Tobacco NtSHN3 gene, encoding protein and application thereof

Abstract

The invention discloses a tobacco NtSHN3 gene, an encoding protein and application of the tobacco NtSHN3 gene. It is found for the first time that the transcription factor NtSHN3 can promote synthesis of tobacco chlorogenic acid and inhibit synthesis accumulation of scopoletin. A genetic material of which the content of chlorogenic acid is increased and the content of scopoletin is reduced due to overexpression of the NtSHN3 gene is obtained by utilizing a transgenic technology. A genetic material of which the function of the NtSHN3 gene is weakened, so that the content of chlorogenic acid is reduced and the content of scopoletin is increased is obtained by utilizing an RNAi technology. And a new way is provided for regulating and controlling beneficial metabolites in tobacco planting and obtaining new varieties.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a tobacco NtSHN3 gene, an encoded protein and an application thereof in regulating scopoletin synthesis. Background Art

[0002] Scopoletin and its glycosylated derivative, scopolin, are important secondary metabolites synthesized by plants in response to various stresses. They belong to the phytoalexin class of coumarins. In plant-pathogen interactions, plants initiate defense mechanisms in response to pathogen infection. These defenses typically utilize signaling pathways (such as salicylic acid, jasmonic acid, and ethylene) and secondary metabolic pathways within the plant body to initiate and transmit defense signals. These defenses are then synthesized to resist infection by foreign pathogens through the synthesis of plant defensins. The synthesis of plant defensins is often an extension of plant secondary metabolic pathways and is positively correlated with plant disease resistance.

[0003] Scopoletin belongs to the coumarin class of compounds, which exhibit a wide range of pharmacological activities, including anti-inflammatory, antibacterial, vasodilatory, anticoagulant, antithrombotic, antipyretic, and sedative properties. In addition to its heat-clearing, analgesic, and antibacterial and anti-inflammatory properties, it also exhibits significant therapeutic effects in treating vascular diseases, such as vasodilatory and anticoagulant effects, and has some anticancer activity. Scopoletin has antioxidant and antifungal properties, effectively inhibiting the growth of Fusarium seminatum. Scopoletin plays a key role in traditional medicine in Africa, Asia, and Europe. The compound and plants containing it have been implicated in the treatment of numerous conditions and diseases, including convulsions, inflammation, rheumatism, and leprosy. Scopoletin can enhance the activation of eosinophils, a key cell type in the pathogenesis of asthma. Scopoletin also exhibits numerous medicinal activities, including hepatoprotective, antioxidant, anti-inflammatory, and anticancer properties.

[0004] Scopoletin (7-hydroxy-6-methoxycoumarin) is an important polyphenolic compound in tobacco, closely related to leaf quality and aroma. It is present in flue-cured and burley tobacco leaves and smoke, with concentrations reaching 300 mg / kg in mature leaves. In plants, scopoletin is synthesized via the phenylpropanoid metabolic pathway, partially overlapping with the flavonoid and lignin biosynthetic pathways. It initiates from phenylalanine and is catalyzed by a two-step cascade, catalyzed by phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaroyl CoA ligase (4CL), to form 4-coumaroyl-CoA (p-Coumaroyl-CoA). Then, under the action of shikimate O-hydroxycinnamoyltransferase (HCT) and coumarate 3-hydroxylase (4-coumarate3-hydroxylase, C3H), p-coumaroylshikimic acid and caffeoylshikimic acid are successively generated. Caffeoylshikimic acid is catalyzed by HCT to produce 4,5-caffeoyl-CoA (Caffeoyl-CoA), which is further catalyzed by caffeoyl-CoA O-methyltransferase (CCoAOMT) to produce feruloyl-CoA. Feruloyl-CoA is an important precursor for the synthesis of scopoletin (Kai, 2008). Then, 6-hydroxyferuloyl-CoA (6′-HydroxyFeruloyl-CoA) is generated under the action of feruloyl-CoA 6′-hydroxylase 1 (F6′H1), and scopoletin is generated through isomerization and lactonization. Scopoletin is further glycosylated by UDP-Glc:glucosyltransferases (UGTs) to produce scopoletin.

