Transcription factor BnaERF5 and application thereof

Abstract

The invention discloses a transcription factor BnaERF5 and application thereof, and belongs to the technical field of gene engineering. The invention provides an application of BnaERF5 in improving the drought resistance of rape and / or improving the anthocyanin content of rape under drought stress. The core transcription factor BnaERF5 is screened out through weighted gene co-expression network analysis (WGCNA) and differential gene clustering, and the core transcription factor BnaERF5 has an important regulation effect on anthocyanin synthesis and drought resistance of rape. By integrating ABA and JA signal pathways, the BnaERF5 synergistically activates osmoregulation related pathways and induces up-regulation expression of key genes CHI, F3H, DFR and FLS in a flavonoid synthesis pathway, the activity of antioxidant enzymes is enhanced, the ROS removal capacity is improved, a large amount of anthocyanin is accumulated, cell osmotic equilibrium is effectively maintained, and the drought resistance is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and particularly relates to a transcription factor. BnaERF5 And its applications. Background Technology

[0002] Rapeseed, as a globally important oilseed crop, occupies a pivotal strategic position in my country's agricultural production and economic development. In recent years, with the intensification of global warming, the frequency and intensity of drought stress have increased significantly, severely impacting the growth, development, yield, and quality of rapeseed, posing a serious challenge to food security. Drought stress triggers a series of physiological responses, including water deficit, damage to the photosynthetic system, and cell membrane lipid peroxidation, leading to an average yield reduction of 20-30% in rapeseed. Therefore, in-depth research into the drought resistance mechanisms of rapeseed and enhancing its drought resistance capacity is of urgent practical significance for ensuring food security.

[0003] Furthermore, rapeseed is not only an important oilseed crop but also possesses high ornamental value. In recent years, with the increasing demand for diversified rapeseed varieties, the cultivation and promotion of colored rapeseed have received growing attention in the agricultural economy, and its industrialization potential is increasingly evident. Purple rapeseed, in particular, plays a vital role in rural revitalization and the tourism economy due to its unique ornamental value. Therefore, studying the synthetic regulatory mechanism of anthocyanins in rapeseed not only contributes to the theoretical development of drought-resistant breeding of colored rapeseed but also promotes the sustainable growth of the colored rapeseed industry and fosters the organic integration of modern agriculture with ecotourism and cultural industries.

[0004] Therefore, there is an urgent need to develop a precise regulation technology that can take into account both the drought resistance of rapeseed and the anthocyanin content of colored rapeseed, so as to break through the current technical bottleneck of industrial development and meet the dual needs of rapeseed industry in terms of food and oil security and economic growth. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a BnaERF5 Application in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress. BnaERF5 It can enhance the activity of antioxidant enzymes in rapeseed, improve the ability to scavenge ROS, accumulate a large amount of anthocyanins, effectively maintain cell osmotic balance, and significantly improve the drought resistance of rapeseed.

[0006] Another object of the present invention is to provide an overexpression BnaERF5 Application of recombinant vectors in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0007] Another objective of this invention is to provide a method for improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0008] Another object of the present invention is to provide a transgenic rapeseed plant.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides BnaERF5 Application in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0010] Preferably, the BnaERF5 Rapeseed drought resistance can be improved by enhancing antioxidant defense, maintaining osmotic balance, and reducing oxidative damage.

[0011] Preferably, the BnaERF5 include BnaERF5 Genes, mRNA, cDNA, or proteins.

[0012] Preferably, the BnaERF5 The gene is located at positions 46719350-46720237 on chromosome A06 of the Brassica napus reference genome, version ZS11.v0.

[0013] This invention also provides an overexpression BnaERF5 Application of recombinant vectors in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0014] Preferably, the original vector of the recombinant vector includes 35S- p CAMBIA1300-sGFP.

[0015] Preferably, the method for constructing the recombinant vector includes: using BamHI and XbaI restriction endonucleases to target 35S- p CAMBIA1300-sGFP was double-digested to obtain a linearized vector; PCR amplification was then performed to obtain... BnaERF5 The CDS target segment; the said BnaERF5 The CDS target fragment was homologously recombined into a linearized vector; the ligation product was transformed into E. coli DH5α competent cells by heat shock, and the plasmid was extracted after correct sequencing.

[0016] Preferably, the primer sequences for the PCR amplification include SEQ ID NO:1 to SEQ ID NO:2.

[0017] This invention also provides a method for improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress, the method comprising: using Agrobacterium-mediated transformation under sterile conditions to overexpress... BnaERF5 The recombinant vector was introduced into rapeseed explants, and transgenic rapeseed plants were obtained through screening and culture.

[0018] The present invention also provides a transgenic rapeseed plant, which is obtained by the method described above.

[0019] The beneficial effects of this invention are: This invention screened core transcription factors through weighted gene co-expression network analysis (WGCNA) and differential gene clustering. BnaERF5 It plays an important regulatory role in anthocyanin synthesis and drought resistance in rapeseed. BnaERF5 Overexpression lines integrate ABA and JA signaling pathways, synergistically activate osmotic regulation pathways, and induce key genes in the flavonoid synthesis pathway. CHI , F3H , DFR and FLS Upregulation of expression enhanced antioxidant enzyme activity, improved ROS scavenging ability, and accumulated large amounts of anthocyanins, effectively maintaining cellular osmotic balance and significantly improving drought resistance. Attached Figure Description

