Application and method of bohy5 gene or protein coded thereby in inhibiting brassica rapa wax synthesis

Abstract

This application relates to the field of genetic engineering technology and provides a... BoHY5 The application and method of a gene or its encoded protein in inhibiting wax synthesis in broccoli, wherein... BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.1. BoHY5 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2. A method for reducing wax synthesis in broccoli, by... BoHY5 Gene overexpression. This invention is the first to reveal gene overexpression in broccoli. BoHY5 A novel function of transcription factors in the negative regulation of wax synthesis. This was achieved through the construction of... BoHY5 Transgenic lines obtained by overexpressing the vector and transforming broccoli showed a significantly lower content of epidermal wax in their leaves compared to the wild type, indicating that... BoHY5 Gene overexpression can effectively inhibit wax synthesis in organs such as stems, leaves, flowers and buds of broccoli.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, specifically to a... BoHY5 Application and methods of genes or their encoded proteins in inhibiting wax synthesis in broccoli. Background Technology

[0002] During the natural growth of broccoli, a layer of plant epidermal wax, composed of very long-chain fatty acids and their derivatives, is secreted and deposited on the surface of its leaves and flower heads. While this wax layer provides some physiological protection against drought, ultraviolet radiation, and pests, in actual agricultural production and the consumer market, an excessively thick wax layer has significant negative impacts on pesticide residues, product washing, and commercial appearance. Therefore, cultivating "bright green" broccoli with vibrant green flower heads and a surface with little or no wax has become an important goal in current broccoli quality breeding.

[0003] With the development of molecular biology, using genetic engineering or molecular marker-assisted breeding to directionally improve the waxy trait of broccoli has become an efficient way to solve the above problems. In-depth exploration and verification of key genes that can effectively inhibit wax synthesis in broccoli, and elucidation of their molecular mechanisms, can not only fill the theoretical gap in the negative regulatory network of wax development in Brassica plants, but also provide core gene resources and molecular breeding targets for cultivating high-quality, bright green (low-wax) broccoli varieties. Summary of the Invention

[0004] This invention aims to solve the problems in the prior art and provide a solution. BoHY5 Application and methods of genes or their encoded proteins in inhibiting wax synthesis in broccoli.

[0005] To achieve the above objectives, the first aspect of this application provides a BoHY5 The application of the gene or its encoded protein in inhibiting broccoli wax synthesis, said BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.1. BoHY5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0006] In the above applications, preferably, the organ from which broccoli wax is synthesized is at least one of the stem, leaf, flower, and bud.

[0007] The second aspect of this application provides a method for reducing wax synthesis in broccoli, thereby reducing the wax content in broccoli. BoHY5 Gene overexpression; the aforementioned BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] The above method, preferably, is used in broccoli. BoHY5 Gene overexpression includes the following steps:

[0009] (1) Constructing a structure containing BoHY5 Plant overexpression vectors for genes; (2) The overexpression vector was introduced into broccoli receptor cells; (3) The introduced cells were cultured and screened to obtain transgenic broccoli plants.

[0010] In the above method, preferably, the plant overexpression vector is a plant expression vector containing the nucleotide sequence shown in SEQ ID NO.1.

[0011] In the above method, preferably, the plant expression vector is the pCBSG015 vector.

[0012] In the above method, preferably, the pCBSG015 vector contains a Basta resistance screening marker.

[0013] In the above method, preferably, in step (2), the method of introduction is Agrobacterium-mediated genetic transformation; the Agrobacterium is strain GV3101; and the recipient cell is broccoli hypocotyl explant cell.

[0014] Preferably, the above method further includes: performing self-pollination or hybridization on the obtained transgenic broccoli plants to screen for genetically stable homozygous varieties.

[0015] Compared with the prior art, this application has the following beneficial effects: (1) This invention discloses for the first time the broccoli BoHY5 A novel function of transcription factors in the negative regulation of wax synthesis. This was achieved through the construction of... BoHY5 Transgenic lines obtained by overexpressing the vector and transforming broccoli showed a significantly lower content of epidermal wax in their leaves compared to the wild type, indicating that... BoHY5 Gene overexpression can effectively inhibit wax synthesis in the stems, leaves, flowers, and buds of broccoli. The above findings of this invention provide novel gene resources and breeding targets for the molecular improvement of waxy traits in broccoli.

[0016] (2) The method for reducing wax synthesis in broccoli provided by this invention can obtain BoHY5Transgenic plants overexpressing this gene exhibit significantly reduced wax content and stable phenotypes, making them suitable as direct breeding intermediates for cultivating bright green broccoli varieties through self-pollination or hybridization. The reduced wax content improves the adhesion and penetration efficiency of pesticides and foliar fertilizers on leaf surfaces, lowering the risk of pesticide residues. It also reduces post-harvest cleaning difficulty and enhances the commercial appearance of the produce. Therefore, this invention provides valuable genetic resources and germplasm materials for cultivating bright green (low-wax), easier-to-clean, and safer broccoli varieties, demonstrating significant food safety implications and promising industrial application prospects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 for BoHY5 Agarose gel electrophoresis diagram of gene identification.