[0005] The synthesis and distribution of flavonoid polyphenols in plants are precisely regulated by a variety of transcription factors. Several major categories of transcription factors have been reported to regulate flavonoid biosynthesis, including MYB, bHLH, WD40, WRKY, bZIP, zinc finger, and MADSbox proteins. MYB, bHLH, and WD40 are the most important. For example, overexpression of the Arabidopsis AtMYB12 gene in tobacco significantly upregulated the expression of multiple genes involved in the phenylpropanoid metabolic pathway, increasing flavonol accumulation severalfold in tobacco leaves. In particular, increased rutin content enhanced resistance of transgenic plants to cotton bollworm. While the structural genes involved in the scopoletin biosynthesis pathway in plants are relatively well characterized, little research has been conducted on the transcription factor genes involved in the transcriptional regulation of scopoletin biosynthesis. Screening and identifying transcription factor genes involved in scopoletin biosynthesis metabolism and further modifying these transcription factors in the scopoletin biosynthesis pathway are important research strategies for regulating scopoletin content in plants.

[0006] The transcription factor WAX INDUCER (WIN) / SHINE (SHN) belongs to the AP2 / ERF (APETALA2 / ethylene response factor) family of proteins. Previous studies have found that transcription factors from the AP2, MYB, bHLH, and HD-ZIP families all play a crucial role in signal transduction and regulation of wax biosynthesis. WAX INDUCER (WIN) / SHINE (SHN) is a key member of the AP2 family, participating in the regulation of wax biosynthesis by modulating the expression of genes involved in wax biosynthesis. In Arabidopsis, there are three important WIN / SHN homologs (SHN1, SHN2, and SHN3). WIN / SHN influences wax biosynthesis by regulating the expression of genes involved in wax biosynthesis. Suppression of the tomato SlSHN3 gene leads to downregulation of multiple genes involved in cutin metabolism. In particular, the expression of genes encoding five transcription factors is significantly reduced in the green-ripe tomato epidermis. Furthermore, SlSHN3 activates the promoters of two transcription factor genes (SlMIXTA and SlG L2a) and two cytochrome P450 genes (SlC YP86A68 and SlCYP86A69). SlSHN3 may directly act on genes regulating cutin biosynthesis and epidermal cell architecture, thereby influencing pericarp formation and epidermal architecture. To date, there have been no reports on the function of the transcription factor SHN3 in scopoletin biosynthesis in plants.

[0007] Chlorogenic acid (CGA), also known as coffee tannin, has a wide range of pharmacological effects, including anti-inflammatory, antibacterial, antiviral, and antidepressant properties. Polyphenols such as chlorogenic acid in tobacco leaves play a decisive role in the color of cured tobacco leaves. Under appropriate curing and aging conditions, polyphenols not only possess a pleasant aroma themselves but also undergo dry distillation, oxidation, and pyrolysis during smoking to produce a series of substances. These degradation products impart an elegant aroma to tobacco and increase the aroma content of tobacco products. The synthesis of chlorogenic acid in plants is tissue-specific and influenced by multiple factors. Its biosynthesis also occurs via the phenylpropanoid metabolic pathway, but the regulation of chlorogenic acid synthesis is currently unclear. Summary of the Invention

[0008] The purpose of the present invention is to provide tobacco NtSHN3 gene, encoding protein, vector, host cell and the like and their application in regulating polyphenol synthesis.

[0009] The present invention discovered a transcription factor NtSHN3 that effectively regulates scopoletin synthesis in tobacco, belonging to the field of plant genetic engineering. The gene is derived from cultivated tobacco. The NtSHN3 gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0010] The NtSHN3 gene is 887 bp long and contains two exons (shown in gray in SEQ ID NO.1) and one intron. The coding region is 645 bp long and encodes 214 amino acids (SEQ ID NO.2). Phylogenetic analysis showed that NtSHN3 has a close evolutionary relationship with AtSHN3, SlSHN3 and other proteins, so it was named NtSHN3. Further amino acid sequence alignment analysis revealed that NtSHN3 contains a typical conserved AP2 / ERF functional domain ( Figure 1 ), indicating that NtSHN3 may be an ERF-type transcription factor protein. Subcellular localization analysis found that NtSHN3-GFP fusion protein was localized in the nucleus ( Figure 2 B), NtSHN3 has transcription factor-like properties.

