded protein in improving the resistance of plants to drought stress. The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0008] The second technical solution of this invention is the application of recombinant vectors, expression cassettes, or transgenic lines containing the STWRKY60 gene in improving plant resistance to drought stress.

[0009] The third technical solution of the present invention is a method to improve the resistance of plants to drought stress by overexpressing the STWRKY60 gene or increasing the level of its encoded protein to improve the resistance of plants to drought stress.

[0010] The fourth technical solution of the present invention is the application of the STWRKY60 gene or its encoded protein in the cultivation of new drought-resistant plant varieties. The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] This invention constructs transgenic potato plants overexpressing StWRKY60 and subjects them to drought stress. The results show that, compared to wild-type plants, the transgenic plants exhibit a slower rate of water loss, higher relative leaf water content, stronger survival rate, and less oxidative damage (such as lower H2O2 content and higher antioxidant enzyme activity). After rehydration following drought stress, the transgenic plants also demonstrate significantly better recovery growth than the wild-type plants.

[0013] The StWRKY60 gene provided by this invention can not only be used to improve the drought resistance of potato varieties, but also be applied to drought resistance breeding of other crops (such as tomatoes, tobacco, rice, etc.) through genetic engineering technology, providing important genetic resources and effective technical means for coping with the global drought environment and ensuring food security. Attached Figure Description

[0014] Figure 1 This is a clone of the potato StWRKY60 gene. Note: M: DL 2000 DNA marker; 1: target fragment.

[0015] Figure 2 Double enzyme digestion validation of the overexpression vector pC2300S-StWRKY60. Note: M1: DL 2000 DNA marker; 1, 2: Kpn I-BamHI digested plasmid; M2: DL 15000 DNA marker.

[0016] Figure 3 The genetic transformation of potatoes. Among them, A: co-culture of "AC142" potato chips; B: "AC142" callus; C: shoot differentiation from overexpressed callus; D: rooting and screening of transformed plants using "AC142".

[0017] Figure 4PCR identification of transgenic plants. Note: M: 2000 marker; −: DNA from “AC142” plant was a negative control; +: pC2300S-GFP empty vector was a positive control; 1-3: StWRKY60 overexpression lines.

[0018] Figure 5 The expression level of the StWRKY60 gene in transgenic plants. Note: WT: wild-type plant; OE-n: overexpression line; error represents standard deviation, *: P<0.05, **: P<0.01.

[0019] Figure 6 Phenotypic analysis of transgenic and wild-type plants treated with PEG6000. Where: A: Growth status of each line under normal conditions, scale bar: 5 cm; B: Growth status of each line under PEG treatment, scale bar: 5 cm; C: Plant height; D: Root length; E: Root fresh weight; F: Root dry weight; G: Stem and leaf fresh weight; H: Stem and leaf dry weight. Error represents standard deviation, *: P < 0.05, **: P < 0.01.

[0020] Figure 7 To analyze the drought tolerance of overexpression lines under normal conditions and after drought treatment. A: Phenotypic observation between StWRKY60 transgenic plants and wild-type plants under drought treatment, scale bar: 10 cm; B: Relative water content of detached leaves; C: Water loss rate of detached leaves. Error represents standard deviation, **: P < 0.01.

[0021] Figure 8 This study analyzed physiological indicators of transgenic and wild-type potato plants under natural drought treatment. A: POD activity; B: SOD activity; C: CAT activity; D: H2O2 content; different lowercase letters indicate significant differences (P < 0.05); n = 3. Detailed Implementation

[0022] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0023] This invention provides the application of the STWRKY60 gene or its encoded protein in improving plant resistance to drought stress. The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0024] In some specific implementations, overexpression of the STWRKY60 gene or increasing the level of its encoded protein can enhance plant resistance to drought stress.

[0025] In some specific implementations, the plant includes potatoes.

