Tobacco nicotine synthesis regulation gene NtbHLH25 as well as encoding protein and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, particularly relates to a tobacco nicotine synthesis regulation gene NtbHLH25 and a tobacco nicotine synthesis regulation protein NtbHLH25 encoded by the same, and further discloses application of the tobacco nicotine synthesis regulation gene NtbHLH25 and the tobacco nicotine synthesis regulation protein NtbHLH25. According to the invention, a new transcription factor gene NtbHLH25 is cloned from cultivated tobacco, the effect of the gene in tobacco synthesis is studied, and detection on nicotine of genetic materials shows that the nicotine content in strains and tobacco leaves with reduced NtbHLH25 gene expression quantity is obviously reduced. The gene NtbHLH25 for regulating and controlling nicotine synthesis and the encoded protein NtbHLH25 identified by the invention are related to nicotine accumulation in tobacco, provide a new genetic target and technical means for regulation and application of tobacco nicotine content, and have important application value in tobacco nicotine regulation and control.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a tobacco nicotine synthesis regulatory gene NtbHLH25 and a tobacco nicotine synthesis regulatory protein NtbHLH25 encoded by the gene, and further discloses the application thereof. Background Art

[0002] Nicotine is an endogenous defensive alkaloid formed by tobacco during its long evolution. It is the most important alkaloid in tobacco, accounting for more than 95% of tobacco alkaloids. As a secondary metabolite, nicotine plays a protective role in tobacco, resisting the invasion of insects and herbivores. When insects or herbivores bite tobacco leaves, nicotine synthesis mediated by jasmonic acid injury-induced signals will immediately start in the roots, and then be transported to the tobacco leaves to deal with various insults. Moreover, the level of nicotine in tobacco is closely related to the quality of tobacco leaves. In addition, due to the powerful biological activity and pharmacological effects of nicotine, it plays an important role in the treatment of Parkinson's disease and various inflammatory diseases.

[0003] Studies have shown that nicotine biosynthesis is a complex regulatory process, which is regulated by a variety of complex factors, including ecological environment, cultivation measures, plant hormones, genetic factors and its own growth and development stage, involving the expression and genetic regulation of a large number of genes. Therefore, the analysis of the molecular mechanism of tobacco nicotine biosynthesis and accumulation is one of the key areas of tobacco biology research.

[0004] With the rapid development of biotechnology and genomics, research on the molecular regulatory mechanism of nicotine synthesis has achieved remarkable results. At present, the key genes of the nicotine biosynthesis metabolic pathway have been identified one after another, and researchers have gradually become clear about the metabolic pathway. For example, ornithine undergoes decarboxylation reaction under the catalysis of ornithine decarboxylase (ODC), or putrescine is generated from arginine under the catalysis of arginine decarboxylase (ADC). S-adenosylmethionine and putrescine generate N-methylputrescine and S-adenosylhomocysteine ​​under the catalysis of putrescine N-methyltransferase (PMT), which is the key rate-limiting enzyme in the nicotine synthesis process. Quinolinate phosphoribosyltransferase (QPT) plays an important regulatory role in the synthesis of pyridine alkaloids. In addition, BBLs and A622 are also considered to be the last two key enzymes in nicotine synthesis. In addition, jasmonic acid plays an important role in plant growth and development and disease and pest defense. Studies have shown that jasmonic acid significantly induces nicotine synthesis and regulates the synthesis and accumulation of nicotine by strongly inducing the expression of ADC, PMT, QPT and transporter MATE1.

[0005] The study also found that nicotine biosynthesis is regulated by a variety of transcription factors. MYC1, a key transcription factor in the jasmonic acid signaling pathway, has been discovered so far. The expression of MYC1a and MYC1b was inhibited by RNA interference (RNAi) technology. When MYC1a and MYC1b were knocked down, the nicotine level in tobacco BY-2 cells was significantly reduced, indicating that MYC1a and MYC1b are positive regulators of nicotine synthesis. In addition, ERF189, a member of the ERF transcription factor subfamily, positively regulates the expression of the putrescine N-methyltransferase (PMT) gene by binding to the GCC box and G-box elements in the gene promoter region, thereby affecting nicotine synthesis. At present, except for the known regulatory factors, no other new nicotine synthesis regulatory genes have been found in cultivated tobacco.