[0011] The present invention not only provides the nucleotide and amino acid sequences of the tobacco NtSHN3 gene.

[0012] The present invention provides an overexpression vector containing the tobacco NtSHN3 gene and an RNAi vector for inhibiting the tobacco NtSHN3 gene.

[0013] The present invention provides a host cell containing the vector.

[0014] The present invention provides the use of the vector or the host cell in regulating the synthesis of scopoletin in plants, especially tobacco.

[0015] Specifically, it promotes the synthesis of tobacco chlorogenic acid while inhibiting the synthesis and accumulation of scopoletin; or it promotes the synthesis of scopoletin while inhibiting the accumulation of chlorogenic acid.

[0016] Furthermore, the overexpression vector resulted in an increase in the chlorogenic acid content and a decrease in the scopoletin content; the RNAi vector resulted in a decrease in the chlorogenic acid content and an increase in the scopoletin content.

[0017] The present invention also provides the use of the host cell in preparing transgenic plants, especially tobacco.

[0018] The present invention also provides a transformed plant containing the tobacco NtSHN3 gene.

[0019] This study, published in the journal Nature Communications, discovered for the first time that the transcription factor NtSHN3 can regulate the synthesis and accumulation of chlorogenic acid and scopoletin in tobacco. Using transgenic technology, genetic material was obtained in which overexpression of the NtSHN3 gene resulted in increased chlorogenic acid content and decreased scopoletin content. Using RNAi technology, genetic material was obtained in which attenuation of the NtSHN3 gene function reduced chlorogenic acid content and increased scopoletin content. This discovery provides a new approach for regulating beneficial metabolites in tobacco cultivation and for obtaining new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .Amino acid sequence alignment of tobacco NtSHN3 with homologous proteins in other species.

[0021] Figure 2 .Analysis of the subcellular localization and expression pattern of tobacco NtSHN3; (A) Intron / exon structure of the tobacco NtSHN3 gene; (B) Subcellular localization analysis of tobacco NtSHN3; NLS is a known nuclear-localized polypeptide, and the position of the NtSHN3-GFP fusion protein completely overlaps with NLS, indicating that the fusion protein is also localized in the cell nucleus; (C) Analysis of the expression levels of NtSHN3 in different tobacco tissues; (D) Analysis of the expression levels of NtSHN3 in tobacco leaves at different developmental stages.

[0022] Figure 3 .Construction of NtSHN3 gene overexpression vector and identification of genetically transformed positive plants: (A) Construction of tobacco NtSHN3 gene plant overexpression vector; (B) Amplification and identification of NtSHN3+GFP target fragment (1145bp) in genetically transformed positive plants; (C) Analysis of NtSHN3 gene expression characteristics in genetically transformed positive plants.

[0023] Figure 4.NtSHN3 regulates the synthesis of polyphenols in tobacco: (AC) The scopoletin content in flowers, leaves and capsules of three independent NtSHN3 overexpression lines was significantly reduced; (D) The chlorogenic acid content in three independent NtSHN3 overexpression lines was significantly increased.

[0024] Figure 5 .NtSHN3 regulates the synthesis of polyphenols in tobacco: (A) The scopoletin content in the leaves of three independent NtSHN3 gene RNAi lines was significantly increased; (B) The chlorogenic acid content in the leaves of three independent NtSHN3 gene RNAi lines was significantly decreased.

[0025] Figure 6 .NtSHN3 regulates the expression of multiple scopoletin synthesis-related genes.

[0026] Figure 7 .Post-translational activation of NtSHN3 significantly changes the expression levels of multiple phenylpropanoid metabolism-related genes: DEX (Dexamethasone) treatment activates NtSHN3-GR fusion protein, and CYC (Cycloheximide) represents a protein synthesis inhibitor.