[0019] Figure 2 for BoHY5 Overexpression of broccoli plants BoHY5 Level of expression.

[0020] Figure 3 The spectrum is for the vector pCBSG015 (Basta).

[0021] Figure 4 For wild-type broccoli plants WT and BoHY5 Overexpression of the plant leaf epidermal wax phenotype.

[0022] Figure 5 For wild-type broccoli plants WT and BoHY5 Scanning electron microscopy (SEM) results of leaf epidermal wax in overexpressing plants: A is an SEM image of WT leaves at 2000× magnification; B is an SEM image of WT leaves at 5000× magnification; C is an SEM image of WT leaves at 10000× magnification; D is an SEM image of OE-3# leaves at 2000× magnification; E is an SEM image of OE-3# leaves at 5000× magnification; F is an SEM image of OE-3# leaves at 10000× magnification; G is an SEM image of OE-5# leaves at 2000× magnification; H is an SEM image of OE-5# leaves at 5000× magnification; I is an SEM image of OE-5# leaves at 10000× magnification.

[0023] Figure 6For wild-type broccoli plants WT and BoHY 5. Overexpression of plant leaf epidermal wax content.

[0024] Figure 7 For wild-type broccoli plants WT and BoHY5 Expression characteristics of key genes for leaf wax synthesis in overexpressed plants: A is transcriptome sequencing results; B is quantitative fluorescence expression results.

[0025] Figure 8 for BoHY5 Results of a yeast one-hybrid experiment involving interaction with the promoter of a key gene for wax synthesis: A is... BoHY5 and CER1 Results of yeast one-hybrid assay for promoters; B is BoHY5 and MYB106 Results of yeast one-hybrid assay for promoters; C is BoHY5 and LACS9 Results of yeast one-hybrid assay for promoters; D is BoHY5 and LACS1 Results of yeast one-hybrid assay for promoters; E is BoHY5 and MYB96 Results of yeast one-hybrid assay for promoters; F is BoHY5 and MAH1 Results of yeast one-hybrid assay for promoters; G is BoHY5 and CER2 Results of yeast one-hybrid assay for promoters; H is BoHY5 and KCS9 Results of yeast one-hybrid assay for promoters; I is BoHY5 and CER3 Results of yeast one-hybrid experiments on promoters.

[0026] Figure 9 To display BoHY5 Results of dual-luciferase assays involving interaction with promoters of key genes for wax synthesis: A is... BoHY5 and CER1 Dual-luciferase assay results for promoter; B is BoHY5 and CER2 Dual-luciferase assay results for promoter; C is BoHY5 and CER3 Dual-luciferase assay results for promoter; D is BoHY5 and LACS1 Dual-luciferase assay results for the promoter; E is BoHY5 and LACS9 Results of dual-luciferase assay for promoter. Detailed Implementation

[0027] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of this application.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The following describes the experiment conducted using the broccoli Bop15-80 material. Broccoli Bop15-80 is a germplasm resource collected by the applicant and preserved in the germplasm resource nursery of the Brassica Sprouts Germplasm Innovation Team at the College of Horticulture, Hunan Agricultural University. The applicant has published this germplasm resource in the paper "Mechanism of LED Blue Light Regulation of Glucosinolates Biosynthesis and Sulforaphane Accumaltion in Broccoli Sprouts Based on Metabolics and Transcriptome".

[0031] The research basis of this application: The applicant previously collected broccoli seedlings of the uniform variety Bop15-80 and treated them with LED blue light (light intensity: 50 μmol / m²). 2 / s; wavelength 449nm; photoperiod: 12h day / 12 / night; duration: 7 days) and a dark control treatment. The applicant found that after treating broccoli plants with LED blue light, the amount of wax deposition on the leaf epidermis was significantly reduced compared to the dark control. Transcriptome sequencing analysis showed that key genes in the light signal transduction pathway were affected after blue light treatment. phyB , CRY2 , COP1 and SPA PhysA expression is upregulated, while phyA expression is downregulated. Broccoli BoHY5 Of the two copies of the gene, only the BolC9t59384H copy showed a significant upregulation after blue light treatment, while the BolC2t06452H copy did not respond significantly. This result indicates that the BolC9t59384H copy is the main transcription factor responding to blue light signals; in subsequent examples, it will be referred to as […]. BoHY5 Based on this, the present invention clones and verifies the functionality of the copy.