[0020] Figure 1 WGCNA analysis of differentially expressed genes in purple-leaf (PL) and green-leaf (GL) Brassica napus under drought and rehydration treatments in Example 1; where A is the hierarchical clustering tree and module partitioning diagram of differentially expressed genes, and B is the GO and KEGG enrichment network diagram of genes in the Yellow module. Figure 2 This is a heatmap of hierarchical clustering analysis of differentially expressed genes in purple-leaf (PL) and green-leaf (GL) Brassica napus varieties under the combined effects of material specificity and drought stress in Example 1. Figure 3 Phylogenetic analysis and amino acid sequence alignment of ERF5 in different species in Example 1; Figure 4 In Example 1 BnaERF5 Gene cloning and subcellular localization analysis; where A is BnaERF5 The CDS amplification electrophoresis image, B is the recombinant vector. pBnaERF5 A schematic diagram of the construction of -sGFP, where C represents... BnaERF5 Subcellular localization, scale bar 10 μm; Figure 5 For the transfer in Example 1 BnaERF5 Identification and screening of gene-positive plants, where AD represents the genetic transformation process in rapeseed, and E represents... BnaERF5 PCR verification of transgenic lines, F is wild-type control WT, and M is DNA Marker; Figure 6 In Example 1 BnaERF5 Relative expression levels in T1 generation lines; Figure 7 As the control and drought stress in Example 1 BnaERF5Phenotypic differences between overexpression lines and wild-type lines, where A represents the control condition and B represents the drought stress condition; Figure 8 For example, the drought stress in Example 1 ERF5 - The effect of OE on the content of chlorophyll a (A), chlorophyll b (B), and anthocyanin (C) in WT; according to the Turkey test, the figure shows... This indicates that p < 0.05. , and The numbers represent p < 0.01, 0.001, and 0.0001, respectively, and ns indicates no significant difference. The same applies to the figures below. Figure 9 For example, under drought stress in Example 1 ERF5 - Differences in relative water content (A), electrical conductivity (B), proline (C), and malondialdehyde (D) content between OE and WT; Figure 10 For example, under drought stress in Example 1 ERF5 Differences in the activities of superoxide dismutase (A), peroxidase (B), catalase (C), and ascorbate peroxidase (D) between OE and WT; Figure 11 For example, under drought stress in Example 1 ERF5 -OE and WT leaf histochemical staining: DAB staining indicates the accumulation of H2O2, and NBT staining indicates the amount of O2. ·— The accumulated amount; Figure 12 Before and after drought stress in Example 2 ERF5 -Analysis of differentially expressed genes (DEGs) between OE and WT, where A is a bar chart of differentially expressed genes between different groups, and B is a Venn diagram analysis of common and specific differentially expressed genes under drought stress; Figure 13 Before and after drought stress in Example 2 ERF5 - GO (A) and KEGG (B) enrichment analysis of differentially expressed genes in OE; Figure 14 In Example 2 BnaERF5 Effects of overexpression and drought stress on anthocyanin synthesis gene expression, where A is a heatmap of differential pathways of anthocyanin synthesis genes among different materials and treatments, and B is a cluster analysis of anthocyanin synthesis gene expression levels among different materials and treatments. Figure 15 For example, the BnERF5 protein in Example 2 and DFR Interaction structure prediction of the (BnaC09G0215200ZS) promoter. Detailed Implementation

[0021] This invention providesBnaERF5 Application in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0022] This invention screened core transcription factors through weighted gene co-expression network analysis (WGCNA) and differential gene clustering. BnaERF5 It plays an important regulatory role in anthocyanin synthesis and drought resistance in rapeseed. BnaERF5 Overexpression lines integrate ABA and JA signaling pathways, synergistically activate osmotic regulation pathways, and induce key genes in the flavonoid synthesis pathway. CHI , F3H , DFR and FLS Upregulation of expression enhanced antioxidant enzyme activity, improved ROS scavenging ability, and accumulated large amounts of anthocyanins, effectively maintaining cellular osmotic balance and significantly improving drought resistance.

[0023] In this invention, the BnaERF5 The drought resistance of rapeseed can be improved by enhancing antioxidant defense, maintaining osmotic balance, and reducing oxidative damage. Meanwhile, research has found that... BnaERF5 Overexpression lines can maintain a basic stress response network and further enhance drought resistance through anthocyanin accumulation.

[0024] In this invention, the BnaERF5 Preferred includes BnaERF5 Genes, mRNA, cDNA, or proteins.

[0025] In this invention, the BnaERF5 The preferred gene is located at position 46719350-46720237 on chromosome A06 of the Brassica napus reference genome version ZS11.v0, with gene ID BnaA06G0417500ZS.

[0026] This invention also provides an overexpression BnaERF5 Application of recombinant vectors in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

[0027] In this invention, the original vector of the recombinant vector preferably includes 35S- p CAMBIA1300-sGFP. The 35S- p CAMBIA1300-sGFP is in psGFP was inserted into CAMBIA1300; the insertion method is not particularly limited and can be conventionally selected according to actual needs. In some embodiments, the insertion method preferably includes: amplifying the sGFP sequence by PCR, adding a Sal I restriction enzyme to the 5' end of the upstream primer, and adding a Pst I restriction enzyme to the 5' end of the downstream primer; digesting the PCR product with Sal I and Pst I and then ligating it into pCAMBIA1300 digested with the same enzymes to obtain 35S- p CAMBIA1300-sGFP.

[0028] In this invention, the preferred method for constructing the recombinant vector includes: double digestion of 35S-pCAMBIA1300-sGFP with BamHI and XbaI restriction endonucleases to obtain a linearized vector; and obtaining the vector by PCR amplification. BnaERF5 The CDS target segment; the said BnaERF5 The CDS target fragment was homologously recombined into a linearized vector; the ligation product was transformed into E. coli DH5α competent cells by heat shock, and the plasmid was extracted after correct sequencing.

[0029] In some embodiments of this invention, the double enzyme digestion reaction system preferably comprises: 2 µL of 10× QuickCut Buffer, 1 µg of 35S- p The CAMBIA1300-sGFP vector DNA, 1 µL of QuickCut BamHI, and 1 µL of QuickCut XbaI were added, and finally, ddH2O was added to bring the total volume to 20 µL. The preferred reaction temperature for the double digestion was 35–40 °C, for example, 35, 36, 37, 38, 39, or 40 °C; the preferred reaction time was 10–20 min, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 min. After the reaction was complete, the temperature was preferably lowered to 4 °C, and the digested products were directly recovered using the FastPure Gel DNA Extraction Mini Kit.

[0030] In some embodiments of this invention, the primer sequences for PCR amplification preferably include SEQ ID NO:1~SEQ ID NO:2. The PCR amplification system preferably includes: 12.5 µL of 2×Master Mix, 1 µL of upstream primer (10 μmol / L), 1 µL of downstream primer (10 μmol / L), 1 µL of cDNA template, and finally, ddH2O to a total volume of 25 µL. The preferred PCR amplification reaction program is: pre-denaturation at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 30 s → annealing at 57 °C for 30 s → extension at 72 °C for 1 min; final extension at 72 °C for 5 min, followed by cooling to 4 °C after the reaction. The annealing temperature can be adjusted appropriately according to the primers used. The PCR products are preferably separated by 1% agarose gel electrophoresis, and the target fragment is recovered using the FastPure Gel DNA Extraction Mini Kit.

