Transcription factor for regulating drought tolerance of brassica napus and application thereof

Abstract

This invention relates to the field of plant molecular biology and discloses a transcription factor that regulates drought resistance in rapeseed and its application, comprising the rapeseed transcription factor gene BnaNAC329, the drought-responsive promoter pBnaNAC329, a recombinant overexpression vector, a recombinant promoter vector, and a host bacterium. This transcription factor regulating drought resistance in rapeseed and its application provide an effective gene resource for the breeding of drought-resistant rapeseed varieties. Based on the experimental results of this invention, haplotypes with high BnaNAC329 expression levels can be screened in natural rapeseed populations for use in the breeding of drought-resistant varieties.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, specifically to a transcription factor that regulates drought resistance in rapeseed and its application. Background Technology

[0002] Climate change alters rainfall patterns and temperatures, bringing a range of abiotic stresses such as nutrient deficiencies, drought, salinity, flooding, and temperature fluctuations, forcing changes, transformations, or even collapses in ecological and agricultural systems (Raza et al. 2019). Rapeseed is an important oilseed crop worldwide, with its seeds containing approximately 61.0% oleic acid and 8.8% linoleic acid. Rapeseed oil is of high nutritional quality and is considered one of the healthiest edible vegetable oils (Afridie et al. 2002). The Yangtze River Basin is my country's main rapeseed producing area, where rainfall is uneven throughout the year, making it prone to seasonal droughts and flooding. Rapeseed-growing areas in southwest and northwest my country also face the impact of drought. Statistics show that autumn droughts can reduce the total rapeseed yield in the Yangtze River Basin by 25%–32% (Dai et al. 2006). However, current research on the impact of drought on crop growth and final yield mainly focuses on several major food crops such as Arabidopsis thaliana and wheat, with few reports on the discovery of drought-resistant genes and related mechanisms in rapeseed. Therefore, in order to avoid the threat of climate change to rapeseed yield, it is necessary to create stable varieties with stronger stress resistance and wider adaptability through biological, genetic and genetic engineering methods to reduce yield loss.

[0003] Transcription factors are key regulators at the gene transcription level, specifically binding to cis-elements in the promoter region upstream of the start codon of target genes to regulate gene transcription (Xion et al. 2005). Under abiotic stress conditions, transcription factors (TFs) act as the core of signal transduction after plants perceive abiotic stress and as molecular switches for the expression of stress-responsive genes, playing a crucial role in stress signal transduction pathways after abiotic stress (Zhang et al. 2022). Therefore, identifying drought-related TFs in rapeseed and elucidating their functions and mechanisms of action is of great significance for understanding the complex spatiotemporal transcriptional behavior of rapeseed genes under drought stress, and also provides important guidance for breeding resistant varieties of rapeseed.

[0004] NAC (NOAPICALMERISTEM (NAM), ARADIBOPSTRANSCRIPTIONACTIVATOR FACTOR1 / 2 (ATAF1 / 2), CUP-SHAPEDCOTYLEDON2 (CUC2)) transcription factors are a class of plant-specific transcription factors (Nuruzzaman et al. 2013). NAC transcription factors contain a conserved NAC domain (approximately 150 amino acids) at the N-terminus and a diverse regulatory domain at the C-terminus, responsible for DNA binding and transcriptional regulation, respectively (Ernste et al. 2004, Olsen et al. 2005). The NAC domain is divided into five subdomains, named A through E. The highly conserved C and D subdomains are essential for DNA interaction, the A subdomain is essential for protein dimerization or heterodimerization, while the highly variable B and E subdomains determine the functional diversity of NAC proteins (Puranike et al. 2012). Studies have shown that NAC transcription factors play a crucial role in plant adaptation to abiotic stresses such as low temperature, drought, and salinity damage. Under drought or high-temperature stress, the rice ONAC023 nucleus can target and promote the expression of multiple genes involved in various processes such as water transport, reactive oxygen species homeostasis, and alternative splicing, including OsPIP2;7, PGL3, OsFKBP20-1b, and OsSF3B1, actively regulating drought and heat tolerance (Change et al. 2024). Tomato SlNAC35 negatively regulates salt tolerance by directly targeting and regulating the olefin oxide cyclase (AOC) SlAOC, inhibiting arsenic acid (JA) biosynthesis (Menget et al. 2025). Foxtail millet SiNAC015 may regulate drought response by directly inhibiting the expression level of the SitPRX gene encoding peroxidase (POD), thereby directly suppressing the expression level associated with reactive oxygen species (ROS) scavenging activity (Xiao et al. 2025). However, there are few reports on the function and role of NAC transcription factors in rapeseed in response to abiotic stress. The BnaNAC329 gene belongs to the NAC family. Cloning the BnaNAC329 gene of rapeseed and studying its potential role under drought stress conditions, as well as elucidating its specific functions, has important theoretical significance and excellent value for breeding superior stress-resistant rapeseed varieties. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a transcription factor that regulates drought resistance in rapeseed and its application. This provides an effective gene resource for the breeding of new drought-resistant rapeseed varieties. Based on the experimental results of this invention, haplotypes with high expression levels of BnaNAC329 can be screened in natural rapeseed populations for the breeding of new drought-resistant varieties, thus solving the problems mentioned in the background technology.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: a transcription factor that regulates the drought resistance of rapeseed, comprising the rapeseed transcription factor gene BnaNAC329, the drought response promoter pBnaNAC329, a recombinant overexpression vector, a recombinant promoter vector, and a host bacterium.