[0026] This invention also provides the application of recombinant vectors, expression cassettes, or transgenic lines containing the STWRKY60 gene in improving plant resistance to drought stress.

[0027] This invention also provides a method for improving plant resistance to drought stress by overexpressing the STWRKY60 gene or increasing the level of its encoded protein.

[0028] In some specific implementations, the plant includes potatoes.

[0029] In some specific embodiments, the nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0030] This invention also provides the application of the STWRKY60 gene or its encoded protein in the breeding of new drought-resistant plant varieties. The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0031] In some specific implementations, the plant includes potatoes.

[0032] In some specific implementations, overexpression of the STWRKY60 gene or increasing the level of its encoded protein can enhance plant resistance to drought stress.

[0033] The invention of the WRKY transcription factor gene StWRKY60 and its applications includes the following steps:

[0034] (1) Cloning of StWRKY60;

[0035] (2) Construction and double enzyme digestion verification of the StWRKY60 overexpression vector;

[0036] (3) Agrobacterium-mediated genetic transformation to obtain overexpressing transgenic plants;

[0037] (4) Identification of overexpressing transgenic plants;

[0038] (5) Analysis of the relative expression level of StWRKY60 in overexpressing transgenic plants;

[0039] (6) Analysis of drought tolerance of transgenic plants treated with 20% PEG-6000;

[0040] (7) Determination of physiological and biochemical indicators of transgenic potatoes under natural drought treatment.

[0041] Example 1

[0042] 1. Experimental Materials

[0043] 1.1 Plant materials

[0044] The tissue culture seedlings of the wild-type potato variety “AC142” were preserved and provided by the Potato Biotechnology Innovation Team of the College of Life Science and Technology, Gansu Agricultural University.

[0045] 1.2 Strains and Vectors

[0046] DH5α Escherichia coli competent cells and GV3101 Agrobacterium competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the expression vector pC2300S was preserved and provided by the Potato Biotechnology Innovation Team of the College of Life Science and Technology, Gansu Agricultural University.

[0047] 2 Experimental Methods

[0048] 2.1 Cultivation of Plant Materials

[0049] Wild-type potato variety “AC142” tissue culture seedlings were inoculated onto MS solid medium (containing 3% sucrose). The culture conditions were (23±2)℃, 16 h light and 8 h dark. After culturing in a light-temperature incubator for 4 weeks, leaves of the tissue culture seedlings were collected, flash-frozen in liquid nitrogen and stored at -80℃ for subsequent experiments.

[0050] 2.2 Cultivation of Aseptic Mini-tubers

[0051] Tissue culture seedlings of the wild-type potato variety "AC142" were inoculated onto MS solid medium (containing 8% sucrose). The culture conditions were set at (23±2)℃, 16 h light, 8 h darkness in a light-temperature incubator for 4 weeks. Then, they were transferred to a completely dark environment at (23±2)℃ for 2-3 months.

[0052] 2.3 Potato RNA Extraction and cDNA Synthesis

[0053] Total RNA was extracted from potato tissue culture seedling leaves using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit, and then reverse transcribed into cDNA using the SweScript All-in-One RT SuperMix for qPCR Reverse Transcription Kit. The cDNA was stored at -20°C for later use.

[0054] 2.4 Cloning of the StWRKY60 gene

[0055] The CDS region of the StWRKY60 gene was cloned using cDNA from leaves of the wild-type potato variety "AC142" as a template. Specific amplification primers for the StWRKY60 gene overexpression vector were designed using the online website Takara Biomed (https: / / www.takarabiomed.com.cn / ). The primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 1.

[0056] Table 1. PCR primers for cloning the StWRKY60 gene.

[0057]

[0058] The PCR reaction system consisted of 10 µL of 2×Mix, 1 µL each of the upstream and downstream primers, 2 µL of cDNA template, and ddH2O to bring the total volume to 20 µL.

[0059] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 15 s, for 34 cycles; final extension at 72℃ for 5 min.

