NtRAX2, a gene regulating nicotine synthesis in tobacco, 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 NtRAX2 and its encoded tobacco nicotine synthesis regulatory protein NtRAX2, and further disclosing its applications. This invention cloned a novel nicotine synthesis regulatory gene NtRAX2 from cultivated tobacco and studied the role of the tobacco NtRAX2 gene in tobacco synthesis. The nicotine synthesis regulatory gene NtRAX2 and its encoded protein NtRAX2 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 important 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 NtRAX2 and its encoded tobacco nicotine synthesis regulatory protein NtRAX2, 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] Therefore, the technical problem to be solved by the present invention is to provide a tobacco nicotine synthesis regulatory gene NtRAX2 and its encoded tobacco nicotine synthesis regulatory protein NtRAX2, 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 NtRAX2 and the tobacco nicotine synthesis regulatory protein NtRAX2 in the field of tobacco cultivation.

[0009] To solve the above-mentioned technical problems, the present invention provides a tobacco nicotine synthesis regulatory gene NtRAX2, whose nucleotide sequence 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, and whose 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 NtRAX2 encoded by the tobacco nicotine synthesis regulatory gene NtRAX2, wherein the protein NtRAX2 has the amino acid sequence shown in SEQ ID NO.2, or, as shown in SEQ ID NO.2, an amino acid sequence having at least one amino acid substituted, deleted, or added and derived from tobacco with nicotine synthesis function.

[0015] The present invention also discloses a recombinant expression vector containing the tobacco nicotine synthesis regulatory gene NtRAX2.

[0016] Specifically, the recombinant expression vector includes pBI121-35S:NtRAX2.

[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 OE_NtRAX2 F and OE_NtRAX2 R for PCR amplification to construct the target sequence fragment of the overexpression vector:

[0019] OE_NtRAX2 F:acgggggactctagaggatcc ATGGACTGCGAACGCCAGACAGCTA;

[0020] OE_NtRAX2 R:cgatcggggaaattcgagctcTTATTCTGTCCTATTTTCATCAAGA;

[0021] (2) pBI121 was digested with BamHI and SacI and ligated with the PCR product of step (1). The product was transformed and positive clones were screened to obtain the recombinant pBI121-35S:NtRAX2 plasmid for overexpressing the tobacco NtRAX2 gene.

[0022] The present invention also discloses a recombinant bacterium containing the tobacco nicotine synthesis regulatory gene NtRAX2.

[0023] The present invention also discloses a transgenic tobacco plant containing the tobacco nicotine synthesis regulatory gene NtRAX2.

[0024] This invention also discloses the application of the tobacco nicotine synthesis regulatory protein NtRAX2, or the tobacco nicotine synthesis regulatory gene NtRAX2, or the recombinant expression vector, or the recombinant bacteria, or the transgenic tobacco plant in the field of tobacco cultivation.

[0025] This invention also discloses the application of the tobacco nicotine synthesis regulatory protein NtRAX2, or the tobacco nicotine synthesis regulatory gene NtRAX2, or the recombinant expression vector, or the recombinant bacteria, or the transgenic tobacco plant in regulating the nicotine content in tobacco.

[0026] 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.

[0027] This invention cloned a novel nicotine synthesis regulating gene, NtRAX2, from cultivated tobacco and investigated its role in tobacco synthesis. Nicotine content analysis of the genetic material showed that overexpression of the NtRAX2 gene significantly increased nicotine content in tobacco leaves, demonstrating that the NtRAX2 gene regulates tobacco nicotine content. Reducing or knocking out NtRAX2 gene expression lowers nicotine content in tobacco leaves; conversely, overexpression of NtRAX2 increases nicotine content. The identified nicotine synthesis regulating gene NtRAX2 and its encoded protein NtRAX2 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 possessing significant application value in tobacco nicotine regulation.

[0028] This invention further investigated the role of the tobacco NtRAX2 gene in tobacco synthesis by constructing the pBI121-35S:NtRAX2 recombinant expression vector. Transforming tobacco plants with this recombinant vector pBI121-35S:NtRAX2 significantly increased the expression level of the NtRAX2 gene, resulting in a marked increase in both the gene and protein expression levels. Genetic materials with significantly increased NtRAX2 gene expression were obtained. Further analysis of the nicotine content in the genetic materials showed that the NtRAX2 overexpression lines exhibited a significant increase in nicotine content in tobacco leaves.

[0029] This invention utilizes the pBI121-35S:NtRAX2 recombinant expression vector to construct transgenic plants. Using Agrobacterium-mediated gene transformation, the pBI121-35S:NtRAX2 expression vector is transformed into Agrobacterium, and engineered bacteria for transformation are screened. These engineered bacteria are then used to infect plant tissue culture tissues. After tissue culture and identification, tobacco genetic material with enhanced NtRAX2 gene expression is obtained, which is of great significance for regulating nicotine content in tobacco. Attached Figure Description

[0030] 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...

