Application of ZFP7 gene in regulating content of glucosinolate in plants

Abstract

This invention discloses ZFP7 The application of genes in regulating the content of glucosinolates in plants relates to the field of biotechnology. ZFP7 Genes are BoaZFP7 Gene or BjuZFP7 Genes; the stated BoaZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; BjuZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.3. This invention has discovered... ZFP7 This gene can regulate the content of glucosinolates in plants, positively regulating the biosynthesis and accumulation of glucosinolates, effectively increasing the content of total glucosinolates, aliphatic glucosinolates, and indole glucosinolates in plants, while comprehensively upregulating the expression levels of key genes in the glucosinolate synthesis pathway. The protein encoded by this gene is located in the cell nucleus and can directly bind to and activate the core gene for glucosinolate side chain modification. AOP2 It regulates synthetic pathways at the transcriptional level, and its mechanism of action is clear and stable.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to... ZFP7 Application of genes in regulating the content of glucosinolates in plants. Background Technology

[0002] Glucosinolates (GS) are a class of sulfur- and nitrogen-rich secondary metabolites widely found in cruciferous plants such as Brassica oleracea. They serve as important defenses against pests and diseases and are also key components influencing flavor. GS also play a significant role in anticancer activity; when plant tissues rupture, GS are rapidly hydrolyzed by myrosinase to form various degradation products with anticancer activity, including thiocyanates, isothiocyanates, and nitriles. GS and their degradation products have attracted widespread attention and hold significant research value and application prospects in crop quality improvement and resistance breeding.

[0003] Chinese kale (Kaikan) is a variety of Brassica oleracea belonging to the Brassicaceae family, and it is currently widely cultivated. The main edible parts of Chinese kale are its flower stalks and young leaves, which have a unique flavor and are very popular with consumers. Mustard greens (Mustard greens) are an important Brassica vegetable, with their leaves as the main edible part, often eaten fresh or pickled. Both Chinese kale and mustard greens are rich in carotenoids, ascorbic acid, and other nutrients, especially glucosinolates, which have anti-cancer properties and participate in plant defense responses and flavor formation. Therefore, increasing glucosinolate content and cultivating high-quality Chinese kale and mustard green varieties has broad development prospects. Thus, finding a method to increase glucosinolate content is essential. Summary of the Invention

[0004] The purpose of this invention is to provide ZFP7 The application of genes in regulating the content of glucosinolates in plants aims to address the problems existing in the prior art. This invention has discovered... ZFP7 Genes can regulate the content of glucosinolates in plants, and can positively regulate the biosynthesis and accumulation of glucosinolates in plants, effectively increasing the content of total glucosinolates, aliphatic glucosinolates and indole glucosinolates in plants.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a ZFP7 protein that regulates the content of glucosinolates in plants, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0006] This invention also provides a method for regulating the content of glucosinolates in plants. ZFP7 Genes, the ones mentioned ZFP7 Genes are BoaZFP7 Gene or BjuZFP7 Genes; the stated BoaZFP7The nucleotide sequence of the gene is shown in SEQ ID NO.1; BjuZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0007] The present invention also provides a recombinant expression vector, comprising the above-described components. ZFP7 Gene.

[0008] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0009] Furthermore, the recombinant host cell is recombinant Agrobacterium.

[0010] The present invention also provides the above-mentioned ZFP7 Application of genes, recombinant expression vectors, or recombinant host cells in increasing glucosinolate content in plants.

[0011] Furthermore, the plant in question is a member of the Brassicaceae family.

[0012] Furthermore, when the plant is kale, the ZFP7 Genes are BoaZFP7 Gene; When the plant is mustard greens, ZFP7 Genes are BjuZFP7 Gene.

[0013] The present invention also provides a method for increasing the glucosinolate content in plants, comprising: […]. ZFP7 Genetic transformation of the plant yields overexpression of the gene. ZFP7 The steps involved in genetically modified plants.

[0014] Furthermore, the plant in question is a member of the Brassicaceae family.

[0015] The present invention discloses the following technical effects: This invention has found that ZFP7 This gene can regulate the content of glucosinolates in plants, positively regulating the biosynthesis and accumulation of glucosinolates, effectively increasing the content of total glucosinolates, aliphatic glucosinolates, and indole glucosinolates in plants, while comprehensively upregulating the expression levels of key genes in the glucosinolate synthesis pathway. The protein encoded by this gene is located in the cell nucleus and can directly bind to and activate the core gene for glucosinolate side chain modification. AOP2 It regulates synthetic pathways at the transcriptional level, with a clear and stable mechanism of action. ZFP7 Overexpression of the gene in plants can yield transgenic plants with stable traits and significantly increased glucosinolate content, and the plants grow and develop normally without adverse phenotypes.

[0016] This invention provides a stable and efficient molecular target for the quality improvement and resistance breeding of cruciferous vegetables, and has broad application prospects in the breeding and improvement of high-quality kale and mustard varieties, especially in the breeding of varieties with high glucosinolate content. Attached Figure Description

