Transcription factor erf113 for enhancing the ability of plants to resist acid rain and application thereof

Abstract

This invention relates to the fields of plant genetic engineering and stress-resistance breeding, specifically disclosing a transcription factor ERF113 that enhances plant resistance to acid rain and its applications. The amino acid sequence of the ERF113 protein is shown in SEQ ID NO:1, and the nucleotide sequence of the nucleic acid molecule encoding this protein is shown in SEQ ID NO:2. This invention introduces a recombinant expression vector containing the nucleic acid molecule shown in SEQ ID NO:2 into plants through genetic transformation, enhancing the expression level or activity of the ERF113 protein, thereby improving the plant's resistance to acid rain stress. Experiments show that overexpression... AtERF113 This invention can reduce leaf necrosis rates in plants under acid rain stress, decrease the accumulation of MDA and H2O2, and mitigate oxidative damage, thereby improving plant tolerance to acid rain stress. This invention provides new gene resources and technical approaches for breeding plants to resist acid rain.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and stress-resistance breeding technology, and in particular to a transcription factor ERF113 that enhances the ability of plants to resist acid rain and its applications. Background Technology

[0002] Acid rain is a major global environmental stressor. It is essentially acidic precipitation (pH below 5.6) formed by the wet deposition of sulfur oxides and nitrogen oxides in the atmosphere. Based on the differences in the main acid-causing ions in the precipitation, acid rain can be divided into sulfuric acid rain and nitric acid rain. When sulfate ions (SO4) in the precipitation... 2- When nitrate ions (NO3) are the main acidifying component, it belongs to the sulfuric acid type of acid rain, and its precursors are mainly sulfur dioxide (SO2) emitted from the combustion of fossil fuels; when nitrate ions (NO3) are the main acidifying component, it belongs to the sulfuric acid type of acid rain. - When nitrogen oxides (NOx) dominate, it is nitric acid-type acid rain, mainly originating from nitrogen oxides (NOx) emitted during motor vehicle exhaust and high-temperature combustion processes. x ).

[0003] Plants have evolved complex response mechanisms to cope with abiotic stresses through long-term adaptation, encompassing signal transduction, physiological metabolism, and gene expression regulation. Existing research has preliminarily confirmed that acid rain stress can induce a series of physiological and biochemical responses in plants, including oxidative damage, photosynthetic inhibition, and metabolic network disruption. However, the molecular regulatory network of plant responses to acid rain stress remains unclear. In particular, the specific targets and regulatory mechanisms of the AP2 / ERF transcription factor family in plant responses to acid rain stress have not yet been reported.

[0004] The APETALA2 / ethylene-responsive element binding factor (AP2 / ERF) family of transcription factors is one of the largest and most functionally diverse transcription factor families in the plant kingdom. Numerous studies have confirmed its core regulatory role in response networks to various abiotic stresses such as drought, high salinity, and low temperature. However, the specific functions and mechanisms of action of this family members in plant responses to acid rain stress still lack systematic and in-depth research. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a transcription factor ERF113 that enhances the acid rain resistance of plants and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Application of ERF113 protein in the preparation of transgenic plants with enhanced resistance to acid rain stress. The amino acid sequence of ERF113 protein is shown in SEQ ID NO:1.

[0007] Preferably, the enhancement of plant acid rain stress resistance is manifested in reducing the accumulation of malondialdehyde and / or hydrogen peroxide in plant leaves under acid rain stress.

[0008] A method for improving plant resistance to acid rain stress includes the step of introducing a recombinant expression vector into the plant to increase the expression level or activity of the ERF113 protein, wherein the recombinant expression vector is a nucleic acid molecule containing the ERF113 protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0009] Preferably, the plant is a cruciferous plant.

[0010] Preferably, the cruciferous plant is Arabidopsis thaliana.

[0011] Preferably, the method for increasing the expression level or activity of ERF113 protein is to introduce a recombinant expression vector containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:2 into a plant.

[0012] Preferably, the recombinant expression vector is the pCAMBIA1300-GFP vector.

