Nornicotine negative regulation nornicotine gene, cloning and application thereof, tobacco and tobacco product

Abstract

The invention discloses a nornicotine negative regulation and control gene and cloning and application thereof, tobacco and a tobacco product, and belongs to the technical field of genetic engineering, the nornicotine negative regulation and control gene is a tobacco NtWRKY51 gene, and can negatively regulate and control nornicotine in tobacco. The cloning method of the nornicotine negative regulation nornicotine gene comprises the following steps: designing a specific primer, amplifying full-length cDNA (complementary deoxyribonucleic acid) and a genome sequence, and realizing PCR (polymerase chain reaction) amplification; and purifying the PCR product. The overexpression vector of the nornicotine negative regulation nornicotine gene is pCHF3. The application of the nornicotine negative regulation and control gene is realized by negatively regulating and controlling accumulation of nornicotine in tobacco leaves by inhibiting expression of CYP82E4 through the nornicotine negative regulation and control gene. The tobacco plant is formed by breeding transgenic tobacco prepared by applying the nornicotine negative regulation nornicotine gene. Through overexpression of the NtWRKY51, the nornicotine content of the plant can be reduced by 40% to the maximum, and the NtWRKY51 has a great application prospect in the aspect of tobacco with low nornicotine content.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a nicotine down-regulation gene, its cloning and application, tobacco and tobacco products. Background Art

[0002] Nicotine is a harmful alkaloid in tobacco. In addition to affecting the aroma and taste of tobacco, it is also a precursor of the potential carcinogen N-nitrosonornicotine (NNN). In some tobacco varieties, the nicotine content accounts for more than 1% of the dry weight. The relatively high nicotine content seriously affects the quality and safety of tobacco. Therefore, it is necessary to reduce the synthesis of nicotine from the source and conduct research on the regulation of nicotine synthesis. Summary of the Invention

[0003] The first object of the present invention is to provide a nicotine down-regulation gene, the second object is to provide a cloning method for the nicotine down-regulation gene; the third object is to provide an overexpression vector for the nicotine down-regulation gene, the fourth object is to provide the application of the nicotine down-regulation gene, the fifth object is to provide a tobacco plant obtained by the application of the nicotine down-regulation gene; the sixth object is to provide a tobacco product obtained by the application of the nicotine down-regulation gene.

[0004] The technical solution adopted by the present invention is as follows.

[0005] The first object of the present invention is achieved as follows. A nicotine down-regulation gene, which is the tobacco NtWRKY51 gene, and it can negatively regulate nicotine in tobacco.

[0006] As a preferred technical solution, the NtWRKY51 is extracted from tobacco plants.

[0007] As a preferred technical solution, the NtWRKY51 is extracted from high-nicotine germplasm tobacco. 。

[0008] The second object of the present invention is achieved as follows. A cloning method for any one of the above nicotine down-regulation genes, comprising the following steps:

[0009] Step 1: Extract total RNA from tobacco plants and reverse transcribe to obtain cDNA;

[0010] Step 2: Design specific primers according to the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database, amplify the full-length cDNA and genomic sequence to achieve PCR amplification; purify the PCR product.

[0011] As a preferred technical solution, in step 1, total RNA is extracted from leaves, roots, stems and flowers; in step 2, after PCR amplification, the product is purified using a FastPure Gel DNA Extraction Mini Kit and cloned into the pMD18-T vector to achieve purification of the PCR product.

[0012] As a preferred technical solution, the high-nornicotine germplasm tobacco of the present invention.

[0013] The third object of the present invention is achieved as follows. An overexpression vector of a nornicotine-negative regulatory gene, and the overexpression vector is pCHF3.

[0014] As a preferred technical solution, by amplifying the coding sequence of NtWRKY51, it is constructed into the pCHF3 vector by homologous recombination.

[0015] The fourth object of the present invention is achieved as follows. The application of any of the above nornicotine-negative regulatory genes, and the nornicotine-negative regulatory gene negatively regulates the accumulation of nornicotine in tobacco leaves by inhibiting the expression of CYP82E4.

[0016] As a preferred technical solution, NtWRKY51 negatively regulates nornicotine in tobacco by overexpression; the relative expression level of NtWRKY51 is not less than 42.4243.

[0017] As a preferred technical solution, the application of the nornicotine-negative regulatory gene includes obtaining transgenic tobacco, and the method for obtaining transgenic tobacco includes the following steps:

[0018] Step 1, screening of high-nornicotine germplasm tobacco;

[0019] Step 2: Cloning, sequence analysis and tissue expression of the NtWRKY51 gene

[0020] Obtain the NtWRKY51 gene and perform PCR amplification; retrieve the amino acid sequences of other related WRKY51 homologs of other species from NCBI; align the NtWRKY51 amino acid sequence with other WRKY homologs from different plant species; construct a phylogenetic tree using the neighbor-joining method in MEGA5.0;

[0021] Design fluorescence quantitative primers for NtWRKY51 and the senescence marker gene NtCP1, and perform qRT-PCR detection using the Actin gene as an internal reference; three biological replicates for each sample; use 2 -ΔΔCt Calculate the relative expression level;

[0022] Step 3: Subcellular localization of NtWRKY51

[0023] The full-length NtWRKY51 was amplified using the primer sequences NtWRKY51-GFP-F / R with added adapters. The subcellular localization vector PBI1305-GFP was linearized with Bgl II restriction endonuclease, and the amplified target fragment was ligated to the pBI1305-GFP vector by homologous recombination; the pBI1305-GFP empty vector was used as a control example; the recombinant plasmid and the empty vector plasmid were respectively transferred into the Agrobacterium competent EHA105; the recombinant plasmid (OD 600 : 0.8 - 1.0) and the nuclear localization marker bacterial solution (OD 600 : 0.5) were mixed at a ratio of 1:1 and injected into the leaves of Nicotiana benthamiana; after 72 hours, observation was carried out through a laser confocal microscope (TCS-SP8 Leica). If green fluorescence was observed in the nucleus, cytoplasm, and cell membrane for 35s::GFP, while the green fluorescence signal of the 35s::GFP::NtWRKY51 fusion vector was only observed in the nucleus, it indicated that NtWRKY51 was localized in the nucleus;

