The tobacco nicotine synthesis regulatory gene NtbHLH25 and its encoded protein and applications

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to a tobacco nicotine synthesis regulatory gene NtbHLH25 and its encoded tobacco nicotine synthesis regulatory protein NtbHLH25, and further disclosing its applications. This invention cloned a novel transcription factor gene, NtbHLH25, from cultivated tobacco and studied its role in tobacco synthesis. Nicotine detection in genetic material showed that lines with reduced NtbHLH25 gene expression had significantly lower nicotine content in tobacco leaves. The nicotine synthesis regulatory gene NtbHLH25 and its encoded protein NtbHLH25 identified in this invention are associated with nicotine accumulation in tobacco, providing new genetic targets and technical means for the regulation and application of tobacco nicotine content, and have significant application value in tobacco nicotine regulation.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a tobacco nicotine synthesis regulatory gene NtbHLH25 and its encoded tobacco nicotine synthesis regulatory protein NtbHLH25, and further disclosing its applications. Background Technology

[0002] Nicotine is an endogenous defensive alkaloid formed in tobacco during its long evolutionary process. It is the most abundant alkaloid in tobacco, accounting for over 95% of the total alkaloids. As a secondary metabolite, nicotine plays a protective role in tobacco, defending against insects and herbivores. When insects or herbivores consume tobacco leaves, nicotine synthesis mediated by jasmonic acid injury signals is immediately initiated in the roots and subsequently transported to the tobacco leaves to combat various attacks. Moreover, the level of nicotine in tobacco is closely related to the quality of tobacco leaves. In addition, due to its strong biological activity and pharmacological effects, nicotine plays an important role in the treatment of Parkinson's disease and various inflammatory diseases.

[0003] Studies show that nicotine biosynthesis is a complex regulated process, influenced by a variety of factors, including the ecological environment, cultivation practices, plant hormones, genetic factors, and the plant's own growth and development stages. It involves the expression and genetic regulation of numerous genes. Therefore, elucidating the molecular mechanisms of nicotine biosynthesis and accumulation in tobacco is one of the key areas of current tobacco biology research.

[0004] With the rapid development of biotechnology and genomics, significant progress has been made in the study of the molecular regulatory mechanisms of nicotine synthesis. Currently, key genes in the nicotine biosynthetic metabolic pathway have been identified, and researchers' understanding of the metabolic pathway is gradually becoming clearer. For example, ornithine undergoes decarboxylation catalyzed by ornithine decarboxylase (ODC), or arginine is converted to putrescine by arginine decarboxylase (ADC). S-adenosylmethionine and putrescine are converted to N-methylputrescine and S-adenosylhomocysteine ​​by putrescine N-methyltransferase (PMT), which is a key rate-limiting enzyme in nicotine synthesis. Quinolinate phosphoribosyltransferase (QPT) plays an important regulatory role in the synthesis of pyridine alkaloids. Furthermore, 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 as well as in the defense against pests and diseases. 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 the transporter protein MATE1.

[0005] Studies have also found that nicotine biosynthesis is regulated by multiple transcription factors. Among the identified factors is MYC1, a key transcription factor in the jasmonic acid signaling pathway. Inhibiting the expression of MYC1a and MYC1b using RNA interference (RNAi) significantly reduces nicotine levels in tobacco BY-2 cells when MYC1a and MYC1b are knocked down, indicating that MYC1a and MYC1b are positive regulators of nicotine synthesis. Furthermore, ERF189, a member of the ERF transcription factor subfamily, positively regulates the expression of putrescine N-methyltransferase (PMT) genes by binding to GCC boxes and G-box elements in gene promoter regions, thereby affecting nicotine synthesis. Currently, no other novel genes regulating nicotine synthesis have been found in cultivated tobacco besides these known regulators.