[0031] Figure 1 The results of PCR detection for pBI121-35S:NtRAX2 positive transformed plants;

[0032] Figure 2 The results of NtRAX2 gene expression analysis in positively transformed plants;

[0033] Figure 3 The results show the nicotine content analysis in NtRAX2 overexpressing plants. Detailed Implementation

[0034] 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.

[0035] In the following embodiments of the present invention, information relating to biomaterials includes:

[0036] Tobacco variety: K326, a common cultivated tobacco. The seeds used in the following examples were provided and preserved by the National Tobacco Gene Research Center.

[0037] Vector: pEASY-T1 Simple vector, purchased from Beijing TransGen Biotech Co., Ltd.;

[0038] pBI121 vector: a conventional plant expression vector, preserved in our laboratory;

[0039] Trans1-T1 chemocompetent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0040] Agrobacterium strain LBA4404 is a commonly used strain in biological experiments and is publicly available.

[0041] In the following embodiments of the present invention, the experimental reagent information includes:

[0042] The RNA extraction kit was purchased from Beijing Codon Biotechnology Co., Ltd.

[0043] Real-time PCR enzyme and PCR amplification enzyme were purchased from Beijing TransGen Biotech Co., Ltd.

[0044] Reverse transcription kit and T4 ligase were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0045] Plant genome extraction kit and DNA purification kit were purchased from QIAGEN.

[0046] Example 1

[0047] This embodiment uses genetic engineering techniques to clone and express the NtRAX2 gene.

[0048] 100 mg of roots from the vigorous long-term tobacco (K326) was taken as a sample, thoroughly ground in liquid nitrogen, and total RNA was extracted according to the instructions of the RNA extraction kit. Then, the RNA was reverse transcribed into cDNA as a cloning template for later use.

[0049] The following primers were designed to amplify the coding sequence of the tobacco NtRAX2 gene:

[0050] NtRAX2-F: 5'-ATGGACTGCGAACGCCAGACAGCTA-3';

[0051] NtRAX2-R: 5'-TTATTCTGTCCTATTTTCATCAAGA-3';

[0052] Using the prepared cDNA as a template, PCR amplification was performed using the primers described above. The reaction system consisted of 25 μL: 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 amplified cDNA was stored at 4℃ for later use, or directly analyzed by electrophoresis.

[0053] Purify the PCR amplified product according to the instructions of the gel extraction kit, and then ligate the purified product into 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°C for 25 min.

[0054] The ligation product was then transformed into competent E. coli cells. The specific transformation process is as follows:

[0055] 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.

[0056] 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;

[0057] 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.

[0058] 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 NtRAX2 gene.

[0059] Sequencing analysis results show that the coding region of the NtRAX2 gene is 903 nucleotides long, as shown in SEQ ID NO.1; analysis of this gene shows that the amino acid sequence of the NtRAX2 protein it encodes is shown in SEQ ID NO.2.

[0060] SEQ ID NO.1:

[0061] ATGGACTGCGAACGCCAGACAGCTACCTCGATATCTCCTACGACGGGGAGAGCTCCTTGTTGTGACAAAGAAAATGTGAAAAGAGGGCCATGGTCTCCTGAAGAAGATGCAAAACTAAAAGAGTATATAGACAAATATGGTACTGGTGGAAACTGGATTGCTCTTCCACAAAAAGCTGGGCTGAGAAGATGTGGAAAGAGCTGCAGATTGAGATGGTTAAACTATCTTAGACCCAACATTAAACATGGAGAGTTTTCAGATGAAGACGACAAAGTAATTTGCAGCCTTTATGCTAAGATTGGAAGCAGATGGTCAATAATAGCTGCTCAGCTACCAGGCAGGACAGGCAATGATATCAAGAACTACTGGAATACCAAGCTCAAGAAGAAACTCATGCCTCTTGTCTCTTCACCTCAACAAATTAGGCCAATTCACCAAACTACATGTCATTCATCAATTTCACCTCTTCAAGCTAATTCATCCCTAGTTTCATCACTATATTCTTCTATATCACCAGCTCTTTCATTAGCAACAACAGCTCATAAATATACAAGTAGCCATTTTTATCCCACTTCAACAGCTACAAATTTCCCAGGCTATGAAACCAATATTATATCTACCCAATCAAGCAGTTTCTTGAGTACTTGTAGTTGTTCAAGTGGAGGTGAAAAATTGGTTAGCTATTTAAATGATGGGGTAGATGAGAAACAGAATAGTTCTAAGTTCTCAATTACAAATGGTAGTACTAATAAATATGCTGAAGAAAAGCCAAGTGGAAAGGGTTGTTATGAAGAGGAAAATCAATTAGAGTGTAGTTTGGAGGAGATTAAGCAGCTAATTAG CACTACTAGCATTTATAACAACTACACCAACTTCTTTCTTGATGAAAATAGGACAGAATAA。