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

[0018] Picture 1 The image shows the subcellular localization detection results of BoaZFP7. Picture 2 Wild-type Chinese kale (WT) plants, empty vector (EV) plants, and overexpression plants BoaZFP7 ( BoaZFP7 Phenotypic observation and gene expression level diagrams of the OE-17 strains (OE2, OE3, OE17); where A is the phenotypic observation diagram of the plants; B is the phenotypic diagram of each plant. BoaZFP7 Gene expression level statistical graph; Picture 3 Wild-type Chinese kale (WT) plants, empty vector (EV) plants, and overexpression plants BoaZFP7 ( BoaZFP7 The graphs show the glucosinolate content of the OE strains (OE2, OE3, OE17); where A represents the total glucosinolate (GSLs) content; B represents the total aliphatic glucosinolate (AGS) content; C represents the total indole glucosinolate (IGS) content; D represents the 3-(methylsulfinyl)propyl glucosinolate (GIB) content; E represents the 4-butyl glucosinolate (GRA) content; F represents the 2-propenyl glucosinolate (SIN) content; and G represents... Content of 3-Butenyl glucosinolate (GNA); H represents the content of 2-hydroxy-3-butenyl glucosinolate (PRO); I represents the content of indole-3-methyl glucosinolate (GBS); J represents the content of 4-hydroxy-indole-3-methyl glucosinolate (4-HGBS); K represents the content of 4-methoxy-indole-3-methyl glucosinolate (4-MGBS); L represents the content of 1-methoxy-indole-3-methyl glucosinolate (NGBS). Picture 4 Wild-type Chinese kale (WT) plants, empty vector (EV) plants, and overexpression plants BoaZFP7 ( BoaZFP7 Figure 1 shows the results of quantitative real-time PCR for glucosinolate-related genes in the OE (OE) strains (OE2, OE3, OE17); where A represents... BoaMYB28 Gene expression level map, B is BoaMYB29Gene expression level map, C is BoaBCAT4 Gene expression level map, D is BoaCYP83A1 Gene expression level map, E is BoaSUR1 Gene expression level map, F is BoaUGT74B1 Gene expression level map, G is BoaFMO GS‑OX5 Gene expression level map, H represents BoaAOP2.1 Gene expression level map, I represents BoaCYP79B2 Gene expression level map, J is BoaCYP79F1 Gene expression level map, K is BoaGSTU20 Gene expression level map, L represents BoaUGT74C1 Gene expression level map, M is BoaCYP83B1 Gene expression level map, N is BoaST5a Gene expression level map, O represents BoaST5b Gene expression level map; Picture 5 For BoaZFP7 direct combination BoaAOP2.1 The experimental verification results of BoaZFP7 and its positive regulation of expression are shown in the figure; where A shows the interaction between BoaZFP7 and... BoaAOP2.1 Figure 1 shows the yeast monohybrid (Y1H) experiment with promoter binding; B represents the effector construct (SK-BoaZFP7) and reporter construct (SK-BoaZFP7) used in the dual-luciferase experiment. BoaAOP2.1pro A schematic diagram of LUC; C is a fluorescence observation diagram of the dual-luciferase transient expression experiment; D is a graph of the relative luciferase activity ratio of the dual-luciferase experiment (represented by the ratio of LUC to REN). Picture 6 The image shows the subcellular localization detection results of BjuZFP7. Picture 7 For wild-type mustard (WT) plants and overexpression BjuZFP7 ( BjuZFP7 Phenotypic observation and gene expression level diagrams of the OE-1 and OE2 lines; where A is the phenotypic observation diagram of mustard plants; B is the phenotypic observation diagram of each mustard plant. BjuZFP7 Gene expression level statistical graph; Picture 8 For wild-type mustard (WT) plants and overexpression BjuZFP7 ( BjuZFP7 The graphs show the glucosinolate content of the OE1 and OE2 strains (OE1 and OE2). A represents the total glucosinolate content, B represents the total aliphatic glucosinolate content, C represents the total indole glucosinolate content, D represents the 2-propenyl glucosinolate content, E represents the 3-butenyl glucosinolate content, F represents the 4-hydroxy-indole-3-methyl glucosinolate content, G represents the indole-3-methyl glucosinolate content, H represents the 4-methoxy-indole-3-methyl glucosinolate content, and I represents the 1-methoxy-indole-3-methyl glucosinolate content. Picture 9 For wild-type mustard (WT) plants and overexpression BjuZFP7 ( BjuZFP7 The image shows the results of quantitative real-time PCR for glucosinolate-related genes in the OE1 and OE2 strains (OE1 and OE2); where A represents... BjuMYB28 Gene expression level map, B is BjuMYB29 Gene expression level map, C is BjuMYB34 Gene expression level map, D is BjuMYB51 Gene expression level map, E is BjuCYP79F1 Gene expression level map, F is BjuCYP83A1 Gene expression level map, G is BjuSUR1 Gene expression level map, H represents BjuUGT74C1 Gene expression level map, I represents BjuFM OGS‑OX2 Gene expression level map, J is BjuAOP2 Gene expression level map, K is BjuCYP79B2 Gene expression level map, L represents BjuCYP83B1 Gene expression level map, M is BjuST5a Gene expression level map, N is BjuCYP81F1 Gene expression level map, O represents BjuCYP81F2 Gene expression level map, P is BjuCYP81F4 Gene expression level map; Picture 10 For BjuZFP7 direct binding BjuAOP2 The experimental verification results of the promoter and its positive regulation of expression are shown in the figure; where A shows the interaction between BjuZFP7 and... BjuAOP2 Y1H promoter binding experimental diagram; B represents the effector construct (SK-BjuZFP7) and reporter construct (SK-BjuZFP7) used in the dual-luciferase experiment. BjuAOP2pro A schematic diagram of LUC; C is a fluorescence observation diagram of the dual-luciferase transient expression experiment; D is a diagram of the relative luciferase activity ratio of the dual-luciferase experiment (represented by the ratio of LUC to REN). Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] BoaZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; BjuZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.4.

[0025] SEQ ID NO.1:ATGACGGAATCTGATGATGCCTCGCGAGAGTTACCAGTCAGAGAAGGAGAAACTTTGTCAAATCAAGATCTATCCAAAACCCAAGCGATCATCCCTGTGTCGCTCGATCTCAAGCTCAAAGACAGTTTCAACGATGAACAAAAGGGAACAAAGCGTGGGTCCAACCCGAGAGTCTTCTCCTGTAACTACTGCAGACGCAAGTTCTATAGCTCTCAGGCTCTTGGAGGCCACCAGAACGCTCACAAGCGAGAACGGACAATGGCGAAAAGAGCAATGTACATGGGAAGAATGTTTGGTCACCATCACAGGCCTTACACATACTCTTCTTCTTCTCTAGGGATGCAAGCTCACTCCGGTCTACTTCACCACACCTTATCCCAGCCTCAAACTCCTCCTCCTCCTCTTGCAAGGTTTCATCAGCAAGGTTATTTTGGTAACACTGTGCCATTGTTCTTTGACTATGATGATGGCAGTGACTTCTTCTGGCCCGGAAGTTTTCGTCAGGCTGTAGAGGAAGTTGAAGCTCCCGTGGTGGTGGCTGTTACAGAGTCAGGTCTTGATCTGAACTCTGCTGCAGCAAATGAAAGTGTAGTAGATAATAATAATAATGGCCTCAAGCCTGATCTTACCTTGAGACTCTAA。