[0027] Figure 8 .NtSHN3 binds to the promoter fragments of NtF6'H1 and NtTOGT1 genes: (A) NtF6'H1 gene promoter and ChIP-qPCR amplification location; (B) NtTOGT1 gene promoter and ChIP-qPCR amplification location; (C) ChIP-qPCR detection of the enrichment level of the NtF6'H1 gene promoter fragment; (D) ChIP-qPCR detection of the enrichment level of the NtTOGT1 gene promoter fragment. DETAILED DESCRIPTION

[0028] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0029] Example 1: Cloning, sequence analysis and expression vector construction of tobacco NtSHN3 gene

[0030] 1. Cloning of the tobacco NtSHN3 gene

[0031] After the tobacco seedlings were harvested, they were immediately frozen with liquid nitrogen. After being fully ground with liquid nitrogen, they were extracted using the Tobacco RNA Rapid Extraction Kit (Gene Answer, RT0110). The total RNA of tobacco leaves was extracted strictly according to the instructions of the kit. During the extraction process, DNase I (Omega, #E1091-01) was added to digest and remove genomic DNA interference. The integrity of the extracted RNA sample was first tested on an agarose gel, and then the concentration of the sample was determined using Nanodrop. Samples of qualified quality were selected as templates for the synthesis of the first chain of cDNA, and PrimeScript was used in the reverse transcription process. TM II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) was used to obtain cDNA samples, which were then used for gene cloning or gene expression analysis after concentration determination. Tobacco genomic DNA was extracted using a new plant genomic DNA rapid extraction kit (Imagene, DE117) in strict accordance with the kit instructions.

[0032] To clone the tobacco (NtSHN3) gene, we first searched the gene and protein sequences of Arabidopsis thaliana AtSHN3 (AT5G25390) in the Chinese Tobacco Genome Database V4.0. The tobacco gene with the highest homology to the Arabidopsis gene was Ntab0611950. Based on the gene sequences in the database, we designed specific primers: NtSHN3-F: ATGGTACAATCAAAGAAGTTC and NtSHN3-R: TCACTTAGAGTTGGAAAAAGAG. PCR amplification was then performed using tobacco genomic DNA and cDNA as templates, respectively. After agarose gel electrophoresis, the target fragments were recovered using the AxyPrep DNA Gel Extraction Kit (Axygen, AP-GX-250G). The ligation system was prepared as follows: 0.1–0.3 pmol of DNA fragment, 1 μl of pMDTM18-T Vector (Takara), 5 μl of Solution I, and up to 10 μl of dd-H2O. Incubate the ligation reaction at 16°C overnight. Add the ligation product to competent E. coli cells for heat shock transformation. Select positive clones for sequencing. Use the positive clones that have been sequenced correctly to extract the plasmid and store at -20°C for subsequent vector construction.

[0033] 2. Sequence Analysis of the Tobacco NtSHN3 Gene

[0034] The full-length CDS sequence of the NtSHN3 gene was sequenced and aligned using DNAMAN software to determine its exon and intron structure. The CDS sequence was then translated into amino acid sequences and compared with homologous proteins in Arabidopsis and rice for phylogenetic analysis. To construct the phylogenetic tree, the amino acid sequences of NtSHN3 were first aligned with those of all Arabidopsis ERF transcription factors using Clustal X, followed by phylogenetic analysis using the Neighbor Joining algorithm in Mega 5 software.

[0035] 3. Construction of tobacco NtSHN3 gene overexpression and RNAi vectors

[0036] Based on the sequence information obtained through sequencing, specific primers containing restriction enzyme cleavage sites were designed to amplify the full-length CDS sequence of the NtSHN3 gene. The primer pair used for overexpression vector construction was: SHN3-OE-F: CGCCTCGAGATGGTACAATCAAAGAAGTTC and SHN3-OE-R: CGGACTAGTCTTAGAGTTGGAAAAAGAGGG. The amplified product was double-digested with enzymes and ligated into the pCAMBIA1301 vector (HonorGene) to generate the recombinant 35S:NtSHN3-GFP plasmid. This plasmid was then transformed into competent Escherichia coli cells. After sequencing and verification, positive clones were selected and the plasmid was extracted. The plasmid was then transformed into competent Agrobacterium tumefaciens cells for tobacco transgenesis. To construct the RNAi vector, specific primers were used to amplify the approximately 206-bp coding region of the NtSHN3 gene, which covers the functional domains of the NtSHN3 protein. The primer sequences used for amplification were NtSHN3-RNAi-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGAGACAGCAGAGGAAGCA and NtSHN3-RNAi-R: GGGGACCACTTTGTACAAGAAAGCTGGGTGGAAGGTGCTGGATCTTTGC. The amplified fragment was ligated into the pHELLSGATE vector (BioVector NTCC) to generate the recombinant RNAi:NtSHN3 plasmid. After sequencing verification, the plasmid was extracted and transformed into Agrobacterium for tobacco transformation.