[0013] Preferably, the method for introducing the recombinant expression vector is Agrobacterium-mediated inflorescence immersion.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides AtERF113 A novel application of transcription factors in regulating plant responses to acid rain stress. Functional validation was achieved through transgenic Arabidopsis thaliana, with overexpression... AtERF113 It can reduce the leaf necrosis rate of plants under simulated acid rain stress, while reducing the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H2O2), indicating that... AtERF113 Improve plant tolerance to acid rain stress by mitigating oxidative damage. AtERF113 The lack of these components leads to increased sensitivity of the plant to acid rain stress. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 Phenotypic analysis of Arabidopsis thaliana seedlings in response to artificially simulated acid rain stress; Figure 2This is a plot showing the differential expression and pathway enrichment analysis of the transcriptome in Arabidopsis thaliana in response to artificially simulated acid rain stress. Figure 2 In the diagram, 'a' represents the Venn diagram of differentially expressed genes. Figure 2 b in the figure represents the KEGG enrichment analysis of differentially expressed genes; Figure 2 In the text c, GO enrichment analysis is performed on significantly enriched genes in the differential co-expression module. Figure 3 Heatmap of hub gene response to artificially simulated acid rain stress; Figure 4 for AtERF113 The diagram shows the subcellular localization and expression levels of the transgenic lines. Figure 4 a in the middle. AtERF113 Subcellular localization analysis; Figure 4 b and Figure 4 c in the text. AtERF113 Expression level analysis in transgenic lines; Figure 5 for AtERF113 Phenotypic and physiological parameters of overexpression lines and mutants in response to acid rain stress were analyzed, among which... Figure 5 a in the middle. AtERF113 Phenotypic analysis of overexpression lines and mutants in response to artificially simulated acid rain stress; Figure 5 b in Figure 5 c in and Figure 5 d in the text. AtERF113 Leaf necrosis rate, MDA and H2O2 content in overexpression and mutant lines; data were obtained using t Statistical significance analysis was performed*. P <0.05, **, P <0.01, ***, P <0.001; Figure 6 T-DNA insertion mutant erf113 Electrophoresis image of three-primer PCR identification. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0018] Example 1: To elucidate the transcriptional regulatory mechanism of Arabidopsis thaliana in response to artificially simulated acid rain stress, this study performed transcriptome sequencing on Arabidopsis thaliana samples treated at pH=2, 3, 4 and the control group at pH=7.

[0019] For Arabidopsis thaliana, in the control group at pH 7, the plants grew well with lush, green leaves. After 3 days of artificial acid rain treatment at pH 4, the leaves curled; after 1 day of artificial acid rain treatment at pH 3, the leaves turned pale; and after 2 hours of artificial acid rain treatment at pH 2, the leaves showed paleness and necrosis, with the degree of necrosis increasing over time. Figure 1 This study also performed a Venn diagram analysis on the interaction of differentially expressed genes in Arabidopsis thaliana in response to artificially simulated acid rain stress. It was found that 1310 differentially expressed genes were co-expressed in Arabidopsis thaliana under pH=2 artificially simulated acid rain stress treatment. Figure 2 (a) KEGG metabolic pathway enrichment analysis was performed on DEGs from the Arabidopsis thaliana pH=2 group. The main KEGG enriched entries are shown. Differentially enriched genes in Arabidopsis thaliana under simulated acid rain stress treatment at pH=2 were significantly enriched in oxidative stress, plant secondary metabolism, and salicylic acid signaling metabolic pathways. Figure 2 (b) Gene expression often exhibits co-regulatory characteristics, with multiple genes involved in the same biological function forming regulatory modules and being jointly regulated. Identifying these regulatory modules not only simplifies mechanistic data analysis but also helps to more comprehensively uncover key regulatory factors in response to artificially simulated acid rain stress. In this study, differentially expressed genes (DEGs) in Arabidopsis thaliana were further standardized and filtered, resulting in 19,594 differentially expressed genes. Subsequently, weighted gene co-expression network analysis (WGCNA) was used to cluster these genes into 34 modules, with the number of characteristic genes in each module ranging from 41 to 3,339. Compared with the mRNA expression levels in the unstressed control samples, the expression levels of characteristic genes in the ME Red, MEGreenyellow, and ME Turquoise modules were significantly upregulated under artificially simulated acid rain stress, suggesting that these modules are activated under artificially simulated acid rain stress. Among these modules, the ME Red module was significantly enriched in GO functional entries related to damage response, abscisic acid (ABA) response, and jasmonic acid (JA) response. Figure 2 The (c) indicates that the characteristic genes in this module collectively provide important support for the artificial simulation of acid rain stress response in Arabidopsis thaliana.