[0024] Step 4: Yeast one-hybrid

[0025] There are 5 WRKY binding sites in the region from -410 to -876 bp upstream of CYP82E4. The fragment from -410 to -876 bp upstream of it was cloned using the specific primers CYP82E4-pAbAi-F / R and was homologously recombined with the pAbAi vector digested with Kpn I and Xho I. The ligation product was transformed into Escherichia coli competent DH5α to obtain the recombinant plasmid pCYP82E4pro-AbAi, which was then digested with Bbs I endonuclease, and finally the linearized plasmid was transformed into the Y1HGold yeast competent cells to obtain positive bait strains; the resuspended bacterial solution was spread on SD / -Ura solid media containing different AbA screening concentrations to observe the growth of yeast, conduct AbA background concentration screening, determine the autoactivation situation according to the growth of colonies on the agar plates, and select the concentration of SD / -Ura + AbA (100 ng / ml) as the final concentration for subsequent experiments;

[0026] The CDS of NtWRKY51 was cloned by adding adapter primers NtWRKY51-AD-F / R); the transcription factor sequence containing Bam HI and XhoI adapters was homologously recombined with the pGADT7 prey vector digested with double enzymes, and Escherichia coli competent DH5α was transformed to obtain the prey vector, and the positive recombinant plasmid NtWRKY51-AD was obtained; the recombinant prey vector plasmid NtWRKY51-AD and the empty pGADT7 (negative control example) were respectively transformed into the bait vector pCYP82E4pro-AbAi yeast competent cells and spread on SD / -Leu and SD / -Leu / AbA100 On solid medium, observe the growth of yeast. Yeast should grow on selective medium containing specific antibiotics or auxotrophs, and its growth rate should be consistent with expectations. Visible colonies usually form within 24 - 48 hours. The colonies should be of uniform size, normal morphology, and without abnormal phenomena such as color changes or irregular shapes.

[0027] Step 5: Dual - luciferase activity assay

[0028] Amplify the coding sequence of transcription factor NtWRKY51 with primer sequences NtWRKY51 - 62 - SK - F / R with specific adapters added, and construct it into the linearized vector pGreenII - 62 - SK digested with Bam HI and Eco RI; amplify the promoter fragment of CYP82E4 with primer CYP82E4 - luc - F / R with specific adapters added, and construct it into the linearized vector pGreenII 0800 - Luc digested with Hind III and Spe I. Using the pGreen II 0800 - Luc empty vector as a control example, transform the recombinant plasmid and the empty vector into the competent Agrobacterium tumefaciens GV3101 containing the pSoup plasmid; mix the bacterial solution of the NtWRKY51 recombinant vector and the bacterial solution of the CYP82E4 promoter in a ratio of 5:1 as the experimental group, and mix the empty - vector bacterial solution and the promoter bacterial solution in a ratio of 5:1 as the control group, and inject the leaves of Nicotiana benthamiana; 72 hours later, detect the luciferase activity using a dual - luciferase reporter gene detection kit (Yeasen Biotechnology). Based on the ratio of F - LUC / R - LUC in the control group and the experimental group, judge the interaction relationship between the transcription factor and the promoter.

[0029] Step 6: Obtaining transgenic and knockout mutants

[0030] Construct NtWRKY51 gene mutants using the CRISPR / Cas9 system. Design specific target gRNA sequences before the functional domain of NtWRKY51, insert the NtWRKY51 - specific target gRNA sequence into the CRISPR / Cas9 vector PDC45 digested with BsaI, and introduce the constructed NtWRKY51 - PDC45 vector into Agrobacterium tumefaciens EHA105, and transform tobacco by Agrobacterium - mediated method.

[0031] Using cDNA as a template, amplify the coding sequence of NtWRKY51 with primer sequences NtWRKY51 - pCHF3 - F / R with Sac I restriction site added, and construct it into the pCHF3 vector by homologous recombination method, and obtain transgenic tobacco by the above method.

[0032] As a preferred technical solution, step 2 further includes extracting total RNA from tobacco plants and reverse-transcribing to obtain cDNA;

[0033] Design specific primers according to the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database, amplify the full-length cDNA and genomic sequence to achieve PCR amplification; purify the PCR product.

[0034] As a preferred technical solution, extract total RNA from leaves, roots, stems, and flowers; after PCR amplification, use the FastPure Gel DNA Extraction Mini Kit to purify the product and clone it into the pMD18-T vector to achieve purification of the PCR product.

[0035] As a preferred technical solution, the tobacco in step 1 is preferably a high-nornicotine tobacco germplasm.

[0036] As a preferred technical solution, the high-nornicotine tobacco germplasm is preferably OX2028.

[0037] The fifth object of the present invention is achieved as follows. A tobacco plant is bred from a transgenic tobacco prepared by using the application of any one of the above nicotine-negative regulatory genes.

[0038] Alternatively, a tobacco plant, wherein the tobacco plant contains any one of the above nicotine-negative regulatory genes, and the nicotine-negative regulatory gene negatively regulates the accumulation of nicotine in tobacco leaves by inhibiting the expression of CYP82E4, and the nicotine content in the tobacco leaves does not exceed 0.4% of its dry weight.

[0039] The sixth object of the present invention is achieved as follows. A tobacco product is prepared from the senescent leaves of any one of the above tobacco plants.

[0040] The beneficial effects of the present invention are as follows: In the invention, the nicotine-negative regulatory gene NtWRKY51 is involved in the biosynthesis of nicotine by negatively regulating the expression of CYP82E4. Overexpression of NtWRKY51 can reduce the nicotine content in plants by up to 40%. Nicotine is a harmful alkaloid in tobacco, which not only affects the quality of tobacco but also has potential toxicity. The present invention can effectively reduce nicotine, and regulating the nicotine content through the tobacco NtWRKY51 gene has great application prospects in the tobacco field. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1It is the sequence and phylogenetic analysis diagram of NtWRKY51. Among them, A: Gene structure of NtWRKY51. B: Sequence alignment of NtWRKY51. The WRKYGKK motif and zinc finger structure are marked, and the shaded areas show that the similarity rates between amino acid sequences reach 100%, 75%, and 50% respectively. C: Phylogenetic analysis of WRKY51 in tobacco and other species.

[0042] Figure 2 It is the expression characteristics of NtWRKY51 and the subcellular localization map in Nicotiana benthamiana. Among them, A: Tissue-specific expression. B: Leaves at different senescence stages. C: Expression levels of NtCP1 in leaves at different senescence stages. D: Expression characteristics of NtWRKY51 in leaves at different senescence stages. E: Subcellular localization of NtWRKY51 in Nicotiana benthamiana leaves. GFP is used as a control.

[0043] Figure 3 It is the nicotine content map in NtWRKY51 overexpression and knockout lines. Among them, A: Target sequence and knockout site of the knockout line. B: Nicotine content in green leaves and fully senescent leaves of the knockout line. C: Overexpression of NtWRKY51 in the overexpression line. D: Nicotine content in green leaves and fully senescent leaves of the overexpression line. Error bars represent the standard deviation of 3 biological replicates, * indicates significant difference at the 0.05 probability level, ** indicates significant difference at the 0.01 probability level, and *** indicates significant difference at the 0.001 probability level.