[0026] SEQ ID NO.2:MTESDDASRELPVREGETLSNQDLSKTQAIIPVSLDLKLKDSFNDEQKGTKRGSNPRVFSCNYCRRKFYSSQALGGHQNAHKRERTMAKRAMYMGRMFGHHHRPYTYSSSSLGMQAHSGLLHHTLSQPQTPPPPLARFHQQGYFGNTVPLFFDYDDGSDFFWPGSFRQAVEEVEAPVVVAVTESGLDLNSAAANESVVDNNNNGLKPDLTLRL 。

[0027] SEQ ID NO.3: ATGACTGAATCTGATGATGCCTCGCGAGAGTTACCAGTCAGAGAAGGAGAAACCTCTTCAAAACAAGATATATCCAAACCCGAAGAGATCATCCCTGTTTCGCTCGATCTCAAGCTCAACGATAGCTTCAACGATGGGCAAAAGGGAACAAAGCGTGAATCCAGCCCTAGAGTCTTCTCCTGTAACTACTGCAAACGCAAGTTCTATAGCTCTCAAGCTCTTGGAGGTCACCAGAACGCTCACAAGCGAGAAAGAACAATGGCTAAACGAGCAATGTACATGGGAAGAATGTTTGGTCACCATCACAGGCCTTACACATACTCCTCTTCTTCTCTAGGGATGCAAGCTCACTCCGGTCTACTTCACCACTCCTTATCCCAGCCTCAGCCTCCTCCTCCTCTTGCAAGGTTTCATCAGCAAGGGTATTTTGGTAACACCGTGCCATTGTTCTTTGACTATGATGATGGCAGTGACTTCTTCTGGCCCGGAAGTTTTCGTCAGGTTGTCGAGGAAGTTGAAGCTCCTGTGGTTGTGGCTGTTACAGAGTCAGGTCTTGATCTGAACTCGGCTGCAGCAAATGAAAGTTTAGTAGATAATAATAATATCTCGAAGCCTGATCTTACTTTGAGACTCTAA。

[0028] SEQ ID NO.4: MTESDDASRELPVREGETSSKQDISKPEEIIPVSLDLKLNDSFNDGQKGTKRESSPRVFSCNYCKRKFYSSQALGGHQNAHKRERTMAKRAMYMGRMFGHHHRPYTYSSSSLGMQAHSGLLHHSLSQPQPPPPLARFHQQGYFGNTVPLFFDYDDGSDFFWPGSFRQVVEEVEAPVVVAVTESGLDLNSAAANESLVDNNNISKPDLTLRL 。

[0029] Example 1 1 Materials and Methods 1.1 Plant Materials This invention uses multigenerational inbred lines of the Chinese kale variety 'Siji Cutiao' as experimental materials, which have a uniform genetic background and stable traits. The Chinese kale tissue culture seedlings were cultured in vitro in a tissue culture room under the following temperature and light conditions: temperature 25℃ (day) / 20℃ (night), relative humidity 75% (day) / 70% (night), and photoperiod 16 h (day) / 8 h (night).

[0030] Tobacco Bunge was grown in a light incubator with a light cycle of 18 h (day) / 6 h (night) and the same temperature and humidity parameters as above. It was ready for use when the seedlings were 4 to 5 weeks old and the leaves were fully expanded.

[0031] All plants are managed with standardized water and fertilizer.

[0032] 1.2 Extraction of total RNA from Chinese kale and synthesis of cDNA Total RNA was extracted from mature leaves of Chinese kale 'Siji Cutiao' using an RNA extraction kit (Nanjing, Novizan), and the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (Japan, TaKaRa).

[0033] 1.3 Chinese kale BoaZFP7 Cloning of genes according to BoaZFP7 nucleotide sequence design of genes BoaZFP7 Specific primers for gene cloning: Upstream primer: 5′-ATGACGGAATCTGATGATGCCT-3′ (SEQ ID NO.5); Downstream primer: 5′-TTAGAGTCTCAAGGTAAGATCAGGCT-3′ (SEQ ID NO.6).

[0034] Using 'Four Seasons Coarse Strip' cDNA as a template, amplification was performed under the following PCR conditions: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 57℃ annealing for 5 s, 72℃ extension for 5 s, for a total of 34 cycles; followed by a 72℃ extension for 1 min. The PCR products were subjected to 1.0% agarose gel electrophoresis, and the target band was recovered. The product was ligated using a pToPo-Blunt Simple cloning kit (Beijing, Adelaide), and then transformed into DH5α competent E. coli cells. Plasmids were extracted, identified by PCR, and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0035] 1.4 BoaZFP7 Subcellular Localization Analysis Subcellular localization was performed using the pC2300-35S-eGFP vector. The CDS sequence of BoaZFP7 from Chinese kale was analyzed, and specific amplification primers containing homologous arms were designed using CEDesign V1.04 software. Upstream primer: 5'-acgggggacgagctcggtaccATGACGGAATCTGATGATGCCT-3' (SEQ ID NO. 9); Downstream primer: 5'-cttgctcaaccatggtgtcgacTTAGAGTCTCAAGGTAAGATCAGG-3' (SEQ ID NO. 10).

[0036] Amplification was performed using the high-fidelity enzyme P520 (Nanjing, Novizan). BoaZFP7 Using restriction endonucleases Kpn I and Sal The target gene fragment and vector were digested with enzyme I and then recovered. After recovery, homologous recombination was performed using the Uniclone One Step Seamless ligase kit (Beijing, Jinsha) to obtain the recombinant vector pC2300-35S-BoaZFP7-eGFP, which was then transformed into DH5α *E. coli* competent cells. Single-colony PCR was performed to select positive clones with correct sequencing, confirming successful vector construction. Plasmids were extracted from the positive bacterial culture using a plasmid extraction kit (Shanghai, Sangon Biotech). The recombinant vector pC2300-35S-BoaZFP7-eGFP and the empty vector pC2300-35S-eGFP were respectively introduced into *Agrobacterium tumefaciens* GV3101 (pSoup-p19). The cells were transiently transformed into *Nicotiana benthamiana* leaf epidermal cells via Agrobacterium-mediated transformation. After 2 days of low-light culture, the subcellular localization of the BoaZFP7 protein was observed using an Olympus laser confocal microscope.