[0037] Example 2: Analysis of subcellular localization and expression pattern of tobacco NtSHN3

[0038] The recombinant 35S:NtSHN3-GFP plasmid was transformed into Agrobacterium tumefaciens GV3101, cultured overnight in LB medium at 28°C, and then collected by centrifugation. Young leaves of Nicotiana benthamiana were infected with the bacterial solution containing the 35S:NtSHN3-GFP plasmid to obtain plants with transient expression of the fusion protein. Fluorescence signals were then collected using a confocal laser scanning microscope (ZEISS LSM 700, Germany). Using the known nuclear localization protein NLS as a reference, if the green fluorescence signal of NtSHN3-GFP overlaps with the red signal of NLS ( Figure 2 B), indicating that the fusion protein is also localized in the nucleus.

[0039] Tobacco root, stem, leaf, axillary bud, bud, flower, capsule and seed tissue samples were collected at the flowering stage and immediately frozen in liquid nitrogen. Total RNA from each tissue was extracted using the Tobacco RNA Rapid Extraction Kit (Gene Answer, RT0110) and PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) was used to obtain cDNA samples from various tissues. The concentration of the cDNA samples was determined by Nanodrop and then used for quantitative PCR to detect the expression level of the NtSHN3 gene. Figure 2 As shown in Figure C, the expression level of tobacco NtSHN3 gene is high in leaves, pistils and seeds, and relatively low in other tissues. The primers used for quantitative PCR are: internal reference primers NtL25-F: CAAAAGTTACATTCCACCG and NtL25-R: TTTCTTCGTCCCATCAGGC. qSHN3-F: AGGAGCCTCATCAAAGCCAT and qSHN3-R:

[0040] GTAGCTCGTCAACCATCTGC. Further analysis of the expression levels of the NtSHN3 gene in leaves at different developmental stages revealed that the expression level of the NtSHN3 gene was high in the early stages of senescence and rapidly decreased in the late stages of senescence ( Figure 2 D).

[0041] Example 3: Tobacco genetic modification experiment

[0042] Tobacco was transformed using the leaf disc method. First, the vigorously growing tobacco leaves were sterilized and cut into 1 cm 2Small pieces of the plant were placed in MS differentiation medium. After pre-incubation for two days at 28°C, 2000 Lx light intensity, and 16 hours of light per day, the plants were inoculated with Agrobacterium containing the 35S:NtSHN3-GFP and RNAi:NtSHN3 plasmids, respectively, for 10-15 minutes. The cultures were shaken several times and then the excess culture was blotted off with sterile filter paper. The plants were then inoculated into MS differentiation medium and co-cultured at 28°C in the dark for 3-5 days. The co-cultured plants were washed three times with sterile water, blotted dry with sterile filter paper, and transferred to MS differentiation medium containing hygromycin and carbenicillin and incubated at a constant temperature. The medium was changed every 10 days. When the adventitious buds reached 1-2 cm in length, the clusters were cut into individual buds and transferred to MS rooting medium containing hygromycin, carbenicillin, and activated charcoal to promote rooting. After the root system is well developed, the tissue culture seedlings are taken out, the culture medium at the roots is washed with clean water, a small number of lower leaves are cut off, and the seedlings are transferred to a pot filled with loose sterile soil and cultured according to conventional management. After obtaining the transgenic positive lines, the leaf DNA samples of each line were first extracted and PCR identification was performed using the gene + GFP specific primer pair. A total of 12 positive seedlings were obtained ( Figure 3 B). RNA was then extracted from 8 seedlings for gene expression analysis. The results showed that the expression level of the NtSHN3 gene in 5 independent lines was significantly higher than that in the wild-type control plants ( Figure 3 C), and the OE#8 strain with the highest expression level was selected for subsequent transcriptome analysis. Example 4: Determination of tobacco polyphenol content