[0006] Therefore, further exploring suitable nicotine synthesis transcription factors has important theoretical significance and practical value for the effective regulation of nicotine content. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a tobacco nicotine synthesis regulatory gene NtbHLH25 and a tobacco nicotine synthesis regulatory protein NtbHLH25 encoded therein, both of which are related to nicotine accumulation in tobacco, and provide new genetic targets and technical means for the regulation and application of tobacco nicotine content;

[0008] The second technical problem to be solved by the present invention is to provide the application of the tobacco nicotine synthesis regulatory gene NtbHLH25 and the tobacco nicotine synthesis regulatory protein NtbHLH25 in the field of tobacco cultivation.

[0009] In order to solve the above technical problems, the tobacco nicotine synthesis regulatory gene NtbHLH25 described in the present invention has a nucleotide sequence having at least one of the following characteristics (a)-(d):

[0010] (a) the nucleotide sequence shown in SEQ ID NO.1;

[0011] (b) a nucleotide sequence in which one or more bases of the nucleotide sequence shown in SEQ ID NO.1 are deleted, substituted or inserted, and the nucleotide sequence is derived from tobacco, and the protein encoded by the nucleotide sequence still has the function or activity of regulating tobacco nicotine synthesis;

[0012] (c) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2;

[0013] (d) an amino acid sequence encoding a protein having nicotine synthesis-related function derived from tobacco, wherein the amino acid sequence shown in SEQ ID NO. 2 is replaced, deleted or added with at least one amino acid.

[0014] The present invention also discloses a tobacco nicotine synthesis regulatory protein NtbHLH25 encoded by the tobacco nicotine synthesis regulatory gene NtbHLH25, wherein the protein NtbHLH25 has an amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence with nicotine synthesis function derived from tobacco after substitution, deletion or addition of at least one amino acid as shown in SEQ ID NO.2.

[0015] The invention also discloses a recombinant expression vector, which contains the tobacco nicotine synthesis regulatory gene NtbHLH25.

[0016] Specifically, the recombinant expression vector includes pBWA(V)KS-RNAi-bHLH25.

[0017] The present invention also discloses a method for constructing the recombinant expression vector, comprising the following steps:

[0018] (1) Design the following recombinant expression vector primer sequences HLH25-F and HLH25-R, perform PCR amplification, and construct the target sequence fragment of the interference vector:

[0019] HLH25-F(+): cagtGGTCTCacaacCCAGATAGAGATAGTCGTCATGCTT;

[0020] HLH25-F(-):cgatGGTCTCacaggCTCATCTACTGCTTCTATTTTACAG;

[0021] HLH25-R(+):cagtGGTCTCagggcCTCATCTACTGCTTCTATTTTACAG;

[0022] HLH25-R(-): cagtGGTCTCatacaCCAGATAGAGATAGTCGTCATGCTT;

[0023] (2) The target fragment was digested with the pBWA(V)KS-RNAi vector by BsaI / Eco31I and ligated with T4_ligase. The ligated product was transformed into competent Escherichia coli, and positive clones were screened and identified to obtain the recombinant expression vector pBWA(V)KS-RNAi-bHLH25.

[0024] The invention also discloses a recombinant bacterium containing the tobacco nicotine synthesis regulating gene NtbHLH25.

[0025] The invention also discloses a transgenic tobacco plant containing the tobacco nicotine synthesis regulating gene NtbHLH25.

[0026] The present invention also discloses the application of the tobacco nicotine synthesis regulatory gene NtbHLH25, or the tobacco nicotine synthesis regulatory protein NtbHLH25, or the recombinant expression vector, or the recombinant bacteria, or the transgenic tobacco plant in the field of tobacco planting.

[0027] The present invention also discloses the application of the tobacco nicotine synthesis regulatory gene NtbHLH25, or the tobacco nicotine synthesis regulatory protein NtbHLH25, or the recombinant expression vector, or the recombinant bacteria, or the transgenic tobacco plant in regulating the nicotine content in tobacco.

[0028] The invention also discloses a method for regulating the nicotine content in tobacco, comprising the steps of transforming tobacco plants with the recombinant expression vector; or, infecting plant tissue culture tissue with the recombinant bacteria.