[0032] Example 1: BoHY5 Gene cloning and sequence identification Using the broccoli reference genome ( Brassica oleracea var. italica Based on the gene sequence information in -CC HDEM-v0.0, specific primers were designed, and PCR amplification was performed using cDNA from the leaves of broccoli Bop15-80 seedlings as a template. Cloning BoHY5 Full-length CDS sequence of the gene. Primer sequences are as follows: BoHY5-F: 5'-TCGGAGGAGAGAGTCAAAGGA-3' (SEQ ID NO.3); BoHY5-R: 5'-TCTCTTGCTTGCTGTGCTGA-3' (SEQ ID NO. 4).

[0033] Total RNA was extracted from the leaves of broccoli seedlings at the two-leaf-one-heart stage using the SteadyPure Plant RNA Extraction Kit. The RNA was then reverse transcribed into cDNA using the TransScript All-in-One First-Strand cDNASynthesis SuperMix for qPCR Kit (Nanjing Novizan Biotechnology Co., Ltd.). The experimental procedures were performed according to the instruction manual.

[0034] The PCR reaction system and conditions were as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 52 ℃ annealing for 30 s, 72 ℃ extension for 2 min, for a total of 35 cycles; final extension at 72 ℃ for 10 min. The PCR amplification products were identified by 1.0% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown (a single specific band is visible at approximately 495 bp). The target band was recovered and purified using a general DNA purification and recovery kit (Shanghai Huiling Biotechnology Co., Ltd.), and then sent to Qingke Biotechnology (Changsha) Co., Ltd. for sequencing.

[0035] Sequence alignment analysis showed that the obtained sequence matched that in the broccoli database ( Brassica oleracea var.italic The a-CC HDEM-v0.0 gene showed no difference. The full-length CDS of this gene is 495 bp, and the nucleotide sequence is shown in SEQ ID NO.1. It encodes 164 amino acids, and the amino acid sequence is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1 ATGCAGGAGCAAACGACTAGCTCTTTACCTGCAAGCTCTCTACCGTCAAGCAGCGAGAGATCCTCAAGCTCTGCTCCTCATTTGGAGATCAAAGAAGGAATTGAAAGTGATGAGGAGATACGGCGAGTTCCGGAGTTTGGAGGAGAAGCTACCGGAAAGGAAATCTCTGGATCGGCGACCGGTCAGGACCAGACACAAATAACGGTCGGAGGAGAGAGTCAAAGGAAGAGAGGGAGGACTCCGGCTGA GAAAGAGACCAAGCGGCTAAAGAGGTTGTTGAGGAACAGAGTTTCAGCACAGCAAGCAAGAGAGGAAGAAAGCTTACTTGGGTGAGTTGGAAAATAGAGTGAAAGACTTGGAGAACAGAAACTCTGAACTTGAAGAGAGACTCTCTACTTTGCAGAACGAGAACCAGATGCTTAGACAGATTCTGAAGAACACAACAGGAAACAAGAGGGGAAGCGGTGGTTCTAACGCTGATGCAAGCCTATGA.

[0037] SEQ ID NO.2 MQEQTTSSLPASSLPSSSERSSSSAPHLEIKEGIESDEEIRRVPEFGGEATGKEISGSATGQDQTQITVGGESQRKRGRTPAEKETKRLKRLLRNRVSAQQARERKKAYLGELENRVKDLENRNSELEERLSTLQNENQMLRQILKNTTGNKRGSGGSNADASL.

[0038] Example 2: BoHY5 Construction of gene overexpression vectors and genetic transformation of broccoli 1. Construction of overexpression vectors The clone obtained in Example 1 BoHY5 Using the gene fragment as a template, the pCBSG015 vector homologous arm sequence (BsaI restriction site recognition sequence) was added to the 5' end of primers BoHY5-F and BoHY5-R, respectively. The target gene fragment with the vector terminal homologous sequence was obtained by PCR amplification.

[0039] The pCBSG015 vector backbone was linearized by digestion with the restriction endonuclease BsaI under the following conditions: 37 ℃ for 2 h; 60 ℃ for 20 min. The target gene fragment with homologous arms was then ligated to the linearized vector backbone using T4 DNA ligase to construct the recombinant vector. The plasmid map of the pCBSG015 vector is shown below. Figure 3 As shown, the vector carries a Basta resistance selection marker.

[0040] The recombinant vector was transformed into *E. coli* DH5α competent cells and plated on LB agar containing 50 μg / mL spectinomycin for screening. Single colonies were picked for PCR verification. Positive clones were sent to a sequencing company for sequencing to confirm that the target gene was correctly inserted into the vector and that the reading frame was correct. The correctly sequenced recombinant plasmid was extracted and transformed into *Agrobacterium* GV3101 competent cells using the freeze-thaw method.