[0060] After the amplification reaction was completed, the amplification products were detected by electrophoresis on a 1.0% agarose gel, and fragments with the same size as the expected target band were recovered. The products were then stored at -20°C for later use.

[0061] The CDS region sequence of the potato StWRKY60 gene is 762 bp long. Using leaf cDNA from the wild-type potato variety "AC142" as a template, the CDS region sequence of the StWRKY60 gene was amplified. The amplified product was detected by 1.0% agarose gel electrophoresis (120V, 25 min), showing a specific band of the expected size at approximately 762 bp. Figure 1 This indicates that the potato StWRKY60 gene has been successfully cloned. The sequence of StWRKY60 is shown in SEQ ID NO.1, and the sequence of the protein it encodes is shown in SEQ ID NO.2.

[0062] SEQ ID NO.1:

[0063] SEQ ID NO.2: MGNKSFFIDLNTNPLLHNINRSPIPRETLDEELSRMRKENKKLATTLTSLCEKYNSLQTHIIELQQKYSTHEEDNSKLLLSRKRKAEEEYCVNNSYINIEEASPKRPREIITNISTVCVKTSPS DQTSVVKDGYNWRKYGQKVTRDNPSPRAYFKCSFAPTCPVKKKVQRSVKDASILVATYEGEHNHPQPSQAEITVPLVNASDPTFLNKFMEDINTNSLQQQYLVEQMASSLSKNPSFAATVATAISGLLF.

[0064] 2.5 Construction of potato StWRKY60 overexpression vector

[0065] 2.5.1 Linearization of the overexpression vector pC2300S

[0066] The pC2300S empty vector strain, frozen at -80℃, was thawed in an ice bath and then inoculated into 50 mL LB liquid medium containing 50 µL Kan (50 µg / mL), and cultured in a shaker at 37℃ and 250 rpm for 12 h. The pC2300S empty vector plasmid was extracted using a plasmid miniprep kit. Double digestion with Kpn I (5' end) and BamHI (3' end) restriction endonucleases was performed. The reaction mixture consisted of: 1 μL Kpn I, 1 μL BamHI, 2 μL 10×Quick Cut Buffer, 4 μL pC2300S plasmid, and ddH2O to a final volume of 20 μL. After preparation on ice, the mixture was briefly centrifuged and then digested in a PCR instrument at 37℃ for 40 min. After separation of the enzyme digestion products by 1.0% agarose gel electrophoresis, the large fragments were collected after gel irradiation and placed in 1.5 mL centrifuge tubes. The target fragments were purified using a DNA gel recovery kit (Beijing Jinsha Biotechnology Co., Ltd.). The recovered products were stored at -20℃ for later use.

[0067] 2.5.2 Homologous recombination of the target gene and the overexpression linearized vector

[0068] The StWRKY60 target gene recovered in section 2.4 and the linearized pC2300S empty vector fragment recovered in section 2.5.1 were subjected to homologous recombination reaction using the Solarbio ClonExpress® Ⅱ One Step Cloning Kit homologous recombination kit. After loading the sample onto ice, the mixture was centrifuged at low speed. The homologous recombination reaction mixture consisted of 5 µL of homologous recombinase, 3 µL of the StWRKY60 target gene, and 2 µL of the linearized pC2300S empty vector. The reaction was incubated at 50°C for 30 min, and then placed on ice for later use.

[0069] 2.5.3 Transformation of Escherichia coli with recombinant plasmid pC2300S-StWRKY60

[0070] Remove DH5α competent E. coli cells from a -80℃ freezer and incubate on ice until partially thawed. Add 10 μL of pC2300S-StWRKY60 recombinant plasmid to 100 μL of DH5α competent cells. First, incubate on ice for 30 min, then heat shock at 42℃ for 45 s, followed by incubation on ice for 2 min. Then, add 700 μL of antibiotic-free LB liquid medium and incubate at 37℃ and 230 rpm for 1 h. After incubation, remove the cells, centrifuge at 6000 rpm for 4 min, discard the 600 µL supernatant, resuspend the cells, and spread 100 μL onto bacterial culture dishes containing LB solid medium containing Kan (50 µg / mL) antibiotic. Incubate upside down at 37℃ for 12–14 h.