[0037] 1.5 BoaZFP7 Construction and transformation of recombinant overexpression vectors for genes Using software design BoaZFP7 Primers for homologous recombination of genes: Upstream primer: 5'-ctcgagggggggcccggtaccATGACGGAATCTGATGATGCCT-3' (SEQ IDNO.13); Downstream primer: 5'-gatctacagcccgggggatccTTAGAGTCTCAAGGTAAGATCAGG-3' (SEQ ID NO. 14).

[0038] Through homologous recombination method BoaZFP7 The gene fragment and the linearized vector pCAMBIA1301 were recombined and transformed into E. coli DH5α. Positive clones were sent for sequencing, and the recombinant vector pCAMBIA1301-35S- was the most accurate for sequencing. BoaZFP7Plasmids were extracted. The obtained overexpression vector plasmid and empty vector plasmid were transformed into Agrobacterium GV3101, respectively, to obtain Agrobacterium positive monoclonal antibodies containing the fusion vector and the empty vector, respectively.

[0039] 1.6 Explant culture and genetic transformation of Chinese kale Aseptic seeds of the Chinese kale variety 'Siji Cutiao' were sown on 1 / 2 MS solid medium. The tissue culture conditions were: temperature 25 / 20℃ (day / night), photoperiod 16 / 8 h (day / night), and light intensity 36 μmol·m⁻¹. -2 ·s -1 After 7 days of culture, cotyledons of Chinese kale with 1-2 mm petioles were cut and used as explants for Agrobacterium-mediated transformation. The explants were pre-cultured in MS medium for 3 days with a photoperiod of 16 h / 8 h (light / dark) and a light intensity of 80 μmol·m⁻¹. -2 ·s -1 The explants were infected with Agrobacterium strain GV3101 transformed with the recombinant vector for 1-2 min, co-cultured in the dark on MS medium for 3 days, and then transferred to delayed selection medium for 7 days of growth. Selection was then performed on resistance selection medium. After 3 months of growth, the explants were transplanted into a substrate of nutrient soil, perlite, and vermiculite in a volume ratio of 3:1:1.

[0040] 1.7 Chinese kale BoaZFP7 Screening of plants with positive gene overexpression Specific primers pM1301-F and pM1301-R (Table 1) were designed for the pCAMBIA1301 vector. PCR amplification was performed using the genomic DNA of each transgenic plant as a template. Positive plants were detected by electrophoresis, and the results were analyzed in conjunction with RT-qPCR. BoaZFP7 Gene expression levels were used to screen for Chinese kale. BoaZFP7 Overexpression-positive plants were named BoaZFP7- OE.

[0041] Table 1. Specific information about the primers 1.8 Chinese kale BoaZFP7 Analysis of glucosinolate content in transgenic plants (1) After the T0 generation transgenic seedlings were transplanted from the tissue culture bottle into the soil, and after about 30 days of normal growth management, tender leaves with the same growth status and leaf position of each line were selected as test samples, with each sample weighing 0.3~0.4 g. The collected leaves were immediately placed in liquid nitrogen for quick freezing, and then stored in an ultra-low temperature freezer at -80℃ for later use.

[0042] (2) Boil the Chinese kale leaves in 5 mL of water for 10 min. Centrifuge (5 min, 7000 × g) and collect the supernatant. Load the extract onto a DEAE-Sephadex A-25 column, wash three times with 1 mL of 0.02% pyridine acetate, and twice with 1 mL of ddH2O. Treat overnight with 100 μL of 0.1% sulfatase to convert glucosinolates to their desulfurized products, and finally wash away the thioglucosinolates with 1 mL of water. Analyze the glucosinolates using HPLC at 30 °C using acetonitrile and water at a flow rate of 1 mL / min in a Waters Spherisorb C10 column. 18 Samples were separated on a chromatographic column. The procedure involved elution with 1.5% acetonitrile for the first 5 minutes, followed by linear gradient elution to 20% acetonitrile for the next 15 minutes, and then elution with 20% acetonitrile for the last 10 minutes. The composition and content of glucosinolates in each transgenic line were determined.

[0043] 1.9 Chinese kale BoaZFP7 Quantitative analysis of glucosinolate-related genes in transgenic plants (1) Sample at the same time as in 1.8, and try to take the two samples from the same leaf.

[0044] (2) Obtain RNA and cDNA according to the method in “1.2 Extraction of total RNA from Chinese kale and synthesis of cDNA”.

[0045] (3) Design of glucosinolate-related genes in Chinese kale ( BoaZFP7 , BoaMYB28 , BoaMYB29 , BoaBCAT4, BoaCYP79F1, BoaCYP79B2, BoaCYP83B1, BoaCYP83A1 , BoaSUR1, BoaUGT74B1, BoaUGT74C1, BoaST5b.11, BoaST5a, BoaFMO GS-OX5 Boa AOP2.1 and BoaGSTU20 Sequence and internal reference gene β-actin Specific primers for gene expression detection were used for quantitative fluorescence detection, and the primers used are shown in Table 2.

[0046] Table 2. Specific information on primers for quantitative analysis of glucosinolate-related genes in Chinese kale plants. (4) Real-time quantitative PCR was performed using TB Green Premix Ex Taq. TM The instructions for the II qPCR Kit (Tli RNaseHPlus, Takara) real-time fluorescence kit are used to detect the expression level of the glucosinolate biosynthesis gene in Chinese kale.

[0047] (5) The formula for calculating the relative transcriptional expression level of the target gene is 2.-ΔΔCt ΔΔCt = (Ct target gene - Ct) Actin Treatment group - (Ct target gene - Ct) Actin Control group.