[0031] In some embodiments of this invention, the homologous recombination reaction system preferably comprises: (0.02 × total base pairs of the vector) ng of linearized vector, (0.04 × total base pairs of the target fragment) ng of target gene fragment, 4 µL of 5 × CEII Buffer, 2 µL of Exnase II, and finally supplemented with ddH2O to a total volume of 20 µL. The preferred reaction temperature for homologous recombination is 35–40 °C, for example, 35, 36, 37, 38, 39, or 40 °C; the preferred reaction time is 20–40 min, for example, 20, 25, 30, 35, or 40 min.

[0032] In some embodiments of the present invention, plasmids are preferably extracted using the TIANprep Mini Plasmid Kit plasmid extraction kit.

[0033] This invention also provides a method for improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress, the method comprising: using Agrobacterium-mediated transformation under sterile conditions to overexpress... BnaERF5 The recombinant vector was introduced into rapeseed explants, and transgenic rapeseed plants were obtained through screening and culture.

[0034] In this invention, the species of Agrobacterium is not specifically limited. In some embodiments, the Agrobacterium includes Agrobacterium GV3101; the Agrobacterium-mediated method utilizes overexpression... BnaERF5 The GV3101 strain of the recombinant vector was introduced into rapeseed explants under aseptic conditions to achieve genetic transformation of rapeseed.

[0035] In this invention, the rapeseed variety is not specifically limited. In some embodiments, the rapeseed includes Brassica napus (Westar). In some embodiments, the explant preferably comprises 6-day-old sterile seedlings.

[0036] The present invention also provides a transgenic rapeseed plant, which is obtained by the method described above.

[0037] Under drought stress BnaERF5 The overexpression lines had significantly higher relative water content than the wild type and increased proline accumulation; MDA content was significantly lower than the wild type, indicating less lipid peroxidation of the cell membrane; changes in the stress response activities of antioxidant enzymes such as SOD, POD, CAT, and APX further support this finding. BnaERF5 The function of protecting cell membrane integrity is inferred by enhancing ROS scavenging efficiency. Regarding the regulation of anthocyanin metabolism under drought stress... BnaERF5 The anthocyanin content of the overexpression lines was significantly increased, and the accumulation of anthocyanins occurred simultaneously with the enhanced ROS scavenging ability. BnaERF5 It may enhance drought resistance by synergistically integrating secondary metabolic and antioxidant pathways. This suggests that... BnaERF5 Drought resistance is enhanced through multiple pathways, including osmotic regulation, antioxidant defense, and anthocyanin accumulation.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1 1. Transcriptome data analysis This study used purple-leaved colored rapeseed (PL) and a green-leaved control (GL) with a similar background. Both were derived from the segregating population of the same rapeseed inbred line and maintained relatively consistent phenotypic characteristics across multiple inbred generations. Both PL and GL were subjected to 0-day (control), 4-day, and 8-day drought treatments, and a 4-day rehydration treatment. RNA sequencing analysis was performed on 24 samples to identify differentially expressed genes. Weighted Gene Co-expression Network (WGCNA) analysis and differential gene clustering analysis were conducted on the 24 samples using the MetWare online analysis platform and IDEP 2.01 (https: / / bioinformatics.sdstate.edu / idepg / ). Hierarchical clustering analysis was performed on DEGs with similar expression patterns across different materials and treatments using IDEP 2.01, employing Pearson correlations and k-means (k=5) algorithms. The clustering results were visualized as heatmaps. The results are shown below. Figure 1 and ​ As shown.

[0042] Different branches of the clustering tree represent different genes. Based on the weighted correlation coefficient of genes, genes are classified into modules, with genes having the same expression pattern grouped into one module. Different colors below represent different modules, such as... ​ As shown in Figure A, the differentially expressed genes were divided into 23 co-expressed gene modules, with high co-expression levels within each module. Screening of these modules using anthocyanin synthesis structural genes revealed that most of these structural genes were concentrated in the Yellow module. All differentially expressed genes within these modules were extracted and subjected to GO and KEGG enrichment analyses, as shown below. ​ As shown in Figure B, these genes were significantly enriched in biological processes, functions, and cellular components such as small molecule metabolic processes, oxidoreductase activity, and chloroplasts. This indicates that these genes may be involved in key biological processes in plants, including metabolic regulation, photosynthesis, energy conversion, and antioxidant defense. Furthermore, KEGG enrichment analysis showed that these genes are mainly concentrated in the synthesis of secondary metabolites, corroborating the screening criteria for the aforementioned modules. This suggests that these genes include not only structural genes related to anthocyanin synthesis but also functional genes responding to drought stress, serving as an important reference for key gene screening.

[0043] Hierarchical cluster analysis revealed a transcription factor belonging to the ERF family. ​ (BnaA06G0417500ZS) and key structural genes for anthocyanin synthesis​ and ​ Clustered in the same cluster ( ​ ).

[0044] 2. Phylogenetic analysis and conserved domain analysis of ERF5 The amino acid sequence of BnaERF5 was obtained from the Brassica napus reference genome (ZS11.v0). BLAST analysis was performed using the NCBI (https: / / ncbi.nlm.nih.gov) and BnIR (https: / / yanglab.hzau.edu.cn / BnIR / ) databases to search for its amino acid composition in Arabidopsis thaliana. ​ ),Chinese cabbage( ​ ), mustard-type rapeseed ( ​ ), cabbage ( ​ Ethiopian mustard () ​ ), black mustard ( ​ ),tomato( ​ ),tobacco( ​ ), rice ( ​ ) and corn ( ​ The orthologous proteins in the protein were analyzed to obtain the corresponding amino acid sequences.

[0045] Cluster analysis of the amino acid sequences of ERF5 from different species was performed using MEGA11 software. Multiple sequence alignment was performed using the MUSCLE program with standard parameter settings. Hierarchical cluster analysis employed the neighbor-joining (ND) method, with the following parameters: Bootstrap method: 1000; Gap / Missing Data Treatment: Pairwise deletion. The amino acid domains of ERF5 from different species were predicted using the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR / ) and the SMART online analysis platform (https: / / embl-heidelberg.de). The results are as follows: ​ As shown.