[0071] White, uniformly grown monoclonal colonies with smooth, intact edges were selected and placed in LB broth containing Kan (50 µg / mL) and cultured at 37°C and 225 rpm for 6 h with shaking. Using the cultured bacterial suspension as a template, the reaction system and conditions were as described in 2.4. After 1.0% agarose gel electrophoresis, positive clones with band sizes matching the expectations were selected and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were compared with the target gene sequence using DNAMAN 9.0 software. Strains with confirmed sequences were stored at -80°C. Recombinant plasmids from the positive strains were extracted using a plasmid miniprep kit and double-digested with Kpn I (5' end) and BamHI (3' end) (reaction system and conditions as described in 2.5.1). The digestion products were detected by 1.0% agarose gel electrophoresis. Plasmids with correct fragment size and length were named the overexpression vector pC2300S-StWRKY60.

[0072] The StWRKY60 gene was amplified using the specific primers designed in section 2.4. Using homologous recombination, a pC2300S-StWRKY60 overexpression vector was constructed using the StWRKY60 gene fragment and the pC2300S linear expression vector. Subsequent verification was performed by bacterial PCR and sequencing by Shanghai Sangon Biotech. The recombinant plasmid was then double-digested with restriction endonucleases Kpn I and BamHI for further validation. Figure 2 The results showed fragments at both ends, with the smaller fragment being approximately 762 bp. The correct recombinant plasmid was named pC2300S-StWRKY60.

[0073] 2.5.4 Recombinant overexpression vector pC2300S-StWRKY60 transformed into Agrobacterium

[0074] Remove GV3101 Agrobacterium competent cells from the -80℃ freezer and incubate on ice until partially thawed. Add 10 μL of pC2300S-StWRKY60 recombinant plasmid to 100 μL of competent cells, gently vortex to mix, and then perform the following steps sequentially: ice incubation for 5 min, liquid nitrogen for 5 min, 29℃ water bath for 5 min, and ice incubation for 5 min. Add 500 μL of antibiotic-free LB liquid medium and incubate at 29℃ and 230 rpm for 2-3 h with shaking. After shaking culture, remove the culture and centrifuge at 6000 rpm for 4 min. Discard 400 μL of supernatant, resuspend the bacterial culture, and spread 100 μL of the bacterial culture onto LB agar plates containing Rif (50 μg / mL) and Kan (50 μg / mL). Incubate at 28℃ upside down for 2-3 h. d; Select white monoclonal colonies with smooth, intact edges and uniform growth, and place them in 10 mL LB liquid medium containing Rif (50 μg / mL) and Kan (50 μg / mL). Incubate at 29°C with shaking for 12–16 h, and then perform PCR identification using the bacterial culture as a template. Verify the target band by 1.0% agarose gel electrophoresis. Positive clones are stored at -80°C.

[0075] 2.5.5 Genetic transformation of the potato StWRKY60 gene

[0076] Add 1 mL of Agrobacterium tumefaciens overexpression vector to 5 mL of LB liquid medium containing 2 μL Rif (50 mg / L) and 5 μL Kan (50 mg / L), and incubate with shaking in a constant temperature shaker at 28℃ and 220 r / min for about 10-12 h. Take a fresh 50 mL LB liquid medium (containing the same hormones), add 1 mL of the above activated Agrobacterium tumefaciens culture, and incubate with shaking in a constant temperature shaker at 28℃ and 220 r / min until OD (excessive oxidative stress) is reached. 600 The value is 0.5; OD 600The bacterial culture with a concentration of 0.5 was transferred to a 50 mL sterile centrifuge tube and centrifuged for 7 min at 4℃ and 5000 r / min. The supernatant was poured into the waste liquid container, and the bacterial precipitate was retained and resuspended in 50 mL of 3% sucrose liquid MS medium.