[0029] The present invention clones a new transcription factor gene NtbHLH25 from cultivated tobacco, and studies the role of the gene in tobacco synthesis. The detection of nicotine in genetic materials shows that the strain with reduced expression of the NtbHLH25 gene has significantly reduced nicotine content in tobacco leaves. Therefore, the NtbHLH25 gene and the NtbHLH25 protein have the function of regulating the nicotine content of tobacco. By reducing or knocking out the expression of the NtbHLH25 gene, the nicotine content in tobacco leaves can be reduced; on the contrary, by overexpressing the NtbHLH25 gene, the nicotine content in tobacco leaves can be increased. The nicotine synthesis regulating gene NtbHLH25 identified by the present invention and its encoding protein NtbHLH25 are related to nicotine accumulation in tobacco, providing new genetic targets and technical means for the regulation and application of nicotine content in tobacco, and have important application value in tobacco nicotine regulation.

[0030] The present invention further studies the role of tobacco NtbHLH25 gene in tobacco synthesis by constructing pBWA(V)KS-RNAi-bHLH25 recombinant expression vector. The present invention constructs genetic materials with reduced expression of NtbHLH25 gene after transforming tobacco plants with the vector, further verifies that NtbHLH25 gene and NtbHLH25 protein have the role of regulating tobacco nicotine content, and reduces or knocks out NtbHLH25 gene expression, which can reduce nicotine content in tobacco leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0032] Figure 1The expression characteristics of NtbHLH25 gene under exogenous methyl jasmonate treatment (50 μmol / L and 100 μmol / L);

[0033] Figure 2 To analyze the expression of NtbHLH25 gene in interference plants;

[0034] Figure 3 These are the results of the analysis of nicotine content in plants affected by NtbHLH25 interference. DETAILED DESCRIPTION

[0035] The present application will be further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments are briefly introduced as follows.

[0036] In the following embodiments of the present invention, the biomaterial information includes:

[0037] Tobacco varieties: Honghua Dajinyuan, a common cultivated tobacco, the seeds used in the examples were provided by the National Tobacco Gene Research Center;

[0038] Vector: pEASY-T1 Simple vector, purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0039] The pBWA(V)KS-RNAi vector was provided by Wuhan Boyuan Biotechnology Co., Ltd.

[0040] Strains:

[0041] Trans1-T1 chemically competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0042] LBA4404 Agrobacterium strain, a commonly used strain in biological experiments, is publicly available;

[0043] In the following embodiments of the present invention, the experimental reagent information includes:

[0044] RNA extraction kit was purchased from Beijing Codon Biotechnology Co., Ltd.;

[0045] Fluorescence quantitative PCR enzyme and PCR amplification enzyme were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0046] Reverse transcription kit and T4 ligase were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

[0047] Plant genome extraction kit and DNA purification kit were purchased from QIAGEN.

[0048] Example 1

[0049] This example clones and expresses the NtbHLH25 gene based on genetic engineering techniques.

[0050] 100 mg of roots of tobacco (Honghua Dajinyuan) in vigorous growth period were taken as samples, and fully ground in liquid nitrogen. Total RNA was extracted according to the instructions of RNA extraction kit, and then reverse transcribed into cDNA as a cloning template for later use.

[0051] The primer sequences designed to amplify the tobacco nicotine transporter gene NtbHLH25 are as follows:

[0052] NtbHLH25-F: 5'-ATGGACAATTCATCTCTAAGGTGGTTTTCA-3';

[0053] NtbHLH25-R: 5'-TTATGCTTGGGTCATTAGCCTCGCAGT-3';

[0054] The above-prepared cDNA was used as a template and the above-mentioned primers were used for PCR amplification. The reaction system was 25 μL, including: 1 μL of each of the above-mentioned upstream and downstream primers, 2 μL of template cDNA, 12.5 μL of FastPfu high-fidelity DNA polymerase, and 8.5 μL of ddH2O. The PCR amplification conditions were: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, for a total of 30 cycles; and 72°C final extension for 10 min; the PCR amplification was stored at 4°C for future use, or directly subjected to electrophoresis detection and analysis.

[0055] Purify the PCR amplification product according to the instructions of the gel recovery kit, and then connect the purified product to the pEASY-T1 vector. The specific connection system is as follows: 6 μL of DNA amplification product, 1 μL of pEASY-T1 vector; after mixing, connect at 25°C for 25 minutes.