[0009] Preferably, the nucleotide sequence of the rapeseed transcription factor gene BnaNAC329 is shown in SEQ ID NO:2.

[0010] Preferably, the amino acid sequence encoded by the rapeseed transcription factor gene BnaNAC329 is shown in SEQ ID NO:3.

[0011] Preferably, the drought response promoter pBnaNAC329 nucleotide sequence is shown in SEQ ID NO:1 and can be activated by drought and ABA stress.

[0012] Preferably, the recombinant promoter vector contains the pBnaNAC329 promoter.

[0013] The application of a transcription factor that regulates drought tolerance in rapeseed: by overexpressing BnaNAC329 in rapeseed, the rate of water loss from leaves is reduced and the number of lateral roots is increased, thereby enhancing the drought tolerance of rapeseed.

[0014] The application of a transcription factor that regulates drought resistance in rapeseed: BnaNAC329 directly binds to and activates the BnaLBD16 and BnaGATA23 promoters, thereby regulating the expression of genes related to lateral root development and positively regulating lateral root development and drought resistance in rapeseed.

[0015] (III) Beneficial Effects

[0016] Compared with existing technologies, this invention provides a transcription factor for regulating drought resistance in rapeseed and its application, which has the following beneficial effects:

[0017] 1. This transcription factor that regulates drought resistance in rapeseed and its application is provided by a drought- and ABA-induced rapeseed promoter pBnaNAC329, the sequence of which is shown in SEQ ID NO.1.

[0018] 2. The transcription factor that regulates drought resistance in rapeseed and its application, by providing a transcription factor gene BnaNAC329 that positively regulates drought resistance in rapeseed and its encoded protein, wherein the nucleotide sequence of the rapeseed transcription factor BnaNAC329 is shown in SEQ ID NO. 2 and the amino acid sequence is shown in SEQ ID NO. 3.

[0019] 3. The transcription factor that regulates drought resistance in rapeseed and its application: By providing the application of BnaNAC329 in improving the drought resistance of rapeseed and breeding new drought-resistant rapeseed varieties. Attached Figure Description

[0020] Figure 1 The following are examples of amplification of pBnaNAC329 and PCR identification of pBnaNAC329::GUS transgenic Arabidopsis thaliana in this invention: A. Cloning electrophoresis image of pBnaNAC329 (M is DNAMaker, 1 is pBnaNAC329); B. Schematic diagram of pBnaNAC329::GUS vector; C. Electrophoresis image of pBnaNAC329::GUS Arabidopsis thaliana for PCR identification (M is DNAMaker, GUS1-3 are T0 generation positive pBnaNAC329::GUS transgenic Arabidopsis thaliana).

[0021] Figure 2 This invention presents the tissue expression pattern analysis of BnaNAC329; A. qRT-PCR results of BnaNAC329 in rapeseed leaves, roots, flowers, siliques, and stems; B. GUS staining results in pBnaNAC329::GUS transgenic Arabidopsis leaves, roots, flowers, siliques, and stems. Errors in the figures represent mean ± SD (Standard Deviation); data significance analysis was performed using Duncan analysis.

[0022] Figure 3 This invention presents the gene expression level of BnaNAC329 under drought stress and the promoter activity analysis of pBnaNAC329 under drought stress; AB. The gene expression level of BanNAC329 in the true leaves of four-leaf stage rapeseed seedlings after drought (A) and ABA (B) treatments; C. GUS staining results of pBnaNAC329::GUS transgenic Arabidopsis thaliana under drought and ABA treatments; D. LUC activity analysis of tobacco leaves infected with Agrobacterium tumefaciens pBnaNAC329::LUC under drought and ABA treatments. Errors in the figures represent mean ± SD (Standard Deviation); data significance analysis was performed using Duncan analysis.

[0023] Figure 4This invention relates to the creation and identification of rapeseed overexpressing BnaNAC329; A. Map of the constructed BnaNAC329 overexpression vector (p35s::BnaNAC329); B. PCR identification of T0 transgenic rapeseed overexpressing BnaNAC329; C. qRT-PCR detection of BnaNAC329 in T3 generation homozygous overexpressing BnaNAC329 rapeseed, with errors in the figure representing mean ± SD (Standard Deviation, SD); data significance analysis was performed using Duncan analysis; D. Seed fluorescence detection of the three lines with the highest BnaNAC329 expression levels (OE1, OE2, and OE3).

[0024] Figure 5 To identify the drought resistance of rapeseed overexpressing BnaNAC329 in this invention; A. Drought treatment of rapeseed overexpressing BnaNAC329; B. Survival rate of rapeseed overexpressing BnaNAC329 and wild-type rapeseed after drought treatment; The significance analysis of the data was performed by Duncan analysis; Error value: mean ± SD.

[0025] Figure 6 For the purposes of this invention, infrared observation of leaves and determination of detached leaf water loss rate of BnaNAC329-overexpressing rapeseed and wild-type rapeseed were performed; A. Far-infrared detection of leaf surface temperature of BnaNAC329-overexpressing rapeseed and wild-type rapeseed seedlings after 3 weeks of growth; B. Statistical analysis of detached leaf water loss rate of BnaNAC329-overexpressing rapeseed and wild-type rapeseed seedlings at the four-leaf stage; Asterisks represent the p-value of t-test for the means of two samples (*: P<0.05; **: P<0.01); Error value: mean ± SD.