[0062] SEQ ID NO.2:

[0063] MDCERQTATSISPTTGRAPCCDKENVKRGPWSPEEDAKLKEYIDKYGTGGNWIALPQKAGLRRCGKSCRLRWLNYLRPNIKHGEFSDEDDKVICSLYAKIGSRWSIIAAQLPGRTGNDIKNYWNTKLKKKLMPLVSSPQQIRPIHQTTCH SSISPLQANSSLVSSLYSSISPALSLATTAHKYTSSHFYPTSTATNFPGYETNIISTQSSSFLSTCSCSSGGEKLVSYLNDGVDEKQNSSKFSITNGSTNKYAEEKPSGKGCYEEENQLECSLEEIKQLISTTSIYNNYTNFFLDENRTE.

[0064] Example 2

[0065] This embodiment further constructs the pBI121-35S:NtRAX2 expression vector with knockdown of the NtRAX2 gene to further verify the function of the tobacco NtRAX2 gene in tobacco nicotine synthesis.

[0066] Based on the NtRAX2 gene coding sequence known in Example 1, the amplified gene coding sequence was subcloned into the pBI121 vector, replacing the original GUS gene in the vector, to form a recombinant plasmid that drives NtRAX2 expression under 35S. The primer sequences for the recombinant expression vector are designed as follows:

[0067] OE_NtRAX2 F:acgggggactctagaggatcc ATGGACTGCGAACGCCAGACAGCTA;

[0068] OE_NtRAX2 R:cgatcggggaaattcgagctcTTATTCTGTCCTATTTTCATCAAGA.

[0069] The PCR reaction in a 25 μl system includes: 1 μL each of the above upstream and downstream primers, 2 μL of template cDNA, 12.5 μL of FastPfu high-fidelity DNA polymerase, and 8.5 μL of ddH2O.

[0070] The PCR reaction conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 10 min. The target fragment was amplified from cDNA to obtain the target sequence fragment for constructing the interference vector.

[0071] Then, pBI121 was digested with BamHI and SacI, purified and recovered, and ligated with recombinase to the PCR product. The ligation product was transformed into competent E. coli cells, and positive clones were screened by identification to obtain the recombinant pBI121-35S:NtRAX2 plasmid for overexpressing the tobacco NtRAX2 gene.

[0072] Example 3

[0073] In this embodiment, the pBI121-35S:NtRAX2 expression vector constructed in Example 2 was used to transform Agrobacterium tumefaciens using electroporation, and then transformed into tobacco plants to create transgenic plants overexpressing the NtRAX2 gene.

[0074] Transformation of Agrobacterium

[0075] Remove Agrobacterium competent cells from the -80℃ freezer, freeze-thaw them on ice, and add 6 μL of the pBI121-35S:NtRAX2 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.

[0076] Adjust the parameters of the electro-polarizer to: voltage 2.5kV, capacitance 25μF, 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;

[0077] Quickly add 800 μL of YEB liquid medium preheated to 28°C, and revive by shaking at 220 rpm and 28°C for 3 hours;

[0078] Then, centrifuge the bacterial suspension at 4500 rpm for 1 min, discard half of the supernatant, resuspend the suspension, and spread it evenly on YEB solid medium containing Rif (100 μg / mL), Str (50 μg / mL), and Kan (50 μg / mL). Incubate at 28°C upside down for about 2 to 3 days until single colonies are formed.

[0079] 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.

[0080] Transform tobacco plants

[0081] 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.

[0082] 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.

[0083] Then, the pre-cultured leaf discs were placed in the bacterial solution and incubated for 10 minutes.

[0084] 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).

[0085] Wash the leaf discs with sterile water containing Cef (400 mg / L) and absorb 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.

[0086] 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.

[0087] After about one 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 using PCR amplification, cloning, and sequencing. The specific identification method was as follows:

[0088] The following detection primers were designed to identify positive transformants:

[0089] NtRAX2-JF: 5'-ATCCTTCGCAAGACCCTTCCTCTAT-3';

[0090] NtRAX2-JR::5'-TAGGAGATATCGAGGTAGCTGTCTG-3';

[0091] PCR amplification was performed using T0 generation transgenic line DNA template. The 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 a final extension at 72℃ for 10 min.