[0006] Therefore, further exploration of suitable nicotine synthesis transcription factors is of great 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 its encoded tobacco nicotine synthesis regulatory protein NtbHLH25, both of which are related to nicotine accumulation in tobacco, providing 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] To solve the above-mentioned technical problems, the present invention provides a tobacco nicotine synthesis regulatory gene NtbHLH25, the nucleotide sequence of which has 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 with one or more bases deleted, substituted or inserted, as shown in SEQ ID NO.1, and the nucleotide sequence is derived from tobacco and the encoded protein still has the function or activity of regulating nicotine synthesis in tobacco.

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

[0013] (d) An amino acid sequence that is derived from tobacco and has the function of a nicotine synthesis-related protein, wherein the amino acid sequence shown in SEQ ID NO.2 has been substituted, deleted or added 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 as shown in SEQ ID NO.2 with at least one amino acid substituted, deleted or added and derived from tobacco having nicotine synthesis function.

[0015] The present invention also discloses a recombinant expression vector containing 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 primer sequences HLH25-F and HLH25-R for recombinant expression vector, 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 obtained target fragment and the pBWA(V)KS-RNAi vector were digested with BsaI / Eco31I and ligated with T4_ligase. The ligation product was transformed into competent E. coli cells, and positive clones were screened by identification to obtain the recombinant expression vector pBWA(V)KS-RNAi-bHLH25.

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

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

[0026] This 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 cultivation.

[0027] This 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 present invention also discloses a method for regulating the nicotine content in tobacco, including the step of transforming the recombinant expression vector into tobacco plants; or the step of infecting plant tissue culture tissues with the recombinant bacteria.

[0029] This invention cloned a novel transcription factor gene, NtbHLH25, from cultivated tobacco and investigated its role in tobacco synthesis. Nicotine content analysis of the genetic material showed that strains with reduced NtbHLH25 gene expression had significantly lower nicotine content in tobacco leaves. Therefore, the NtbHLH25 gene and its protein regulate tobacco nicotine content. Reducing or knocking out NtbHLH25 gene expression lowers nicotine content in tobacco leaves; conversely, overexpressing NtbHLH25 increases nicotine content. The nicotine synthesis-regulating gene NtbHLH25 and its encoded protein NtbHLH25 identified in this invention are associated with nicotine accumulation in tobacco, providing a new genetic target and technical means for the regulation and application of tobacco nicotine content, and have significant application value in tobacco nicotine regulation.

[0030] This invention further investigated the role of the tobacco NtbHLH25 gene in tobacco synthesis by constructing the pBWA(V)KS-RNAi-bHLH25 recombinant expression vector. By transforming this vector into tobacco plants, this invention obtained genetic material with decreased NtbHLH25 gene expression, further verifying that the NtbHLH25 gene and NtbHLH25 protein regulate nicotine content in tobacco. Reducing or knocking out NtbHLH25 gene expression can decrease the nicotine content in tobacco leaves. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

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

[0033] Figure 2 To interfere with the analysis of NtbHLH25 gene expression levels in plants;

[0034] Figure 3 The results of the analysis of nicotine content in plants affected by NtbHLH25 interference are presented. Detailed Implementation

[0035] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the following is a brief introduction to some biological materials, experimental reagents, experimental equipment and other situations involved in the embodiments.

[0036] In the following embodiments of the present invention, information related to biomaterials includes:

[0037] Tobacco variety: Red Flower Dajinyuan, a common cultivated tobacco. The seeds used in the examples were provided and preserved by the National Tobacco Gene Research Center.

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

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

[0040] Strains:

[0041] Trans1-T1 chemocompetent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0042] LBA4404 Agrobacterium strain is a commonly used strain in biological experiments and is publicly available.

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

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

[0045] Real-time PCR enzyme and PCR amplification enzyme were purchased from Beijing TransGen Biotech Co., Ltd.

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

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

[0048] Example 1

[0049] This embodiment uses genetic engineering techniques to clone and express the NtbHLH25 gene.

[0050] Take 100mg of the root of the long-term tobacco (Honghua Dajinyuan) as a sample, grind it thoroughly in liquid nitrogen, extract total RNA according to the instructions of the RNA extraction kit, and then reverse transcribe it into cDNA as a cloning template for later use.