[0044] Figure 4 It is the schematic diagram of NtWRKY51 binding to the CYP82E4 promoter and inhibiting CYP82E4. A: Yeast one-hybrid assay to detect the binding of NtWRKY51 to the CYP82E4 promoter motif. B: Dual-luciferase reporter gene assay to detect the binding of NtWRKY51 to the CYP82E4 promoter and inhibit CYP82E4 transcription. Error bars represent the standard deviation of 3 biological replicates, ** indicates significant difference at the 0.01 probability level.

[0045] Figure 5 It is the expression level map of CYP82E4 in green leaves and senescent leaves of NtWRKY51 overexpression lines and knockout lines. Error bars represent the standard deviation of each biological replicate, * indicates significant difference at the 0.05 probability level, and ** indicates significant difference at the 0.01 probability level. Specific implementation manners

[0046] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0047] Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0048] Example 1. The nornicotine-negative regulatory gene is the tobacco NtWRKY51 gene, which can negatively regulate nornicotine in tobacco. The NtWRKY51 is extracted from tobacco plants. The NtWRKY51 is extracted from tobacco with high nornicotine germplasm 。 The tobacco variety with high nornicotine germplasm is OX2028 (Nicotiana tabacum cv. OX2028), Nicotiana benthamiana, which is cultured in a phytotron at a temperature of 26 °C, a humidity of RH80%, and a photoperiod of 16 h / d.

[0049] Nornicotine is a harmful alkaloid in tobacco, which not only affects the quality of tobacco but also has potential toxicity. Nornicotine mainly accumulates in senescent tobacco leaves, and the content of nornicotine in green leaves is very low. As the degree of senescence increases, the content of nornicotine continues to increase. Although CYP82E4 is known to be a key enzyme gene for nornicotine synthesis and its expression is induced by senescence, the regulatory genes in this process are still unclear. Nornicotine is synthesized from nicotine in tobacco. After nicotine is synthesized in the roots of tobacco, it is transported to the leaves through the xylem and accumulates in the vacuoles of mesophyll cells. Nicotine is further demethylated at the N-methyl group by the catalysis of nicotine demethylase (NND) to form nornicotine. Currently, four nicotine demethylase-encoding genes, CYP82E4, CYP82E5, CYP82E10, and CYP82E21, have been identified. These genes have specific expression sites and expression periods: CYP82E10 is expressed in roots, CYP82E21 is expressed in flowers, CYP82E5 is expressed in developing green leaves, and CYP82E4 is highly expressed in senescent leaves. Since nornicotine mainly accumulates in large amounts in senescent leaves, and the content of nornicotine is very low in other parts and when the leaves are not senescent, CYP82E4 is the key gene regulating the content of nornicotine in leaves. When CYP82E4 is silenced by RNAi, or mutated, the nornicotine content of the RNAi plants or mutants is reduced by more than 95%, from 1% to less than 0.05%.

[0050] The expression of CYP82E4 is induced by senescence. The content of nornicotine in non-senescent green leaves is very low. As the degree of senescence increases, its expression level gradually increases. However, it is still unclear which genes are involved in the transcriptional process of CYP82E4 induced by senescence. We sequenced the transcriptomes of leaves before and after senescence and found that NtWRKY51 is induced by senescence. We found that NtWRKY51 plays a negative feedback role in the synthesis of nornicotine induced by senescence. Therefore, the biosynthesis of nornicotine can be reduced by using the method of negatively regulating the accumulation of nornicotine in tobacco leaves by inhibiting the expression of CYP82E4.

[0051] In the present invention, NtWRKY51 is involved in the biosynthesis of nornicotine by negatively regulating the expression of CYP82E4. NtWRKY51 belongs to the WRKYII subfamily, is a nuclear protein, and is highly expressed in the leaves and stems of tobacco and is induced by senescence. Knocking out NtWRKY51 significantly increased the nornicotine content in senescent leaves, while the nornicotine content in the overexpressing plants of NtWRKY51 was reduced by up to 40%. Yeast one-hybrid (Y1H) and dual-luciferase assay (DLA) showed that NtWRKY51 inhibited the activity of CYP82E4 by binding to the promoter region of CYP82E4. Quantitative fluorescence expression of CYP82E4 in green leaves and senescent leaves of the knockout lines and overexpressing lines also indicated that NtWRKY51 significantly affected the expression of CYP82E4. NtWRKY51 is the first negative regulatory gene for nornicotine biosynthesis and plays a negative feedback role in senescence-induced nornicotine synthesis. The negative regulatory gene of nornicotine and its application in the present invention provide a new method for the regulation mechanism of nicotine biosynthesis, provide new gene resources for the regulation of nornicotine in tobacco, and provide new targets for reducing the nornicotine content in tobacco.

[0052] Example 2. The method for cloning the negative regulatory gene of nornicotine as described in Example 1 is characterized by comprising the following steps:

[0053] Step 1: Extract total RNA from tobacco plants and reverse transcribe to obtain cDNA;

[0054] Step 2: Design specific primers according to the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database, amplify the full-length cDNA and genomic sequence to achieve PCR amplification; purify the PCR product.

[0055] In Step 1, total RNA is extracted from leaves, roots, stems, and flowers; in Step 2, after PCR amplification, the product is purified using a FastPure Gel DNA Extraction Mini Kit and cloned into the pMD18-T vector to achieve purification of the PCR product.

[0056] Example 3. The overexpression vector of the negative regulatory gene of nornicotine as described in Example 1, and the overexpression vector is pCHF3.

[0057] By amplifying the coding sequence of NtWRKY51, it is constructed into the pCHF3 vector by homologous recombination method.

[0058] Example 4. The application of the nicotine-downregulating gene for negative regulation, characterized in that: the nicotine-downregulating gene for negative regulation negatively regulates the accumulation of nicotine in tobacco leaves by inhibiting the expression of CYP82E4. NtWRKY51 achieves negative regulation of nicotine in tobacco by overexpression; the relative expression level of NtWRKY51 is not less than 42.4238.