[0026] Figure 7 The effect of mannitol osmotic stress on the root phenotype of wild-type (WT) and overexpression lines (OE1 / 2 / 3). A. Root growth phenotypes of wild-type (WT) and overexpression lines (OE1, OE2, OE3) under normal conditions (Control) and 200 mM mannitol osmotic stress treatment. Scale bar = 2 cm. B. Statistical analysis of primary root length (PRlength) (B) and lateral root number (LRnumber) of the above materials under control and 200 mM mannitol treatment. Error in the figure represents mean ± SD (Standard Deviation); the significance analysis method was Duncan analysis. Different lowercase letters indicate significant differences between groups (P < 0.05).

[0027] Figure 8The figures show the expression levels of genes related to the number of lateral roots in the roots of BnaNAC329-overexpressing rapeseed and wild-type rapeseed grown hydroponically for 12 days. Error in the figures represents mean ± SD (Standard Deviation); data significance analysis was performed using Duncan's analysis.

[0028] Figure 9 To verify the interaction and transcriptional activation of BnaNAC329 with BnaLBD16 and BnaGATA23: A. Yeast one-hybrid (Y1H) experiment to verify the binding ability of BnaNAC329 to the promoters of BnaLBD16 and BnaGATA23. Positive: positive control; Negative: negative control. B. Dual-luciferase reporter gene (LUC) experiment to verify the transcriptional activation activity of BnaNAC329 on the promoters of BnaLBD16 and BnaGATA23. The color bar on the right is the fluorescence intensity scale. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Please see Figure 1-9 A transcription factor that regulates drought resistance in rapeseed includes the rapeseed transcription factor gene BnaNAC329, the drought response promoter pBnaNAC329, a recombinant overexpression vector, a recombinant promoter vector, and a host bacterium.

[0031] The nucleotide sequence of the rapeseed transcription factor gene BnaNAC329 is shown in SEQ ID NO:2.

[0032] The amino acid sequence encoded by the rapeseed transcription factor gene BnaNAC329 is shown in SEQ ID NO:3.

[0033] The drought response promoter pBnaNAC329 nucleotide sequence is shown in SEQ ID NO:1 and can be activated by drought and ABA stress.

[0034] The recombinant promoter vector contains the pBnaNAC329 promoter.

[0035] The application of a transcription factor that regulates drought tolerance in rapeseed is characterized by: reducing the rate of water loss from leaves and increasing the number of lateral roots by overexpressing BnaNAC329 in rapeseed, thereby enhancing the drought tolerance of rapeseed.

[0036] The application of a transcription factor that regulates drought resistance in rapeseed is characterized by the fact that BnaNAC329 directly binds to and activates the BnaLBD16 and BnaGATA23 promoters, thereby regulating the expression of genes related to lateral root development and positively regulating lateral root development and drought resistance in rapeseed.

[0037] In summary, this transcription factor that regulates drought resistance in rapeseed and its application are currently under investigation.

[0038] (1) Gene and promoter cloning

[0039] This invention cloned the following from Brassica napus: pBnaNAC329 promoter: sequence as shown in SEQ ID NO:1, length 1718bp, significantly induced by drought and ABA; BnaNAC329 gene: CDS sequence as shown in SEQ ID NO:2, full length 903bp, protein sequence as shown in SEQ ID NO:3, total 300 amino acids.

[0040] (2) Carrier construction

[0041] Construct vectors for analyzing the activity of pBnaNAC329::GUS and pBnaNAC329::LUC promoters;

[0042] A 35S::BnaNAC329 overexpression vector was constructed for rapeseed genetic transformation.

[0043] (3) Genetic transformation

[0044] Using Agrobacterium-mediated hypocotyl infection, 35S::BnaNAC329 was introduced into rapeseed to obtain homozygous overexpression lines OE1, OE2, and OE3.

[0045] (4) Functional verification

[0046] Phenotypic identification: Under drought stress, the BnaNAC329 overexpression lines exhibited slower leaf water loss, higher leaf temperature, and a significantly increased number of lateral roots. Their survival rate after rehydration was significantly higher than that of the wild type.

[0047] Gene expression: In overexpression lines, genes related to lateral root development and auxin pathways, such as BnaYUCCA4, BnaAUX1, BnaARF5 / 19, BnaGATA23, and BnaLBD16 / 18 / 29, were significantly upregulated.

[0048] Molecular mechanism: Yeast one-hybrid and dual-luciferase experiments confirmed that BnaNAC329 can directly bind to the BnaLBD16 and BnaGATA23 promoters and activate their transcription, making it a key upstream factor regulating lateral root development.

[0049] This invention is the first to demonstrate that BnaNAC329 is a positive regulator of drought in rapeseed, and its expression is induced by drought and ABA. It elucidates the function of BnaNAC329 in improving drought resistance through a dual pathway of reducing leaf water loss and promoting lateral root development. It reveals a new pathway by which BnaNAC329 directly targets BnaLBD16 and BnaGATA23 to regulate root architecture. This invention can be directly used for transgenic breeding, gene editing, and molecular marker-assisted selection to cultivate new drought-resistant rapeseed varieties.

[0050] To enable researchers in the field to better understand the technical solution of this experiment, a detailed description is provided below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the technical methods in the embodiments are conventional methods and conditions well known to those skilled in the art. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments can be obtained commercially.