[0056] The ligation product was transformed into E. coli competent cells. The specific transformation process is as follows:

[0057] Take out the competent cells from the -80℃ refrigerator, place them on ice to dissolve, add the ligation product to 50μL of Trans1-T1 competent cells, flick to mix, and place on ice for 30min;

[0058] Heat shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes; add 250 μL of LB (without antibiotics) equilibrated to room temperature, and culture at 37°C with shaking for 1 hour;

[0059] Take 8 μL of 500 mM IPTG and 40 μL of 20 mg / mL X-gal, mix them and evenly spread them on a LB solid plate (containing 60 μg / μL ampicillin), invert the culture dish, and culture at 37°C overnight.

[0060] After the white spots were selected for amplification and culture, the plasmid DNA was extracted, the recombinant plasmid was identified by plasmid PCR amplification, and the corresponding positive clones were sent for sequencing to obtain the coding sequence of the NtbHLH25 gene.

[0061] The results of sequencing analysis showed that the length of the coding region of the NtbHLH25 gene was 1017 nucleotides, as specifically shown in SEQ ID NO.1; after analyzing the gene, it was found that the amino acid sequence of the NtbHLH25 protein encoded by it was shown in SEQ ID NO.2.

[0062] SEQ ID NO.1:

[0063] ATGGACAATTCATCTCTAAGGTGGTTTTCAAGAACGGGGGAGGCTGGAGCCTATTACTCAAATGAGAAGAAATCTGTTGACGATTTCACCACTCAAAAATCCACAATTTCAGAGGATGGAGAAATTAGAGAAGTATCTCTATCTCCAGATAGAGATAGTCGTCATGCTTATTCTCGTTCGAATTATTTCAATCAGGAAAACAAAAGTAAAGTCGCCAAGTTCGATTTTTCCCTCATTACCCCTTTGGAAAATCAGCTAAAATGAATACTAATATGCTTTCATTTACAAATAATGTGGATTCGGTCCCTAGTCAGCAAGTCTGTAAAATAGAAGCAGTAGATGAGATGTACTTGTGTGATTCAGATCACAAGTATTTTCAATTTCTGAATTCTAAAAGTGGCTACGATCAAAAGGATAGCCAAGCCACGAAATTGGGACAAGGTCAGAAAAGAGAGAGACTTCCTTCAG TTGATCAACATCACATTATTGCAGAGAGGAAGCGCCGAGAAAAACTCAGCCAGAGGTTCGTCACACTTTCAGCAATTCTTCCGGGTCTTAAAAAGGTGGACAAAGCTTCAATTCTTGAGCAAGCAATCAGACATGTGAAAGAGCTTAAGGAAAAAGTGCAACAATTGGAAGAACAGGAAAAGACAAGGTCCGAGAAGTCTGTTATGTTTGTGAATAAATCTCAACTGGAAGAAGATGATCATTCCTCTTCTTCATCTGCTGAAAACAATAATTGCTCCTCTGGCGACGCAGATATTGAAGTAAGATTCTCAGATAAAAATGTATTGATCAGAATAACCTGTGAGAGGCGCAATGCCTTCGTCCAAAACATCCATAACGAAATAGAGAAACTTCATCTTTCTATTGTTCAAAGCTGTATGTCATTTGGGAAACATGCCATTGATGTCACTGTTGTCGCTCAGATGGATGAAAGTTTCTGCATGACGCTAAAGGATCTTGTGAAGCATGTTCGAATTGTCACTGCGAGGCTAATGACCCAAGCATAA.

[0064] SEQ ID NO.2:

[0065] MDNSSLRWFSRTGEAGAYYSNEKKSVDDFTTQKSTISEDGEIREVSLSPDRDSRHAYSRSNYFNQENKSKVAKFDFSLITPLENQPKMNTNMLSFTNNVDSVPSQQVCKIEAVDEMYLCDSDHKYFQFLNSKSGYDQKDSQATKLGQGQKRERSVDQHHIIAERKRR EKLSQRFVTLSAILPGLKKVDKASILEQAIRHVKELKEKVQQLEEQEKTRSEKSVMFVNKSQLEEDDHSSSSSAENNNCSSGDADIEVRFSDKNVLIRITCERRNAFVQNIHNEIEKLHLSIVQSCMMSFGKHAIDVTVVAQMDESFCMTLKDLVKHVRIVTARLMTQA.