[0051] The primer sequences designed for amplifying the tobacco nicotine transport protein gene NtbHLH25 are as follows:

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

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

[0054] Using the prepared cDNA as a template, PCR amplification was performed using the aforementioned primers. The reaction volume was 25 μL, comprising: 1 μL each of the 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℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 30 cycles; followed by a final extension at 72℃ for 10 min. PCR amplification was stored at 4℃ for later use, or directly analyzed by electrophoresis.

[0055] Purify the PCR amplification product according to the instructions of the gel extraction kit, and then ligate the purified product to the pEASY-T1 vector. The specific ligation system is as follows: 6 μL of DNA amplification product and 1 μL of pEASY-T1 vector; after mixing, ligate at 25℃ for 25 min.

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

[0057] Remove competent cells from the -80℃ freezer, place them on ice to thaw, add the ligation product to 50 μL of Trans1-T1 competent cells, gently tap to mix, and incubate on ice for 30 min.

[0058] Heat shock in a 42℃ water bath for 30 seconds, then immediately place on ice for 2 minutes; add 250 μL of LB (antibiotic-free) equilibrated to room temperature, and incubate at 37℃ 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 spread them evenly on an LB solid plate (containing 60 μg / μL ampicillin). Invert the culture dish and incubate at 37°C overnight.

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

[0061] Sequencing analysis results show that the coding region of the NtbHLH25 gene is 1017 nucleotides long, as shown in SEQ ID NO.1; analysis of this gene shows that the amino acid sequence of the NtbHLH25 protein it encodes is shown in SEQ ID NO.2.

[0062] SEQ ID NO.1:

[0063] ATGGACAATTCATCTCTAAGGTGGTTTTCAAGAACGGGGGAGGCTGGAGCCTATTACTCAAATGAGAAGAAATCTGTTGACGATTTCACCACTCAAAAATCCACAATTTCAGAGGATGGAGAAATTAGAGAAGTATCTCTATCTCCAGATAGAGATAGTCGTCATGCTTATTCTCGTTCGAATTATTTCAATCAGGAAAACAAAAGTAAAGTCGCCAAGTTCGATTTTTCCCTCATTACCCCTTTGGAAAATCAGCCTAAAATGAATACTAATATGCTTTCATTTACAAATAATGTGGATTCGGTCCCTAGTCAGCAAGTCTGTAAAATAGAAGCAGTAGATGAGATGTACTTGTGTGATTCAGATCAAGTATTTTCAATTTCTGAATTCTAAAAGTGGCTACGATCAAAAGGATAGCCAAGCCACGAAATTGGGACAAGGTCAGAAAAGAGAGAGACTTCCTTCAG TTGATCAACATCACATTATTGCAGAGAGGAAGCGCCGAGAAAAACTCAGCCAGAGGTTCGTCACACTTTCAGCAATTCTTCCGGGTCTTAAAAAGGTGGACAAAGCTTCAATTCTTGAGCAAGCAATCAGACATGTGAAAGAGCTTAAGGAAAAAGTGCAACAATTGGAAGAACAGGAAAAGACAAGGTCCGAGAAGTCTGTTATGTTTGTGAATAAATCTCAACTGGAAGAAGATGATCATTCCTCTTCTTCATCTGCTGAAAACAATAATTGCTCCTCTGGCGACGCAGATATTGAAGTAAGATTCTCAGATAAAAATGTATTGATCAGAATAACCTGTGAGAGGCGCAATGCCTTCGTCCAAAACATCCATAACGAAATAGAGAAACTTCATCTTTCTATTGTTCAAAGCTGTATGATGTCATTTGGGAAACATGCCATTGATGTCACTGTTGTCGCTCAGATGGATGAAAGTTTCTGCATGACGCTAAAGGATCTTGTGAAGCATGTTCGAATTGTCACTGCGAGGCTAATGACCCAAGCATAA.