[0059] The application of the nicotine-downregulating gene for negative regulation includes obtaining transgenic tobacco, and the method for obtaining transgenic tobacco includes the following steps:

[0060] Step 1, screening of tobacco germplasm with high nicotine content;

[0061] Step 2: Cloning, sequence analysis and tissue expression of the NtWRKY51 gene

[0062] Obtain the NtWRKY51 gene and perform PCR amplification; retrieve the amino acid sequences of other related WRKY51 homologs of other species from NCBI; align the NtWRKY51 amino acid sequence with other WRKY homologs from different plant species; construct a phylogenetic tree using the neighbor-joining method in MEGA5.0;

[0063] Design fluorescence quantitative primers for NtWRKY51 and the senescence marker gene NtCP1 (Table 1), and perform qRT-PCR detection using the Actin gene as an internal reference; three biological replicates for each sample; use 2 -ΔΔCt Calculate the relative expression level;

[0064] Step 3: Subcellular localization of NtWRKY51

[0065] Amplify the full length of NtWRKY51 using the primer sequences with adapters (NtWRKY51-GFP-F / R), linearize the subcellular localization vector PBI1305-GFP with Bgl II restriction endonuclease, and ligate the amplified target fragment onto the pBI1305-GFP vector using homologous recombination; use the pBI1305-GFP empty vector as a control example; transfer the recombinant plasmid and the empty vector plasmid into the Agrobacterium competent EHA105 respectively; mix the recombinant plasmid (OD 600 : 0.8 - 1.0) and the marker bacterial solution for nuclear localization (OD 600 : 0.5) at a ratio of 1:1 and inject the leaves of Nicotiana benthamiana; after 72 hours, observe through a laser confocal microscope (TCS-SP8 Leica). If green fluorescence is observed in the nucleus, cytoplasm and cell membrane for 35s::GFP, while the green fluorescence signal of the 35s::GFP::NtWRKY51 fusion vector is only observed in the nucleus, it indicates that NtWRKY51 is localized in the nucleus;

[0066] Step 4: Yeast one-hybrid

[0067] There are 5 WRKY binding sites in the region of -410 to -876 bp upstream of CYP82E4. The fragment of -410 to -876 bp upstream of it was cloned with specific primers CYP82E4-pAbAi-F / R and was homologous recombined with the pAbAi vector digested with Kpn I and Xho I. The ligation product was transformed into Escherichia coli competent DH5α to obtain the recombinant plasmid pCYP82E4pro-AbAi. Then it was digested with Bbs I endonuclease, and finally the linearized plasmid was transformed into Y1HGold yeast competent cells to obtain positive bait strains; The resuspended bacterial solution was spread on SD / -Ura solid medium containing different AbA screening concentrations to observe the growth of yeast, and the AbA background concentration was screened. According to the growth of colonies on the agar plate, the auto-activation situation was determined, and the concentration SD / -Ura + AbA (100 ng / ml) was selected as the final concentration and used for subsequent experiments;

[0068] The CDS of NtWRKY51 was cloned by adding adapter primers NtWRKY51-AD-F / R; The transcription factor sequence containing Bam HI and Xho I adapters was homologous recombined with the digested pGADT7 prey vector, and Escherichia coli competent DH5α was transformed to obtain the prey vector, and the positive recombinant plasmid NtWRKY51-AD was obtained; The recombinant prey vector plasmid NtWRKY51-AD and the empty vector pGADT7 (negative control example) were respectively transformed into the bait vector pCYP82E4pro-AbAi yeast competent cells and spread on SD / -Leu and SD / -Leu / AbA 100 solid medium to observe the growth of yeast. Yeast should grow on selective medium containing specific antibiotics or auxotrophs, and its growth rate should be consistent with the expectation. Usually, visible colonies are formed within 24 - 48 hours, and the colonies should be of uniform size and normal morphology, without abnormal phenomena such as color change or irregular morphology;

[0069] Step 5: Dual-luciferase activity assay

[0070] The coding sequence of the transcription factor NtWRKY51 was amplified with the primer sequences NtWRKY51-62-SK-F / R added with specific adapters and constructed in the linearized vector pGreenII-62-SK digested with BamHI and EcoRI; the promoter fragment of CYP82E4 was amplified with the specific adapter primer CYP82E4-luc-F / R (Table 1) and constructed in the linearized vector pGreenII 0800-Luc digested with HindIII and SpeI. Taking the pGreen II 0800-Luc empty vector as a control example, the recombinant plasmid and the empty vector were transformed into the competent Agrobacterium tumefaciens GV3101 containing the pSoup plasmid; the bacterial solution of the NtWRKY51 recombinant vector and the bacterial solution of the CYP82E4 promoter were mixed at a ratio of 5:1 as the experimental group, and the empty vector bacterial solution and the promoter bacterial solution were mixed at a ratio of 5:1 as the control group, and the Nicotiana benthamiana leaves were injected; 72 hours later, the luciferase activity was detected with a dual-luciferase reporter gene detection kit (Yeasen Biotechnology), and the interaction relationship between the transcription factor and the promoter was judged according to the ratio of F-LUC / R-LUC in the control group and the experimental group;

[0071] Step 6: Obtaining transgenic and knockout mutants

[0072] The NtWRKY51 gene mutant was constructed using the CRISPR / Cas9 system. A specific target gRNA sequence was designed before the NtWRKY51 functional domain, and the NtWRKY51 specific target gRNA sequence was inserted into the CRISPR / Cas9 vector PDC45 digested with BsaI. The constructed NtWRKY51-PDC45 vector was introduced into Agrobacterium tumefaciens EHA105, and tobacco was transformed by the Agrobacterium tumefaciens-mediated method;

[0073] Using cDNA as a template, the coding sequence of NtWRKY51 was amplified with the primer sequences NtWRKY51-pCHF3-F / R (Table 1) added with Sac I restriction enzyme sites, and constructed in the pCHF3 vector by homologous recombination method, and transgenic tobacco was obtained by the above method.

[0074] Step 2 also includes extracting total RNA from tobacco plants and reverse transcribing to obtain cDNA;

[0075] Specific primers were designed based on the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database to amplify the full-length cDNA and genomic sequences, achieving PCR amplification; the PCR products were purified. Specifically, total RNA was extracted from leaves, roots, stems, and flowers; after PCR amplification, the products were purified using a FastPure Gel DNA Mini Kit and cloned into the pMD18-T vector to achieve purification of the PCR products.

[0076] The tobacco described in step 1 is preferably a tobacco germplasm with high nicotine reduction. The tobacco germplasm with high nicotine reduction is preferably OX2028.

[0077] A tobacco plant, bred from a transgenic tobacco prepared by the application of the nicotine reduction negative regulatory gene described above.

[0078] Example 4. The application of the nicotine reduction negative regulatory gene described in Example 1, characterized in that: the nicotine reduction negative regulatory gene negatively regulates the accumulation of nicotine in tobacco leaves by inhibiting the expression of CYP82E4. NtWRKY51 achieves negative regulation of nicotine in tobacco through overexpression; the relative expression level of NtWRKY51 is not lower than 42.4238.