[0066] Example 2

[0067] In this example, tobacco seedlings were further treated with methyl jasmonate, RNA was extracted and reverse transcribed, and the expression pattern of the NtbHLH25 gene was analyzed by fluorescent quantitative PCR.

[0068] Tobacco seeds were placed in a culture dish and cultured with Hoagland solution (light / dark=18 / 6h, 23℃~28℃). After germination for 2 weeks, the seedlings were transplanted and immersed in a solution containing methyl jasmonate, and methyl jasmonate of corresponding concentration was sprayed on the leaves for 5h. The samples were collected, quickly frozen with liquid nitrogen, and placed in a -80℃ refrigerator. After quick freezing with liquid nitrogen, they were placed in a -80℃ refrigerator and stored for later use.

[0069] In this embodiment, the specific treatment method for the experimental group is as follows: 50 μmol / L and 100 μmol / L of methyl jasmonate MeJA; at the same time, a control group is set up, and the tobacco seedlings in the control group are treated with 1% (v / v) DMSO solution.

[0070] The treated tobacco seedling materials were placed in 1.5 mL centrifuge tubes, quick-frozen in liquid nitrogen, and stored at -80°C for later use.

[0071] Extract RNA from the stored materials, synthesize cDNA using a reverse transcription kit (refer to the kit instructions for operation), and perform fluorescence quantitative PCR detection using the tobacco NtGAPDH gene as an internal reference. During the detection, the primer sequences are designed as follows:

[0072] Fluorescence quantitative primers for detecting NtbHLH25 gene, the primer sequences are:

[0073] NtbHLH25-qF: 5'-GAAAAACTCAGCCAGAGGTTCGTCA-3',

[0074] NtbHLH25-qR: 5'-CTCGGACCTTGTCTTTTCCTGTTCT-3';

[0075] Tobacco NtGAPDH gene primers are:

[0076] NtGAPDH-F: 5'-TGGGTGTCAACGAGAAGGAA-3',

[0077] NtGAPDH-F: 5'-TCTGGGTGGCAGTAAGGGA-3';

[0078] The specific fluorescence quantitative PCR reaction system is 15 μL, including 1 μL of upstream and downstream primers, 2 μL of template cDNA, 7.5 μL of qPCR SuperMix, and ddH 2 O 3.5 μL. The reaction conditions were as follows: first step pre-denaturation, 95°C for 10 s; second step PCR reaction, 95°C for 5 s, 60°C for 30 s, 39 cycles; third step melting curve.

[0079] Each sample was biologically replicated three times. -△△CT The relative gene expression differences were analyzed by the method. The results are shown in the attached Figure 1 The analysis results showed that, compared with the control group, the relative expression of NtbHLH25 gene was up-regulated by jasmonic acid.

[0080] Above Figure 1 The results of fluorescence quantitative analysis showed that both 50uM MeJA and 100MeJA could significantly induce the up-regulation of tobacco NtbHLH25 gene. Existing research results show that MeJA treatment can induce the expression of nicotine synthesis genes and the synthesis of nicotine.

[0081] Example 3

[0082] In order to further verify the function of tobacco NtbHLH25 gene in tobacco nicotine synthesis, this example further constructed a pBWA(V)KS-RNAi-bHLH25 expression vector for knocking down the NtbHLH25 gene.

[0083] According to the NtbHLH25 gene coding sequence known in Example 1, according to the RNAi principle, a target site of 201 bp in length was selected in the coding region of the NtbHLH25 gene, and the primer sequence of the recombinant expression vector was designed as follows:

[0084] bHLH25-F(+): cagtGGTCTCacaacccagatagagatagtcgtca;

[0085] bHLH25-F(-): cgatGGTCTCacaggctcatctactgcttctattt;

[0086] loop(+): cgatGGTCTCacctgcaggtctagtttttct;

[0087] loop(-): cgatGGTCTCagcccgggctctgtaactatc;

[0088] bHLH25-R(+): cagtGGTCTCagggcctcatctactgcttctattt;

[0089] bHLH25-R(-): cagtGGTCTCatacaccagatagagatagtcgtca.