[0051] Example 1: Cloning of the promoter sequence of the drought response-related gene BnaNAC329 from Brassica napus and obtaining pBnaNAC329::GUS transgenic Arabidopsis thaliana.

[0052] 1. Primer design

[0053] The sequence of the promoter pBnaNAC329, 2000 bp upstream of the start codon of the BnaNAC329 gene in the reference genome ZS11.V0, was obtained from the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR). Primers containing a 15-20 bp homologous sequence of the vector backbone were designed for cloning pBnaNAC329 and constructing the pBnaNAC329::GUS vector through homologous recombination. The primer sequences are as follows:

[0054] pN329-GUS-F2:

[0055] tggctgcaggtcgacggatccTTACCGTTTGACTATCTGGGGATA

[0056] pN329-GUS-R2:

[0057] tcttagaattcccggggatccTCTTCAGCAGACAAAACAAT

[0058] 2. Cloning of pBnaNAC329

[0059] Genomic DNA was extracted from ZS11 rapeseed using the CTAB method. Using ZS11 genomic DNA as a template, pBnaNAC329 was amplified using primers pN329-GUS-F2 and pN329-GUS-R2. Figure 1After sequencing verification, the total length of the amplified pBnaNAC329 nucleotide sequence was 1718 bp. The amplification system is shown in Table 1.

[0060] Table 1. PCR system for pBnaNAC329 amplification

[0061] Reagent volume (μl)

[0062] KODOne™ PCRMasterMix-Blue25

[0063] pN329-GUS-F2 (10μM) 1.5

[0064] pN329-GUS-R2 (10μM) 1.5

[0065] ZS11 genomic DNA (100 ng / μl) 1

[0066] ddH2O21

[0067] PCR amplification program: 98℃ for 3 min; 98℃ for 15 s, 58℃ for 15 s, 68℃ for 30 s, 32 cycles, then 68℃ for 5 min.

[0068] Following the method described in the DNA Gel Recovery Kit (Safe and Convenient Type) (Catalog No.: TSP602-200) from Qingke Biotechnology Co., Ltd., the PCR products were recovered for the construction of the pBnaNAC329::GUS vector.

[0069] 3. Enzyme digestion of pCAMBIA-1391 vector

[0070] The pCAMBIA-1391 vector plasmid was digested according to the method and system described in the Thermo Fisher Scientific BamHI (10 U / μL) (catalog number: ER0051) instructions. The digestion products were recovered according to the method described in the Qingke Biotechnology DNA Gel Recovery Kit (Safe and Convenient Type) (catalog number: TSP602-200) for the construction of the pBnaNAC329::GUS vector.

[0071] 4. Homologous recombination and E. coli transformation

[0072] The pBnaNAC329 vector was ligated to the BamHI-linearized pCAMBIA-1391 vector using the method and system described in the instructions of Nanjing Novizan Biotechnology Co., Ltd.'s ClonExpressUltraOneStepCloningKitV3 (catalog number: C117-01). Figure 1Following the instructions for use of DH5α competent cells (catalog number CC96102) from Shanghai Tolo Harbor Biotechnology Co., Ltd., the homologous recombination product was transformed into *E. coli*. Single colonies that tested positive by PCR were selected and sent for further testing, confirming their correctness and yielding the pBnaNAC329::GUS vector.

[0073] 5. Obtaining pBnaNAC329::GUS transgenic Arabidopsis thaliana

[0074] Following the instructions for GV3101 (PSoup) competent cells (catalog number: SP8140) from Protech Biotechnology Co., Ltd., the pBnaNAC329::GUS plasmid, which had been verified by sequencing, was transformed into GV3101. Single colonies that tested positive by colony PCR were selected and inoculated into YEP liquid medium (50 mg / L rifampin and 50 mg / L kanamycin), and cultured at 28°C and 220 rpm on a shaker until OD600 = 0.8-1.2. After centrifugation at 5000 rpm, the colonies were resuspended in an equal volume of infection medium (1 / 2 MS, 5% sucrose, 0.05% silwet L-77, pH 5.7). Unopened flower buds of Arabidopsis thaliana Col-0 were immersed in the infection medium solution for 30-40 seconds, kept moist, and incubated in the dark for 24 hours. Then, the colonies were continued under normal conditions (25°C, 16 hours light, 8 hours dark) until seed harvest.

[0075] 6. Positive identification of pBnaNAC329::GUS transgenic Arabidopsis thaliana

[0076] After harvesting and disinfecting infected Arabidopsis seeds with 0.1% mercuric chloride solution (containing 0.05% Tween 20), the seeds were spotted onto 0.61 / 2 MS plates (containing 10 mg / L HYG) to screen for positive seedlings. The selected positive plants were then transplanted into soil, and DNA was extracted for PCR identification (results are shown in the figure). Figure 2 As shown), positively identified Arabidopsis thaliana samples were cultured under normal conditions (25°C, 16 hours light, 8 hours darkness) until seed harvest. Figure 3 ).

[0077] Example 2: Analysis of BnaNAC329 tissue expression patterns and pBnaNAC329 promoter activity.