[0079] The application of the nicotine reduction negative regulatory gene includes obtaining transgenic tobacco, and the method for obtaining transgenic tobacco and index testing includes the following steps.

[0080] 1. Materials and methods.

[0081] 1.1 Experimental materials and growth conditions

[0082] The tobacco germplasm with high nicotine reduction OX2028 (Nicotiana tabacum cv. OX2028) and Nicotiana benthamiana were cultured in a phytotron at a temperature of 26°C, a humidity of RH80%, and a photoperiod of 16 h / d.

[0083] 1.2 Cloning, sequence analysis, and tissue expression of the NtWRKY51 gene

[0084] Total RNA was extracted from the leaves, roots, stems, and flowers of tobacco, and cDNA was obtained by reverse transcription. Specific primers were designed based on the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database (https: / / solgenomics.net / ) (the specific specific primers are shown in the AmplifyNtWRKY51 part of Table 1).

[0085] Table 1 Primer sequences

[0086]

[0087] Amplify the full-length cDNA and genomic sequences. After PCR amplification, the products were purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China) and cloned into the pMD18-T vector (TaKaRa, China) for sequencing at Sangon Biotech (Qingdao, China).

[0088] Retrieve the amino acid sequences of other related WRKY51 homologs from other species in NCBI (https: / / www.ncbi.nlm.nih.gov / ). Align the NtWRKY51 amino acid sequence with other WRKY homologs from different plant species using DNAMAN software. Construct a phylogenetic tree using the neighbor-joining method in MEGA5.0.

[0089] Design fluorescence quantitative primers for NtWRKY51 and the senescence marker gene NtCP1 (specific primers are shown in the qRT-PCR section of Table 1), and use the Actin gene as an internal reference for qRT-PCR detection. Each sample has three biological replicates, and use 2 -ΔΔCt Calculate the relative expression level.

[0090] 1.3 Subcellular localization of NtWRKY51

[0091] Amplify the full length of NtWRKY51 using the primer sequences with adapters (NtWRKY51-GFP-F / R). Linearize the subcellular localization vector PBI1305-GFP with Bgl II restriction endonuclease, and ligate the amplified target fragment onto the pBI1305-GFP vector by homologous recombination. Use the pBI1305-GFP empty vector as a control example. Transfer the recombinant plasmid and the empty vector plasmid into the Agrobacterium competent EHA105 respectively. Using the transient transformation technique, mix the recombinant plasmid (OD600: 0.8 - 1.0) with the nuclear localization marker bacterial solution (OD600: 0.5) at a ratio of 1:1 and inject it into the leaves of Nicotiana benthamiana. After 72 hours, observe through a laser confocal microscope (TCS-SP8 Leica).

[0092] 1.4 Yeast one-hybrid

[0093] Bioinformatics analysis showed that there were 5 WRKY binding sites in the interval of -410 to -876 bp upstream of CYP82E4

[17] , the fragment of its upstream -410 to -876 bp was cloned with the specific primers CYP82E4-pAbAi-F / R (Y1H part in Table 1), and was homologously recombined with the pAbAi vector digested by Kpn I and Xho I. The ligation product was transformed into Escherichia coli competent DH5α to obtain the recombinant plasmid pCYP82E4pro-AbAi. Then it was digested with the Bbs I endonuclease, and finally the linearized plasmid was transformed into the Y1HGold yeast competent cells to obtain the positive bait strain. The resuspended bacterial solution was spread on the SD / -Ura solid medium containing different AbA screening concentrations, and the yeast growth was observed for AbA background concentration screening.

[0094] The CDS of NtWRKY51 was cloned with the primers NtWRKY51-AD-F / R (Table 1) adding adapters. The transcription factor sequence containing Bam HI and Xho I adapters was homologously recombined with the prey vector pGADT7 digested by double enzymes, and transformed into Escherichia coli competent DH5α to obtain the prey vector, and the positive recombinant plasmid NtWRKY51-AD was obtained. The recombinant prey vector plasmid NtWRKY51-AD and the empty vector pGADT7 (negative control example) were respectively transformed into the bait vector pCYP82E4pro-AbAi yeast competent cells, and spread on the SD / -Leu and SD / -Leu / AbA 100 solid medium, and the yeast growth was observed.

[0095] 1.5 Dual-Luciferase Activity Assay

[0096] The coding sequence of the transcription factor NtWRKY51 was amplified with the primer sequence NtWRKY51-62-SK-F / R (LUC part in Table 1) adding specific adapters, and was constructed into the pGreenII-62-SK linearized vector digested by Bam HI and Eco RI; the promoter fragment of CYP82E4 was amplified with the primer CYP82E4-luc-F / R adding specific adapters (LUC part in Table 1), and was constructed into the pGreenII 0800-Luc linearized vector digested by Hind III and Spe I. Taking the pGreenII 0800-Luc empty vector as the control example, the recombinant plasmid and the empty vector were transformed into the Agrobacterium competent GV3101 containing the pSoup plasmid. Using the transient transformation technology, the bacterial solution of the NtWRKY51 recombinant vector and the bacterial solution of the CYP82E4 promoter were mixed at a ratio of 5:1 as the experimental group, and the empty vector bacterial solution and the promoter bacterial solution were mixed at a ratio of 5:1 as the control example group, and the Nicotiana benthamiana leaves were injected. After 72 hours, the luciferase activity was detected with the Dual-Luciferase Reporter Assay Kit (Yeasen Biotechnology).

[0097] 1.6 Obtaining transgenic and knockout mutants

[0098] The CRISPR / Cas9 system was used to construct NtWRKY51 gene mutants. A specific target gRNA sequence was designed before the functional domain of NtWRKY51. The NtWRKY51 specific target gRNA sequence (see the generate editing part in Table 1) was inserted into the CRISPR / Cas9 vector PDC45 digested with BsaI alone. The constructed NtWRKY51-PDC45 vector was introduced into Agrobacterium tumefaciens EHA105, and tobacco was transformed by Agrobacterium-mediated method.

[0099] Using cDNA as a template, the coding sequence of NtWRKY51 was amplified with the adapter primers NtWRKY51-pCHF3-F / R containing Sac I restriction sites (see the over-expression part in Table 1), and was constructed into the pCHF3 vector by homologous recombination method. Transgenic tobacco was obtained by the above method. The nicotine content and gene expression analysis were carried out on the green leaves and fully senescent leaves of overexpression lines and knockout lines. All experiments were repeated three times biologically.