[0046] The results showed that BnaERF5 (BnaA06G0417500ZS) had a higher sequence similarity to BjuERF5 (BjuA024881) from rapeseed, possibly originating from the A subgenome of a common ancestor. Domain analysis revealed that all ERF5 proteins possessed the AP2 / ERF domain (PF00847). Particularly in cruciferous plants, the amino acid sequence of the AP2 domain (positions 154-204) was identical, indicating high evolutionary homology and highly conserved functional core regions, providing a theoretical reference for subsequent gene function analysis.

[0047] 3. Subcellular localization analysis (1) Extraction of DNA and RNA and synthesis of cDNA DNA was extracted using a high-efficiency plant genomic DNA extraction kit (DP350, Tiangen), and total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (AG21022). Extraction methods were performed according to the respective kit instructions. Total RNA was extracted from purple-leaved colored rapeseed leaf samples. cDNA synthesis utilized the extracted RNA, employing the HiScript II 1st Strand cDNA Synthesis Kit (R212-01, Vazyme) to synthesize long-fragment cDNA templates for subsequent CDS sequence cloning. The reverse transcription system and reaction conditions were as per the kit instructions.

[0048] (2) Enzymatic digestion and recovery of the vector Using the laboratory-preserved 35S-pCAMBIA1300-sGFP vector, ​ I and ​ I restriction endonuclease (QuickCut) TM Linearization was achieved by double digestion with Takara enzyme. The reaction mixture consisted of 2 µL of 10× QuickCut Buffer, 1 µg of vector DNA, and 1 µL of QuickCut Buffer. ​ I, 1µL QuickCut ​ First, add ddH2O to bring the total volume to 20µL. Mix well and react at 37℃ for 15min. After the reaction is complete, cool to 4℃. The enzyme digestion product is directly recovered using the FastPure Gel DNA Extraction Mini Kit (DC301-01, Vazyme) and stored at -20℃ for later use.

[0049] (3) Acquisition of gene sequences and synthesis of CDS sequences Based on the reference genome of Brassica napus (ZS11.v0) ​ The CDS sequence information of (gene ID: BnaA06G0417500ZS), combined with 35S- pBased on the upstream and downstream sequences of the restriction enzyme sites on the CAMBIA1300-sGFP vector, specific amplification primers for homologous recombination were designed using the Vazyme online primer design platform (https: / / crm.vazyme.com / cetool.html) (Table 1). Using the synthesized cDNA as a template, high-fidelity enzyme 2×Phanta MaxMaster Mix (Dye Plus) was used to... ​ The CDS sequence was amplified using a PCR system consisting of 12.5 µL of 2×MasterMix, 1 µL of upstream primer (10 μmol / L), 1 µL of downstream primer (10 μmol / L), 1 µL of cDNA template, and finally, ddH2O to a total volume of 25 µL. The reaction program was as follows: pre-denaturation (95 °C, 3 min), 35 cycles of denaturation (95 °C, 30 s) → annealing (57 °C, 30 s) → extension (72 °C, 1 min), final extension (72 °C, 5 min), followed by cooling to 4 °C (the specific annealing temperature can be adjusted according to the primers used). The obtained PCR products were separated by 1% agarose gel electrophoresis. The gel containing the target fragment was excised and the target fragment was recovered using the FastPure Gel DNA Extraction Mini Kit (DC301-01, Vazyme).

[0050] Table 1 Primers for homologous recombination fragment amplification

[0051] The results of 1% agarose gel electrophoresis showed that ( ​ (A) ​ The amplified product band was approximately 900 bp, which matches the 888 bp sequence length in the reference genome.

[0052] (4) Construction of the carrier Using the linearized vector after enzyme digestion and the obtained CDS target fragment, the ClonExpress MultiS OneStep Cloning Kit (C113-01, Vazyme) was used to... ​ Homologous recombination to 35S- p The recombinant plasmid constructed on the CAMBIA1300-sGFP vector is denoted as ​ -sGFP ( ​ (Medium B). The reaction system consisted of: [0.02 × total base pairs of the vector] ng of linearized vector, [0.04 × total base pairs of the fragment] ng of the target gene fragment, 4 µL of 5 × CE II Buffer, 2 µL of Exnase II, and finally, ddH2O to a total volume of 20 µL. After mixing, the mixture was incubated at 37 °C for 30 min and then cooled on ice.

[0053] The ligation product was transformed into *E. coli* DH5α competent cells (Shanghai Weidi Biotechnology) using a heat shock method. Randomly selected positive colonies were verified by colony-mediated PCR. Products with the correct band length were sequenced, and the amplified CDS sequence was found to be completely identical to the reference genome sequence. Plasmids were extracted using the TIANprep Mini Plasmid Kit (Tiangen). ​ -sGFP, refer to the kit instructions for specific steps.

[0054] After extraction and testing to ensure that the concentration and quality meet the standards... ​ -sGFP plasmid was transformed into Agrobacterium GV3101 competent cells (Shanghai Weidi Biotechnology) using the freeze-thaw method. After the positive colonies were confirmed to have bands by PCR, they were mixed with 50% glycerol at a 1:1 volume ratio and stored at -80℃ for later use.

[0055] (5) Subcellular localization analysis Subcellular localization of the BnaERF5 protein was observed using an Agrobacterium-mediated transient expression system in tobacco epidermal cells. Flat leaves with a deep green color from 3-4 week old tobacco plants were selected and transiently transformed using preserved Agrobacterium. The transient expression resuspension (OD600=1.0) contained 10 mM / L MgCl2, 10 mM / L MES, and 0.1 mM / L acetylsylcholine. Tobacco samples were collected 24-48 h after injection of the transient expression resuspension and incubated in 2 µg / mL DAPI solution in the dark for 15 min for nuclear staining. Fluorescence signals were observed using a laser confocal microscope (Nikon AI-SHS, Japan).