[0043] The content of polyphenols was determined by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry. The specific operation was as follows: 50 mg of tobacco leaf sample was transferred into 1.5 mL of ethanol-water extract (internal standard: umbelliferone 75 ng / mL), ultrasonicated at room temperature for 1 hour, and then centrifuged at 14,000 rpm to obtain the supernatant for detection. The analytical conditions used were as follows: chromatographic conditions: BEH Phenyl column (2.1×150 mm, 1.7 μm), mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B); elution gradient: phase B increased from 5% to 15% within 0-2 min, phase B maintained at 15% from 2-10 min, and phase B increased to 100% from 10.01-15 min; flow rate: 0.3 mL / min, column temperature: 35°C, injection volume: 1 μl; mass spectrometry conditions: electrospray ionization source ionization, capillary voltage: 4 kV in positive ion ionization mode, nebulizer gas pressure: 40 psi, drying gas flow: 12 L / min, drying gas temperature: 290°C, sheath gas flow: 11 L / min, sheath gas temperature: 200°C, and real-time multiple reaction monitoring (dMRM) mode scanning was used. The results are as follows: Figure 4As shown in Figure 2, in three independent NtSHN3 overexpression lines, the content of scopoletin in tobacco flowers, leaves and capsules was significantly lower than that in wild-type control plants. In contrast, the content of chlorogenic acid in flowers, leaves and capsules of three independent NtSHN3 overexpression lines was significantly higher than that in control plants ( Figure 4 The results of content determination showed that NtSHN3 could promote the synthesis of chlorogenic acid in tobacco leaves and inhibit the accumulation of scopoletin.

[0044] In three independent NtSHN3 gene-encoded RNAi lines, the scopoletin content in leaves was significantly higher than that in wild-type control plants, while the chlorogenic acid content in leaves was significantly lower than that in control plants ( Figure 5 The results also showed that NtSHN3 could promote the synthesis of chlorogenic acid in tobacco leaves and inhibit the accumulation of scopoletin.

[0045] Example 5: Transcriptome Sequencing and Data Analysis

[0046] Total RNA was extracted from tobacco leaf tissue using Trizol reagent (Invitrogen). After quality assessment, sequencing libraries were constructed using the Illumina TruSeq RNA Sample PreKit. Sequencing was performed using the Illumina HiSeq 2500 sequencing platform, and the quality of mRNA sequence reads was assessed using the FastQC method (http: / / www.bioinformatics.bbsrc.ac.uk / projects / FastQC / ). Low-quality (<20 bases) and adapter sequences at the 5' and 3' ends were removed using Trimmomatic (v0.30). The reference genome of cultivated tobacco (Nicotiana tabacum) was obtained from ftp: / / ftp.solgenomics.net / genomes / Nicotiana_tabacum / , and reads were mapped to this genome using HISAT2 (v2.1.0). Cufflinks (v2.2.1) was used to calculate gene expression levels, and Cuffdiff software was used to identify differentially expressed genes (DEGs) with a log2 ratio ≥1.5 or ≤-1.5 (FDR < 0.05). The results showed that NtHCT, NtF3'H, NtGT3, and NtANS genes were significantly upregulated in the NtSHN3 overexpression line, while NtF6'H1 and NtTOGT1 genes were significantly downregulated in the NtSHN3 overexpression line (Table 1 and Figure 6 ).

[0047] Table 1. Expression levels of phenylpropanoid metabolism-related genes in NtSHN3 overexpressing plants detected by RNA-seq

[0048]

[0049] Example 6: Post-transcriptional activation of NtSHN3

[0050] 35S:NtSHN3-GR positive seedlings were treated with DMSO (control), DEX (10 μM), CYC (5 μM), and DEX+CYC for 1 hour and 4 hours, respectively. The seedlings were collected and quickly frozen in liquid nitrogen for RNA extraction. Subsequently, reverse transcription of cDNA was performed for gene expression level detection. Figure 7 As shown, activation of NtSHN3-GR protein by DEX treatment significantly altered the expression levels of the differentially expressed genes mentioned above, further demonstrating that NtSHN3 regulates the transcription of these genes. In the presence of the protein synthesis inhibitor CYC, DEX administration still significantly altered the expression levels of NtTOGT1 and NtF6'H1, indicating that activation of NtSHN3 can still regulate the expression of these two genes when protein synthesis is inhibited. In other words, NtSHN3's regulation of the transcription levels of these two genes does not require the synthesis of new proteins and may be a direct regulatory effect. However, in the presence of CYC, DEX administration had no significant effect on the expression levels of NtHCT, NtF3H, NtGT3, and NtANS, suggesting that NtSHN3's regulation of the expression levels of these genes may be indirect and require the involvement of other protein factors.

