Tobacco nicotine synthesis regulation gene NtRAX2 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 NtRAX2 and a tobacco nicotine synthesis regulation protein NtRAX2 coded by the same, and further discloses application of the tobacco nicotine synthesis regulation gene NtRAX2 and the tobacco nicotine synthesis regulation protein NtRAX2. According to the invention, a novel nicotine synthesis regulating gene NtRAX2 is cloned from cultivated tobacco, and the effect of the tobacco NtRAX2 gene in tobacco synthesis is researched. The gene NtRAX2 for regulating and controlling nicotine synthesis and the encoded protein NtRAX2 of the gene NtRAX2 are related to nicotine accumulation in tobacco, a new genetic target and a new technical means are provided for regulation and control and application of tobacco nicotine content, and the gene NtRAX2 and the encoded protein NtRAX2 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 NtRAX2 and a tobacco nicotine synthesis regulatory protein NtRAX2 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] To this end, the technical problem to be solved by the present invention is to provide a tobacco nicotine synthesis regulatory gene NtRAX2 and a tobacco nicotine synthesis regulatory protein NtRAX2 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 NtRAX2 and the tobacco nicotine synthesis regulatory protein NtRAX2 in the field of tobacco cultivation.

[0009] In order to solve the above technical problems, the tobacco nicotine synthesis regulatory gene NtRAX2 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 NtRAX2 encoded by the tobacco nicotine synthesis regulatory gene NtRAX2. The protein NtRAX2 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 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 to perform 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 double-digested with BamHI and SacI, and ligated with the PCR product of step (1), transformed and positive clones were screened to obtain the recombinant pBI121-35S:NtRAX2 plasmid for overexpressing the tobacco NtRAX2 gene.

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

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

[0024] The present 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 planting.

[0025] The present 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 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.

[0027] The present invention clones a new nicotine synthesis regulating gene NtRAX2 from cultivated tobacco, and studies the role of the tobacco NtRAX2 gene in tobacco synthesis. The detection of nicotine in genetic materials shows that the nicotine content of tobacco leaves in the strain overexpressing the tobacco nicotine synthesis regulating gene NtRAX2 is significantly increased, which can prove that the tobacco NtRAX2 gene has the function of regulating the nicotine content of tobacco. By reducing or knocking out the expression of the NtRAX2 gene, the nicotine content in tobacco leaves can be reduced; on the contrary, by overexpressing the NtRAX2 gene, the nicotine content in tobacco leaves can be increased. The nicotine synthesis regulating gene NtRAX2 identified by the present invention and its encoding protein NtRAX2 are related to nicotine accumulation in tobacco, provide new genetic targets and technical means for the regulation and application of tobacco nicotine content, and have important application value in tobacco nicotine regulation.

[0028] The present invention further studies the role of tobacco NtRAX2 gene in tobacco synthesis by constructing pBI121-35S:NtRAX2 recombinant expression vector. The present invention can improve the expression level of NtRAX2 gene in tobacco after transforming tobacco plants with the recombinant vector pBI121-35S:NtRAX2, so that the expression amount of NtRAX2 gene and protein in the transformed plants is significantly increased, and genetic materials with significantly increased expression amount of NtRAX2 gene are constructed. Further nicotine detection of the genetic materials shows that the nicotine content of tobacco leaves of the NtRAX2 overexpression strain is significantly increased.

[0029] The present invention utilizes the pBI121-35S:NtRAX2 recombinant expression vector to construct transgenic plants, utilizes an Agrobacterium-mediated gene transformation method to transform the pBI121-35S:NtRAX2 expression vector into Agrobacterium, screens and obtains engineered bacteria for transformation, utilizes the engineered bacteria to infect plant tissue culture tissues, and obtains tobacco genetic materials with improved tobacco NtRAX2 gene expression through tissue culture and identification, which is of great significance for regulating tobacco nicotine content. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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:

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

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

[0033] Figure 3 The results of nicotine content analysis in NtRAX2 overexpressing plants. DETAILED DESCRIPTION

[0034] 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.