[0064] SEQ ID NO.2:

[0065] MDNSSLRWFSRTGEAGAYYSNEKKSVDDFTTQKSTISEDGEIREVSLSPDRDSRHAYSRSNYFNQENKSKVAKFDFSLITPLENQPKMNTNMLSFTNNVDSVPSQQVCKIEAVDEMYLCDSDHKYFQFLNSKSGYDQKDSQATKLGQGQKRERSVDQHHIIAERKRR EKLSQRFVTLSAILPGLKKVDKASILEQAIRHVKELKEKVQQLEEQEKTRSEKSVMFVNKSQLEEDDHSSSSSAENNNCSSGDADIEVRFSDKNVLIRITCERRNAFVQNIHNEIEKLHLSIVQSCMMSFGKHAIDVTVVAQMDESFCMTLKDLVKHVRIVTARLMTQA.

[0066] Example 2

[0067] In this embodiment, tobacco seedlings were treated with methyl jasmonate, RNA was extracted and reverse transcribed, and the expression pattern of the NtbHLH25 gene was analyzed using quantitative real-time PCR.

[0068] Tobacco seeds were placed in petri dishes 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. The leaves were also sprayed with the corresponding concentration of methyl jasmonate for 5 hours. Samples were collected, flash-frozen in liquid nitrogen, and then stored in a -80℃ freezer for later use.

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

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

[0071] RNA was extracted from the preserved material, and cDNA was synthesized using a reverse transcription kit (following the kit instructions). The tobacco NtGAPDH gene was used as an internal control for quantitative real-time PCR detection. The primer sequences for detection are as follows:

[0072] The primers for quantitative real-time detection of the NtbHLH25 gene are as follows:

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

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

[0075] The primers for the tobacco NtGAPDH gene are:

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

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

[0078] The specific reaction system for quantitative real-time PCR was 15 μL, including 1 μL each of forward and reverse primers, 2 μL of template cDNA, 7.5 μL of qPCR SuperMix, and 3.5 μL of ddH2O. The reaction conditions were as follows: Step 1: Pre-denaturation, 95℃ for 10 s; Step 2: PCR reaction, 95℃ for 5 s, 60℃ for 30 s, 39 cycles; Step 3: Melting curve.

[0079] Each sample was biologically replicated three times, using 2 -△△CT The method analyzed the relative differences in gene expression, and the results are shown in the appendix. Figure 1 As shown in the figure. The analysis results show that, compared with the control group, the relative expression level of the NtbHLH25 gene was upregulated by jasmonic acid.

[0080] The above Figure 1 The quantitative fluorescence analysis results showed that both 50 μM MeJA and 100 μM MeJA significantly induced the upregulation of the tobacco NtbHLH25 gene. Existing research indicates that MeJA treatment can induce the expression of nicotine synthesis genes and the synthesis of large amounts of nicotine.

[0081] Example 3

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

[0083] Based on the NtbHLH25 gene coding sequence known in Example 1, and according to the principles of RNAi, a 201 bp target site was selected in the coding region of the NtbHLH25 gene, and the primer sequences for the recombinant expression vector were 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] Using the three primer pairs described above, three 50 μl PCR reactions were performed, each consisting of: 1 μL of each of the 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℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 30 cycles; followed by a final extension at 72℃ for 10 min. The target fragment was amplified to construct the interference vector. After transforming into competent *E. coli* cells and confirming successful vector construction by PCR, the plasmid was extracted to obtain the recombinant pBWA(V)KS-RNAi-bHLH25 expression vector for reducing NtbHLH25 in tobacco.

[0091] Example 4

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

[0093] Transformation of Agrobacterium

[0094] Remove Agrobacterium competent cells from the -80°C freezer, freeze-thaw them on ice, and add 6 μL of the pBWA(V)KS-RNAi-bHLH25 expression vector prepared in Example 3 when they are about to thaw. Gently mix the mixture and place it in a pre-cooled electric transfer cup on ice for 5 min.

[0095] Adjust the parameters of the electro-polarizer to: voltage 2.5kV, capacitance 25μF, and resistance 200Ω; then use absorbent paper to remove the water droplets from the outer wall of the electro-polarizer cup, place the electro-polarizer cup into the electro-polarizer tank, and electro-polarize for 5ms.