[0041] 2. Genetic transformation of broccoli Agrobacterium-mediated genetic transformation was conducted using the broccoli variety Bop15-80 as the material. BoHY5 The specific steps for gene overexpression genetic transformation assays are as follows: Prepare blank MS medium. Take an appropriate amount of seeds, wrap them in gauze, soak them in 50 ℃ warm water for 30 min, and rinse them in cool water for 30 min. Disinfect the seeds with 75% alcohol for 30 s, then disinfect them with 0.1% mercuric chloride for 8 min. After rinsing with pure water, use tweezers to gently place the seeds onto the MS medium. Calculate the seedling age from the time the seeds show white sprouts after sowing. Generally, use the hypocotyl of a healthy seedling (when the cotyledons first appear) with a seedling age of 10 days as the explant. Cut the hypocotyl of the broccoli seedlings 0.5 cm, make a "trapezoidal" oblique cut, and place it in the pre-medium (4.42 g / L MS + 20 g / L sucrose + 2.2 g / L plant gel + 0.01 mg / L NAA + 3 mg / L 6-BA) and culture it under light for 3 days. Correctly identified positive Agrobacterium (GV3101) cells were cultured with shaking in YEB medium containing the appropriate antibiotic until the logarithmic growth phase. The cells were collected by centrifugation, resuspended in liquid pre-medium (containing 100 μM acetylsyringone AS and 1 mM MES), and the OD was adjusted. 600After reaching a temperature of 0.5-0.6, the cultured hypocotyl explants were incubated at 28 °C for 3 h before inoculation. The pre-cultured hypocotyl explants were immersed in Agrobacterium tumefaciens solution for 10 min, gently agitated occasionally. After inoculation, the explants were cultured in the dark for 2 days in pre-medium, then transferred to sterile culture medium (4.42 g / L MS + 20 g / L sucrose + 2.2 g / L plant gel + 0.01 mg / L NAA + 3 mg / L 6-BA + 300 mg / L Tim) for 4 days, followed by differentiation culture (4.42 g / L MS + 20 g / L sucrose + 2.2 g / L plant gel + 0.01 mg / L NAA + 3 mg / L 6-BA + 5 mg / L PPT + 200 mg / L Tim). After the seedlings have rooted, transfer them to a seedling tray containing sterile substrate (peat moss: perlite: vermiculite = 2:1:1) and cover the tray to harden them off. After one week, open the tray cover daily for ventilation. After 15 days, the tray cover can be completely opened.

[0042] When the transformed seedlings reached the two-leaf stage, total RNA was extracted from the broccoli leaves using the SteadyPure Plant RNA Extraction Kit, and quantitative PCR was performed using the SYBR Green method to detect RNA in the transformed seedlings. BoHY5 Gene expression levels were measured in three biological replicates. Primers used for quantitative real-time PCR are shown in Table 1.

[0043] Table 1: qPCR primer sequences

[0044] The results of expression level measurement are as follows Figure 2 As shown, BoHY5 Overexpression lines OE-3# and OE-5# BoHY5 The gene expression level was significantly upregulated compared to wild-type WT, indicating that it had been acquired. BoHY5 Overexpressing transgenic positive plants. The positive plants were propagated, self-crossed, and screened to obtain homozygous transgenic lines.

[0045] Example 3: BoHY5 Determination of the epidermal wax phenotype and content of transgenic broccoli leaves 1. Observation of wax morphology using scanning electron microscopy (SEM) Take the wild type (WT) obtained in Example 2 and BoHY5 Mature leaves at the same leaf position from the overexpressing transgenic lines (OE-3# and OE-5#) were cut into approximately 10 mm × 5 mm sections with waxy epidermis using a blade. These sections were then attached to the sample stage of a field emission scanning electron microscope (FET) using conductive adhesive. A platinum film was deposited using an ion sputtering coating system, and observation and photography were performed at an accelerating voltage of 15 kV. The magnifications were 2000x, 5000x, and 10000x, respectively.

[0046] Observation results as follows Figure 4 and Figure 5 As shown: Compared to the leaves of wild-type plants, BoHY5 The overexpression lines OE-3# and OE-5# showed less wax accumulation in the leaf epidermis and a sparse distribution of the wax layer. This result indicates that... BoHY5 Overexpression significantly inhibited the accumulation of waxy substances in the epidermis of broccoli leaves.

[0047] 2. Determination of wax content and composition by gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) (1) Wax extraction: Wild-type and BoHY5 Leaves from the same leaf position of the overexpressing transgenic lines (OE-3#, OE-5#) were accurately weighed and placed in glass test tubes containing 10 mL of chloroform for 2 min to extract the surface wax. 30 μL of n-tetracosane was added to the extract as an internal standard for quantitative calculation.