[0020] A co-expression network was constructed based on the characteristic genes of the ME Red, ME Greenyellow, and ME Turquoise modules. Six candidate core genes were identified through visualization. AtERF111 , AtERF113 , AtERF114 , AtERF115 , AtRAP 2.6L and AT2G33710These candidate core genes may play an important role in the response of Arabidopsis thaliana to artificially simulated acid rain stress by transcriptionally regulating the expression of a large number of stress-response genes. Heatmap analysis of the expression patterns of these six core genes in response to artificially simulated acid rain stress revealed that… AtERF111 Primarily induced under artificially simulated acid rain stress at pH=4. AtRAP 2.6L It was primarily induced to a high level during treatment at different pH levels for 3 days, with lower expression levels at other time points. AtERF113 At different pH levels and time points, artificially simulated acid rain stress significantly induced ( Figure 3 Therefore, this study will AtERF113 These are key genes in Arabidopsis thaliana's response to artificially simulated acid rain stress.

[0021] Specifically: Plant materials and treatment: According to SO4 levels in historical precipitation in Hangzhou, Zhejiang Province 2- With NO3 - The proportions (data from the Zhejiang Provincial Department of Ecology and Environment website, 2004-2024 precipitation data) were used to prepare a stock solution by mixing concentrated sulfuric acid and concentrated nitric acid at a molar ratio of 3:1. Sterile water was added to adjust the pH to 4.0, 3.0, and 2.0, respectively. The control group was treated with sterile water at pH 7. 21-day-old Arabidopsis seedlings were selected, and artificial acid rain was sprayed onto the leaves using a sprayer, with 5 mL sprayed per plant. Treatment times were 0 h, 2 h, 1 d, 3 d, and 5 d. Each treatment had three biological replicates. Leaves were selected for RNA sequencing. The pH gradient was set according to the actual pH range of precipitation in most cities in Zhejiang Province (4.0-5.0). pH=3 represented mild acid rain treatment, and a strong acid gradient of pH=2 was added to screen for differentially expressed genes responding to acid rain stress. The highest treatment time was determined to be 5 days, because preliminary experiments showed that plant leaves would completely die after 7 days of pH=2 stress treatment, making subsequent sample collection and testing impossible.

[0022] RNA extraction, library construction, and sequencing: Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system. During the synthesis of the second strand, dTTPs in dNTPs were replaced by dUTPs. Following this, cDNA end repair, A-tailing, ligation of sequencing adapters, and length screening were performed. Then, the U-containing second strand of cDNA was degraded using USER enzyme before PCR amplification to obtain the library. Strand-specific libraries offer numerous advantages, such as obtaining more effective information with the same amount of data; more accurate gene quantification, localization, and annotation information; and providing expression levels of antisense transcripts and individual exons within each isoform. The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® was used for library construction. After library acceptance, different libraries were pooled according to their effective concentration and target sequencing data volume requirements before Illumina sequencing. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers were added to the sequencing flow cell for amplification. During the extension of the complementary strand of each sequencing cluster, the addition of each fluorescently labeled dNTP released corresponding fluorescence. The sequencer captured the fluorescence signal and converted it into a sequencing peak using computer software, thereby obtaining the sequence information of the target fragment. All sequencing library construction and sequencing experiments were commissioned to Tianjin Jizhi Technology Co., Ltd.

