Tobacco NtWIN1 gene, encoding protein and application of tobacco NtWIN1 gene

Abstract

The invention discloses a tobacco NtWIN1 gene, an encoding protein and application of the tobacco NtWIN1 gene. It is found for the first time that the transcription factor NtWIN1 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 NtWIN1 gene is obtained by utilizing a transgenic technology. A genetic material of which the function of the NtWIN1 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 application belongs to the field of genetic engineering, and particularly relates to a tobacco NtWIN1 gene, a coding protein and application thereof in regulating synthesis of scopoletin. BACKGROUND

[0002] When plants encounter stresses such as drought, salt, cold, wound, virus / bacteria or fungal infection, they will synthesize a variety of low molecular weight compounds, which are called phytoalexins. Phytoalexins can have various structural types, including sesquiterpenes, isoflavones, stilbenes, polyacetylenes and phenolic coumarins. Scopoletin is a phenolic coumarin, which is an important member of the plant phytoalexin group isolated from many plants. Scopoletin plays a key role in traditional medicine in Africa, Asia and Europe, and the compound and the plants containing it are involved in the treatment of many symptoms and diseases, such as convulsions, inflammation, rheumatic pain and leprosy. In addition, scopoletin is also involved in cardiovascular and neuromuscular effects and anticonvulsant processes. Scopoletin can enhance the activation of eosinophils, which are one of the key cells in the pathogenesis of asthma. Scopoletin can also act as an effective antihypertensive drug and a non-specific antispasmodic (neuromuscular blocking) agent. At the same time, scopoletin also has various medicinal activities such as protecting liver, antioxidant, anti-inflammatory, anticancer and the like. In tobacco leaves, scopoletin is one of the main polyphenols, which has an important influence on the color of tobacco leaves, cigarette aroma and smoke, and is one of the important indicators for measuring the quality of tobacco. Therefore, the research on the synthesis regulation of tobacco scopoletin has important medicinal health and application value.

[0003] Scopoletin is derived from phenylpropanoid metabolic pathway, and its precursor is phenylalanine. Phenylalanine is formed by the combination of phosphoenolpyruvate (PEP) from glycolytic pathway (EMP) and erythrose-4-phosphate (E4P) from pentose phosphate pathway (PPP), and then converted by intermediate shikimic acid. 4-coumaroyl-CoA (p-Coumaroyl-CoA) is formed by two-step cascade catalysis of phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumaroyl CoA ligase (4CL). Then, p-coumaroylshikimic acid and caffeoylshikimic acid are formed by shikimate O-hydroxycinnamoyltransferase (HCT) and 4-coumarate 3-hydroxylase (C3H), respectively. Caffeoyl-CoA is formed by HCT catalysis of caffeoylshikimic acid, and further catalyzed by caffeoyl CoA O-methyltransferase (CCoAOMT) to form Feruloyl-CoA, which is an important precursor of Scopoletin synthesis. 6’-HydroxyFeruloyl-CoA is formed by feruloyl-CoA 6’-hydroxylase 1 (F6’H1), and then Scopoletin is formed by isomerization and lactonization. Scopoletin is further glycosylated by UDP-Glc: glucosyltransferases (UGTs) to form Scopolin (Hino et al., 1982). So far, various transcription factors have been confirmed to be involved in the regulation of plant phenylpropanoid metabolism, including MYB, bHLH, WD40, WRKY, bZIP, zinc finger and MADS box proteins, etc. For example, MYB4 can affect the synthesis of lignin, flavonoids, chlorogenic acid and other phenylpropanoid compounds by regulating the expression of PAL, C4H and other genes. The structural genes in the biosynthetic pathway of Scopoletin in plants are relatively clear, but there are few reports on the transcription factor genes involved in the transcriptional regulation of Scopoletin biosynthesis.

[0004] Transcription factor WAX INDUCER1 (WIN1) belongs to AP2 / ERF class (APETALA2 / ethylene response factor) protein, and previous studies have found that WIN1 can directly bind to the promoter region of LACS2 (long-chain acyl-CoA synthetase) gene, promote the transcription of the latter, and then promote the synthesis of cutin in Arabidopsis, and the synthesis and accumulation of cutin can enhance the resistance of plants to various adverse environments. However, as of now, there is no report on the function of transcription factor WIN1 in the synthesis process of plant scopoletin.