[0048] (2) Derivatization: An appropriate amount of anhydrous sodium sulfate was added to the extract to remove residual water. After filtration, the filtrate was transferred to a new glass tube and dried using a nitrogen blower. 80 μL of N,O-bis(trimethylsilyl)trifluoroacetamid (BSTFA, Sigma) was added, and the mixture was incubated at 60 °C for 40 min for derivatization. After the reaction was complete, the mixture was dried again using a nitrogen blower, and 1 mL of chloroform was added to reconstitute the solution. The solution was then filtered through a 0.22 μm syringe-type microporous membrane into a glass inner tube for GC-TOFMS analysis. Each sample was tested in triplicate (technical and biological). Data are expressed as mean ± standard deviation, and Student's t-test was used for significance analysis.

[0049] GC-TOFMS analysis results are as follows Figure 6 As shown: BoHY5 The contents of very long chain fatty acids (VLCFAs, ≥C20) and their derivatives in the leaf epidermis of the overexpression lines OE-3# and OE-5# were 8755 ng / g and 5335 ng / g, respectively, which were significantly lower than the 17064 ng / g of the wild type. Figure 6 ).

[0050] Example 4: BoHY5 Screening of downstream target genes Based on the findings of Example 3, the applicant used wild-type (WT) and BoHY5 Leaf samples from the overexpression lines were used as material, and transcriptome sequencing technology was employed for analysis. BoHY5 Effects of overexpression on the expression of genes related to the wax synthesis pathway in broccoli leaves.

[0051] Total RNA was extracted from 100 mg of sample using an RNA extraction kit (Tiangen Biotech, Beijing). The purity and integrity of the RNA were assessed using a NanoDrop One spectrophotometer and an Agilent 2100 bioanalyzer. High-quality RNA samples (total ≥ 1 μg, concentration ≥ 100 ng·μL) were selected. -1 (RIN ≥ 7.0) cDNA libraries were constructed using the NEBNext® Ultra™ RNA Library Preparation Kit (NEB, USA). After the libraries passed quality control, sequencing was performed using the Illumina NovaSeq 6000 platform (Illumina, USA). Clean reads were obtained by removing adapters, poly-N sequences, and low-quality reads from the raw data. The clean reads were then aligned to the broccoli reference genome using HISAT2 (v2.2.2.0) software. Brassica oleracea var. italica -CC HDEM-v0.0). Gene expression levels were normalized using FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Principal component analysis (PCA) was used to assess biological reproducibility. Differentially expressed genes (DEGs) were screened using the DESeq2 R package, with the screening criteria being |log2(Fold Change)| ≥ 1 and adjust p-value < 0.05.

[0052] Transcriptome sequencing results ( Figure 7 A) shows that, compared to the wild type, BoHY5 Overexpression of key genes in the wax synthesis pathway in plant leaves LACS1, LACS9, KCS13, KCS2, KCS9, CER1, CER2, CER3, WSD1, FAR3, MAH1 and major regulatory genes MYB30, MYB96, MYB106 The expression abundance was significantly downregulated compared to the wild type.

[0053] To further verify the reliability of the transcriptome sequencing results, the expression levels of the above-mentioned genes were verified using real-time quantitative PCR (RT-qPCR). Using the broccoli internal reference gene as a control, SYBR Green was employed. Pro Taq The experiment was conducted using HS. Reaction system (10 μL): 2× SYBR Green Pro Taq HS Premix 5 μL, 10 μmol·L -10.2 μL each of forward and reverse primers, 3 μL of cDNA template, and ddH2O to make up the volume. Amplification program: 95 ℃ for 30 s pre-denaturation; 95 ℃ for 5 s, 60 ℃ for 30 s, for a total of 40 cycles. Primers were designed for the specific sequences of each target gene (see Table 2). Each sample was tested in triplicate, using 2^... -ΔΔC Relative expression levels were calculated using the t-method, and the difference was analyzed for significance using Student's t-test. RT-qPCR results ( Figure 7 B) shows that the above-mentioned wax synthesis-related genes are in BoHY5 The expression levels in the overexpression lines were significantly lower than those in the wild type, consistent with the trend observed in transcriptome sequencing results.

[0054] Table 2: qPCR primer sequences

[0055] The above screening results indicate that, LACS1, LACS9, KCS13, KCS2, KCS9, CER1, CER2, CER3, WSD1, FAR3, MAH1, MYB30, MYB96, MYB106 may be BoHY5 Downstream target genes that negatively regulate wax synthesis in broccoli leaves.