[0077] Remove the induced potatoes from the laminar flow hood, remove the skin and eyes of the induced potatoes with a sterile scalpel, and cut them into potato slices with a thickness of about 2 mm and a radius of 5 mm. Then transfer the cut potato slices to 50 mL of liquid MS medium with a sucrose content of 3% containing bacteria for infection. During the infection process, shake continuously in the same direction for 6-7 minutes. Pour the infected potato slices onto filter paper, gently wipe the surface of the potato slices dry with sterile filter paper, and place them on solid MS medium with a sucrose content of 3% supplemented with 100 μL 1 mg / L IAA, 20 μL 0.2 mg / L GA3, 50 μL 0.5 mg / L 6-BA, and 100 μL 2 mg / L ZT. Co-culture at 28°C in the dark for 2 days.

[0078] After co-culturing, the Agrobacterium on the surface of the potato chips was wiped clean and transferred to MS differentiation medium with a sucrose content of 3% (100 μL 1 mg / L IAA, 20 μL 0.2 mg / L GA3, 50 μL 0.5 mg / L 6-BA, 100 μL 2 mg / L ZT, and 75 μL 200 mg / L Cef). The medium was placed in a constant temperature incubator at (23±2)℃, light intensity of 2500 Lx, with a 16-h light-8-h dark cycle. The medium was changed according to the growth of the potato chips. After callus formation, the culture was continued until the differentiated shoots reached 1.5-2 cm in length. The differentiated shoots were then cut off using a sterile scalpel and aseptically transferred to rooting medium supplemented with 20 μL Hyg (50 mg / L) and 50 μL Cef (200 mg / L) for root selection. Rooting selection was performed in approximately 7-10 days. If, after d, the differentiated buds are observed to be able to take root, it can be preliminarily determined that the transgenic plant has been successfully cultivated.

[0079] Induced potatoes obtained from tissue culture seedlings of the wild-type potato variety "AC142" were skinned and had their eyes removed in a clean bench. The induced potatoes were then sliced ​​into 1-2 mm thick slices. The slices were then inoculated with Agrobacterium tumefaciens solution containing the pC2300S-StWRKY60 overexpression vector for 7 min, and then transferred to a co-culture medium and cultured in the dark for 2 days. Figure 3 (A) After wiping the bacterial solution off the surface of the potato tubers, place them on the differentiation medium for further culture. Figure 3 (B) Wait for the potato chips to form callus tissue and continue culturing until sprouts differentiate ( Figure 3(C) When the differentiated buds grow to 1.5-2 cm, the differentiated buds are gently cut off and inoculated into a rooting selection medium containing hygromycin and thiazomycin for rooting selection. Plants that have rooted after three selections are preliminarily identified as transgenic plants.

[0080] 2.5.6 Identification of transgenic plants

[0081] The NPT II (neomycin phosphotransferase) screening marker gene on the plant expression vector pC2300S was used as the identification gene. The universal primer sequences for the NPT II screening marker gene are: NPT II-F: GCTATGACTGGGCACAACAG, NPT II-R: ATACCGTAAAGCACGAGGAA. Genomic DNA was extracted from transgenic plants and wild-type plants that maintained normal rooting ability after three consecutive generations of antibiotic selection using a plant genomic DNA extraction kit. Wild-type plant genomic DNA was used as a negative control template, and pC2300S empty vector plasmid DNA was used as a positive control template for subsequent PCR detection. A NPT II band of approximately 676 bp was amplified using specific primers NPT II-F and NPT II-R. The extracted DNA was used as a template, and the reaction system and conditions were as described in 2.4. The PCR amplification products were detected by 1% agarose gel electrophoresis. Plants with a bright fluorescent band at 676 bp were named OE-n. The successfully identified transgenic plants overexpressing StWRKY60 were preserved and propagated for subsequent experiments.