[0048] 1.10 Yeast One-Hybrid Experiment (1) Construction of yeast one-hybrid bait and capture vectors clone BoaAOP2.1 promoter sequence ( BoaAOP2.1 -pro)(SEQ ID NO.89), using homologous recombination, the cloned promoter fragment is ligated into the pABAi vector, and BoaZFP7 The CDS sequence was ligated into the pGADT7 vector. After DH5α positivism screening and sequencing, the target strain was cultured and plasmids were extracted to obtain the bait vector pABAi- BoaAOP2.1 -pro and capture vector pGADT7-BoaZFP7.

[0049] SEQ ID NO.89: .

[0050] (2) Transformation and screening of Yeast Y1H competent cells Using pABAi- as a bait carrier BoaAOP2.1 Using -pro as a template, BstB I was digested at 65℃ for 1 h, and the digested product was purified and recovered. Following the TAKARA Y1H Gold yeast competent cell transformation instructions, the single enzyme plasmid was transformed into Y1H Gold cells. After culturing on SD / -Ura solid medium at 30℃ for 3-5 days, single-clone colony PCR identification was performed. Positive clones were mixed with 50% glycerol at a 1:1 ratio, flash-frozen in liquid nitrogen, and stored at -80℃.

[0051] (3) Screening of AbA concentration in bait yeast strains The activated yeast positive clone culture was adjusted to OD using 0.9% NaCl. 600 =1, and successively diluted 10 times, 100 times, and 1000 times. 5 μL of each solution was dropped onto SD / -Ura solid medium with AbA concentrations of 0 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 400 ng / mL, and 500 ng / mL. The cultures were incubated at 30℃ for 3-5 days to screen for the minimum inhibitory concentration of AbA in the bait yeast strain.

[0052] (4) Verification of co-conversion and droplet plate interaction Yeast competent cells were prepared according to the TAKARA instructions. The empty pGADT7 plasmid and the capture vector plasmid pGADT7-BoaZFP7 were transformed into the competent yeast cells, and the cells were cultured upside down at 30°C for 3-5 days on SD / -Leu and SD / -Leu / AbA solid media. Single colonies were picked and plated on SD / -Leu and SD / -Leu plates containing the minimum inhibitory concentration of AbA for verification. The growth of yeast cells on solid media containing AbA was observed to determine whether the target protein and the target gene promoter interact.

[0053] 1.11 Dual-luciferase assay (1) Construction of dual-luciferase reporter vector and effector vector Using homologous recombination, cloned... BoaAOP2.1 The -pro fragment was ligated into the pGreenⅡ 0800-LUC vector, BoaZFP7 The CDS was ligated into the pGreenII 62-SK vector, transformed into DH5α positive screening and sequenced, and the target strain was cultured and plasmids were extracted to obtain the reporter vector pGreenII 0800- BoaAOP2.1 -pro-LUC and the effector pGreenII 62-SK-BoaZFP7.

[0054] (2) Agrobacterium GV3101 (pSoup-p19) competent transformation Refer to the transformation instructions for Agrobacterium GV3101 (pSoup-p19) from Angyu Biotechnology. 1 μg of effector vector pGreenII62-SK-BoaZFP7 and reporter vector pGreenII 0800- were added. BoaAOP2.1 -pro-LUC, empty vector pGreen-62-SK, and empty vector pGreenll 0800-LUC were transformed into Agrobacterium, respectively. The bacterial culture was detected by PCR using universal primers for pGreenll 0800-LUC and pGreen-62-SK. Positive clones were picked and inoculated into 700 μL of liquid LB medium containing kanamycin (Kan) and rifampicin (Rif) resistance, and activated by shaking at 220 rpm for about 36 h at 28°C.

[0055] (3) Instantaneous contamination of tobacco Take the above activated bacterial solution and add it to 50 mL of LB liquid resistant medium. Incubate at 28°C with shaking at 220 rpm until OD reaches the target value. 600 =0.8~1.0. After the OD value reaches the required level, centrifuge the bacterial culture at 4°C and 6000 rpm for 5 min. Discard the supernatant and resuspend the Agrobacterium in infection buffer until the OD value reaches 0.8~1.0. 600 =Approximately 0.6, let stand at room temperature for 2 hours. Add pGreenII 0800- BoaAOP2.1 A 1:1 mixture of *Agrobacterium* containing *pro-LUC* and *pGreenII* 62-SK-BoaZFP7 / pGreen 62-SK vectors was used as the experimental group; 1:1 mixtures of *pGreenII* 0800-LUC and *pGreenII* 62-SK-BoaZFP7 vectors were used as negative controls. One month after bud break, *Nicotiana benthamiana* plants with similar growth and good development were selected as plant materials. The mixed bacterial solution was injected into the underside of tobacco leaves using a needleless syringe. The injected tobacco plants were marked and cultured in the dark for 24 h followed by light for 24 h.

[0056] (4) Fluorescence intensity and imaging detection The dual-luciferase assay was performed according to the instructions of the Yisheng Company kit. Fresh leaves were smeared with the substrate D-luciferin potassium salt, and the fluorescence imaging of the leaves was observed. The leaf material (sampled using a perforator) was then ground. 100 μL of lysis buffer was added, and the mixture was incubated in an ice-water bath for 5 min, followed by centrifugation (4℃, 12000 rpm, 10 min). The supernatant and substrate were added to the microplate, and the ratio of the two luciferases in the tobacco leaves (LUC / REN) was detected and calculated.

[0057] 2. Experimental Results 2.1 Sequence characteristics and subcellular localization analysis of BoaZFP7 The full-length cDNA of BoaZFP7 is 642 bp, encoding a protein composed of 213 amino acids. Subcellular localization results indicate that the BoaZFP7 protein is mainly located in the cell nucleus (…). Picture 1 ).