[0078] 1. Design of primers for BnaNAC329qRT-PCR

[0079] The CDS sequence of the BnaNAC329 gene in the reference genome ZS11.V0 was obtained from the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR), and primers were designed for the quantification of BnaNAC329 clones. The primer sequences are as follows:

[0080] N329C07-qRT-F1:

[0081] TCATTTCTCTCTCCAGGTC

[0082] N329C07-qRT-R1:

[0083] GTCTCTCGGGCTAAAGAAATA

[0084] 2. BnaNAC329 tissue expression pattern

[0085] Total RNA was extracted from roots, true leaves, bolting stems, flowers, and siliques of Brassica napus ZS11 at the four-leaf stage, according to the method described in the RNASteady PureRNA Extraction Kit (catalog number AG21024) from Hunan Aikerui Biotechnology Co., Ltd. The total RNA was converted to cDNA using a two-step method with the EvoM-MLV Reverse Transcription Premixed Kit (catalog number AG11728) from Aikerui Biotechnology Co., Ltd. The BnaACTIN gene from Brassica napus was used as an internal reference gene in the SYBR GreenProTaq HS Premixed qPCR Kit (AG11701) purchased from Hunan Aikerui Biotechnology Co., Ltd. The expression level of the BnaNAC329 gene in leaves, roots, stems, flowers, and siliques of ZS11 Brassica napus was detected using a Roche LightCycler® 480 real-time quantitative PCR instrument. The results showed that BnaNAC329 had a high expression level in leaves and roots of Brassica napus. Figure 2 ).

[0086] Leaves, roots, flowers, stems, and siliques of pBnaNAC329::GUS transgenic Arabidopsis thaliana were immersed in GUS staining solution (100 μL GUS staining solution formulation: 1 mL X-Gluc 0.1 g gin DMF, 49 mL 0.1 M phosphate buffer (Na2HPO4·12H2O 2.18 g, NaH2PO4·2H2O 0.61 g), 1 mL potassium ferricyanide (5 mM), 1 mL potassium ferrocyanide (5 mM), 100 μL Triton X-100), and incubated at 37°C in the dark. The staining time was adjusted appropriately according to the tissue type and GUS activity intensity. After staining, the GUS staining solution was aspirated, and the tissues were repeatedly rinsed with 75% ethanol until the negative control tissues were completely free of chlorophyll and other background pigments. The location where the GUS signal appeared was the gene expression site; subsequent observation was performed directly under a stereomicroscope. GUS staining results showed that GUS signals were present in all tissues examined, with the strongest signals observed in leaves and roots, indicating that BnaNAC329 has a high expression level in leaves and roots. Figure 2 ).

[0087] Four-leaf stage Brassica napus ZS11 was treated with 30 μMABA and dehydration. Leaf samples were collected at 0 h, 1 h, 3 h, 6 h, 9 h, and 12 h after treatment. Total RNA was extracted and reversed to cDNA. The expression level of the BnaNAC329 gene in the samples was quantitatively detected. The results showed that BnaNAC329 in rapeseed leaves was induced by 30 μMABA and dehydration treatment. Figure 3 ).

[0088] Four-leaf stage pBnaNAC329::GUS transgenic Arabidopsis thaliana was treated with 10 μM MABA and 250 mM mannitol. Nine hours after treatment, samples were immersed in GUS staining solution and incubated at 37°C in the dark. After staining, the GUS staining solution was removed, and the samples were repeatedly rinsed with 75% ethanol and observed directly under a stereomicroscope. GUS staining results showed that after treatment with 10 μM MABA and 250 mM mannitol, the GUS signal in the leaves of pBnaNAC329::GUS transgenic Arabidopsis thaliana was stronger than that in the untreated control material, indicating that BnaNAC329 was induced by ABA and mannitol treatment. Figure 3 ).

[0089] 3. pBnaNAC329 promoter activity analysis

[0090] The pBnaNAC329 was ligated into the pGreenII0800-LUC reporter vector and transformed into Escherichia coli DH5α. The plasmid pBnaNAC329::LUC, which was verified as a positive monoclonal strain by kanamycin (50 mg / ml) and sequencing, was extracted and introduced into the GV3101 strain carrying the helper plasmid pSoup using the freeze-thaw method. The monoclonal strains that were identified as positive by colony PCR were inoculated into 3 mL of YEP liquid medium containing the corresponding antibiotic and cultured at 28 °C and 220 rpm until the OD600 reached 0.6–0.8. The cells were centrifuged at 5000 rpm for 5 min and the supernatant was discarded. The bacterial pellet was resuspended in tobacco transient transformation resuspension buffer (100 mL formula: 2 mL LME S (0.5 M, pH 5.7), 500 µL MgCl2 (2 M, pH 7.0), 200 µL LAS, 92.3 mL ddH2O) and the OD600 was adjusted to 0.8–1.0. The suspension was then incubated at room temperature for 2–3 h. Select healthy tobacco seedlings and gently infuse the lower epidermis of the leaves with a sterile syringe needle, slowly injecting the resuspended bacterial solution into the leaf tissue through the wound. Incubate the injected plants in the dark for 12 hours, then transfer them to normal light conditions (16 hours light / 8 hours dark) for another 48 hours. Dehydrate the leaves and treat them with 30 μMABA. Take untreated leaves, leaves 6 hours after dehydration, and leaves 9 hours after ABA treatment. Measure the LUC enzyme activity using the Dual Luciferase (LUC) reporter gene assay kit from Yisheng Biotechnology (Shanghai) Co., Ltd. All enzyme activity assays were performed in the dark; all reagents were equilibrated to room temperature before use; and all assays were performed in at least three biological replicates. Results showed that the pBnaNAC329 promoter activity was significantly higher after dehydration and ABA treatment than the control, indicating that pBnaNAC329 is a drought- and ABA-induced promoter.