[0023] Transcriptome data analysis: This study used gene expression levels in each sample as the basis for screening and identifying genes with a fold change ≥ 2 and a significance p < 0.05 as differentially expressed genes (DEGs). Transcriptome sequencing data were analyzed using the Kangce Bio Cloud Platform for bioinformatics. Pearson correlation coefficient (PCC) analysis was performed using the platform's correlation heatmap module; principal component analysis (PCA) was performed using the platform's PCA tool; GeneOntology (GO) functional enrichment analysis was conducted using the platform's enrichment analysis module; and Venn diagram visualization was created using the platform's Venn diagram module for sample intersection analysis. To elucidate the genetic association patterns among different samples and screen for gene sets with research value and highly synergistic expression, this study constructed a co-expression regulatory network of differentially expressed genes using the Lianchuan Bio Weighted Correlation Network Analysis (WGCNA) online platform. The visualization of the gene co-expression network was completed using Cytoscape software. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of differentially expressed genes was performed using TBtools software.

[0024] Example 2: AtERF113 Gene cloning and subcellular localization analysis: 1) Gene cloning and vector construction According to the TAIR website AtERF113 The coding sequence (SEQ ID NO: 2) was obtained from the annotation information. Full-length primers were designed using Primer 6.0, and restriction enzyme sites were introduced at their 5' and 3' ends, respectively. The primer sequences used in this study are shown in Table 1.

[0025] Table 1. Primer sequences used in this study Arabidopsis cDNA was used as a template for PCR amplification using KOD One (KOD One™ PCR Master Mix, TOYOBO, catalog number KMM-101) polymerase. The PCR system consisted of 1 μl template DNA, 2 μl each of forward and reverse primers, 25 μl KOD One, and ddH2O added to a final volume of 50 μl. The reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 60 s, for a total of 35 cycles; 72℃ for 5 min.

[0026] The PCR products were detected and purified by 1% agarose gel electrophoresis. The purified target fragment was ligated into the Sal I-linearized pCAMBIA1300-GFP vector. The ligation system consisted of 2 μl of linearized vector, 3 μl of insert fragment, and 5 μl of 2×CEMix V (catalog number C117-01 Novizan). The reaction was carried out at 37°C for 30 min. The ligation product was transformed into E. coli, screened with kanamycin, and single clones were selected for PCR identification and sequencing verification. Positive clone plasmids were extracted.

[0027] 2) Subcellular localization A transient transformation method mediated by Agrobacterium was used in tobacco leaves. The recombinant vector and the empty vector control were transformed into Agrobacterium GV3101, respectively, and plated on LB agar plates containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubated at 28°C for 2–3 days. Single colonies were picked and cultured overnight in 3 mL of liquid LB agar, and the next day, the culture was scaled up at a 1:100 ratio to OD. 600 The concentration was 0.6–0.8. The bacterial cells were collected by centrifugation and resuspended in tobacco infection solution to an OD value of 0.6–0.8. 600 Add acetylsuccinone (AS) to a final concentration of 150 μM, and let stand at room temperature in the dark for 2-3 hours. Inject the bacterial solution into the back of the tobacco leaves using a needleless syringe. After culturing for 36-48 hours, peel off the leaf epidermis and observe the fluorescence signal using a laser confocal microscope (Zeiss LSM880).

[0028] The AP2 / ERF protein family possesses unique structural characteristics. Its molecules contain one to two classic AP2 domains, each composed of 60 to 70 highly conserved amino acid residues. These residues exhibit extremely high sequence conservation throughout evolution. This domain is typically composed of three β-sheets and one α-helix. This spatial structure endows AP2 / ERF transcription factors with the core ability to bind to DNA and precisely regulate the expression of downstream genes. Figure 4 As shown in Figure a, the transient transformation system of tobacco leaves mediated by Agrobacterium was used for... AtERF113 Subcellular localization expression analysis revealed that 35S:: was observed only in the cell nucleus. AtERF113 The green fluorescence of the -GFP recombinant, while the fluorescence signal of the empty vector can be detected throughout the cell, indicates that... AtERF113 It is a nuclear-localized transcription factor.