[0035] In the following embodiments of the present invention, the information related to the biological material includes:

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

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

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

[0039] Trans1-T1 chemically competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

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

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

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

[0043] Fluorescence quantitative PCR enzyme and PCR amplification enzyme were purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

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

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

[0046] Example 1

[0047] This example clones and expresses the NtRAX2 gene based on genetic engineering techniques.

[0048] 100 mg of roots of vigorous tobacco (K326) were taken as samples, fully ground in liquid nitrogen, and total RNA was extracted according to the instructions of the RNA extraction kit, and then reverse transcribed into cDNA as a cloning template for later use;

[0049] The primers designed to amplify the coding sequence of the tobacco NtRAX2 gene are as follows:

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

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

[0052] 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: 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.

[0053] Purify the PCR amplified 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 includes: 6 μL of DNA amplification product and 1 μL of pEASY-T1 vector. After mixing, connect at 25°C for 25 minutes.

[0054] Continue to transform the ligation product into E. coli competent cells. The specific transformation process is as follows:

[0055] 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;

[0056] 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;

[0057] 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.

[0058] After the white spots were picked and amplified and cultured, 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 NtRAX2 gene.

[0059] The results of sequencing analysis showed that the length of the coding region of the NtRAX2 gene was 903 nucleotides, as specifically shown in SEQ ID NO.1; after analyzing the gene, it was found that the amino acid sequence of the NtRAX2 protein encoded by it was 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] In this example, the pBI121-35S:NtRAX2 expression vector for knocking down the NtRAX2 gene was further constructed to further verify the function of the tobacco NtRAX2 gene in tobacco nicotine synthesis.

[0066] According to the NtRAX2 gene coding sequence known in Example 1, the amplified gene coding sequence was subcloned into the pBI121 vector to replace the original GUS gene in the vector to form a recombinant plasmid for 35S-driven NtRAX2 expression. The primer sequences of the recombinant expression vector were designed as follows:

[0067] OE_NtRAX2 F:acgggggactctagaggatcc ATGGACTGCGAACGCCAGACAGCTA;

[0068] OE_NtRAX2 R:cgatcggggaaattcgagctcTTATTCTGTCCTATTTTCATCAAGA.

[0069] The 25 μl PCR reaction system includes: 1 μl of the upstream and downstream primers mentioned above, 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°C pre-denaturation for 4 min; 94°C denaturation for 30 s, 60°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 target fragment was amplified from the cDNA to obtain the target sequence fragment for constructing the interference vector.

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

[0072] Example 3

[0073] This example further utilizes the pBI121-35S:NtRAX2 expression vector constructed in Example 2, transforms into Agrobacterium by electroporation, and then transforms tobacco plants to create transgenic plants overexpressing the NtRAX2 gene.

[0074] Agrobacterium transformation

[0075] Take out the Agrobacterium competent cells from the -80°C refrigerator, freeze and thaw on ice, add 6 μL of the pBI121-35S:NtRAX2 expression vector prepared in Example 3 when about to thaw, and flick to mix; place the mixture in a pre-cooled electroporation cup and place on ice for 5 minutes;

[0076] Adjust the parameters of the electroporator to: voltage 2.5kV, capacitance 25μF, resistance 200Ω; then use absorbent paper to remove the water droplets on the outer wall of the electroporator cup, put the electroporator cup into the electric shock tank, and shock for 5ms;

[0077] Quickly add 800 μL of YEB liquid medium preheated to 28°C, shake and resuscitate at 220 rpm and 28°C for 3 h;

[0078] Then the bacterial solution was centrifuged at 4500 rpm for 1 min, half of the supernatant was discarded, and the suspension was resuspended and evenly applied on YEB solid medium containing Rif (100 μg / mL), Str (50 μg / mL) and Kan (50 μg / mL), and inverted culture was carried out at 28°C for about 2 to 3 days until a single colony was formed;

[0079] 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.