[0051] Example 7: Chromatin Immunoprecipitation (ChIP)

[0052] Chromatin immunoprecipitation (ChIP) experiments were performed using the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (Epigentek, New York, USA). Tobacco seedlings were fixed with 1.0% formaldehyde for 10 minutes under vacuum infiltration. Glycine solution (final concentration 0.125 M) was then added for cross-linking under vacuum infiltration for an additional 5 minutes. Tissues were ground into a fine powder in liquid nitrogen and lysed using the lysis buffer provided by the kit. Nuclei were pelleted by centrifugation, resuspended, and sheared by sonication. Sheared DNA should be between 200 and 1000 bp in length. The pelleted nuclear solution was then added to a strip well containing anti-GFP (Abcam, ab290) to pull down the NtSHN3-GFP fusion protein bound to its putative target DNA fragment. The collected DNA fragments were then reverse-transcribed and purified for subsequent qRT-PCR analysis. Enrichment of the TUB (tubulin β chain, XP_016456097.1) genomic fragment served as a negative control. The results are as follows Figure 8As shown, among the DNA fragments enriched by ChIP, the promoter fragments of NtF6'H1 and NtTOGT1 genes were significantly enriched, further confirming that NtSHN3 directly binds to the promoter regions of these two genes.

[0053] In summary, the present invention discovered that the tobacco NtSHN3 transcription factor can regulate the expression of the NtF6'H1 and NtTOGT1 genes by directly binding to their promoter fragments. The enzyme encoded by NtF6'H1 catalyzes the key reaction in scopoletin synthesis. NtSHN3 significantly inhibits the synthesis and accumulation of scopoletin in tobacco by directly inhibiting the expression of the NtF6'H1 gene. Therefore, the present invention confirms that NtSHN3 can regulate the content of scopoletin in tobacco.

[0054] SEQ ID NO.1

[0055]

[0056]

[0057] SEQ ID NO.2

[0058] MVQSKKFKGVRQRQWGSWVSEIRHPLLKKRIWLGTFETAEEAARAYDEAAILMNGQVAKTNFPIV

[0059] KENHVNNSNDKKFPLSSSSTLSTVLNAKLRKCCKDPAPSMTCLRLDNDNCHIGVWQKRSGKNSGS

[0060] NWITKIELGKKEEPHQSHESMNSWSLSSSSGSTSSSSSELGISKPLDEENRAAMQMVDELLYWN

[0061] SPFSSAPISDLSPSFSNSK.

Claims

1. Tobacco NtSHN3 gene, the sequence of which is shown in SEQ ID NO.

1.

2. Tobacco NtSHN3 protein, the sequence of which is shown in SEQ ID NO.

2.

3. An overexpression vector containing the tobacco NtSHN3 gene according to claim 1.

4. An RNAi vector containing the tobacco NtSHN3 gene according to claim 1. A host cell containing the vector according to claim 3 or 4.

6. Use of the vector according to claim 3 or 4, or the host cell according to claim 5, in regulating scopoletin synthesis in plants, in particular tobacco.

7. The use according to claim 6, characterized in that Specifically, it promotes the synthesis of tobacco chlorogenic acid while inhibiting the synthesis and accumulation of scopoletin; or it promotes the synthesis of scopoletin while inhibiting the accumulation of chlorogenic acid.

8. The use according to claim 7, characterized in that The overexpression vector of claim 3 results in increased chlorogenic acid content and decreased scopoletin content.

9. The use according to claim 7, characterized in that The RNAi vector of claim 4 results in a decrease in chlorogenic acid content and an increase in scopoletin content.

10. Use of the vector according to claim 3 or 4, or the host cell according to claim 5, in preparing a transgenic plant, in particular tobacco.

Images