[0056] The results showed that the fluorescence signal of BnaERF5-sGFP was concentrated in the cell nucleus, indicating the nuclear localization characteristics of BnaERF5. ​ (C). The negative control, using an empty plasmid, showed uniform fluorescence distribution throughout the cell, without nuclear-specific aggregation. (Note: This likely refers to a specific cell type or parameter.) ​ The proteins of the gene are all located in the cell nucleus, consistent with their functional characteristics as transcription factors.

[0057] 4. Screening and validation of genetically transformed lines (1) Genetic transformation of rapeseed The same method as described in the above experiment was used to construct and obtain ​ The -sGFP plasmid was then transformed into Agrobacterium GV3101 competent cells using the same method to obtain cells carrying the plasmid. ​ The GV3101 strain carrying the -sGFP plasmid was used via Agrobacterium-mediated transformation. ​The GV3101 strain containing the -sGFP plasmid was used for genetic transformation of rapeseed under sterile conditions.

[0058] The Westar inbred line, a common variety of Brassica napus, was selected as the genetic transformation recipient material. Seeds were rigorously screened and stored in a dry environment at 4℃ for later use. The aseptic seedling culture conditions were: temperature 25℃, relative humidity 60%–70%, no light during the dark culture stage, and a light intensity of 60 μmol / m² during the light culture stage. -2 s -1 The photoperiod is 16 hours of light / 8 hours of darkness. The genetic transformation process is as follows: a. Seed sterilization and aseptic seedling culture: Take 200 Westar seeds and place them in a 50mL centrifuge tube. Add 75% ethanol and soak for about 1 minute, gently shaking during the process. After discarding the ethanol, add 1.5% HgCl2 solution and soak for about 15 minutes. Rinse 4 times with sterile water. Sow the sterilized seeds evenly on M0 medium, about 20 seeds per dish, and culture at 24℃ in the dark for 6 days.

[0059] b. Agrobacterium activation and bacterial culture preparation: The preserved strain was streaked onto LB agar containing the appropriate antibiotic and incubated at 28°C for 48 hours. A single colony was picked and inoculated into a 10 mL centrifuge tube containing 4 mL of liquid LB (containing 50 mg / L Kan and 50 mg / L Rif), and incubated at 28°C with shaking at 200 rpm for 14–16 hours until OD (Organic Dioxide) was reached. 600 ≈0.6; Take 4 mL of bacterial culture into a sterile centrifuge tube, centrifuge at 3000 rpm for 3 min, discard the supernatant, resuspend twice in DM medium, and finally dilute to OD0.6. 600 =0.6~0.8, keep at 4℃.

[0060] c. Explant preparation and infection: Take 6-day-old sterile seedlings, cut off the hypocotyl and cut it into small segments of 0.8~1.0cm, place them in a culture dish with DM medium to keep them moist; immerse the explants in the prepared Agrobacterium suspension for 30min, gently shaking them several times during the process; after removing them, use sterile filter paper to absorb the bacterial solution on the surface of the explants, transfer them to M1 medium, and incubate at 24℃ in the dark for 48h.

[0061] d. Co-culture and screening for differentiation: After co-culture, the explants were transferred to medium containing M2 and cultured under light for 3 weeks to induce callus formation; the callus was transferred to medium M3 and subcultured every 2-3 weeks to screen for differentiated green shoots; when the green shoots grew to 2-3 cm, the complete growing point was cut off and transferred to medium M4 to induce rooting.

[0062] e. Identification and screening of positive plants: DNA was extracted from the transgenic plants screened in hygromycin-resistant medium. The target gene was amplified by PCR according to the method in the above experiment "(3) Acquisition of gene sequence and synthesis of CDS sequence" (primer sequences are shown in Table 1). After band detection by 1% agarose gel electrophoresis and sequencing identification, the positive seedlings were transplanted to the greenhouse for cultivation. After flowering, they were bagged and self-pollinated for seed collection.

[0063] During the tissue culture stage, Agrobacterium-transformed explants were inoculated onto selection medium containing 50 mg / L hygromycin (Hyg). Continuous selection was performed to ensure that the resulting explants exhibited Hyg resistance, thus ensuring the transfer of the vector plasmid. ​ (A and B). Next, the green shoots were transferred to a rooting medium for cultivation. Once roots had developed and the seedlings were relatively robust, they were transferred to substrate soil and cultivated in a greenhouse. ​ (C). After the seedlings have adapted to the soil cultivation environment and are growing normally ( ​ (D), DNA was extracted from young leaf tissue and analyzed using 35S- p PCR amplification of the target gene was performed using specific primers on the CAMBIA1300-sGFP vector. 1% agarose gel electrophoresis showed... ​ All transgenic plants amplified the target band, and the fragment length was consistent with the corresponding sequence length, while WT showed no specific product. ​ (EF). PCR products were sequenced, and sequence consistency was confirmed by BLAST alignment. Plants with completely correct sequences were selected for further culture. After flowering, artificial self-pollination was performed using a bagging method to prevent cross-pollination from contaminating the genetic background. T1 generation seeds were harvested after maturity, dried, and stored at 4℃ for subsequent generation propagation and phenotypic analysis.

[0064] (2) Screening of genetically transformed lines Using the self-pollinated progeny lines of the selected positive seedlings as experimental materials, and the Westar inbred line of the conventional rapeseed variety as a wild-type control (WT), physiological indicators under drought stress were determined. When the seedlings grew to 2-3 true leaves, RNA was extracted from the seedling leaves to determine the expression level of the target gene. Primers were designed using Primer Premier 5.0 based on the sequencing results in the above experiment "(4) Vector Construction" combined with the gene sequence of the reference genome (Table 2). Two lines with high expression of the target gene were selected and transferred to soil for culture (nutrient soil: vermiculite = 1:1) for subsequent physiological indicator determination.

[0065] Table 2 Primers for Quantitative Fluorescence

[0066] To screen transgenic lines with stable and high expression of target genes, total RNA was extracted from young leaves of T1 generation seedlings, and the expression levels of key genes were analyzed by qRT-PCR, such as... ​ As shown, in the T1 generation transgenic lines ​ The expression level was significantly higher than that of wild-type (WT), and it was finally selected. ​ -OE1、 ​ -OE2 was used as the material for subsequent experiments. By analyzing and measuring phenotypes and physiological indicators under drought stress, the regulatory role and possible mechanisms of key transcription factors in the response to drought stress were elucidated.