[0100] 1.7 Determination of alkaloid content

[0101] The nicotine content was determined by HPLC. 50 mg of tobacco leaf powder was dried at 60 °C and ground into powder. 50 mg of tobacco leaf powder was weighed, extracted with absolute ethanol containing 0.05% NaOH for 30 min, centrifuged at 13,000 rpm for 30 min, and the supernatant was filtered, and then subjected to HPLC analysis. HPLC analysis was performed using a 4.6×250 mm Welch Ultimate HILIC silica gel column. The mobile phase for elution was water containing 0.2% acetic acid and 4 mM ammonium acetate (A) and acetonitrile containing 0.2% acetic acid (B). Each sample was repeated twice.

[0102] 2 Results and analysis

[0103] 2.1 Identification and bioinformatics analysis of NtWRKY51

[0104] The open reading frame (ORF) of NtWRKY51 is 576 bp in length, encoding 191 amino acids. The predicted protein pI is 6.521 and the molecular weight is 22.08 kDa. Comparative analysis of the coding sequence and genomic sequence of NtWRKY51 showed that it consists of 3 exons and 2 introns ( Figure 1 as shown in part A). Multiple alignment of NtWRKY51 with other homologous proteins showed that NtWRKY51 contains a WRKYGKK core sequence and a C2H2 (C-X 4-5- C-X23- H-X 1- H) zinc finger motif and belongs to the WRKYII subfamily. Phylogenetic analysis showed that NtWRKY51 has high sequence homology with homologous proteins such as AtWRKY51 in Arabidopsis thaliana, SsWRKY51 in Solanum tuberosum, and SlWRKY51 in Solanum lycopersicum ( Figure 1 as shown in part C).

[0105] 2.2 Tissue expression, senescence induction and subcellular localization of NtWRKY51

[0106] NtWRKY51 has tissue expression specificity, specifically showing relatively high expression in leaves and stems, while relatively low expression in roots and flowers ( Figure 2 part A). At the same time, the expression of NtWRKY51 is induced by senescence. Tobacco leaves at different senescence stages were sampled ( Figure 2 part B), and the senescence marker gene NtCP1 was used as a reference for the degree of senescence ( Figure 2 part C). It can be seen that the expression of NtWRKY51 is closely related to the degree of senescence. The expression level is relatively low before leaf senescence, rises sharply after the start of senescence, and continues to rise with the aggravation of the senescence degree ( Figure 2 part D).

[0107] The 35s::GFP::NtWRKY51 fusion vector and 35s::GFP were respectively injected into Nicotiana benthamiana. The results showed that green fluorescence was observed in the nucleus, cytoplasm and cell membrane of 35s::GFP, while the green fluorescence signal of the 35s::GFP::NtWRKY51 fusion vector was only observed in the nucleus. This indicates that NtWRKY51 is localized in the nucleus ( Figure 2 part E).

[0108] 2.3 NtWRKY51 negatively regulates the biosynthesis of nornicotine

[0109] To study whether NtWRKY51 is involved in the regulation of nornicotine synthesis, targets were designed on the first exon before the WRKY domain, and a total of 9 knockout lines were obtained. Sequencing showed that two lines were homozygous mutants. NtWRKY51-CR24 (CR-24) inserted base A, and NtWRKY51-CR27 (CR-27) inserted base T ( Figure 3 A). The nornicotine contents of the mutant green leaves and fully senescent leaves were measured. The results showed that the nornicotine content in the green leaves was not much different from that of the control example (WT), but the nornicotine content in the fully senescent leaves was 1.6 times and 2.4 times that of the control example ( Figure 3 B).

[0110] At the same time, NtWRKY51 was overexpressed in tobacco, and 24 independent overexpression lines were obtained. Among them, the overexpression multiples of NtWRKY51-OE4 (OE-4), NtWRKY51-OE14 (OE-14), and NtWRKY51-OE23 (OE-23) were 113 times, 42 times, and 1064 times that of the control example, respectively ( Figure 3 C). Analysis of the nornicotine content in the green leaves and fully senescent leaves of the overexpression lines showed that there was no significant difference in the nornicotine content of OE-4, OE-14, and OE-23 in the green leaves compared with the control example, while in the senescent leaves, it was significantly lower than that of the control example. Among them, the nornicotine content in the senescent leaves of OE-23 was reduced by 40% compared with the control example ( Figure 3 D), indicating that NtWRKY51 negatively regulates the biosynthesis of nornicotine.

[0111] 2.4 NtWRKY51 binds to the CYP82E4 promoter

[0112] Yeast one-hybrid analysis was performed on the NtWRKY51 and the CYP82E4 promoter region. The empty vector pGADT7-AD could grow normally in the SD / -Leu medium, but could not grow normally in the SD / -Leu / AbA100 medium; while the recombinant prey vector containing NtWRKY51 could grow normally on both the SD / -Leu and SD / -Leu / AbA100 media ( Figure 4 A), indicating that the NtWRKY51 protein can directly bind to the CYP82E4 promoter.

[0113] Furthermore, the regulation of the CYP82E4 promoter by NtWRKY51 was verified by the dual-luciferase experiment. Compared with the control example, when the NtWRKY51 protein was present, the activity of the CYP82E4 promoter was significantly down-regulated, and the LUC / REN ratio was reduced by 79% ( Figure 4 B), indicating that the NtWRKY51 protein binds to the CYP82E4 promoter and inhibits the transcription of CYP82E4.

[0114] 2.5 NtWRKY51 inversely regulates the expression of CYP82E4

[0115] Quantitative fluorescence expression analysis of CYP82E4 in the green leaves and senescent leaves of the knockout lines and overexpression lines was performed. The results showed that the relative expression level of CYP82E4 in the overexpression lines was significantly lower than that of the control example, while the relative expression level of the knockout lines was significantly higher than that of the control example ( Figure 5 ). Thus, it can be seen that NtWRKY51 participates in the regulation of nornicotine synthesis by negatively regulating the expression of CYP82E4.

[0116] Nornicotine is an adverse alkaloid in tobacco. Besides affecting the aroma, taste and flavor of tobacco, it also has a significant impact on the safety of tobacco. In this study, NtWRKY51 negatively regulates the biosynthesis of nornicotine by inhibiting the expression of CYP82E4, thereby reducing the content of nornicotine in tobacco leaves. Therefore, NtWRKY51 has important utilization value for reducing the nornicotine content in tobacco leaves.