[0090] The above three pairs of primers were used to perform three 50 μl PCR reactions, including: 1 μL of each of the above upstream and downstream primers, 2 μL of DNA template, 25.5 μL of FastPfu high-fidelity DNA polymerase, and 20.5 μL of ddH2O. The PCR amplification conditions were: 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min, for a total of 30 cycles; and 72°C final extension for 10 min; to amplify the target fragment to construct the interference vector. The competent Escherichia coli was transformed, and after the vector construction was confirmed by PCR, the plasmid was extracted to obtain the recombinant pBWA(V)KS-RNAi-bHLH25 expression vector for reducing tobacco NtbHLH25.

[0091] Example 4

[0092] The pBWA(V)KS-RNAi-bHLH25 expression vector constructed in Example 3 was transformed into Agrobacterium by electroporation, and then transformed into tobacco plants to create transgenic plants with down-regulated NtbHLH25 gene.

[0093] Agrobacterium transformation

[0094] Take out the Agrobacterium competent cells from the -80°C refrigerator, freeze and thaw on ice, and add 6 μL of the pBWA(V)KS-RNAi-bHLH25 expression vector prepared in Example 3 when about to thaw, and gently tap to mix; place the mixture in a pre-cooled electroporation cup and place on ice for 5 minutes.

[0095] The parameters of the electroporator were adjusted to: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω; then, water droplets on the outer wall of the electroporator cup were cleaned off with absorbent paper, and the electroporator cup was placed in the electric shock tank for 5 ms.

[0096] Quickly add 800 μL of YEB liquid culture medium preheated to 28°C, and shake at 220 rpm and 28°C for 3 h to recover.

[0097] Then centrifuge the bacterial solution at 4500rpm for 1min, discard half of the supernatant, resuspend and evenly apply on YEB solid medium containing Rif (100μg / mL), Str (50μg / mL) and Kan (50μg / mL), and culture inverted at 28℃ for about 2-3 days until a single colony is formed.

[0098] Pick a single colony, expand it, and perform PCR identification on the bacterial solution. The correct positive clone strain is the correctly transformed engineered bacteria.

[0099] Transformed tobacco plants

[0100] Leaves of sterile tobacco seedlings grown for about one month were taken and processed into leaf disks with a diameter of 0.5 cm using a hole puncher. The processed leaf disks were pre-cultured on MS solid culture medium for 3 days.

[0101] The transformed Agrobacterium engineered bacteria prepared above were cultured to about OD600=0.6, centrifuged at 4000 rpm for 5 min to collect the bacteria, and then suspended in 30 mL of MS liquid culture medium.

[0102] Then the pre-cultured leaf discs were placed in the bacterial solution and infected for 10 minutes.

[0103] Use sterile filter paper to absorb the excess bacterial liquid around the leaf disc after infection, and culture it in the dark on a solid culture medium of MS+6-BA (2 mg / L)+NAA (0.5 mg / L) for 3 days.

[0104] The leaf disc was washed with sterile water containing Cef (400 mg / L), and the excess liquid was removed with sterile filter paper. The leaf disc was transferred to MS solid screening medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L) and Kan (50 mg / L) and cultured at 28°C under illumination.

[0105] When the adventitious buds grew to 0.5 cm, they were transferred to MS solid medium containing Cef (200 mg / L) and Kan (50 mg / L) for rooting.

[0106] After about one month of growth, take a small amount of leaves and extract DNA according to the instructions of the plant genome extraction kit. Use PCR amplification, cloning and sequencing methods to detect positive transgenic strains.

[0107] In this embodiment, the specific identification method is:

[0108] The following detection primers were designed to identify positive transformed plants:

[0109] NtHLH25-JF:5'-GACGCACAATCCCACTATCC-3';

[0110] NtHLH25-JR:5'-CTCATCTACTGCTTCTATTTTACAG-3'.

[0111] PCR amplification was performed using the DNA template of the T0 transgenic strain (the system was the same as in Example 1); the PCR conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 40 s, for a total of 30 cycles; and final extension at 72°C for 10 min;

[0112] The PCR amplification was performed on agarose gel electrophoresis to detect the target detection fragment of the recombinant pBWA(V)KS-RNAi-bHLH25 vector, which was determined to be a positive transformed plant, and then different transgenic lines with down-regulated NtbHLH25 gene were obtained.

[0113] Example 5

[0114] This example is based on the positive transformed plants determined in the above Example 4, and tests are performed on different transgenic lines R1-R5 in which the NtbHLH25 gene is down-regulated.