[0029] In this embodiment, the recombinant vector used for subcellular localization analysis is pCAMBIA1300-35S:: AtERF113 -GFP, its expression cassette structure is: CaMV 35S promoter— AtERF113The coding sequence (SEQ ID NO:2) — coding sequence for green fluorescent protein (GFP) — Nos terminator. This vector carries the hygromycin phosphotransferase gene ( hpt ) as a plant selection marker, using the kanamycin resistance gene ( nptII ) was used as a selection marker for E. coli. Overexpression lines (OE2, OE4, OE6) used for functional validation were constructed using the same vector and transformed into Arabidopsis thaliana, driven by the 35S promoter. AtERF113 - Expression of GFP fusion protein.

[0030] Example 3: Arabidopsis genetic transformation and screening of homozygous lines: Wild-type Arabidopsis thaliana (Col-0) seeds were disinfected with 75% alcohol for 10 min, washed, and then sown on 1 / 2 MS solid medium. After vernalization at 4℃ for 48 h, the seeds were transferred to a culture room. Once the seedlings had developed two true leaves, they were transplanted into the substrate for further cultivation.

[0031] Genetic transformation was performed using the floral dip method. Agrobacterium GV3101 bacterial culture containing the target gene was expanded to OD. 600 The bacterial cells were collected by centrifugation at 3000 rpm for 20 min to a concentration of 0.6–0.8, resuspended in Arabidopsis thaliana infection solution, and Silwet L-77 (Yisheng Biotechnology, catalog number 41008ES10) was added to a final concentration of 0.02% (v / v). The Arabidopsis thaliana infection solution was prepared as follows: 0.22 g of 1 / 2 MS medium powder, 5 g of sucrose, 0.2033 g of MgCl2·6H2O, and ddH2O was added to a final volume of 100 mL. The solution was sterilized before use. Arabidopsis thaliana inflorescences at the initial flowering stage were selected, immersed in the Arabidopsis thaliana infection solution for 10–20 s, cultured in the dark for 24 h, and then resumed normal culture. Seeds were harvested to obtain the T0 generation.

[0032] After sterilization, T0 generation seeds were sown on 1 / 2 MS selection medium containing 50 mg / L hygromycin. Positive T1 generation seedlings with distinct taproots were selected and transplanted into soil. Seeds were harvested and screened generation by generation to obtain homozygous T3 generation. DNA was extracted from T3 generation lines for transgenic identification, and expression levels were analyzed using qRT-PCR. Lines with significant differences in expression levels (e.g., #2, #4, #6) were selected as overexpression materials, and T-DNA insertion mutants were also developed. erf113 As a comparison.

[0033] T-DNA insertion mutant erf113 (Purchased from the Airosha website) Homozygous identification was performed using a three-primer PCR method. The primer sequences used for identification are: ERF113-LP: CTTCGATTTCTAAGCCTGAATGTG (SEQ ID NO: 7) ERF113-RP: CTTCCTCTAAAGCGAAGTGCTG (SEQ ID NO: 8) LBb1.3 (BP):ATTTTGCCGATTTCGGAAC (SEQ ID NO:9) Following PCR amplification, the LP+BP primer pair amplified a specific band (T-DNA insertion fragment) in the mutant genome, while the LP+RP primer pair amplified a specific band in the wild-type genome. Lines that were positive only for LP+BP amplification but not for LP+RP amplification were identified as homozygous mutants. See Appendix. Figure 6 , Figure 6 T-DNA insertion mutant erf113 Electrophoresis diagram of three-primer PCR identification; M is DL2000 DNA marker; 1-9 are amplification products of LP+BP primer pair; 10-18 are amplification products of LP+RP primer pair.