[0117] WRKY is a class of transcription factors unique to plants and has very rich biological functions. According to the number of WRKY domains and the characteristics of zinc finger motifs, WRKY proteins are divided into three subfamilies. Subfamily I has two WRKY domains and a C2H2-type zinc finger motif (C-X4-5-C-X22-23-H-X1-H). Subfamily II has one WRKY domain and a C2H2-type zinc finger motif, while subfamily III has a C2HC-type zinc finger motif (C-X7-C-X23-H-X1-C). NtWRKY51 contains a WRKYGKK core sequence and a C2H2 (C-X 4-5- C-X 23- H-X 1- H) zinc finger motif and belongs to the WRKYII subfamily( Figure 1 B). In other plants, WRKY51 is mainly involved in the biosynthesis of hormones such as ethylene and salicylic acid, and inhibits the biosynthesis of jasmonic acid, and has biological functions of regulating lateral root growth and promoting plant immunity. In this study, we found that the nornicotine content in NtWRKY51 overexpressing plants was lower than that in the control group, while the nornicotine content in plants with NtWRKY51 knockout increased significantly( Figure 3 ), indicating that NtWRKY51 is involved in the biosynthesis process of nornicotine. Therefore, this study further enriches the biological functions of WRKY51.

[0118] The senescence of plant leaves is accompanied by physiological changes such as the disassembly of chloroplasts and the weakening of photosynthetic function, as well as biochemical changes such as the orderly decomposition of macromolecules, the increase of membrane ion leakage, and the continuous accumulation of fructose and glucose

[25] , and the physiological and biochemical changes of plant cells caused by senescence further affect the synthesis and decomposition of other plant metabolites as stimulating factors

[26] . In tobacco, senescence induces the expression of CYP82E4, thereby promoting the synthesis and accumulation of nornicotine. In this process, NtWRKY33 participates in the synthesis of nornicotine as a positive regulatory factor

[16] . However, in this study, we found that NtWRKY51 is induced by senescence. After leaf senescence, the expression level of NtWRKY51 increased by more than 30 times( Figure 2 B), but NtWRKY51 negatively regulates the transcription of CYP82E4 by binding to the CYP82E4 promoter, thereby inhibiting the biosynthesis of nornicotine(Figure 3 , 4), so NtWRKY51 plays a negative feedback role in the regulation process of senescence-NtWRKY51-CYP82E4, and counteracts the process of senescence-induced nornicotine synthesis by negatively regulating the expression of CYP82E4. It can be seen that the synthesis conditions of nornicotine during senescence are achieved through at least two pathways. It can not only promote the transcription of CYP82E4 by inducing the expression of NtWRKY33, positively regulating the synthesis of nornicotine, but also induce the expression of NtWRKY51 to inhibit the synthesis of nornicotine, forming a negative feedback loop, making the synthesis and accumulation of nornicotine during plant senescence more precise and balanced.

[0119] Although overexpression or knockout of NtWRKY51 significantly changed the expression of CYP82E4 in green leaves ( Figure 5 ), it had little effect on the content of nornicotine in green leaves ( Figure 3 ). This is related to the low expression of NtWRKY51 and CYP82E4 in green leaves. The expression level of NtWRKY51 in green leaves is very low. When NtWRKY51 is knocked out, the change in the expression level of CYP82E4 is small, so the content of nornicotine has no significant change; although overexpression of NtWRKY51 significantly inhibits the expression of CYP82E4, the expression level of CYP82E4 in green leaves is very low, and CYP82E4 is not a contributor to the synthesis of nornicotine in green leaves

[10] , so the decrease in the expression of CYP82E4 caused by overexpression of NtWRKY51 has little effect on the content of nornicotine in green leaves.

[0120] A tobacco plant, which contains the nornicotine negative regulation gene of this example. The nornicotine negative regulation gene negatively regulates the accumulation of nornicotine in tobacco leaves by inhibiting the expression of CYP82E4. After detection, the content of nornicotine in the tobacco leaves is 0.4% of its dry weight. This example identified the function of tobacco NtWRKY51 in the synthesis of nornicotine. NtWRKY51 belongs to the WRKY II subfamily, is localized in the nucleus and is induced by senescence. The content of nornicotine in the NtWRKY51 knockout mutant increased significantly, while the content of nornicotine decreased significantly after overexpression of NtWRKY51, and the highest reduction was 40% compared with the control example. It can be seen that NtWRKY51 directly interacts with the CYP82E4 promoter in vitro, negatively regulates the expression of CYP82E4, plays an obvious negative feedback role in the process of senescence-induced nornicotine synthesis, and has great application prospects in cultivating tobacco with reduced nornicotine content.

[0121] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, making equivalent replacements or changes should be covered within the protection scope of the present invention.

Claims

1. The nicotine-lowering gene is negatively regulated, characterized in that: The gene is the tobacco NtWRKY51 gene, which can negatively regulate nornicotine in tobacco.

2. The nicotine-downregulating gene with negative regulation as described in claim 1, wherein: The NtWRKY51 is extracted from tobacco plants.

3. The nicotine-downregulating gene as claimed in claim 2, wherein: The NtWRKY51 is extracted from high-nornicotine germplasm tobacco.

4. The cloning method of the nicotine-lowering negative regulatory gene as described in any one of claims 1 to 3, characterized in that, It includes the following steps: Step 1: Extract total RNA from tobacco plants and reverse transcribe to obtain cDNA; Step 2: Design specific primers according to the tobacco NtWRKY51 gene sequence in the SolGenomicsNetwork database, amplify the full-length cDNA and genomic sequence to achieve PCR amplification; Purify the PCR product.

5. The cloning method of the nicotine-downregulating gene as described in claim 4, characterized in that: In Step 1, total RNA is extracted from leaves, roots, stems, and flowers; in Step 2, after PCR amplification, the product is purified using a FastPure Gel DNA Extraction Mini Kit and cloned into the pMD18-T vector to achieve purification of the PCR product.

6. The cloning method of the nicotine-downregulating gene as described in claim 5, characterized in that: The high-nornicotine germplasm tobacco of the said tobacco.

7. The overexpression vector for negatively regulating the expression of the nicotine-degrading gene according to any one of claims 1 to 3, characterized in that: The overexpression vector is pCHF3.

8. The overexpression vector for negatively regulating the expression of the nicotine-degrading gene according to claim 7, characterized in that: By amplifying the coding sequence of NtWRKY51, it is constructed in the pCHF3 vector using homologous recombination.

9. Use of the nicotine-downregulating gene for negatively regulating genes as described in any one of claims 1 to 3, characterized in that: The nornicotine-negative regulatory gene negatively regulates the accumulation of nornicotine in tobacco leaves by inhibiting the expression of CYP82E4.

10. The application of the nicotine-lowering negative regulatory gene as claimed in claim 7, wherein: NtWRKY51 achieves negative regulation of nornicotine in tobacco through overexpression; the relative expression level of the said NtWRKY51 is not less than 42.42 times that of the control.