[0092] PCR amplification was performed and detected by agarose gel electrophoresis. The target fragment of the recombinant pBI121-35S:NtRAX2 vector was detected, confirming the plant as a positive transformant. Results for different lines of the same batch of transgenic plants are shown below. Figure 1 As shown.

[0093] Example 4

[0094] In this embodiment, RNA was extracted from positive plants from different lines of the same batch of transgenic plants obtained in Example 3. After reverse transcription into cDNA, the expression level of the tobacco NtRAX2 gene in the positive plants was analyzed by real-time quantitative PCR.

[0095] In this embodiment, the tobacco NtGAPDH gene was used as an internal control for quantitative real-time PCR detection. The primer sequences for quantitative real-time PCR detection were designed as follows:

[0096] q-NtRAX2 F: 5'-AGCCATTTTTATCCCACTTCAAC-3',

[0097] q-NtRAX2 R: 5'-CCACTTGGCTTTTTCTTCAGCATA-3';

[0098] The primers for the tobacco NtGAPDH gene were designed as follows:

[0099] NtGAPDH-F: 5'-TGGGTGTCAACGAGAAGGAA-3',

[0100] NtGAPDH-F: 5'-TCTGGGTGGCAGTAAGGGA-3';

[0101] The conditions for quantitative real-time PCR are as follows: Step 1: pre-denaturation, 95℃ for 10s; Step 2: PCR reaction, 95℃ for 5s, 60℃ for 30s, 39 cycles; Step 3: melting curve.

[0102] Each sample was biologically replicated three times, using 2 -△△CT Methods were used to analyze relative gene expression differences. Results from different lines of the same batch of transgenic plants are shown in the appendix. Figure 2 The analysis results showed that, compared with wild-type tobacco, the expression level of the tobacco NtRAX2 gene was significantly increased in positively transformed plants.

[0103] Wild-type and transgenic T0 generation plants were transplanted into pots and cultured in a greenhouse until their vigorous growth period. Leaves were then collected, and nicotine content was detected using ultra-high performance liquid chromatography (UHPLC). Results for different lines from the same batch of transgenic plants are attached. Figure 3 The analysis results showed that the nicotine content in the leaves of overexpressing plants was significantly higher than that in wild-type plants, indicating that tobacco NtRAX2 can positively regulate nicotine synthesis.

[0104] In summary, this invention can regulate the nicotine content of tobacco by controlling the expression level of the NtRAX2 gene, providing technical means and theoretical basis for the regulation of tobacco nicotine content and quality improvement.

[0105] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made 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. A gene regulating tobacco nicotine synthesis NtRAX2 Its characteristics are, Its nucleotide sequence is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.

2.

2. A tobacco nicotine synthesis regulatory gene according to claim 1 NtRAX2 Its characteristics are, Its nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.

1.

3. A tobacco nicotine synthesis regulatory gene as described in claim 1 or 2 NtRAX2 The encoded tobacco nicotine synthesis regulatory protein NtRAX2 is characterized by, The protein NtRAX2 has the amino acid sequence shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the tobacco nicotine synthesis regulatory gene as described in claim 1. NtRAX2 .

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector includes pBI121-35S:NtRAX2.

6. A method for constructing a recombinant expression vector as described in claim 4 or 5, characterized in that, Includes the following steps: (1) Design the following primer sequences OE_NtRAX2 F and OE_NtRAX2 R for PCR amplification to construct the target sequence fragment of the overexpression vector: OE_NtRAX2 F: acgggggactctagaggatccATGGACTGCGAACGCCAGACAGCTA; OE_NtRAX2 R:cgatcggggaaattcgagctcTTATTCTGTCCTATTTTCATCAAGA; (2) pBI121 was digested with BamHI and SacI and ligated with the PCR product from step (1). The PCR product was then transformed and positive clones were screened to obtain the product for overexpression of tobacco. NtRAX2 Recombinant gene pBI121-35S:NtRAX2 plasmid.

7. A gene containing the tobacco nicotine synthesis regulatory gene as described in claim 1 or 2 NtRAX2 Recombinant bacteria.

8. The tobacco nicotine synthesis regulatory gene as described in claim 1 or 2 NtRAX2 The application of the tobacco nicotine synthesis regulatory protein NtRAX2 as described in claim 3, the recombinant expression vector as described in claim 4 or 5, or the recombinant bacteria as described in claim 7 in upregulating the nicotine content in tobacco.

9. A method for increasing the nicotine content in tobacco, characterized in that, The method includes the step of transforming tobacco plants with the recombinant expression vector of claim 4 or 5; or the step of infecting plant tissue culture tissues with the recombinant bacteria of claim 7.

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