[0056] Example 5: BoHY5 Interaction with downstream target gene promoters and verification of transcriptional regulation Example 4 above obtained the results through transcriptome sequencing and quantitative real-time PCR screening. LACS1, LACS9, KCS13, KCS2, KCS9, CER1, CER2, CER3, WSD1, FAR3, MAH1, MYB30, MYB96, MYB106 A total of 14 BoHY5 Candidate downstream target genes that negatively regulate wax synthesis. This embodiment further utilizes yeast single-hybrid assays and dual-luciferase reporter gene assays to verify the binding relationship between BoHY5 and the promoters of candidate downstream target genes.

[0057] 1. Yeast single-hybrid experiment (1) Construction and verification of the carrier The clone obtained in Example 1 BoHY5 Using the gene fragment as a template, homologous arm sequences of the pGADT7 vector (recognition sequences of restriction enzyme sites XhoI and EcoRI) were added to the 5' ends of primers BoHY5-F and BoHY5-R, respectively. The target gene fragment with the vector terminal homologous sequences was then obtained by PCR amplification. Similarly, candidate target genes were cloned using wild-type broccoli DNA as a template. CER1 (SEQ ID NO.43) CER2 (SEQ ID NO.44) CER3 (SEQ ID NO.45) LACS1 (SEQ ID NO.46) LACS9 (SEQ ID NO.47) MAH1 (SEQ ID NO.48) ,MYB96 (SEQ ID NO.49) MYB106 (SEQ ID NO.50) KCS9 The (SEQ ID NO.51) promoter was used to add the pAbAi vector homologous arm sequence (HindIII and XhoI restriction sites) to the target gene fragment with the vector terminal homologous sequence by PCR amplification.

[0058] The pGADT7 vector backbone was double-digested with restriction endonucleases XhoI and EcoRI, and the pAbAi vector backbone was double-digested with HindIII and XhoI to linearize it. The reaction conditions were: 37 ℃ for 2 h; 60 ℃ for 20 min. The target gene fragment with homologous arms was ligated to the linearized vector backbone using T4 DNA ligase to construct the recombinant prey vector pGADT7-BoHY5 and the bait vector pAbAi-Promoter.

[0059] The recombinant vector was transformed into *E. coli* DH5α competent cells and plated on LB agar containing 100 μg / mL ampicillin for screening. Single colonies were picked for PCR verification. Positive clones were sent to a sequencing company for sequencing to confirm that the target gene was correctly inserted into the vector and that the reading frame was correct. The recombinant plasmid that was correctly sequenced was extracted.

[0060] (2) Construction of bait yeast strains and detection of self-activation (AbA concentration screening) The correctly sequenced pAbAi-Promoter bait plasmid was linearized by restriction endonuclease BstbI, and the linearized fragment was recovered by agarose gel electrophoresis. The linearized bait vector was transformed into Y1H Gold yeast competent cells using a LiAc / PEG-mediated yeast transformation method. The transformation product was plated on SD / -Ura solid plates and incubated at 30°C for 3-5 days until yeast clones appeared. Single clones were selected for yeast colony PCR verification, yielding positive bait yeast strains that successfully integrated the target gene promoter.

[0061] After culturing the above-mentioned positive yeast strains overnight in liquid YPDA medium, the bacterial concentration (e.g., OD) was adjusted. 600=0.002), and spread onto SD / -Ura solid medium containing different concentration gradients of Aureobasidin A (AbA) (0, 50, 100, 150, 200, 250, and 300 ng / mL). After incubation at 30 ℃ for 3-5 days, yeast growth was observed, and the AbA concentration range that inhibited the growth of this yeast strain (e.g., 50-100 ng / mL) was recorded. Different AbA concentration gradients (e.g., 55, 60, 65, 70, 75, 80, 85, 90, and 95 ng / mL) were then set within this range to obtain the lowest AbA concentration that completely inhibited the growth of this yeast strain. This is the critical AbA inhibition concentration for this promoter, used to eliminate self-activation.

[0062] (3) Yeast one-hybrid (Y1H) interaction verification Competent cells of Y1H Gold-positive yeast strain containing the pAbAi-Promoter bait vector were prepared. The prey vector pGADT7-BoHY5 (experimental group) and the empty pGADT7 vector (negative control group) were transformed into these competent yeast cells, respectively. The transformation products were plated on SD / -Leu agar plates and incubated at 30 °C for 3-5 days to screen for yeast clones successfully transformed with the prey vector. Single clones that grew on SD / -Leu plates from both the experimental and control groups were picked and inoculated into liquid YPDA medium, and cultured on a shaker until the logarithmic growth phase. The bacterial concentration was standardized using sterile double-distilled water (OD). 600 =0.5), and then perform gradient dilution (100, 10). -1 10 -2 10 -3 Different dilution gradients of bacterial suspensions were dropwise onto SD / -Leu solid medium containing a pre-selected critical concentration of AbA. The cultures were incubated upside down at 30 °C for 3-5 days, and the growth status of the yeast colonies was observed and recorded. If the experimental group grew normally while the negative control group was inhibited, it proved... BoHY5 It can specifically bind to the promoter of candidate target genes.