[0082] Genomic DNA was extracted from potato variety “AC142” and transgenic plants using a plant genomic DNA extraction kit. Using the extracted DNA as a template, “AC142” plant DNA as a negative control, and the pC2300S empty vector as a positive control, reporter genes were amplified. NPT II. Results as follows Figure 4 As shown, the genomic DNA of the transgenic plant was successfully amplified to a fragment approximately 676 bp in size. NPT Band Ⅱ was observed, while the genomic DNA of the "AC142" plant showed no band. Based on the order from left to right, 1, 2, and 3 were named OE-1, OE-2, and OE-3, respectively.

[0083] 2.5.7 qRT-PCR expression analysis of StWRKY60 in overexpressing plants

[0084] Transgenic and wild-type plants were cultured in 3% MS medium for 3 weeks. Total RNA was extracted from both plants and reverse transcribed into cDNA. qPCR primers were designed using NCBI Primer-BLAST (primer sequences are shown in Table 2). Using wild-type plants as a control, the relative expression level of the StWRKY60 gene in transgenic potato plants was detected by qRT-PCR.

[0085] Table 2 qPCR primers

[0086]

[0087] 20 μL reaction system: 10 μL of 2×Universal Blue SYBR Green qPCR Master Mix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template, and 7.2 μL of Nuclease-Free Water.

[0088] qRT-PCR reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, for 40 cycles.

[0089] Each treatment was configured with three biological replicates, using the potato StEF1α gene as an internal control gene, and employing 2 -△△Ct The expression level of StWRKY60 in potatoes was calculated using a method.

[0090] To investigate the expression differences of the StWRKY60 gene in wild-type and transgenic plants, wild-type plants and StWRKY60 overexpressing transgenic lines were used as materials. Total RNA was extracted from the plants and reverse transcribed into cDNA. The transcriptional level of the StWRKY60 gene in wild-type and transgenic plants was analyzed using qRT-PCR. Quantitative analysis results are as follows: Figure 5 As shown, the transcriptional level of StWRKY60 in the overexpressing transgenic lines exhibited a significant upregulation trend, with the relative expression levels in the three transgenic lines reaching 5.3-fold, 3.3-fold, and 6.9-fold compared to the wild type, respectively. These results indicate that plants overexpressing the StWRKY60 gene were successfully obtained.

[0091] 2.5.8 Analysis of drought tolerance in transgenic plants treated with 20% PEG-6000

[0092] 2.5.8.1 Treatment of plant materials

[0093] (1) PEG treatment: The selected OE-n transgenic lines and wild-type plants containing one axillary bud were inoculated into MS liquid medium with a sucrose content of 3% and placed in a temperature of (23±2)℃, a light intensity of 2500 Lx, and a light-dark condition of 16 h for 8 h for one week. All the original liquid medium was poured out and replaced with new MS liquid medium with a sucrose content of 3% containing 20% ​​PEG-6000. After three weeks of stress culture, the phenotypic analysis of the tissue culture seedlings was performed.

[0094] (2) Natural drought treatment: The screened OE-n transgenic lines and wild-type plants were placed in an incubator with a temperature of (23±2)℃, a light intensity of 2500 Lx, and a light-to-dark cycle of 16 h for 8 h for three weeks. Three tissue culture seedlings with good growth and uniform height from each line were selected for natural drought treatment. The tissue culture seedlings were removed from the conical flasks, and the culture medium remaining on the roots was rinsed off. Peat moss and vermiculite were mixed at a ratio of 3:1, and the relative soil moisture content was kept above 75%. The mixture was then divided into 5 cm × 5 cm flower pots and grown to about 10 cm in an environment of (23±2)℃. The seedlings were then transplanted into 15 cm × 15 cm flower pots and grown at room temperature. Watering was done every two days with 200 mL of water each time. Watering was stopped when the plant height reached about 35 cm, and the relative soil moisture content was kept at (25±5)%.