11. The application of the nicotine-lowering negative regulatory gene as claimed in claim 9, characterized in that, The application of the nornicotine-negative regulatory gene includes obtaining transgenic tobacco, and the method for obtaining transgenic tobacco includes the following steps: Step 1, screening of high-nornicotine germplasm tobacco; Step 2: Cloning, sequence analysis, and tissue expression of the NtWRKY51 gene Obtain the NtWRKY51 gene and perform PCR amplification; retrieve the amino acid sequences of other related WRKY51 homologs from other species in NCBI; align the NtWRKY51 amino acid sequence with other WRKY homologs from different plant species; construct a phylogenetic tree using the neighbor-joining method in MEGA5.0; design fluorescence quantitative primers for NtWRKY51 and the senescence marker gene NtCP1, and perform qRT-PCR detection using the Actin gene as an internal reference; three biological replicates for each sample; use 2 –ΔΔCt Calculate the relative expression level; Step 3: Subcellular localization of NtWRKY51 Amplify the full length of NtWRKY51 using the primer sequences NtWRKY51-GFP-F / R with added adapters, linearize the subcellular localization vector PBI1305-GFP with Bgl II restriction endonuclease, and ligate the amplified target fragment to the pBI1305-GFP vector using homologous recombination; use the pBI1305-GFP empty vector as a control example; transfer the recombinant plasmid and the empty vector plasmid into the Agrobacterium competent EHA105 respectively; mix the recombinant plasmid and the nuclear localization marker bacterial solution at a ratio of 1:1 and inject the leaves of Nicotiana benthamiana; after 72 hours, observe through a laser confocal microscope; Step 4: Yeast one-hybrid There are 5 WRKY binding sites in the -410 to -876 bp region upstream of CYP82E4. The fragment of -410 to -876 bp upstream of it was cloned with specific primers CYP82E4-pAbAi-F / R and was homologously recombined with the pAbAi vector digested with Kpn I and Xho I. The ligation product was transformed into Escherichia coli competent DH5α to obtain the recombinant plasmid pCYP82E4pro-AbAi, and then digested with Bbs I endonuclease. Finally, the linearized plasmid was transformed into Y1HGold yeast competent cells to obtain positive bait strains; the resuspended bacterial solution was spread on SD / -Ura solid medium containing different AbA screening concentrations to observe the growth of yeast and perform AbA background concentration screening; The CDS of NtWRKY51 was cloned with primers NtWRKY51-AD-F / R with added adapters; the transcription factor sequence containing Bam HI and Xho I adapters was homologously recombined with the digested pGADT7 prey vector, and Escherichia coli competent DH5α was transformed to obtain the prey vector, and the positive recombinant plasmid NtWRKY51-AD was obtained; the recombinant prey vector plasmid NtWRKY51-AD and the empty vector pGADT7 were respectively transformed into the bait vector pCYP82E4pro-AbAi yeast competent cells and spread on SD / -Leu and SD / -Leu / AbA100 solid media to observe the growth of yeast; Step 5: Dual-luciferase activity assay The coding sequence of the transcription factor NtWRKY51 was amplified with primer sequences NtWRKY51-62-SK-F / R with added specific adapters and was constructed in the pGreenII-62-SK linearized vector digested with Bam HI and Eco RI; the promoter fragment of CYP82E4 was amplified with specific adapter primers CYP82E4-luc-F / R and was constructed in the pGreenII 0800-Luc linearized vector digested with Hind III and Spe I. Taking the pGreen II 0800-Luc empty vector as a control example, the recombinant plasmid and the empty vector were transformed into Agrobacterium tumefaciens competent GV3101 containing the pSoup plasmid; the bacterial solution of the NtWRKY51 recombinant vector and the bacterial solution of the CYP82E4 promoter were mixed at a ratio of 5:1 as the experimental group, and the empty vector bacterial solution and the promoter bacterial solution were mixed at a ratio of 5:1 as the control example group, and Nicotiana benthamiana leaves were injected; 72 hours later, the luciferase activity was detected with a dual-luciferase reporter gene detection kit (Yeasen Biotechnology); Step 6: Obtaining of transgenic and knockout mutants Construct a mutant of NtWRKY51 gene using the CRISPR / Cas9 system. Design a specific target gRNA sequence before the functional domain of NtWRKY51, insert the NtWRKY51-specific target gRNA sequence into the CRISPR / Cas9 vector PDC45 digested with BsaI alone, introduce the constructed NtWRKY51-PDC45 vector into Agrobacterium tumefaciens EHA105, and transform tobacco by the Agrobacterium-mediated method; Using cDNA as a template, amplify the coding sequence of NtWRKY51 with the adapter primers NtWRKY51-pCHF3-F / R with Sac I restriction sites added, construct it in the pCHF3 vector by homologous recombination, and obtain transgenic tobacco by the above method.

12. Use of the nicotine-downregulating gene for negative regulation as described in claim 11, characterized in that: Step 2 also includes extracting total RNA from tobacco plants and reverse transcribing to obtain cDNA; Design specific primers according to the tobacco NtWRKY51 gene sequence (Nitab4.5_0000047g0140) in the SolGenomicsNetwork database, amplify the full-length cDNA and genomic sequence to achieve PCR amplification; Purify the PCR product.

13. Use of the nicotine-lowering negative regulatory gene according to claim 12, characterized in that: Extract total RNA from leaves, roots, stems, and flowers; after PCR amplification, use the FastPure Gel DNA Extraction Mini Kit to purify the product and clone it into the pMD18-T vector to achieve purification of the PCR product.

14. Use of the nicotine-downregulating gene for negative regulation as described in claim 11, characterized in that: The tobacco in step 1 is preferably a tobacco germplasm with high nicotine reduction.

15. The application of the nicotine-lowering negative regulatory gene as described in claim 14, characterized in that: The tobacco germplasm with high nicotine reduction is OX2028.

16. Tobacco plant, characterized in that: It is bred from transgenic tobacco prepared by using the application of the nicotine reduction negative regulatory gene described in any one of claims 11-15.

17. Tobacco plant, characterized in that: The tobacco plant contains the nicotine reduction negative regulatory gene described in any one of claims 1-3. The nicotine reduction negative regulatory gene negatively regulates the accumulation of nicotine in tobacco leaves by inhibiting the expression of CYP82E4, and the nicotine content in the tobacco leaves does not exceed 0.4% of its dry weight.

18. A tobacco product, characterized in that: It is prepared from the senescent leaves of the tobacco plant described in claim 16 or 17.

Images