[0005] Chlorogenic acid (CGA), also known as coffee tannin, has a wide range of pharmacological effects such as anti-inflammatory, antibacterial, antiviral, and antidepressant. Polyphenols such as chlorogenic acid in tobacco leaves play a decisive role in the color of modulated tobacco leaves, and under reasonable modulation and aging conditions, polyphenols not only have a pleasant aroma, but also generate a series of substances through dry distillation, oxidation and cracking during smoking, which can impart elegant aroma to tobacco and increase the aroma amount of tobacco products. The synthesis of chlorogenic acid in plants has certain tissue specificity and is affected by many factors, and its biosynthesis also occurs through the phenylpropanoid metabolic pathway, but the current research on the regulation of chlorogenic acid synthesis is not very clear.

[0006] The present application proves that tobacco NtWIN1 transcription factor can regulate the synthesis of scopoletin and chlorogenic acid in tobacco leaves through a series of experiments, and therefore the patent protection is applied for. SUMMARY

[0007] The purpose of the present application is to provide tobacco NtWIN1 gene, coding protein and its application in regulating polyphenol synthesis.

[0008] The transcription factor NtWIN1 discovered by the present application, which effectively regulates the synthesis of scopoletin in tobacco, belongs to the field of plant genetic engineering, and the gene is derived from cultivated tobacco. The NtWIN1 gene sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2.

[0009] The full length of tobacco NtWIN1 gene is 2,048 bp, containing 3 exons (shown as the gray part in SEQ ID NO. 1) and 2 introns. The full length of the coding region is 618 bp, and it encodes 205 amino acids (SEQ ID NO. 2). Evolutionary analysis finds that the gene has a close evolutionary relationship with Arabidopsis AtWIN1 (At1g15360) (C), so it is named NtWIN1. Subcellular localization analysis finds that the NtWIN1-GFP fusion protein is located in the nucleus (C), so it is named NtWIN1. Figure 1 Figure 2 ​A), NtWIN1 has the transcription factor characteristic attribute.

[0010] The present application not only provides tobacco NtWIN1 gene nucleotide and amino acid sequence.

[0011] The present application provides overexpression vector containing the tobacco NtWIN1 gene; and RNAi vector containing the tobacco NtWIN1 gene.

[0012] The present application provides host cell containing the vector.

[0013] The present application provides the application of the vector or the host cell in regulating plant anisatin synthesis, especially tobacco.

[0014] Specifically, promoting the synthesis of chlorogenic acid, while inhibiting the synthesis and accumulation of anisatin; or promoting the synthesis of anisatin, while inhibiting the accumulation of chlorogenic acid.

[0015] Further, the overexpression vector leads to the increase of chlorogenic acid content and the decrease of anisatin content; the RNAi vector leads to the decrease of chlorogenic acid content and the increase of anisatin content.

[0016] The present application also provides the application of the host cell in preparing transgenic plants, especially tobacco.

[0017] The present application also provides transformed plants containing tobacco NtWIN1 gene.

[0018] The present application first discovers that transcription factor NtWIN1 can regulate the synthesis and accumulation of tobacco chlorogenic acid and anisatin. Overexpression of NtWIN1 gene by transgenic technology leads to genetic material with high chlorogenic acid content and low anisatin content. RNAi technology is used to weaken the function of NtWIN1 gene, which leads to genetic material with low chlorogenic acid content and high anisatin content. This provides a new way for tobacco planting and obtaining new varieties. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Cloning and sequence analysis of tobacco NtWIN1 gene; (A) Cloning of NtWIN1 gene and CDS full-length sequence; (B) Intron / exon structure of NtWIN1 gene; (C) Evolutionary analysis of NtWIN1 and Arabidopsis ERF class transcription factors; (D) Amino acid sequence alignment of NtWIN1 and homologous proteins in other species.

[0020] Figure 2.Analysis of the subcellular localization and expression pattern of tobacco NtWIN1; (A) Analysis of the subcellular localization of tobacco NtWIN1; NLS is a known nuclear-localized polypeptide, and the position of the NtWIN1-GFP fusion protein completely overlaps with NLS, indicating that the fusion protein is also localized in the cell nucleus; (B) Analysis of the expression levels of NtWIN1 in different tobacco tissues; (C) Analysis of the expression levels of NtWIN1 in tobacco leaves at different developmental stages.