[0034] Tests confirmed that, compared with wild-type plants, the OE2, OE4, and OE6 lines... AtERF113 The mRNA expression level was significantly upregulated ( Figure 4 (b) Meanwhile, this study was obtained through the Airosha website. AtERF113 T-DNA insertion mutant ( erf113 The mutant was obtained using a three-primer identification method. erf113 This further validates the function of the gene; compared to the wild type, erf113 mutant AtERF113 The mRNA expression level decreased significantly by 45%~60% ( Figure 4 (c in the text)

[0035] qRT-PCR detection: Total RNA was extracted from Arabidopsis leaves, reverse transcribed into cDNA, and then subjected to quantitative real-time PCR using SupRealQ Purple universal SYBR qPCR premix. AtACTIN2 (At3g18780) is the internal reference gene, and the internal reference primer sequence is: ACTIN2 (At3g18780)-F:GGTAACATTGTGCTCAGTGGTGG (SEQ ID NO:10) ACTIN2 (At3g18780)-R:AACGACCTTAATCTTCATGCTGA (SEQ ID NO:11) RNA was extracted from Arabidopsis leaves, reverse transcribed into cDNA, and then used for real-time quantitative PCR with SupRealQ Purple universal SYBR qPCR premix. The real-time quantitative PCR reaction system is shown in Table 2 below.

[0036] Table 2. Real-time quantitative PCR reaction system Example 4: Simulated Acid Rain Treatment and Phenotypic Analysis: According to the precipitation data of Hangzhou City, Zhejiang Province over the past five years (based on precipitation data from 2004-2024 on the official website of the Zhejiang Provincial Department of Ecology and Environment), SO4 content... 2- With NO3 - The concentrated sulfuric acid and concentrated nitric acid were mixed in a molar ratio of 3:1 to prepare a mother liquor. The pH was adjusted to 4.0, 3.0 and 2.0 respectively with RO water, and the RO water with pH 7.0 was used as the control group.

[0037] 21-day-old Arabidopsis thaliana seedlings were selected, and artificially simulated acid rain was sprayed evenly onto the leaves using a sprayer, with 5 mL sprayed per plant. Treatment time points were set at 0h, 2h, 1d, 3d, and 5d. The treated plants were placed in an isolated culture room to avoid cross-contamination.

[0038] Phenotypic observation: Leaves were photographed, and the area of ​​visible necrotic area and total leaf area were calculated using Adobe Photoshop CS2 software. The necrosis rate (necrotic area / total leaf area × 100%) was calculated, with 25 leaves per group as replicates.

[0039] Physiological index measurement: MDA content determination: (1) The plant malondialdehyde (MDA) assay kit (microplate method, catalog number: A003-3-1) of Nanjing Jiancheng Bioengineering Institute was used. The specific operation is as follows: accurately weigh the plant tissue sample and add the reagent five application extract (diluted with double-distilled water at a ratio of 1:9) according to the mass-volume ratio of 1:9. After cutting the sample into pieces, place it in an ice-water bath environment and homogenize it using an internal cut homogenizer. The homogenization speed is set to 8000-10000 r / min, and the homogenization time is 10-15 s each time. Repeat the operation after an interval of 30 s, for a total of 3-5 times. After homogenization, transfer it to a centrifuge tube and centrifuge at a speed of 3500-4000 r / min for 10 min. Collect the supernatant for later detection. (2) Make a small hole in the surface of the centrifuge tube cap with a needle, seal the cap, and mix thoroughly on a vortex mixer. Then heat in a water bath at 95 °C or above for 20 min. After removing, cool to room temperature with running water. Set the detection wavelength to 530 nm. First, perform a blank reading on the empty plate of the microplate. Then, accurately pipette 0.25 mL of the reaction solution from each tube into the 96-well plate and measure the absorbance value of each well using a microplate reader. Calculation formula: MDA content (nmol / g) = (A 测定 -A 空白 ) / (A 标准 -A 空白 )*C 标准 *V 提 / W C represents the standard concentration: 10 nmol / mL; W represents the plant tissue weight (g); V 提 The total volume (mL) of the extract added.