[0058] 2.2 Creation BoaZFP7 Stable overexpression of Chinese kale material This invention utilizes an Agrobacterium-mediated genetic transformation system to obtain... BoaZFP7 Transgenic positive plants were identified, and the genetically modified positive plants were tested for... BoaZFP7 Gene expression levels were obtained from 3 strains. BoaZFP7 Overexpression plants ( Picture 2 (A). Among wild-type and uncultivated plants BoaZFP7 There was no difference in the relative expression levels of OE2, OE3, and OE17 in Chinese kale overexpressing plants. BoaZFP7 Expression levels were significantly upregulated, with overexpression rates of 2.27-fold, 2.30-fold, and 1.97-fold compared to WT, respectively. Picture 2 (B)

[0059] 2.3 Chinese kale BoaZFP7 Analysis of glucosinolate components and contents in transgenic plants This invention uses HPLC for determination. BoaZFP7 The composition and content of glucosinolates in leaves of overexpressing transgenic plants (OE2, OE3, OE17), wild-type plants (WT), and empty vector plants (EV) were investigated. The results showed that... BoaZFP7The total glucosinolate content in transgenic plants was significantly higher than that in wild-type and unexpressed plants. Compared with WT and EV, the three overexpression lines showed a significant increasing trend in the content of three aliphatic glucosinolates: 3-(methylsulfinyl)propylglucosinolate (GIB), 4-butylglucosinolate (GRA), and 3-butenylglucosinolate (GNA). GNA, the most abundant aliphatic glucosinolate component in Chinese kale, had an average content of 0.40 μmol / g FW in the overexpression lines, which was 4.06 times that of WT and 3.26 times that of EV. The content of indole glucosinolates was increased to varying degrees in the overexpression lines. As the main component of indole glucosinolates, indole-3-methylglucosinolate (GBS) accounts for 78.77-88.71% of the total indole glucosinolates. The average GBS content in the overexpression lines was 0.69 μmol / gFW, which is 2.12 times the average GBS content in WT and EV. In addition, the contents of 4-hydroxy-indole-3-methylglucosinolate (4-HGBS), 4-methoxy-indole-3-methylglucosinolate (4-MGBS), and 1-methoxy-indole-3-methylglucosinolate (NGBS) also showed an increasing trend. Picture 3 ).

[0060] In summary, BoaZFP7 Overexpression of the gene promoted the accumulation of glucosinolates in Chinese kale plants.

[0061] 2.4 Chinese kale BoaZFP7 Analysis of glucosinolate-related gene expression levels in transgenic plants This invention uses qPCR assays to... BoaZFP7 The expression levels of glucosinolate-related genes were detected in overexpressing transgenic plants (OE2, OE3, OE17), wild-type plants (WT), and empty vector plants (EV). The results showed that in the aliphatic glucosinolate synthesis pathway, related transcription factors... BoaMYB28 and BoaMYB29 exist BoaZFP7 The expression levels in transgenic plants were significantly upregulated, with average expression levels 1.13 and 2.35 times that of WT, respectively. Upstream gene BoaBCAT4 The expression was significantly upregulated in overexpression lines, reaching a maximum up to 4.46 times that of WT. This gene is responsible for the formation of the core structure of aliphatic glucosinolates. BoaGSTU20 , BoaSUR1 , BoaUGT74B1 The relative expression level of [a specific gene] was significantly upregulated. [This gene is responsible for secondary modification of the aliphatic glucosinolate side chain.] BoaAOP2.1 The relative expression level was significantly upregulated, reaching 1.51 times that of WT. Furthermore, BoaZFP7 In overexpressing transgenic plants, the core structure responsible for the formation of indole glucosinolates is formed. BoaCYP79B2 and BoaCYP83B1 The relative expression level was also significantly upregulated. Picture 4 ).

[0062] In summary, BoaZFP7 Overexpression of genes can positively regulate the expression of glucosinolate-related genes in Chinese kale, thereby promoting the accumulation of glucosinolates.

[0063] 2.5 The effect of transcription factor BoaZFP7 on BoaAOP2.1 transcriptional regulatory mechanisms This invention first uses a yeast one-hybrid assay to detect the relationship between BoaZFP7 and... BoaAOP2.1 Promoter binding capacity. Results showed that co-transformation of pGADT7-BoaZFP7 and pABAi- BoaAOP2.1 pro The yeast cells were able to grow normally on the selective culture medium, while the control group could not grow, indicating that BoaZFP7 can interact with... BoaAOP2.1 Promoter interaction ( Picture 5 (A). Based on the results of the yeast single hybridization, a dual-luciferase reporter assay was further used to detect the effect of BoaZFP7 on... BoaAOP2 Does it have a regulatory effect? BoaAOP2.1-pro The BoaZFP7 gene was constructed into the reporter vector and effector gene vector, respectively. Picture 5 BoaZFP7 (B1) was transfected with Agrobacterium and then injected into Nicotiana benthamiana. Fluorescence observation using a plant in vivo imaging system showed that BoaZFP7 promoted the growth of [a specific growth factor]. BoaAOP2.1 The expression ( Picture 5 (C); LUC and REN values ​​were detected using an ELISA reader, and the LUC / REN ratio was calculated. The results showed that the dual fluorescence ratio of the experimental group was significantly higher than that of the control group, being 1.98 times that of the control group. Picture 5 (D). This indicates that in a dual-luciferase reporter system, the BoaZFP7 protein can directly interact with... BoaAOP2.1 Promoter interaction, facilitate BoaAOP2.1 Gene activity.

[0064] In summary, the transcription factor BoaZFP7 can directly bind to the aliphatic glucosinolate side chain to modify major genes. BoaAOP2.1, And positively activate its transcription.

[0065] Example 2 1. Materials and Methods 1.1 Plant materials This invention uses multi-generation inbred lines of the mustard variety 'Yongan Xiaoye' as experimental materials, which have a uniform genetic background and stable traits. Mustard tissue culture seedlings are cultured in vitro in a tissue culture room, with temperature and light conditions controlled as follows: temperature 25℃ (day) / 20℃ (night), relative humidity 75% (day) / 70% (night), and photoperiod 16 h (day) / 8 h (night).

[0066] 1.2 Extraction of total RNA from mustard greens and synthesis of cDNA The method is the same as in Example 1.

[0067] 1.3 Mustard greens BjuZFP7 Cloning of genes according to BjuZFP7 nucleotide sequence design of genes BjuZFP7 Specific primers for gene cloning: Upstream primer: 5′-ATGACTGAATCTGATGATGCCTC-3′ (SEQ ID NO.7); Downstream primer: 5′-TTAGAGTCTCAAAGTAAGATCAGGC-3′ (SEQ ID NO.8).