[0096] Quickly add 800 μL of YEB liquid culture medium preheated to 28°C, and revive by shaking at 220 rpm and 28°C for 3 hours.

[0097] The bacterial suspension was then centrifuged at 4500 rpm for 1 min, half of the supernatant was discarded, and the suspension was resuspended and evenly spread onto YEB solid medium containing Rif (100 μg / mL), Str (50 μg / mL), and Kan (50 μg / mL). The medium was then incubated upside down at 28°C for approximately 2–3 days until single colonies formed.

[0098] Select a single colony, expand it, and then perform PCR identification on the bacterial culture. The correctly identified positive clone strain is the engineered strain that has been successfully transformed.

[0099] Transform tobacco plants

[0100] Take leaves from sterile tobacco seedlings that have grown for about one month, and use a punch to process the leaves into leaf discs with a diameter of 0.5 cm. Pre-culture the processed leaf discs on MS solid medium for 3 days.

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

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

[0103] Use sterile filter paper to blot away excess bacterial solution around the leaf disc after infection, and then incubate in the dark for 3 days on MS medium containing 6-BA (2 mg / L) and NAA (0.5 mg / L).

[0104] Wash the leaf discs with sterile water containing Cef (400 mg / L), and blot away excess liquid with sterile filter paper. Transfer the leaf discs to MS solid selection medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L), and Kan (50 mg / L), and incubate at 28°C under light.

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

[0106] After about a month of growth, a small number of leaves were taken, and DNA was extracted according to the instructions of the plant genome extraction kit. Positive transgenic lines were detected by PCR amplification, cloning, and sequencing.

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

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

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

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

[0111] Using T0 generation transgenic line DNA template, PCR amplification was performed (system same as in Example 1); PCR conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 30 cycles; and then 72℃ final extension for 10 min.

[0112] PCR amplification was performed and detected by agarose gel electrophoresis. The target detection fragment of the recombinant pBWA(V)KS-RNAi-bHLH25 vector was detected, and the plants were identified as positive transgenic plants. Different transgenic lines with downregulated NtbHLH25 gene were then obtained.

[0113] Example 5

[0114] This embodiment is based on the positive transformant plants determined in the aforementioned embodiment 4, and tests are performed on the different transgenic lines R1-R5 with downregulated NtbHLH25 gene.

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

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

[0117] In summary, this invention can regulate the nicotine content of tobacco by controlling the expression level of the NtbHLH25 gene, providing a technical means and theoretical basis for the regulation of tobacco nicotine content and quality improvement. Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Reduce the regulatory genes for nicotine synthesis in tobacco. NtbHLH25 The application of the expression of [a specific ingredient] in reducing nicotine content in tobacco is characterized by [a specific ingredient]. The tobacco nicotine synthesis regulatory gene NtbHLH25 The nucleotide sequence is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.

1.

3. A gene for reducing nicotine synthesis as described in claim 1 NtbHLH25 The application of the encoded tobacco nicotine synthesis regulatory protein NtbHLH25 in reducing nicotine content in tobacco is characterized by, The amino acid sequence of the protein NtbHLH25 is shown in SEQ ID NO.

2.

4. The application of a recombinant expression vector in reducing nicotine content in tobacco, characterized in that, The recombinant expression vector contains the tobacco nicotine synthesis regulatory gene as described in claim 1. NtbHLH25; The recombinant expression vector is pBWA(V)KS-RNAi-bHLH25.

5. The application according to claim 4, characterized in that, The method for constructing the recombinant expression vector includes the following steps: (1) Design the following primer sequences HLH25-F and HLH25-R for recombinant expression vector, 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 obtained target fragment and pBWA(V)KS-RNAi vector were digested with BsaI / Eco31I and ligated with T4_ligase. The ligation product was transformed into competent E. coli cells. Positive clones were screened by identification to obtain the recombinant expression vector pBWA(V)KS-RNAi-bHLH25.

6. A method for reducing the nicotine content in tobacco, characterized in that, The step includes transforming the recombinant expression vector of claim 4 into tobacco plants.

Images