[0021] Figure 3 .Identification of plants overexpressing the NtWIN1 gene: (A) Amplification and identification of the NtWIN1+GFP target fragment (1260bp) in genetically transformed positive plants; (B) Analysis of the expression characteristics of the NtWIN1 gene in genetically transformed positive plants.

[0022] Figure 4 .NtWIN1 regulates the synthesis of polyphenols in tobacco; (AC) The scopoletin content in flowers, leaves, and capsules of three independent NtWIN1 overexpression lines was significantly reduced; (D) The chlorogenic acid content in three independent NtWIN1 overexpression lines was significantly increased.

[0023] Figure 5 .NtWIN1 regulates the expression of multiple scopoletin synthesis-related genes.

[0024] Figure 6 .Post-translational activation of NtWIN1 significantly changes the expression levels of multiple phenylpropanoid metabolism-related genes; DEX (Dexamethasone) treatment will activate NtWIN1-GR fusion protein, and CYC (Cycloheximide) represents a protein synthesis inhibitor.

[0025] Figure 7 .NtWIN1 binds to the promoter fragments of the NtF6'H1 and NtCCoAMT genes; (A) NtF6'H1 and NtCCoAMT gene promoters and ChIP-qPCR amplification locations; (B) ChIP-qPCR detection of the enrichment level of each gene promoter fragment; (C) Analysis of NtWIN1's transcriptional regulation of the NtF6'H1 and NtCCoAMT genes; (D-E) Detection of LUC signal intensity in transcriptional activation experiments.

[0026] Figure 8 .NtWIN1 regulates scopoletin synthesis in tobacco through NtF6'H1; (A)-(C) Identification of NtWIN1, NtF6'H1, and NtWIN1 / NtF6'H1 RNAi plants (representing inhibition of both genes); (D) Scopoletin content in different plant materials. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1: Cloning, sequence analysis and expression vector construction of tobacco NtWIN1 gene

[0029] 1. Cloning of the Tobacco NtWIN1 Gene

[0030] 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.

[0031] To clone the tobacco NtWIN1 gene, we first searched the gene and protein sequences of Arabidopsis thaliana AtWIN1 (AT1G15360) in the Chinese Tobacco Genome Database V4.0. We found that the tobacco gene with the highest homology to the Arabidopsis gene was Ntab0284400. Based on the gene sequences in the database, we designed specific primers: NtWIN1-F: ATGGTACATTCAAAGAAGTTC and NtWIN1-R: TTAAATATCAAATGAAGGGCAAG. PCR amplification was then performed using tobacco genomic DNA and cDNA as templates. After agarose gel electrophoresis ( Figure 1A), the target fragment was recovered by AxyPrep DNA gel recovery kit (Axygen, AP-GX-250G). The ligation system was prepared: DNA fragment 0.1-0.3 pmol, pMDT 18-T Vector (Takara) 1 μl, Solution I 5 μl, dd-H2O up to 10 μl. The ligation reaction was performed at 16°C overnight, and the ligation product was added to the competent cells of Escherichia coli, heat-shocked and transformed, and the positive clones were selected for sequencing. The plasmid was extracted from the positive clones with correct sequencing, and stored at -20°C for subsequent vector construction.

[0032] 2. Sequence analysis of tobacco NtWIN1 gene

[0033] According to the CDS sequence of the full-length NtWIN1 gene obtained by sequencing, the exon and intron structure of the gene were determined by alignment with DNAMAN software. Figure 1 B). Then the CDS sequence was translated into amino acid sequence, and the homologous proteins in Arabidopsis and rice were subjected to phylogenetic analysis. When constructing the phylogenetic tree, first, the amino acid sequences of NtWIN1 and all Arabidopsis ERF-type transcription factors were aligned by Clustal X, and then the phylogenetic analysis was performed by the Neighbor Joining algorithm of Mega 5 software. The analysis results showed that NtWIN1 was closely related to Arabidopsis AtWIN1 (At1g15360) Figure 1 C), and contained typical AP2 / ERF family domains and CMV-1 and CMV-2 functional domains of SHINE (SHN) subgroup Figure 1 D).