[0067] (3) ​ Phenotypic characteristics of overexpression lines under drought stress right ​ -OE1、 ​ Phenotypic observations and physiological index measurements were performed on OE2 and wild-type (WT) materials under drought stress. When seedlings reached 3-4 true leaves, a 10-day cessation of watering was used as the drought stress treatment condition, with normal conditions serving as the control. The experiment was divided into 6 treatment groups: (a) ERF5-OE1_CK: 20 plants were randomly selected ​ Seedlings with uniform appearance phenotype in gene-high expression line 1 were cultured under normal conditions; (b) ERF5-OE1_D: 20 plants were randomly selected. ​ In the gene-high expression line 1, seedlings with uniform appearance and phenotype were kept dry for 10 days; (c) ERF5-OE2_CK: 20 plants were randomly selected ​ Seedlings with identical appearance phenotypes in gene-high expression line 2 were cultured under normal conditions; (d) ERF5-OE2_D: 20 plants were randomly selected. ​ Stop watering seedlings with uniform appearance in the gene-high expression line 2 for 10 days; (e) WT_CK: 20 seedlings with identical appearance phenotypes were randomly selected and cultured under normal conditions; (f) WT_D: Randomly select 20 seedlings of wild type material with the same appearance and phenotype and stop watering for 10 days; Take photos to document the growth on the tenth day after stopping watering. For example... ​ As shown in Figure A, there was no significant difference in growth between the overexpression material and the wild-type material under control conditions. However, significant phenotypic differences were observed under drought stress treatment. ​ -OE (hereinafter referred to as OE) ​ -OE) exhibits stronger drought resistance, compared to the wild type where leaves wilt and older leaves turn yellow. ​-OE only shows slight curling and wrinkling of the leaf margins and slight bending of the petiole, and overall shows a relatively good growth condition. ​ (B)

[0068] (4) Under drought stress ​ Physiological phenotype analysis of -OE and WT Measurements were taken under drought stress. ​ -OE1、 ​ Physiological indicators such as photosynthetic pigment content, anthocyanin content, osmotic regulation capacity, ROS accumulation, and antioxidant enzyme activity of -OE2 and wild-type WT were used to investigate ​ Possible mechanisms involved in regulating the drought stress response of rapeseed.

[0069] In the determination of photosynthetic pigment content, ​ The chlorophyll a content of the -OE2 strain was significantly lower than that of WT and under control conditions. ​ -OE1, however, its content was significantly upregulated under drought stress, and the chlorophyll a content of the different materials showed no significant difference under drought stress. ​ (A). Chlorophyll b content also showed significant differences under control conditions, specifically, the content in WT was significantly higher than that in control conditions. ​ -OE two lines, however, after drought stress ​ The content of -OE1 increased by about 4 times, while ​ The increase in -OE2 was 4.2 times, compared to 2.36 times in WT, and the increase in ERF5-OE was more significant, especially under drought stress. ​ -OE1 chlorophyll b content was significantly higher than WT ( ​ (B) Regarding anthocyanin content, there was no significant difference among the materials under control conditions, but under drought stress... ​ The content of -OE increased sharply, significantly higher than that of WT ( ​ (C)

[0070] From the perspectives of moisture regulation and oxidative damage, physiological indicators such as relative water content (RWC), electrical conductivity (EL), proline (Pro), and malondialdehyde (MDA) content were measured to assess [the effects of these factors]. ​ -Differences in drought stress response between OE and WT.

[0071] The decrease in RWC under drought stress was more significant in WT materials, decreasing by 40% compared to the control condition. ​ -OE1 and ​ -OE2 decreased by 26% and 29% respectively under drought treatment. ​ The relative water content in OE1 is significantly higher than that in WT. ​ (A). In terms of conductivity, WT is comparable to... ​-OE exhibited greater electrolyte leakage under drought stress, indicating that WT cell membrane damage was more significant. ​ -OE is more severe ( ​ (B) As a key solute for osmotic regulation, although there was no statistically significant difference under control conditions. ​ The Pro content in -OE is already slightly higher than that in WT. Under drought stress ​ The Pro content in -OE1 is significantly higher than that in WT. ​ -OE2 still shows no significant difference from WT. ​ (C). From the perspective of assessing oxidative damage to cell membranes, such as... ​ As shown in Figure D, under control and drought stress treatments, ​ The MDA content in -OE is lower than that in WT, among which ​ -OE2 levels under drought stress were significantly lower than those under total WT, indicating lower cell membrane lipid peroxidation and stronger drought stress adaptability.

[0072] Regarding antioxidant enzyme activity, it showed [significance] under control conditions. ​ -OE activity was lower than WT, and under drought stress ​ There was no significant difference between -OE and WT, because SOD activity was... ​ -OE especially ​ -OE2 was significantly upregulated by drought stress ( ​ (A). The activity of POD showed significant differences under both control and drought conditions. ​ The activity of -OE was significantly higher than that of WT under control conditions. ​ This advantage in -OE1 persists under drought stress, while ​ -OE2 content increased less due to drought-induced factors than WT, and its content under drought stress was not significantly different from WT. ​ (B). Similar characteristics exist in CAT, under control conditions. ​ The content of -OE was significantly higher than that of WT, while it was only [missing information] after drought stress treatment. ​ -OE2 and WT showed a highly significant difference in content, while ​ The content of -OE1 did not differ significantly among treatments. ​ (C). In the APX activity assay, although the ERF5-OE content was higher than WT under control conditions, the difference was not significant. However, after drought stress treatment... ​ The content of -OE1 increased sharply, significantly higher than that of WT, while ​ -OE2 showed no significant difference from WT. ​(D). In general, although there were some differences in content among strains, the antioxidant enzyme activity generally showed improvement under drought stress. BnaERF5 Significantly elevated levels in overexpression lines. BnaERF5 By regulating the antioxidant enzyme network, a key physiological and biochemical basis for enhanced drought resistance is provided.