[0068] Using cDNA from the mustard 'Yongan Xiaoye' variety as a template, PCR amplification was performed under the following conditions: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 57℃ annealing for 5 s, 72℃ extension for 5 s, for a total of 34 cycles; followed by a final extension at 72℃ for 1 min. The PCR products were analyzed by 1.0% agarose gel electrophoresis, and the target band was recovered. The product was then ligated using a pToPo-Blunt Simple cloning kit (Beijing, Adley), and subsequently transformed into DH5α competent E. coli cells. Plasmids were extracted, identified by PCR, and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0069] 1.4 Subcellular localization analysis of BjuZFP7 The method is the same as in Example 1. The specific amplification primers containing homologous arms, designed based on the CDS sequence of BjuZFP7 from mustard greens, are as follows: Upstream primer: 5'-acgggggacgagctcggtaccATGACTGAATCTGATGATGCCTC-3' (SEQ ID NO. 11); Downstream primer: 5'-cttgctcaaccatggtgtcgacTTAGAGTCTCAAAGTAAGATCAGGC-3' (SEQ ID NO. 12).

[0070] 1.5 BjuZFP7 Construction and transformation of recombinant overexpression vectors for genes The specific method is the same as in Example 1. BjuZFP7 The primers for homologous recombination of genes are as follows: Upstream primer: 5'-ctcgaggggggcccggtaccATGACTGAATCTGATGATGCCTC-3' (SEQ IDNO.15); Downstream primer: 5'-gatctacagcccgggggatccTTAGAGTCTCAAAGTAAGATCAGGC-3' (SEQ ID NO. 16).

[0071] 1.6 Explant culture and genetic transformation of mustard The material used was the mustard variety 'Yongan Xiaoye', and the specific method is described in Example 1.

[0072] 1.7 Mustard greens BjuZFP7 Screening of plants with positive gene overexpression The method is the same as in Example 1, and mustard greens are obtained through screening. BjuZFP7 Overexpression-positive plants were named BjuZFP7- OE.

[0073] 1.8 Mustard greens BjuZFP7 Analysis of glucosinolate content in transgenic plants The method is the same as in Example 1.

[0074] 1.9 Mustard greens BjuZFP7 Quantitative analysis of glucosinolate-related genes in transgenic plants (1) Sample at the same time as in 1.8, and try to take the two samples from the same leaf.

[0075] (2) RNA and cDNA were obtained according to the method in "1.2 Extraction of total RNA from mustard and synthesis of cDNA"; (3) Design of mustard glucosinolate-related genes ( BjuZFP7 , BjuMYB28 , BjuMYB29 , BjuMYB34 , BjuMYB51, BjuCYP79F1, BjuCYP83A1, BjuSUR1, BjuUGT74C1 , BjuFMO GS-OX2 BjuAOP2, BjuCYP79B2, BjuCYP83B1, BjuST5a, BjuCYP81F1, BjuCYP81F2 and BjuCYP81F4 Sequence and internal reference gene BjuActin Specific primers for gene expression detection were used for quantitative fluorescence detection, and the primers used are shown in Table 3.

[0076] Table 3. Information on primers for quantitative analysis of glucosinolate-related genes in mustard plants. (4) Real-time quantitative PCR was performed using TB Green Premix Ex Taq. TM The instructions for the II qPCR Kit (Tli RNaseHPlus, Takara) real-time fluorescence kit are used to detect the expression level of the glucosinolate biosynthesis gene in mustard. (5) The formula for calculating the relative transcriptional expression level of the target gene is 2. -ΔΔCtΔΔCt = (Ct target gene - Ct) Actin Treatment group - (Ct target gene - Ct) Actin Control group.

[0077] 1.10 Yeast One-Hybrid Experiment The method is as described in Example 1. BjuAOP2 promoter sequence ( BjuAOP2 -pro) as shown in SEQ ID NO.90.

[0078] SEQ ID NO.90: .

[0079] 1.11 Dual-luciferase assay The method is described in Example 1.

[0080] 2. Experimental Results 2.1 BjuZFP7 Sequence characteristics and subcellular localization analysis BjuZFP7 The full-length cDNA is 633 bp, encoding a protein composed of 211 amino acids. Subcellular localization results indicate that the BjuZFP7 protein is mainly located in the cell nucleus (…). Picture 6 ).

[0081] 2.2 Creation BjuZFP7 Stable overexpression of mustard materials This invention utilizes an Agrobacterium-mediated genetic transformation system to obtain... BjuZFP7 Transgenic positive plants were identified, and the genetically modified positive plants were tested for... BjuZFP7 Gene expression levels were measured, and two mustard plants were obtained. BjuZFP7 Plants with high gene expression levels (OE1 and OE2) Picture 7 (A). Mustard overexpression of OE1 and OE2 in plants BjuZFP7 Expression levels were significantly upregulated, by 2.42 times and 3.61 times that of WT, respectively. Picture 7 (B)

[0082] 2.3 Mustard greens BjuZFP7 Analysis of glucosinolate components and contents in transgenic plants This invention uses HPLC for determination. BjuZFP7 The composition and content of glucosinolates in the leaves of overexpressing transgenic plants (OE1 and OE2) and wild-type (WT) plants. A total of 6 glucosinolates were detected in mustard leaves, including 2 aliphatic and 4 indole glucosinolates. BjuZFP7 In the overexpression plants, the contents of total aliphatic glucosinolates, total indole glucosinolates, and total glucosinolates were all significantly higher than those in the WT plants. In this study, the aliphatic glucosinolates in mustard leaves were mainly 2-propenylglucosinolates (SIN) and GNA. The results showed that the SIN content in the two overexpression lines OE1 and OE2 was significantly increased to 2.11 and 3.05 times that of WT, respectively; the GNA content was significantly increased to 14.97 and 3.47 times that of WT, respectively. The four indole glucosinolates detected in mustard leaves in this invention were 4-HGBS, GBS, 4MGBS, and NGBS, among which 4-HGBS was the most abundant component, accounting for more than 45% of the total indole glucosinolates. The results showed that the 4-HGBS content of the two overexpression lines OE1 and OE2 was significantly increased to 2.65 and 3.67 times that of the WT; the GBS content of OE2 was significantly increased to 3.08 times that of the WT; the 4MGBS content of OE1 and OE2 was significantly increased to 2.21 and 2.09 times that of the WT; and the NGBS content of OE1 and OE2 was significantly increased to 2.39 and 2.65 times that of the WT. Picture 8 ).