[0080] Transformed tobacco plants

[0081] 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.

[0082] 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.

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

[0084] 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.

[0085] The leaf discs were washed with sterile water containing Cef (400 mg / L), and the excess liquid was removed with sterile filter paper. The leaf discs were 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 light.

[0086] 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.

[0087] After growing for about one month, take a small amount of leaves and extract DNA according to the instructions of the plant genome extraction kit. Detect positive transgenic strains through PCR amplification, cloning and sequencing. The specific identification method is as follows:

[0088] The following detection primers were designed to identify positive transformed plants, specifically:

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

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

[0091] PCR amplification was performed using DNA template of T0 transgenic strains; 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.

[0092] The PCR amplification was tested by agarose gel electrophoresis. The target detection fragment of the recombinant pBI121-35S:NtRAX2 vector was detected, and the positive transformed plants were determined. The results of different strains of the same batch of transgenic plants are as follows: Figure 1 shown.

[0093] Example 4

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

[0095] In this example, the tobacco NtGAPDH gene was used as an internal reference to perform fluorescence quantitative PCR detection, and the fluorescence quantitative detection primer sequences were designed as follows:

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

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

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

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

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

[0101] The conditions of fluorescence quantitative PCR were as follows: the first step was pre-denaturation at 95°C for 10 s; the second step was PCR reaction at 95°C for 5 s, 60°C for 30 s, and 39 cycles; the third step was melting curve.

[0102] Each sample was biologically replicated three times. -△△CT The results of relative gene expression analysis of different lines of the same batch of transgenic plants are shown in the attached Figure 2 The analysis results showed that the expression level of tobacco NtRAX2 gene was significantly increased in the positive transformed plants compared with the wild-type tobacco.

[0103] The T0 generation plants of the wild type and transgenic lines were transplanted into pots and cultured in the greenhouse until the vigorous growth period, and the leaves were collected. The nicotine content was detected by ultra-high performance liquid chromatography. The results of different lines of the same batch of transgenic plants are shown in the attached Figure 3 The analysis results showed that the nicotine content in the leaves of the overexpressed plants was significantly higher than that of the wild type, indicating that tobacco NtRAX2 can positively regulate the synthesis of nicotine.

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

[0105] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A tobacco nicotine synthesis regulatory gene NtRAX2, 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 the function of nicotine synthesis-related protein 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 NtRAX2 encoded by the tobacco nicotine synthesis regulatory gene NtRAX2 according to claim 1, characterized in that: The protein NtRAX2 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 NtRAX2 according to claim 1.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector includes pBI121-35S:NtRAX2.

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 primer sequences OE_NtRAX2 F and OE_NtRAX2 R to perform PCR amplification to construct the target sequence fragment of the overexpression vector: OE_NtRAX2 F:acgggggactctagaggatcc ATGGACTGCGAACGCCAGACAGCTA; OE_NtRAX2 R:cgatcggggaaattcgagctcTTATTCTGTCCTATTTTCATCAAGA; (2) pBI121 was double-digested with BamHI and SacI, and ligated with the PCR product of step (1), transformed and positive clones were screened to obtain the recombinant pBI121-35S:NtRAX2 plasmid for overexpressing the tobacco NtRAX2 gene.

6. A recombinant bacterium containing the tobacco nicotine synthesis regulatory gene NtRAX2 according to claim 1.

7. A transgenic tobacco plant containing the tobacco nicotine synthesis regulatory gene NtRAX2 according to claim 1.

8. Use of the tobacco nicotine synthesis regulatory gene NtRAX2 according to claim 1, or the tobacco nicotine synthesis regulatory protein NtRAX2 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 NtRAX2 according to claim 1, or the tobacco nicotine synthesis regulatory protein NtRAX2 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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