[0034] 3. Construction of tobacco NtWIN1 gene overexpression and RNAi vectors

[0035] 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 NtWIN1 gene. The primer pair used for overexpression vector construction was: WIN1-OE-F: ATACACCAAATTGACTCTAGAATGGTACATTCAAAGAAGTTC and WIN1-OE-R: GCCCTTGCTCACCATGGTACCTTAAATATCAAATGAAGGGCAAG. The amplified product was double-digested with enzymes and ligated into the pCAMBIA1301 vector (HonorGene) to form a recombinant 35S:NtWIN1-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 318-bp coding region of the NtWIN1 gene, which covers the functional domains of the NtWIN1 protein. The primer sequences used for amplification were WIN1-RNAi-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTAAGAAGGGTATGGCTTGGCA and WIN1-RNAi-R: GGGGACCACTTTGTACAAGAAAGCTGGGTTTCGAGTCAGAAGAAGGGCC. The amplified fragment was ligated into the pHELLSGATE vector (BioVector NTCC) to generate the recombinant RNAi:Nt WIN1 plasmid. After sequencing verification, the plasmid was extracted and transformed into Agrobacterium for tobacco transformation.

[0036] Example 2: Analysis of subcellular localization and expression pattern of tobacco NtWIN1

[0037] The recombinant 35S:NtWIN1-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:NtWIN1-GFP plasmid to obtain plants transiently expressing 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 NtWIN1-GFP overlaps with the red signal of NLS ( Figure 2 A), indicating that the fusion protein is also localized in the nucleus.

[0038] 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 TMII 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) to obtain cDNA samples of each tissue, and the concentration of cDNA samples was determined by Nanodrop before being used for qPCR quantitative detection of NtWINl gene expression level. The results are shown in Figure 2 B. The expression level of tobacco NtWINl gene in stem, sepals, pistil, leaf and seed is relatively high, and the expression level in other tissues is relatively low. Subsequently, the expression of NtWINl gene in tobacco leaves at different development stages was detected, and it was found that the expression level gradually decreased from seedling to full flowering stage, was significantly up-regulated in pre-senescence leaves, and then significantly decreased in mature and senescent leaves Figure 2 C). The primers used for quantitative PCR are: reference primers NtL25-F: CAAAAGTTACATTCCACCG and NtL25-R: TTTCTTCGTCCCATCAGGC. qWINl-F: AAGTTCAGAGGTGTCAGGCA and qWINl-R: AGTTGGTCTTTGCATTGCGG.

[0039] Example 3: Tobacco transgenic experiment

[0040] Leaf disc method was used to transform tobacco. First, the growing tobacco leaves were sterilized and cut into 1 cm 2 pieces, which were placed in MS differentiation medium; under the conditions of 28°C, light intensity of 2000Lx, and light time of 16h / d, pre-cultured for 2 days; then infected with Agrobacterium containing the target plasmid for 10-15 min, during which the bacterial solution was shaken several times, and then the excess bacterial solution was absorbed with sterile filter paper; transferred to MS differentiation medium, and cultured at 28°C in the dark for a total of 3-5 days. The plant bodies after co-culture were washed with sterile water for 3 times, and then dried with sterile paper, and transferred to MS differentiation medium containing hygromycin and carbenicillin for constant temperature culture. The medium was replaced every 10 days. When the adventitious shoots grew to 1-2 cm, the clumps were cut into single shoots and transferred to MS rooting medium containing hygromycin, carbenicillin and activated carbon to promote root development. After the root system developed well, the tissue culture seedlings were taken out, the root medium was washed with water, a small amount of lower leaves were cut off, and then transferred to a flowerpot containing loose sterile soil for normal management and culture.

[0041] After obtaining the positive transgenic lines, the leaf DNA samples of each seedling were first extracted, and PCR identification was performed using specific primers of NtWINl+GFP, and 12 positive seedlings were obtained Figure 3 A). Subsequently, the RNA of 8 seedlings was extracted for gene expression level analysis, and the results showed that the expression level of NtWINl gene in 5 independent lines was significantly higher than that of wild type control plants Figure 3B), from which the OE#5 line was selected for subsequent transcriptome analysis based on the expression level.