[0083] In summary, BjuZFP7 Overexpression of the gene promotes the accumulation of glucosinolates in mustard plants.

[0084] 2.4 BjuZFP7 Analysis of glucosinolate-related gene expression levels in transgenic plants This invention uses qPCR assays to... BjuZFP7 The expression levels of glucosinolate-related genes were detected in overexpressing plants OE1 and OE2 and wild-type plants WT. BjuZFP7 Overexpression of aliphatic glucosinolates in plants regulates major transcription factors BjuMYB28 and BjuMYB29 Expression levels were significantly increased to 2.87-3.29 times and 1.53-2.42 times that of WT, respectively. Indole glucosinolates regulate transcription factors. BjuMYB51 Expression levels significantly increased to 1.75-1.86 times that of the WT. Among the core structural synthesis genes, the expression levels of genes in aliphatic pathways mostly showed an upward trend. BjuCYP79F1 The expression level was significantly increased to 1.33-1.54 times that of WT; BjuCYP83A1 Expression levels were significantly increased to 1.86-3.33 times that of WT; BjuSUR1 The expression level was significantly increased to 2.39-3.29 times that of WT; BjuUGT74C1 Expression levels were significantly increased to 3.52-5.16 times that of WT; BjuFMO GS-OX2 Expression levels were significantly increased to 1.67-2.32 times that of WT; BjuAOP2 Expression levels were significantly increased to 5.64-8.46 times that of WT. Upstream structural genes in the indole pathway. BjuCYP83B1 Expression levels were significantly increased to 1.79-1.95 times that of WT; downstream side-chain modification genes of the indole pathway. BjuCYP81F1 The expression level was significantly increased to 1.33-1.48 times that of the WT. BjuCYP81F2 and BjuCYP81F4 The expression levels increased to 1.16-1.43 times and 1.22-1.28 times that of WT, respectively. Picture 9 ).

[0085] In summary, BjuZFP7 Overexpression of genes can positively regulate the expression of glucosinolate-related genes in mustard, thereby promoting the accumulation of glucosinolates.

[0086] 2.5 Transcription factor BjuZFP7 on BjuAOP2 transcriptional regulatory mechanisms This invention first uses a yeast one-hybrid assay to detect the relationship between BjuZFP7 and... BjuAOP2 Promoter binding affinity. Results showed that co-transformation of pGADT7-BjuZFP7 and pABAi- BjuAOP2-pro Yeast cells from the control group were able to grow normally on selective culture media, while those from the control group could not, indicating that BjuZFP7 can interact with... BjuAOP2 Promoter interaction ( Picture 10 (A). Based on the results of yeast single-hybridization, a dual-luciferase reporter assay was further used to detect the effect of BjuZFP7 on... BjuAOP2Does it have a regulatory effect? BjuAOP2-pro The BjuZFP7 gene was constructed into the reporter vector and effector gene vector, respectively. Picture 10 B12 (B22) was transfected with Agrobacterium and then injected into Nicotiana benthamiana. Fluorescence observation using a plant in vivo imaging system showed that B22ZFP7 promoted the growth of [a specific growth factor]. BjuAOP2 The expression ( Picture 10 (C); LUC and REN values ​​were detected using an ELISA reader, and the LUC / REN ratio was calculated. The results showed that the dual fluorescence ratio of the experimental group was significantly higher than that of the control group, being 12.35 times that of the control group. Picture 10 (D). This indicates that in a dual-luciferase reporter system, the BjuZFP7 protein can directly interact with... BjuAOP2 Promoter interaction, facilitate BjuAOP2 Gene activity.

[0087] In summary, transcription factor BjuZFP7 can directly bind to the aliphatic glucosinolate side chain to modify major genes. BjuAOP2, And positively activate its transcription.

[0088] In summary, it can be seen that overexpression is used in specific embodiments of the present invention. BoaZFP7 and BjuZFP7 The gene can significantly increase the glucosinolate content in plants and positively regulate the expression of glucosinolate-related genes. The two genes directly bind and activate the major gene that modifies the side chain of aliphatic glucosinolates. AOP2 Therefore, it can be seen that Chinese kale BoaZFP7 Genes and mustard BjuZFP7 Genes can effectively increase the glucosinolate content of plants and have promising applications in breeding and improving high-quality cruciferous plant varieties, especially in breeding Chinese kale and mustard greens with high glucosinolate content.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A ZFP7 protein that regulates the content of glucosinolates in plants, characterized in that, The amino acid sequence of the ZFP7 protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. A method for regulating the content of glucosinolates in plants. ZFP7 Genes, characterized by, The ZFP7 Genes are BoaZFP7 Gene or BjuZFP7 Genes; the stated BoaZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.1; BjuZFP7 The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that, Including the claims 2 ZFP7 Gene.

4. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 3.

5. The recombinant host cell according to claim 4, characterized in that, The recombinant host cell is recombinant Agrobacterium.

6. A device as described in claim 2 ZFP7 The application of genes, the recombinant expression vector of claim 3, or the recombinant host cell of claim 4 or 5 in increasing the glucosinolate content in plants.

7. The application according to claim 6, characterized in that, The plant in question is a member of the Brassicaceae family.

8. The application according to claim 7, characterized in that, When the plant is Chinese kale, the ZFP7 Genes are BoaZFP7 Gene; When the plant is mustard greens, ZFP7 Genes are BjuZFP7 Gene.

9. A method for increasing the glucosinolate content in plants, characterized in that, Including the one described in claim 2 ZFP7 Genetic transformation of the plant yields overexpression of the gene. ZFP7 The steps involved in genetically modified plants.

10. The method according to claim 9, characterized in that, The plant in question is a member of the Brassicaceae family.

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