[0073] Visualization of ROS accumulation in leaves using DAB and NBT staining further confirmed the results of the antioxidant enzyme activity assay. Picture 11 It can be observed that under drought stress ERF5 - The blue spots stained with NBT in OE leaves are more dispersed and lighter in color compared to WT, indicating that O2 ·— exist ERF5 -OE accumulates less and is more dispersed, indicating a less severe threat of oxidative stress. Similarly, after DAB staining, compared to the darker and more widely distributed brown spots in WT leaves, ERF5 The -OE contains only small, light-colored brown precipitates, indicating low H2O2 accumulation under drought stress. Based on the above results, BnaERF5 Overexpression lines significantly reduce ROS accumulation by enhancing antioxidant enzyme activity, effectively alleviating drought-induced oxidative damage and thus conferring stronger drought resistance to the plants.

[0074] Example 2 1. ERF5 Differential gene expression analysis between OE and WT In order to investigate BnaERF5 The study investigated the function of plant responses to drought and anthocyanin synthesis, selecting the example from Example 1. BnaERF5 -OE2 strain (hereinafter referred to as ERF5 Transcriptome analysis was performed on samples from wild-type controls (OE) and wild-type controls (WT) under normal conditions and under drought stress treatment with 10 days of withheld irrigation. Results are as follows: Picture 12 As shown in Figure A.

[0075] Under drought stress ERF5 -OE and WT detected 14,034 and 13,939 DEGs, respectively, with upregulated genes accounting for 67.6% (9,491) and 66% (9,132), respectively, indicating that both materials exhibit a response pattern dominated by gene activation. It is noteworthy that although the total number of DEGs is similar between the two materials, their regulatory direction and functional gene composition may differ to some extent. Furthermore, under normal conditions, ERF5 There are 4646 constitutive DEGs between -OE and WT, of which 57.9% (2690) are genes in -OE. ERF5 -OE expression was suppressed; however, after drought stress, the number of DEGs between the two materials decreased to 2448, and ERF5 The proportion of highly expressed genes in ERF5-OE increased from 42.1% to 53.6% (1312 genes). This shift suggests that drought stress may partially offset the constitutive differences between ERF5-OE and WT by activating common stress response pathways. BnaERF5 It may enhance the transcriptional activation of downstream target genes by integrating conserved drought signaling pathways.

[0076] Through the ERF5 Venn diagram analysis was performed on differentially expressed genes between -OE and WT under drought stress. Picture 12 In the study (B), 8916 common DEGs (46.79% of the total DEGs) were found between the two materials, indicating that a significant portion of the response mechanisms are the same between the two materials under drought conditions, which may involve basic drought resistance pathways. ERF5 -OE and WT have 5118 (26.86%) and 5023 (26.36%) specific DEGs, respectively. These specific genes may be involved in unique regulatory networks or biological processes, ultimately leading to differences in drought stress response and anthocyanin synthesis between the two.

[0077] 2. Under drought stress ERF5 Differential gene enrichment analysis between OE and WT To further explore ERF5 The mechanism of the difference in drought stress response between -OE and WT, ERF5 -OE was also used in GO and KEGG enrichment analysis of DEGs before and after drought stress. Picture 13 The results of GO enrichment analysis showed a high degree of similarity to the significantly activated pathways in WT, only in order. Under drought stress, both pathways activated core drought-resistance pathways such as ABA signaling, JA signaling, and thylakoid function, indicating that... ERF5 -OE responds to water loss through conservative mechanisms such as stomatal closure, hormonal synergy, and photosynthetic protection. Picture 13 (A). Although KEGG analysis also showed a large enrichment in the same pathway, some pathways related to stress response protein synthesis and membrane stability, such as the ribosome pathway, ether lipid metabolism, and zeatin biosynthesis, were specifically enriched in ERF5-OE. Picture 13 (Middle B). Of particular note is the role of flavonoid biosynthesis. ERF5 The significant activation of -OE in the OE partly explains the effects of drought stress. ERF5 -The physiological phenotype of significantly increased anthocyanin content in OE. Given... ERF5The similarity of activation pathways between -OE and WT under drought stress suggests that the large accumulation of flavonoids with antioxidant activity, such as anthocyanins, under drought stress is the cause of... ERF5 -OE has higher drought resistance than WT.

[0078] 3. Under drought stress ERF5 Expression analysis of anthocyanin structural genes in -OE and WT To further explore ERF5 To investigate the regulatory mechanisms of anthocyanin synthesis under drought stress, 18 differentially expressed anthocyanin structural genes were screened, including: 3 chalcone isomerase (CHI) genes, 4 flavanone 3-hydroxylase (F3H) genes, 3 flavanone 3'-hydroxylase (F3'H) genes, 4 flavonol synthase (FLS) genes, 2 dihydroflavonol reductase (DFR) genes, and 1 anthocyanin synthase (ANS) gene and 1 UDP-flavonoid glucosyltransferase (UFGTs) gene. Based on their respective positions in the anthocyanin synthesis pathway and the changes in their expression levels in different comparison groups, pathway heatmaps were created for visual visualization. ERF5 Effects of drought stress on the expression of anthocyanin synthesis structural genes ( Picture 14 (A). They also mostly responded to drought stress by adjusting upwards. Two of them FLS Genes (BnaC09G0565100ZS, BnaA06G0169300ZS) and two DFR Genes (BnaC09G0215200ZS, BnaA09G0187400ZS) showed increased activity under drought stress in both materials and treatments. ERF5 Upregulated expression in -OE may be ERF5 Strong candidates for downstream target genes. It is noteworthy that under control conditions, ANS The (BnaA03G0469300ZS) gene is in ERF5 -OE expression was downregulated compared to WT, and further downregulated after drought stress compared to control, showing an expression trend different from other structural genes. Further reference was made to cluster analysis of differentially expressed genes (…). Picture 14 (B) was found to be an early synthetic gene. CHI , F3H Late-stage synthetic genes DFR and flavonol synthase gene FLS It showed ERF5 The significant high expression of -OE genes suggests they may be key structural genes in the flavonoid, especially anthocyanin, pathway in response to drought stress. Furthermore, changes in their expression levels may explain, to some extent, the effects of drought. ERF5 - Physiological phenotype of significant anthocyanin accumulation in OE.