[0042] Example 4: Determination of tobacco polyphenol content

[0043] The polyphenol content was determined by ultra-high performance liquid-triple quadrupole tandem mass spectrometry. Specifically, 50 mg of tobacco leaf sample was transferred into 1.5 mL of ethanol-water extraction solution (internal standard: umbelliferone 75 ng / mL), and then ultrasonicated at room temperature for 1 h. The supernatant was obtained by centrifugation at 14000 rpm and then detected. The analysis conditions were as follows: chromatographic condition: BEH Phenyl column (2.1 x 150 mm, 1.7 μm), mobile phase: 0.1% formic acid water (A) and 0.1% formic acid methanol (B); elution gradient: B phase from 5% to 15% within 0-2 min, B phase remained 15% within 2-10 min, B phase from 100% within 10.01-15 min; flow rate: 0.3 mL / min, column temperature: 35°C, injection volume: 1 μl; mass spectrometry condition: electrospray ionization source ionization, capillary voltage: 4 kV in positive ionization mode, nebulization gas pressure: 40 psi, dry gas flow rate: 12 L / min, dry gas temperature: 290°C, sheath gas flow rate: 11 L / min, sheath gas temperature: 200°C, real-time multiple reaction monitoring (dMRM) mode scanning. The results are shown in Table 1. Figure 4 As shown in Figures 4A, 4B and 4C, the scopoletin content in the flowers, leaves and capsules of the three independent NtWIN1 overexpression lines was significantly lower than that of the wild type control plants. Conversely, the chlorogenic acid content in the flowers, leaves and capsules of the three independent NtWIN1 overexpression lines was significantly higher than that of the control plants Figure 4 D). The content determination results showed that NtWIN1 could promote the synthesis of chlorogenic acid in tobacco leaves, while inhibit the accumulation of scopoletin.

[0044] Example 5: Transcriptome sequencing and data analysis

[0045] 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 HiSeq2500 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). Gene expression levels were calculated using Cufflinks (v2.2.1), and differentially expressed genes (DEGs) with a log2 ratio ≥1.5 or ≤-1.5 (FDR < 0.05) were identified using Cuffdiff software. The results showed that Nt4CL, NtCCoAMT, NtGT, and NtCYP71A1 genes were significantly upregulated in the NtWIN1 overexpression line, while NtF6'H1 and NtTOGT1 genes were significantly downregulated in the NtWIN1 overexpression line (Table 1 and Figure 5 ).

[0046] Table 1. Expression levels of phenylpropanoid metabolism-related genes in NtWIN1 overexpressing plants detected by RNA-seq

[0047]

[0048] Example 6: Post-transcriptional activation of NtWIN1

[0049] DMSO ( Figure 6 After treatment of 35S:NtWIN1-GR positive seedlings with DEX (10 μM), CYC (5 μM), and DEX+CYC for 1 and 4 hours, 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 6The expression levels of the above differentially expressed genes were significantly changed after the activation of NtWIN1-GR protein by DEX treatment, which again proved that NtWIN1 regulated the transcription of these genes. The expression levels of NtCCoAMT and NtF6'H1 genes were still significantly changed by DEX treatment in the presence of CYC, which indicated that the activation of NtWIN1 could regulate the expression of these two genes in the absence of new protein synthesis. In other words, the regulation of NtWIN1 on the transcription levels of these two genes did not require the participation of new protein synthesis, which might be a direct regulation. However, the expression levels of Nt4CL, NtGT, NtCYP71A1 and NtTOGT1 genes were not significantly affected by DEX treatment in the presence of CYC, which indicated that the regulation of NtWIN1 on the expression levels of the above genes might be an indirect action, which required the participation of other protein factors.

[0050] Example 7: Chromatin immunoprecipitation (ChIP)

[0051] Chromatin immunoprecipitation (ChIP) experiment was performed by EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (Epigentek, New York, USA). The tobacco seedlings were fixed with 1.0% formaldehyde for 10 min under vacuum infiltration, and then cooled for 5 min under vacuum infiltration by adding glycine solution (final concentration 0.125 m). The tissues were ground into fine powder in liquid nitrogen, and tissue lysis was performed with the lysis buffer provided by the kit. The pellet nuclei were centrifuged and then resuspended, and DNA shearing was performed by ultrasonic wave. The length of sheared DNA should be between 200-1000 bp. Then the pellet nuclei solution was added to the strip well with anti-GFP (Abeam, ab290) to pull down the NtWIN1-GFP fusion protein bound to its putative target DNA fragments. Then the collected DNA fragments were reversed and purified for subsequent qRT-PCR analysis. The enrichment of TUB (tubulin beta chain, XP_016456097.1) genomic fragment was used as a negative control. The results are shown in Figures 7A and 7B. Figure 7 As shown in Figures 7A and 7B, the promoter fragments of NtCCoAMT and NtF6'H1 genes were significantly enriched in the ChIP-enriched DNA fragments, which further confirmed that NtWIN1 directly binds to the promoter segments of these two genes.