[0063] Experimental results are as follows Figure 8 As shown: All co-transformed yeast strains grew normally and uniformly on SD / -Leu medium without selection pressure (without AbA), indicating that the prey and bait vectors were successfully co-transformed into yeast cells. When spotted onto SD / -Leu select plates containing the corresponding critical inhibitory concentration of AbA, the co-transformed pGADT7-BoHY5 and... CER1 (100 ng / mL AbA) CER2 (20 ng / mL AbA) CER3 (30 ng / mL AbA) LACS1 (25 ng / mL AbA) LACS9 (200 ng / mL AbA) MAH1 (40 ng / mL AbA) ,MYB96 (25 ng / mL AbA) MYB106 (20 ng / mL AbA) KCS9 The yeast in the experimental group with the 20 ng / mL AbA promoter bait vector was able to overcome the inhibitory effect of AbA and grow obvious colonies, while the growth of the corresponding co-transformed pGADT7 empty negative control group was inhibited.

[0064] In summary, these results indicate that BoHY5 can specifically and directly bind in vitro. CER1 , CER2, CER3 LACS1, LACS9, MAH1, MYB96, MYB106, KCS9 Gene promoters generate interactions.

[0065] 2. Dual-luciferase reporter gene assay (1) Construction of effect carriers and report carriers Candidate target genes CER1, CER2, CER3, LACS1, LACS9, MAH1, MYB96, MYB106, KCS9 The promoter of the pGreenII-0800-LUC dual-luciferase reporter vector homologous arm sequence (KpnI and BamHI restriction sites) was added to the vector. After purification, the sequence was inserted upstream of the firefly luciferase (LUC) gene in the vector. The vector itself carries Renida luciferase (REN) driven by the 35S promoter as an internal control.

[0066] amplification using cDNA as a template BoHY5 The CDS region fragment of the gene was added with the homologous arm sequence (HindIII and KpnI recognition sequences for restriction enzyme sites) of the pGreenII-62-SK vector, and ligated into the pGreenII-62-SK expression vector, positioned downstream of the strong promoter CaMV 35S, to construct the effector vector. An empty vector was retained as a negative control. All constructed recombinant plasmids were verified by sequencing.

[0067] (2) Agrobacterium transformation and infection solution preparation The correctly sequenced reporter and effector vector plasmids were transformed into Agrobacterium GV3101-pSoup competent cells. Positive clones were screened on LB agar containing 50 μg / mL kanamycin and 100 μg / mL rifampin, and further identified by colony PCR.

[0068] Select single clones and inoculate them into liquid LB containing the appropriate antibiotic, and culture at 28 ℃ with shaking at 200 rpm until the logarithmic growth phase (OD). 600The concentration was approximately 0.6-0.8. Agrobacterium cells were then collected by centrifugation at 5000 rpm for 10 min, and resuspended in freshly prepared Agrobacterium infiltration buffer (containing 10 mM MES, 10 mM MgCl2, and 200 μM acetylsuccinone AS). After centrifugation again, the final concentration of all bacterial suspensions was adjusted to the same OD value using the same buffer. 600 =0.8. After incubating the resuspended solution at room temperature for 2-3 hours, inject tobacco.

[0069] (3) Tobacco injection and in vivo imaging The *Agrobacterium* suspension containing the 0800-Promoter reporter vector and the *Agrobacterium* suspension containing the 62SK-BoHY5 effector vector were mixed at a volume ratio of 1:1 to form the experimental group. The control groups consisted of: ① a 1:1 mixture of *Agrobacterium* suspension containing the pGreenII-62-SK empty vector and the pGreenII-0800-LUC empty vector; ② a 1:1 mixture of *Agrobacterium* suspension containing the 62SK-BoHY5 effector vector and the pGreenII-0800-LUC empty vector; ③ a 1:1 mixture of *Agrobacterium* suspension containing the 0800-Promoter reporter vector and the pGreenII-62-SK empty vector. Control groups ① and ② served as negative controls to exclude false positives and were injected into the upper left and upper right regions of tobacco leaves. Control group ③ served as a background control representing the transcriptional activity of the promoter itself.