[0079] 4. Analysis of promoter cis-regulatory elements of differentially expressed genes in the anthocyanin synthesis pathway To further explore the molecular mechanisms of anthocyanin response to drought stress and identify downstream target genes of key genes, the cis-regulatory elements of the promoters of these structural genes were analyzed. Cis-regulatory elements of the 2000 bp sequences of the promoters of the 19 differentially expressed structural genes enriched above were analyzed using the PlantCARE online analysis software. Visualization using TBtool revealed the widespread presence of ERF binding sites (such as the ethylene response element ERE) and various hormone and stress response elements (such as ABRE, DRE, highly repetitive TC sequences, G-boxes, etc.) on the promoters of anthocyanin synthesis structural genes, suggesting that their expression may be synergistically regulated by multiple signaling pathways. Specifically, ERF transcription factors can respond to ethylene (ET) and jasmonic acid (JA) signals by binding to the ERE element. Furthermore, the enrichment of ABRE and DRE elements in the promoters suggests that ABA signaling and non-ABA-dependent dehydration response signals may be regulated through the ARB / ABF-DREB / CBF transcription factor network, while ROS signaling may indirectly affect the expression of these structural genes by activating factors such as NAC or WRKY and binding stress response elements such as TC-rich repeats. Therefore, it is hypothesized that key transcription factors may synergistically activate the expression of these structural genes by integrating multiple signals such as ABA, JA, and ROS. This partially explains why a large number of structural genes are upregulated under drought stress; this multi-level regulatory mechanism enables anthocyanin synthesis genes to respond rapidly under drought stress. Simultaneously, structural genes with a large number of ERE elements in their promoters may be strong candidates for downstream genes of the key transcription factor ERF5.

[0080] 5. Prediction of downstream target genes of BnERF5 Based on the PlantTFDB-based Retrieve Regulation module, for key transcription factors BnaERF5 Predicting downstream target genes, BnaERF5 The target gene prediction results included two CHI The genes (BnaC08G0351800ZS, BnaA09G0510300ZS) share a common binding site CCAACGCCTGCGCCT (SEQ ID NO:9), but their expression levels differ under drought stress. ERF5 - It was upregulated in both OE and WT, and there was no difference in expression between materials, therefore it was excluded as a... ERF5 Candidate downstream target genes.

[0081] The prediction results of PlantTFDB were further validated and supplemented using the online prediction platform JASPAR database. ERF5 An individual analysis of 18 structural genes that showed differential expression in -OE and WT revealed... DFR There are two binding sites with relative scores >0.8 on (BnaC09G0215200ZS): CCTCCTCCTCCTTTG (SEQ ID NO:10) / TTACCTCCTCCTCCT (SEQ ID NO:11), which have been verified to be present in the promoter sequence of the reference genome. Furthermore, in Picture 14 The expression level of the DFR (BnaC09G0215200ZS) gene differed significantly between materials and treatments, therefore it was included as... ERF5 Downstream target gene candidates will be further validated.

[0082] Based on the above prediction results, the BnERF5 protein and its derivatives were predicted using AlphaFold and HDOCK servers. DFR The structural model and interaction sites of the (BnaC09G0215200ZS) promoter (1500bp) were analyzed. The model with the highest confidence was submitted to PyMOL software for visualization. The gray chain represents the protein sequence, the green chain represents the candidate target gene promoter sequence, and the hydrogen bonds between them are indicated by red dashed lines. Picture 15 The directional and specific interactions between proteins and their ligands rely primarily on hydrogen bonds, which play a crucial role in protein recognition of specific DNA sequences. For example... Picture 15 As shown, BnaERF5 can interact with DFR The formation of hydrogen bonds (BnaC09G0215200ZS) indicates that there may be interaction sites between these DNAs and proteins, further increasing the possibility that these structural genes are downstream target genes of key transcription factors.

[0083] The above results indicate that BnaERF5 By integrating the ABA and JA signaling pathways, key genes in the osmotic regulation pathway and the flavonoid pathway are synergistically activated. CHI , F3H , DFR and FLS These substances enhance antioxidant enzyme activity, ROS scavenging capacity, and anthocyanin accumulation, effectively maintaining osmotic balance and significantly improving drought resistance. Key structural genes... CHI , F3H , DFR and FLSThe promoter contains numerous ERF binding sites and hormone and stress response elements. It is synergistically regulated by multiple signals such as transcription factor integration ABA, JA, and ROS, and is a key structural gene in the anthocyanin synthesis pathway of Brassica napus in response to drought stress. BnaERF5 Overexpression can maintain the basic stress response network and further enhance drought resistance through anthocyanin accumulation. Anthocyanin-mediated drought resistance depends on the complex gene regulatory network behind it, rather than simply an increase in content.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. BnaERF5 Application in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

2. The application according to claim 1, characterized in that, The BnaERF5 Rapeseed drought resistance can be improved by enhancing antioxidant defense, maintaining osmotic balance, and reducing oxidative damage.

3. The application according to claim 1, characterized in that, The BnaERF5 include BnaERF5 Genes, mRNA, cDNA, or proteins.

4. The application according to claim 3, characterized in that, The BnaERF5 The gene is located at positions 46719350-46720237 on chromosome A06 of the Brassica napus reference genome, version ZS11.v0.

5. Overexpression BnaERF5 Application of recombinant vectors in improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress.

6. The application according to claim 5, characterized in that, The original vector for the recombinant vector included 35S-pCAMBIA1300-sGFP.

7. The application according to claim 6, characterized in that, The method for constructing the recombinant vector includes: using BamHI and XbaI restriction endonucleases to target 35S- p CAMBIA1300-sGFP was double-digested to obtain a linearized vector; PCR amplification was then performed to obtain... BnaERF5 The CDS target segment; the said BnaERF5 The CDS target fragment was homologously recombined into a linearized vector; the ligation product was transformed into E. coli DH5α competent cells by heat shock, and the plasmid was extracted after correct sequencing.

8. The application according to claim 7, characterized in that, The primer sequences for the PCR amplification include SEQ ID NO:1~SEQ ID NO:

2.

9. A method for improving the drought resistance of rapeseed and / or increasing the anthocyanin content of rapeseed under drought stress, characterized in that, The method includes: under sterile conditions, using Agrobacterium-mediated transformation to overexpress... BnaERF5 The recombinant vector was introduced into rapeseed explants, and transgenic rapeseed plants were obtained through screening and culture.

10. A transgenic rapeseed plant, characterized in that, The plant is obtained by the method of claim 9.

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