[0040] H2O2 content determination: First, clean the plant tissue with PBS, then dry it with absorbent paper, cut it into small pieces and put it into a mortar, grind it into powder with liquid nitrogen, weigh the plant powder, add 9 times the volume of PBS at a mass (g): volume (mL) ratio of 1:9, vortex for 1 min, centrifuge at 12000 rpm / min for 10 min, and take the supernatant for testing; The hydrogen peroxide assay kit (microplate method, catalog number: A064-2-1) from Nanjing Jiancheng Bioengineering Institute was used. Specific procedures were performed according to the kit instructions, following the sample addition system as follows: Add 10 µL of distilled water and 100 µL of reagent one to the blank wells; add 10 µL of 163 mmol / L H₂O₂ standard working solution and 100 µL of reagent one to the standard wells; add 10 µL of sample and 100 µL of reagent one to the assay wells. Gently shake the plate and incubate at room temperature for 3-5 min, then read the absorbance value A1 at 405 nm using a microplate reader. Subsequently, add 100 µL of reagent two to each of the blank, standard, and assay wells, gently shake the plate, incubate at room temperature for 3-5 min, and read the absorbance value A2 at 405 nm using a microplate reader. Calculate ΔA = A2 - A1. Calculation formula: (ΔA measurement - ΔA blank) / (ΔA standard - ΔA blank) * C 标准 *V 样总 / W C represents the standard concentration: 163 mmol / L; W represents the weight of the plant tissue (g); V represents the total volume of homogenizing medium added before sample processing (L).

[0041] right AtERF113 Overexpression lines ( AtERF113 -OE) and erf113 The mutants were subjected to artificially simulated acid rain stress treatment. The experiment was set up in two groups: one group was treated with foliar spraying of artificially simulated acid rain at pH=2, and the other group was sprayed with RO water at pH=7 as a no-stress control. After the treatment, the plant phenotype was observed and recorded. Figure 5 (a) in the text. The results showed that compared with wild-type plants, AtERF113The -OE strain showed significantly improved tolerance to artificially simulated acid rain stress and significantly reduced leaf damage; erf113 The mutant showed a significantly reduced tolerance to artificially simulated acid rain stress, with more severe leaf necrosis and curling compared to the wild type, further confirming... AtERF113 The gene plays a positive regulatory role in the response of Arabidopsis thaliana to artificially simulated acid rain stress. All experiments were performed in triplicate to ensure the reliability of the results. Further verification at the physiological and biochemical levels is needed. AtERF113 This study investigates the regulatory role of genes in plant tolerance to artificially simulated acid rain stress, overcoming the subjective limitations of morphological observation and providing quantitative support for gene regulation mechanisms. It also studies the effects of genes on wild-type and... AtERF113 Overexpression lines and mutants were subjected to artificial acid rain stress at pH 2. After stress treatment, key physiological indicators were measured. Leaf necrosis rate analysis ( Figure 5 b) shows that under pH=2 treatment, erf113 The leaf necrosis rate of the mutant was significantly higher than that of Col-0, while AtERF113 The leaf necrosis rate of the overexpression lines was significantly lower than that of Col-0. Under normal conditions, the H2O2 and MDA contents of all lines remained at low levels. However, under pH=2 treatment, the H2O2 and MDA contents of all lines increased significantly, indicating that artificially simulated acid rain stress induced reactive oxygen species bursts and membrane lipid peroxidation. Compared with Col-0, AtERF113 H2O2 content of overexpression lines ( Figure 5 c) and MDA content ( Figure 5 d) in the data were all significantly reduced, while erf113 Both indicators were significantly elevated in the mutant lines. This indicates overexpression AtERF113 It effectively alleviated the oxidative damage induced by artificially simulated acid rain stress, while AtERF113 The loss of function exacerbates oxidative stress. These oxidative stress indicators are completely consistent with the changing trends of leaf necrosis rate, further confirming that... AtERF113 The positive role of overexpression in enhancing the tolerance of Arabidopsis thaliana to artificially simulated acid rain stress. In summary, overexpression AtERF113 It can significantly reduce ROS accumulation, membrane lipid peroxidation and leaf necrosis rate in Arabidopsis thaliana under artificial simulated acid rain stress, thereby enhancing the plant's tolerance to artificial simulated acid rain stress.