[0091] Example 3: Cloning of the CDS sequence of the drought response-related gene BnaNAC329 in Brassica napus and obtaining Brassica napus overexpressing BnaNAC329.

[0092] 1. Primer design, cloning of BnaNAC329 and construction of the p35s::BnaNAC329 vector

[0093] The CDS sequence of the BnaNAC329 gene in the reference genome ZS11.V0 was obtained from the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR), and primers containing 15-20 bp homologous sequences of the vector backbone were designed. The primer sequences are as follows:

[0094] BnaNAC329-F1:

[0095] GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGGTGTTAGAGAAATGGA

[0096] BnaNAC329-R1:

[0097] GGGGACCACTTTGTACAAGAAAGCTGGGTATGCCTAAACTCGAATGTTT

[0098] Using cDNA from rapeseed ZS11 leaves as a template, BnaNAC329 was amplified using primers BnaNAC329-F1 and BnaNAC329-R1. Sequencing confirmed that the amplified BnaNAC329CDS nucleotide sequence (with stop codon removed) was 900 bp in length. BnaNAC329 was ligated into the pEarleyGate103-RFP vector using Gateway recombination technology. Figure 4 The recombinant overexpression vector p35s::BnaNAC329 was successfully obtained by transforming *E. coli* DH5α competent cells and screening with kanamycin (50 mg / ml) and sequencing. The successfully constructed overexpression plasmid p35s::BnaNAC329 was then transformed into GV3101 cells according to the instructions for use with Prydex Biotechnology Co., Ltd. GV3101 (PSoup) competent cells (catalog number: SP8140).

[0099] 2. Obtaining rapeseed with BnaNAC329 overexpression

[0100] Sterilized rapeseed seeds were spotted onto MS medium and incubated in the dark for 16 / 8 hours for 5 days. Single colonies of p35s::BnaNAC329, identified positive by PCR, were selected and inoculated into YEP liquid medium (50 mg / L rifampin and 50 mg / L kanamycin). The culture was shaken at 220 rpm until the OD600 reached 0.8, and the colonies were collected by centrifugation at 5000 rpm for 5 min. The colonies were resuspended in 1 ml of liquid MS medium (containing 100 μmol / L acetosyringone). The hypocotyls of the rapeseed cultured in the dark for 5 days were cut into segments approximately 8 mm long after removing the growing point. The cut hypocotyls were placed in 20 ml of liquid MS medium (containing 100 μmol / L acetosyringone), and Agrobacterium containing the resuspended p35s::BnaNAC329 plasmid were added. The mixture was incubated in the dark for 15-20 min, with continuous shaking to ensure thorough mixing. Remove the hypocotyl and place it on sterile filter paper to blot off the surface bacterial solution. Then transfer it to a culture dish containing co-culture medium and incubate in the dark at 25°C for 36-48 hours. Transfer the co-cultured hypocotyl to sterile culture medium and incubate at 25°C for 16 / 8 hours, light / dark for 20 days. Transfer the sterile cultured hypocotyl to differentiation selection medium and incubate at 25°C for 16 / 8 hours, light / dark. Subculture every 2-3 weeks until green shoot differentiation occurs. Continue culturing under normal conditions until seed harvest.

[0101] 3. Positive identification and BnaNAC329 expression level identification in rapeseed overexpressing BnaNAC329

[0102] Genomic DNA was extracted from the obtained transgenic T0 rapeseed and wild-type rapeseed plants and identified by PCR. The identification results are as follows: Figure 2 As shown, six positive plants were selected for further growth. RNA was extracted from these six transgenic T0 plants and wild-type rapeseed, reverse transcribed, and identified by qRT-PCR. During the T1-T3 propagation process, positive seeds with red fluorescent endosperm were selected under a stereofluorescence microscope after germination and sown. Using this method, homozygous overexpression lines OE1, OE2, and OE3 with high upregulation of BnaNAC329 were finally screened. Figure 4 ).

[0103] Example 4: Analysis of drought resistance in rapeseed overexpressing BnaNAC329

[0104] This embodiment systematically evaluates the effects of BnaNAC329 overexpression on drought resistance, leaf water retention capacity, and root phenotype in rapeseed, clarifying its drought resistance function.

[0105] 1. Identification of drought sensitivity in transgenic rapeseed overexpressing BnaNAC329

[0106] Wild-type rapeseed seedlings (WT) and BnaNAC329 transgenic overexpression plants (three lines, OE1, OE2, and OE3) of uniform length, grown hydroponically for 18 days (four-leaf stage), were transplanted into soil. After one week of normal cultivation, watering was stopped and drought treatment was carried out for 12 days, followed by 4 days of rehydration. Phenotypic characteristics were observed and survival rates were recorded. The results showed that after drought treatment, WT plants suffered severe wilting and leaf drying, while the overexpression lines showed less wilting and retained more green leaves. After rehydration, the survival rate of OE lines was significantly higher than that of WT, and the BnaNAC329 transgenic overexpression material exhibited stronger drought tolerance. Figure 5 ).