[0070] Using a 1 mL sterile medical syringe (needle removed), gently inject the mixed bacterial solution into the intercellular spaces of the mesophyll cells from the underside of the tobacco leaf, ensuring the infiltration solution spreads evenly throughout the injection area. Mark the injection sites on the leaves using a marker (injecting the experimental group on the lower right side of the same leaf to eliminate individual differences). After injection, incubate the tobacco plants in darkness for 24 hours, then transfer them to normal light conditions (16 hours light / 8 hours darkness, temperature 22-25 °C) and continue incubation for 48 hours to allow for sufficient expression of transcription factors and reporter genes.

[0071] Before in vivo imaging, a 1 mM firefly luciferase substrate (D-Luciferin potassium salt aqueous solution) was evenly sprayed onto the surface of the injected tobacco leaves and incubated in a dark room for 5 min to allow the substrate to fully penetrate and react with the enzyme. Subsequently, the leaves were placed in a plant in vivo fluorescence imaging system, and LUC luminescence images were acquired with a 2-min exposure time. The images were then observed and compared. BoHY5 The activation / inhibition of luminescence for each promoter.

[0072] The results of the dual-luciferase assay are as follows: Figure 9As shown, the results indicated that no obvious fluorescence signal was observed in the negative control area at the top of the tobacco leaf, ruling out background interference. Comparison of the luminescence in the lower part of the leaf revealed that, compared to the background control injected with empty vector, the co-expression... BoHY5 Effect carriers and CER1, LACS9 The region of the promoter reporter vector showed a change in LUC luminescence signal compared to the background control; when BoHY5 respectively with CER2, CER3 and LACS1 When co-expressed with the promoter-reporter vector, the fluorescence intensity of these three regions, which originally had strong background luminescence signals, showed a significant decrease or even disappearance. These imaging results clearly demonstrate that the transcription factor BoHY5 can transcribe and activate... CER1, LACS9 Upregulate expression, but suppress CER2、CER3、LACS1 Expression, indication BoHY5 It may be through transcriptional repression CER2、CER3、LACS1 The expression level of this substance negatively regulates the synthesis of leaf epidermal waxes.

[0073] Based on the combined results of yeast one-hybrid and dual-luciferase experiments and BoHY5 Phenotypic evidence for the negative regulation of wax synthesis in broccoli leaves indicates BoHY5 Transcription factors can directly bind CER2 , CER3 and LACS1 The promoter region of the gene is targeted, and the expression levels of these genes are inhibited through transcription, thereby negatively regulating the biosynthesis of epidermal wax in broccoli leaves. Dual-luciferase assays show... BoHY5 It has the ability to directly bind and activate in in vitro systems. CER1 and LACS9 The potential of promoters, and in overexpression BoHY5 In the transgenic plants, the wax content and expression of these target genes were significantly downregulated. This phenotypic reversal indicates that in the complex internal environment of plants, BoHY5 The regulation of wax synthesis is not a simple linear activation; it may activate higher-level negative regulators in vivo or trigger substrate competition and negative feedback regulatory networks.

[0074] >CER1 promoter (SEQ ID NO.43):

[0075] >MYB106 promoter (SEQ ID NO.50):

[0076] >LACS9 promoter (SEQ ID NO.47):

[0077] >LACS1 promoter (SEQ ID NO.46):

[0078] >MYB96 promoter (SEQ ID NO.49):

[0079] MAH1 promoter (SEQ ID NO.48):

[0080] >CER2 promoter (SEQ ID NO.44):

[0081] KCS9 promoter (SEQ ID NO.51):

[0082] >CER3 promoter (SEQ ID NO.45):

[0083] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

Claims

1. BoHY5 The application of a gene or its encoded protein in inhibiting broccoli wax synthesis, characterized in that, The BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. BoHY5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The organs in which broccoli produces wax are at least one of the stem, leaves, flowers, and buds.

3. A method for reducing wax synthesis in broccoli, characterized in that, In broccoli BoHY5 Gene overexpression; the aforementioned BoHY5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, In broccoli BoHY5 Gene overexpression includes the following steps: (1) Constructing a structure containing BoHY5 Plant overexpression vectors for genes; (2) The overexpression vector was introduced into broccoli receptor cells; (3) The introduced cells were cultured and screened to obtain transgenic broccoli plants.

5. The method according to claim 4, characterized in that, The plant overexpression vector is a plant expression vector containing the nucleotide sequence shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, The plant expression vector is the pCBSG015 vector.

7. The method according to claim 6, characterized in that, The pCBSG015 vector contains a Basta resistance selection marker.

8. The method according to claim 4, characterized in that, In step (2), the method of introduction is Agrobacterium-mediated genetic transformation; the Agrobacterium is strain GV3101; the recipient cell is broccoli hypocotyl explant cell.

9. The method according to claim 4, characterized in that, The method further includes: performing self-pollination or hybridization on the obtained transgenic broccoli plants to screen for genetically stable homozygous varieties.

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