[0042] Table 3 shows the results of leaf necrosis rate, MDA content and H2O2 content of each strain under artificial acid rain stress treatment at pH=2.

[0043] Table 3. Physiological parameters of Arabidopsis thaliana lines under simulated acid rain stress at pH=2 (Mean±SD) Note: In Table 3, "No necrosis was observed in the leaves of the control group" is marked as "—".

[0044] SEQ ID NO: 1 is the amino acid sequence of the ERF113 protein: MVSMLTNVVSGETEPSASATWTMGHKREREEFSLPPQPLITGSAVTKECESSMSLERPKKYRGVRQRPWGKWAAEIRDPHKATRVWLGTFETAEAAARAYDAAALRFRGSKAKLNFPENVGTQTIQRNSHFLQNSMQPSLTYIDQCPTLLSYSRCMEQQQPLVGMLQPTEEENHFFEKPWTEYDQYNYSSFG SEQ ID NO:2 is ERF113 The nucleotide coding sequence: ATGGTGTCTATGCTGACTAATGTTGTCTCTGGTGAGACCGAACCCTCGGCATCTGCGACATGGACGATGGGTCATAAGAGAGAAAGAGAAGGTTTTCTTTGCCTCCTCAACCATTGATTACCGGTTCAGCTGTGACTAAAGAA TGTGAAAGCTCAATGTCCTTGGAGAGGCCAAAAAAATATAGAGGAGTAAGGCAACGACCATGGGGAAAATGGGCGGCGGAGATTCGAGACCCACACAAGGCGACACGTGTATGGCTTGGGACATTCGAGACAGCCGAGGCCGCCG CAAGAGCCTATGATGCGGCAGCACTTCGCTTTAGAGGAAGCAAAGCAAAGCTTAATTTCCCCGAAAATGTTGGAACTCAGACGATTCAACGAAATTCTCATTTCTTGCAAAACTCTATGCAACCTTCTCTGACATACATCGATCA ATGTCCAACTCTATTATCTTACTCTCGATGTATGGAGCAACAACAACCATTAGTAGGCATGTTGCAGCCAACAGAAGAGGAAAATCACTTTTTCGAAAAACCATGGACCGAATATGATCAATACAATTACTCCTCTTTTGGTTAA The foregoing has shown and described the basic principles, main features, and advantages of this application. Those skilled in the art should understand that this application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this application. Various changes and modifications can be made to this application without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the claims of this application.

Claims

1. The application of ERF113 protein in the preparation of transgenic plants with enhanced resistance to acid rain stress, characterized in that, The plant in question is Arabidopsis thaliana, and the amino acid sequence of the ERF113 protein is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The plant in question is Arabidopsis thaliana, and the enhancement of the plant's acid rain stress resistance is manifested by reducing the accumulation of malondialdehyde and / or hydrogen peroxide in the plant leaves under acid rain stress.

3. A method for improving plant resistance to acid rain stress, characterized in that, The plant is Arabidopsis thaliana, and the procedure includes the step of introducing a recombinant expression vector into the plant to increase the expression level or activity of the ERF113 protein. The recombinant expression vector is a nucleic acid molecule containing the ERF113 protein, and its nucleotide sequence is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that, The method for increasing the expression level or activity of ERF113 protein is as follows: introducing a recombinant expression vector containing a nucleic acid molecule with nucleotide sequence SEQ ID NO:2 into plants.

5. The method according to claim 4, characterized in that, The recombinant expression vector is the pCAMBIA1300-GFP vector.

6. The method according to claim 5, characterized in that, The method for introducing the recombinant expression vector is Agrobacterium-mediated inflorescence immersion.

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