[0095] 2.5.8.2 Phenotypic Analysis

[0096] (1) Phenotypic identification of transgenic lines treated with 20% PEG-6000. Three plants of uniform growth status were randomly selected from each line, and the following parameters were measured: plant height (cm) and root length (cm) were measured using vernier calipers; biomass indicators: fresh weight of stems and leaves (g) and fresh weight of roots (g) were weighed using an electronic balance. Before measurement, the plants were pretreated. The tissue culture seedlings were completely removed from the conical flasks, and the culture medium attached to the roots was gently rinsed with distilled water. The surface moisture was absorbed with clean filter paper, and the fresh weight was weighed immediately. The plants were cut short from the base, and the fresh weight of stems and leaves, fresh weight of roots, dry weight of stems and leaves, and dry weight of roots were weighed using an electronic balance (after weighing, the stems and leaves and roots were placed in marked foil bags and dried in an oven at 60℃ until constant weight). This experiment was repeated three times.

[0097] (2) When the plants grow to about 35 cm in the flowerpot, the wild-type plants and the transgenic plants (three lines) are divided into two groups. The growth status of different plants is recorded by taking pictures. Then, watering is stopped. The first group is the control group, and the second group is subjected to natural drought treatment for 14 days. The phenotypic changes of the transgenic plants before and after treatment are observed.

[0098] 2.5.8.3 Determination of physiological and biochemical indicators of transgenic potatoes under natural drought treatment:

[0099] (1) Determination of water loss rate of detached leaves: The fourth to fifth layer of leaves of transgenic lines and wild-type control plants were selected. Three leaves were collected from each line and their fresh weight (W1) was quickly measured. The weight of the leaves after water loss (W2) was measured every hour for 8 hours. Water loss rate of detached leaves = W1-W2 / W1×100%.

[0100] (2) Determination of relative moisture content: The third to fourth layers of leaves were collected from transgenic plants and wild-type potato plants undergoing natural drought experiments, with 3 leaves collected from each line. Fresh weight (FW) determination: The initial weight (g) of fresh leaves was weighed immediately; Saturated fresh weight (SW) determination: The leaves were completely immersed in deionized water, left to stand for 7 h, and then the surface moisture was absorbed with filter paper and weighed (g); Dry weight (DW) determination: The water-saturated leaves were sealed in pre-labeled aluminum foil bags and placed in a 70℃ oven for continuous drying for 48 h until constant weight, and the dry weight (g) was weighed. RWC calculation: The relative moisture content was calculated according to the formula: RWC (%) = (SW-DW) / (FW-DW) × 100%.

[0101] (3) Determination of peroxidase (POD) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, and hydrogen peroxide (H2O2) content: Leaves were collected from the control group and potted seedlings used in the natural drought experiment and PEG experiment. The second and third layers of whole leaves were collected from each plant, with 3 leaves collected from each line. The determination of POD activity, SOD activity, CAT activity, and H2O2 content were all performed according to the instructions of the test kit (Shanghai Youxuan Biotechnology Co., Ltd.).

[0102] Analysis of drought tolerance in overexpressing plants under 20% PEG-6000 treatment: This invention simulated drought stress in transgenic lines overexpressing StWRKY60 and wild-type control groups using 20% ​​PEG-6000. Phenotypic observation showed that under normal culture conditions, there were no significant phenotypic differences between the transgenic lines and wild-type plants; however, under drought stress conditions, they showed obvious morphological differences. Figure 6 (A and B). Systematic measurement of plant morphological parameters revealed that, compared to wild-type plants, the overexpression of StWRKY60 transgenic lines showed greater increases in plant height and root extension. Figure 6 (C, D). Furthermore, the roots and stems / leafs of the plants were weighed, and the results showed that the fresh weight and dry weight of the roots of the overexpressing plants were both higher than those of the wild-type plants. Figure 6 In the middle E and F groups, the fresh weight and dry weight of stems and leaves of the overexpressing plants were also higher than those of the wild-type plants. Figure 6 G, H).