[0107] 2. Determination of water loss rate and leaf temperature under drought stress in detached leaves of transgenic rapeseed overexpressing BnaNAC329

[0108] Two cotyledons and two true leaves of T3 generation transgenic rapeseed overexpressing BnaNAC329, grown hydroponically for 18 days (four-leaf stage), were collected to determine the water loss rate of detached leaves. Initial fresh weight was measured, and the plants were left to stand naturally indoors. Weight was measured every 5 minutes to calculate the water loss rate. Results showed that compared to wild-type rapeseed (WT), the water loss rate of detached leaves from BnaNAC329-overexpressing rapeseed was slower, indicating that BnaNAC329 can enhance the water retention capacity of leaves. Figure 6 The rate of leaf transpiration determines a plant's drought tolerance to a certain extent. Transpiration leads to a decrease in leaf temperature, with faster water loss resulting in lower leaf temperatures compared to slower loss. We further measured leaf temperature in rapeseed overexpressing BnaNAC329 under drought stress. The results showed that the leaf temperature of the OE line was significantly higher than that of the WT line, further demonstrating that it experienced less transpiration and stronger water retention. These results indicate that BnaNAC329 positively regulates rapeseed drought tolerance by influencing leaf water loss.

[0109] 3. Root phenotypic analysis of transgenic rapeseed overexpressing BnaNAC329 under drought stress

[0110] Seeds from uniformly sized and plump wild-type rapeseed WT and BnaNAC329 transgenic overexpression plants (3 lines) were selected. After sterilization with 0.1% mercuric chloride solution (containing 0.05% Tween 20), the seeds were spotted on 0.8 1 / 2 MS plates and 0.8 1 / 2 MS plates containing 200 mM mannitol. After germination, the plants were vertically cultured for 14 days, and the root phenotypes of each genotype were analyzed. The results showed that, under both normal and mannitol treatment conditions, the taproot length of BnaNAC329 overexpression plants was not significantly different from that of the wild type. Figure 7 However, regardless of whether under normal conditions or mannitol treatment, the number of lateral roots in BnaNAC329-overexpressing rapeseed was significantly lower than that in wild-type rapeseed. Figure 7These results indicate that BnaNAC329 can positively regulate the development of lateral roots in rapeseed under drought stress.

[0111] Example 5: Interaction and transcriptional activation verification of BnaNAC329 with BnaLBD16 and BnaGATA23

[0112] 1. Quantitative analysis of genes related to the number of lateral roots in wild-type rapeseed and rapeseed overexpressing BnaNAC329.

[0113] Wild-type (WT) and BnaNAC329 overexpression lines (OE1, OE2, OE3) hydroponically cultured rapeseed seedlings aged 10 days were selected. RNA was extracted from root tissues and reverse transcribed into cDNA. Based on the rapeseed genome database sequence, specific qRT-PCR primers for genes related to lateral root development and auxin signaling pathway (BnaPIN3, BnaYUCCA4, BnaAUX1, BnaIAA28, BnaARF5, BnaARF19, BnaGATA23, BnaLBD16, BnaLBD18, BnaLBD29) were designed using Primer 5.0 software, with BnaActin7 as an internal reference gene. Using cDNA as a template, qRT-PCR amplification was performed on a real-time PCR instrument using a 2×SYBR Green qPCR Mix system. The reaction program was: 95℃ pre-denaturation for 30 s; 40 cycles of 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s (fluorescence signal acquisition); and finally, 95℃ denaturation for 15 s, 60℃ annealing for 1 min, and 95℃ extension for 15 s to plot melting curves. The relative expression levels of each gene were calculated using the 2⁻ΔΔCT method, and expression level bar charts were plotted using GraphPadPrism software. One-way ANOVA combined with Duncan's multiple comparison method was used to analyze the significance of differences between groups (P<0.05). The results showed that the above genes were significantly upregulated in the BnaNAC329-OE line, indicating that BnaNAC329 can activate the core gene network for lateral root development. Figure 8 ).

[0114] 2. Yeast one-hybrid (Y1H) of BnaNAC329 promoters with BnaLBD16 and BnaGATA23 promoters.

[0115] The full-length CDS sequence of BnaNAC329 was ligated into the pGADT7 vector to construct the effect plasmid pGADT7-BnaNAC329. The promoter sequences of BnaLBD16 and BnaGATA23 (approximately 2000 bp upstream of the ATG) were cloned and ligated into the pAbAi vector to construct the bait plasmids pAbAi-BnaLBD16 and pAbAi-BnaGATA23, respectively. All vectors were verified by sequencing. The linearized bait plasmids and the empty pAbAi vector were transformed into Y1HGold yeast competent cells, plated on SD / -Ura solid medium, and cultured at 30°C for 3–5 days. Positive clones were verified by PCR. AbA background testing was performed on positive clones to determine the minimum AbA concentration for inhibiting yeast growth as 100 ng / mL. The effect plasmid pGADT7-BnaNAC329 and the empty pGADT7 vector were transformed into Y1HGold yeast competent cells containing the bait plasmids, plated on D / -Leu medium, and positive transformants were screened. Select positive yeast clones and adjust the OD of the bacterial culture with 0.9% NaCl solution. 600 Up to 0.6, perform 10 0 Yeast was serially diluted 10⁻¹ and 10⁻² times, and 2 μL was spotted onto SD / -Ura / -Leu (growth control) and SD / -Ura / -Leu (selection medium) containing 100 ng / mL AbA plates. Yeast growth was observed after incubation at 30℃ for 3–5 days. p53-AbAi+pGADT7-p53 was used as a positive control, and empty pAbAi+pGADT7-BnaNAC329 was used as a negative control. Results showed that yeast co-transformed with pGADT7-BnaNAC329, pAbAi-BnaLBD16, and pAbAi-BnaGATA23 could grow normally on SD / -Ura / -Leu medium containing 100 ng / mL AbA, while the negative control could not. This indicates that BnaNAC329 can bind to the promoters of BnaLBD16 and BnaGATA23. Figure 9 )

[0116] 3. Experiments on dual-luciferase reporter (LUC) genes using BnaNAC329, BnaLBD16, and BnaGATA23 promoters.