[0115] RNA was extracted from positive plants R1-R5, reverse transcribed into cDNA, and then the expression level of tobacco NtbHLH25 gene in positive plants was analyzed by real-time quantitative PCR. The results are shown in the attached figure. Figure 2The analysis results showed that the expression level of tobacco NtbHLH25 gene was significantly reduced in RNAi plants compared with wild-type tobacco.

[0116] The T0 plants of wild type and transgenic lines R2, R4, and R5 were transplanted into pots and cultured in the greenhouse for about 8 days. The leaves were collected and analyzed for nicotine content using ultra-high performance liquid chromatography. The results are shown in the attached figure. Figure 3 The results showed that the nicotine content in the leaves of RNAi plants was lower than that of wild-type plants. This result also showed that reducing the expression level of tobacco NtbHLH25 gene can effectively reduce the nicotine content of tobacco.

[0117] In summary, the present invention can regulate the nicotine content of tobacco by regulating the expression level of the NtbHLH25 gene, providing technical means and theoretical basis for the regulation of the nicotine content of tobacco and quality improvement. Obviously, the above embodiments are only examples for clear explanation, and are not limitations on the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. And the obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. A tobacco nicotine synthesis regulatory gene NtbHLH25, characterized in that: The nucleotide sequence has at least one of the following characteristics (a)-(d): (a) the nucleotide sequence shown in SEQ ID NO.1; (b) a nucleotide sequence in which one or more bases of the nucleotide sequence shown in SEQ ID NO.1 are deleted, substituted or inserted, and the nucleotide sequence is derived from tobacco, and the protein encoded by the nucleotide sequence still has the function or activity of regulating tobacco nicotine synthesis; (c) a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; (d) an amino acid sequence encoding a protein having nicotine synthesis-related function derived from tobacco, wherein the amino acid sequence shown in SEQ ID NO. 2 is replaced, deleted or added with at least one amino acid.

2. A tobacco nicotine synthesis regulatory protein NtbHLH25 encoded by the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1, characterized in that: The protein NtbHLH25 has an amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence as shown in SEQ ID NO.2 with substitution, deletion or addition of at least one amino acid and derived from tobacco and having nicotine synthesis function.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector includes pBWA(V)KS-RNAi-bHLH25.

5. A method for constructing a recombinant expression vector as claimed in claim 3 or 4, characterized in that: The steps include: (1) Design the following recombinant expression vector primer sequences HLH25-F and HLH25-R, perform PCR amplification, and construct the target sequence fragment of the interference vector: HLH25-F(+): cagtGGTCTCacaacCCAGATAGAGATAGTCGTCATGCTT; HLH25-F(-):cgatGGTCTCacaggCTCATCTACTGCTTCTATTTTACAG; HLH25-R(+):cagtGGTCTCagggcCTCATCTACTGCTTCTATTTTACAG; HLH25-R(-): cagtGGTCTCatacaCCAGATAGAGATAGTCGTCATGCTT; (2) The target fragment was digested with the pBWA(V)KS-RNAi vector by BsaI / Eco31I and ligated with T4_ligase. The ligated product was transformed into competent Escherichia coli, and positive clones were screened and identified to obtain the recombinant expression vector pBWA(V)KS-RNAi-bHLH25.

6. A recombinant bacterium containing the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1.

7. A transgenic tobacco plant containing the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1.

8. Use of the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1, or the tobacco nicotine synthesis regulatory protein NtbHLH25 according to claim 2, or the recombinant expression vector according to claim 3 or 4, or the recombinant bacteria according to claim 6, or the transgenic tobacco plant according to claim 7 in the field of tobacco cultivation.

9. Use of the tobacco nicotine synthesis regulatory gene NtbHLH25 according to claim 1, or the tobacco nicotine synthesis regulatory protein NtbHLH25 according to claim 2, or the recombinant expression vector according to claim 3 or 4, or the recombinant bacteria according to claim 6, or the transgenic tobacco plant according to claim 7 in regulating the nicotine content in tobacco.

10. A method for regulating nicotine content in tobacco, characterized in that: The method comprises the steps of transforming tobacco plants with the recombinant expression vector of claim 3 or 4; or the step of infecting plant tissue culture tissue with the recombinant bacteria of claim 6.

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