[0103] Determination of physiological and biochemical indicators of transgenic plants under natural drought treatment: To further understand the function of the StWRKY60 gene under drought conditions, one-month-old StWRKY60 overexpressing plants and wild-type potted seedlings were subjected to natural drought treatment for 14 days. Before drought treatment, there was no significant difference in the growth status of StWRKY60 overexpressing plants and wild-type plants. However, after 14 days of drought treatment and 2 days of rehydration, the leaves of StWRKY60 overexpressing plants grew well, while wild-type plants showed varying degrees of leaf wilting and more dead leaves. Figure 7 (A). Measurements of leaf relative water content after 7 days of drought treatment revealed that the RWC of wild-type plants was 76.85%, while the RWC of StWRKY60-overexpressing plants were 92.5%, 94.38%, and 96.05%, respectively. The RWC of StWRKY60-overexpressing plants was significantly higher than that of wild-type plants. Figure 7 (B). Furthermore, by measuring the water loss rate of detached leaves, we found that the water loss rate of detached leaves from StWRKY60-overexpressing plants was lower than that from wild-type plants (B). Figure 7 (C). The results showed that StWRKY60 overexpression in plants increased the relative water content of leaves, thereby improving the drought resistance of the plants.

[0104] To elucidate the potential mechanisms leading to phenotypic differences, further analysis was conducted using physiological methods. Four key physiological parameters—POD, SOD, CAT activities, and H2O2 content—were systematically measured under untreated, natural drought stress, and 20% PEG-6000 simulated drought stress conditions. Comparison of these physiological and biochemical parameters revealed that under untreated conditions, there were no significant differences in POD, SOD, and CAT activities between transgenic and wild-type plants. However, under drought stress conditions, the activities of all three antioxidant enzymes in the transgenic lines were increased to varying degrees compared to the wild type, while H2O2 accumulation was decreased. These results reveal the stress tolerance mechanism of the transgenic plants from the perspective of redox homeostasis.

[0105] In summary, this invention, by constructing transgenic potato plants overexpressing StWRKY60 and subjecting them to drought stress, found that the transgenic plants exhibited a slower rate of water loss, higher relative leaf water content, stronger survival rate, and less oxidative damage (such as lower H2O2 content and higher antioxidant enzyme activity) compared to wild-type plants. After rehydration following drought stress, the transgenic plants also demonstrated significantly better recovery growth than the wild-type plants.

[0106] The StWRKY60 gene provided by this invention has broad application prospects: It can not only be used to improve the drought resistance of potato varieties, but also be applied to drought resistance breeding of other crops (such as tomatoes, tobacco, rice, etc.) through genetic engineering technology, providing important genetic resources and effective technical means to cope with the global drought environment and ensure food security.

[0107] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of the STWRKY60 gene or its encoded protein in improving plant resistance to drought stress, characterized in that, The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; Overexpression of the STWRKY60 gene or increase in the level of its encoded protein can improve plant resistance to drought stress. The plant in question is a potato.

2. The application of recombinant vectors, expression cassettes, or transgenic lines containing the STWRKY60 gene in improving plant resistance to drought stress; the nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1; the plant is potato.

3. A method for improving plant resistance to drought stress, characterized in that, Overexpression of the STWRKY60 gene or increase in the level of its encoded protein can improve plant resistance to drought stress. The plant in question is a potato; The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

4. The application of the STWRKY60 gene or its encoded protein in the breeding of new drought-resistant plant varieties, characterized in that, The nucleotide sequence of the STWRKY60 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The plant in question is a potato; Overexpression of the STWRKY60 gene or increasing the level of its encoded protein can enhance plant resistance to drought stress.

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