[0117] The promoter sequences of BnaLBD16 and BnaGATA23 were cloned into the pGreenII0800-LUC vector to construct reporter plasmids (pBnaLBD16::LUC, pBnaGATA23::LUC); the CDS sequence of BnaNAC329 was cloned into the pGreenII62-SK vector to construct the overexpression effect plasmid p35S::BnaNAC329, with an empty pGreenII62-SK vector as a negative control (Empty). The constructed reporter and effect plasmids were transformed into Agrobacterium GV3101 (containing pSoup helper plasmid) and cultured at 28℃ with shaking at 200 rpm until OD. 600 =0.6~0.8, centrifuge at 5000 rpm for 10 min to collect bacterial cells, resuspend in infection buffer (10 mM NES, 10 mM MgCl2, 150 μM NAS, pH 5.6) and adjust OD 600 To 0.8. Mix the effector bacterial suspension and reporter bacterial suspension at a 1:1 volume ratio and let stand at room temperature in the dark for 2-3 hours. Select 4-6 week old tobacco leaves and inject the bacterial suspension into the lower epidermis using a needleless syringe. Inject 3-5 leaves per combination. After injection, the tobacco leaves are placed in a 25℃, 16h light / 8h dark culture chamber for 48-72 hours. Prepare 100mM fluorescein substrate, dilute it to 1mM with 1×PBS, and spray it evenly on the surface of tobacco leaves. Incubate in the dark for 5-10 minutes. Use a live fluorescence imaging system to acquire LUC fluorescence signal images. Use the right-hand color scale to quantify the fluorescence intensity, reflecting the transcriptional activation activity of BnaNAC329 on the promoters of target genes. The results show that co-expression of BnaNAC329 can significantly enhance the fluorescence intensity of LUC, indicating that BnaNAC329 can bind to the promoters of BnaLBD16 and BnaGATA23 and activate their expression. Figure 9 ).

[0118] It should be noted that since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. However, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention. Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of this invention. Thus, if these modifications and variations of this invention fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

[0119] sequence list

[0120] SEQ ID No. 1

[0121]

[0122] SEQID No.2

[0123] ATGGGTGTTAGAGAAATGGATCCGTTAGCCCAGTTGAGCTTACCACCGGGTTTCAGATTTTACCCGACAGATGAAGAGCTTCTTGTTCAGTATCTCTGTCGGAAAGTTGCAGGCTATCATTTCTCTCTCCAGGTCATCGGAGACATCGATCTCTACAAGTTCGATCCTTGGGATTTGCCAAGTAAGGCCTTGTTTGGGGAGAAGGAATGGTATTTCTTTAGCCCGAGAGACCGGAAATATCCAAACGGGTCAAGACCCAATAGAGTAGCCGGGTCGGGTTATTGGAAGGCTACGGGTACCGACAAAATCATCACGTCGGATGGCCACCGTGTCGGGATTAAAAAAGCTCTGGTTTTCTACGCCGGAAAAGCTCCGAAAGGAACCAAAACGAACTGGATCATGCACGAGTATCGCCTCGTGGAGCATTCTCGCAGCCATGGAAGCTCCAAGTTGGATGATTGGGTGTTGTGTCGAATCTACAAGAAGACGTCGGGGTCTCAGAGACAAGCCGTTCCTCCGGTTCAACCTTGCCGCGAAGAGCACAGCACGAACGGGTCGTCGTCGTCTTCTTCGTCACATCACGACGACGTTCTTGACTCGTTCCCGGAGATGAACGATCGGTCTTTCAATCTTCCTCGGGTGAATTCTCTGAGGACGCTTCTCAACGGGAACTTCGACTGGGCGAGCTTAGCGGGTCTTAAACCTATCCCGGAGCTAGCTCCGGCGAGCAACGGTTACGGAGGTTACGACGCGTTTAGAGCGGCGGAGGGAGAGGCGGAGAGCGGGTTGAGGAATTTGCAGATGAACTCGAGCGAGTTGACTCAGAGTTACGGGTACAGCTCGAGCGGGTTGAGTAGTGGTGGGTTCGGGCTTTCGGGTCAAACATTCGAGTTTAGGCATTAA

[0124] SEQID No.3

[0125] MGVREMDPLAQLSLPPGFRFYPTDEELLVQYLCRKVAGYHFSLQVIGDIDLYKFDPWDLPSKALFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKIITSDGHRVGIKKALVFYAGKAPKGTKTNWIMHEYRLVEHSRSHGSSK LDDWVLCRIYKKTSGSQRQAVPPVQPCREEHSTNGSSSSSSSHHDDVLDSFPEMNDRSFNLPRVNSLRTLLNGNFDWASLAGLKPIPELAPASNGYGGYDAFRAAEGEAESGLRNLQMNSSELTQSYGYSSSGLSSGGFGLSGQTFEFRH

[0126] It should be noted that the term "comprising" or any other variation thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0127] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

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