[0052] Example 8: Dual-luc transcription activation

[0053] The promoter fragments of the NtCCoAMT and NtF6'H1 genes were cloned into pGreenII 0800-LUC to construct a fusion reporter vector. The full-length CDS of the NtWIN1 gene was connected to the pGreenII 62-SK vector to form an effector vector. The reporter vector and effector vector were introduced into the Agrobacterium strain GV3101 (pSoup-p19), and the Agrobacterium was suspended in 10mL of infiltration medium (10mM MgCl2, 0.5μM acetosyringone, 10mmol / L 2-N-morpholineethanolsulfonic acid / MES) to OD600≈0.2 and incubated at room temperature for 2 hours. The Agrobacterium mixture containing the reporter vector and the effector vector was co-injected into the back of Nicotiana benthamiana leaves. After 3 days, the tobacco leaves were treated with 1 M D-luciferin (Sigma, L9504) and kept in the dark for 5 minutes to extinguish the fluorescence. Images of luciferase (LUC) activity were captured using a CCD imaging device. The results are shown in Figure 2. Figure 7 As shown in C, 7D and 7E, NtWIN1 can significantly promote the transcription of LUC gene driven by NtCCoAMT gene promoter, and significantly inhibit the transcription of LUC gene driven by NtF6'H1 gene promoter.

[0054] Example 9: Identification and detection of RNAi plants

[0055] After transformation, the transgenic materials were identified by quantitative PCR, and three independent NtWIN1-RNAi lines were obtained. The expression levels of NtWIN1 gene in the plants were reduced by 55% to 80% ( Figure 8 A). Two independent NtF6'H1-RNAi lines and one NtWIN1-RNAi / NtF6'H1-RNAi line were obtained ( Figure 8 BC). The results of scopoletin content assay showed that the scopoletin content in NtWIN1-RNAi was significantly higher than that in the wild-type control, but the scopoletin content in NtWIN1-RNAi / NtF6'H1-RNAi was not significantly different from that in NtF6'H1-RNAi, and both were significantly lower than that in the control plants ( Figure 8 D) This indicates that NtWIN1 regulates scopoletin synthesis in tobacco through NtF6'H1.

[0056] In summary, the present application discovers that tobacco NtWINl transcription factor can regulate the expression of NtCCoAMT and NtF6'Hl genes by directly binding to the promoter fragments of the two genes. NtF6'Hl encodes an enzyme that catalyzes the key reaction of scopoletin synthesis, and NtWINl significantly inhibits the synthesis and accumulation of scopoletin in tobacco by directly inhibiting the expression of NtF6'Hl gene. Therefore, the present application confirms that NtWINl can regulate the content of scopoletin in tobacco.

[0057] SEQ ID NO. 1

[0058]

[0059]

[0060] SEQ ID NO. 2

[0061] MVHSKKFRGVRQRHWGSWVSEIRHPLLKRRVWLGTFDTAEEAARAYDQAAILMSGRNAKTNFPIT

[0062] QDSDNKELKAKDQEFSSIPSPKALSEILYAKLRKCSKVPSPSLTCLRLDIENSHIGVWQKRAGPS

[0063] SDSKWVMTVELQKKNNVNAAIEGELNSSSTSSSRNCGEIVEGISIRSEMDEEERVALQMIEELLQINSPCPSFDI.

Claims

1. A tobacco NtWIN1 gene, the sequence of which is shown as SEQ ID NO.

1.

2. A tobacco NtWIN1 protein, the sequence of which is shown as SEQ ID NO.

2.

3. An overexpression vector containing the tobacco NtWIN1 gene of claim 1.

4. An RNAi vector containing the tobacco NtWIN1 gene of claim 1.

5. A host cell containing the vector of claim 3 or 4.

6. Use of the vector of claim 3 or 4, or the host cell of claim 5 in modulating the synthesis of scopoletin in a plant, particularly tobacco.

7. Use according to claim 6, characterized in that, Specifically, to promote the synthesis of chlorogenic acid while inhibiting the synthesis and accumulation of scopoletin; or to promote the synthesis of scopoletin while inhibiting the accumulation of chlorogenic acid.

8. Use according to claim 7, characterized in that, The overexpression vector of claim 3 results in an increase in chlorogenic acid content and a decrease in scopoletin content.

9. 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 of claim 3 or 4, or the host cell of claim 5 in preparing a transgenic plant, particularly tobacco.

Images