Method for heterologous synthesis of nicotine in solanaceae plants through agrobacterium tumefaciens mediation

Abstract

The invention relates to a method for heterologous synthesis of nicotine in solanaceae plants through agrobacterium tumefaciens mediation. Comprising the following steps: activating agrobacterium engineering bacteria, preparing an infection solution, pretreating solanaceae plant leaves, performing vacuum infection, and performing co-culture to complete nicotine heterologous synthesis. The nicotine synthesis key genes comprise an NtAO2 gene, an NtQS gene and an NtQPT2 gene of an NAD (Nicotinamide Adenine Dinucleotide) pathway, an NtODC2 gene, an NtPMT1 gene and an NtMPO1 gene of a polyamine pathway, and an A622 gene and an NtBBL gene of a two-ring condensation pathway, wherein the NtBBL gene is an NtBBLa gene or an NtBBLb gene. The method is high in efficiency and low in cost, and a practical path is provided for nicotine industrial heterologous synthesis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and molecular biology technology, specifically relating to a method for heterologous synthesis of nicotine in Solanaceae plants mediated by Agrobacterium. Background Technology

[0002] Nicotine is a pyridine alkaloid unique to the genus *Nicotiana* in the Solanaceae family. It possesses multiple core values ​​in medicine, agriculture, and industry: In the pharmaceutical field, it is a key active ingredient in smoking cessation patches and nicotine replacement therapy (NRT) drugs, and can also be used as an adjunct treatment for neurological diseases such as Alzheimer's and Parkinson's; In the agricultural field, nicotine derivatives, due to their strong targeting of pests and safety to natural enemies, have become an important research and development direction for green pesticides; In the new tobacco industry, nicotine is a core raw material for products such as e-cigarettes and heated cigarettes, with a global annual demand exceeding 100,000 tons.

[0003] At present, the production of nicotine mainly relies on extraction from tobacco leaves, but this method has significant limitations: (1) In addition to nicotine, tobacco also contains harmful components such as tar and polycyclic aromatic hydrocarbons, which need to be separated by complex processes such as multiple column chromatography and solvent extraction, resulting in extraction costs accounting for more than 60% of the total cost, and the purity is difficult to break through, which cannot meet the pharmaceutical grade requirements; (2) Tobacco planting is limited by natural conditions such as region and climate, and the yield is unstable, making it difficult to match the continuous supply requirements of industrial production; (3) As a nicotine crop, the planting area of ​​tobacco is strictly controlled, making it difficult to meet the demand for nicotine in industrial production.

[0004] To overcome the aforementioned bottlenecks, synthetic biology-driven heterologous synthesis of nicotine has become a core research and development direction in the industry. Nicotine biosynthesis is a complex metabolic pathway involving multiple pathways and multi-gene collaboration, see... Figure 1 First, quinolinic acid is synthesized through the NAD pathway, then putrescine is synthesized through the polyamine pathway, and finally nicotine is formed through the bicyclic condensation pathway. Therefore, to achieve heterologous synthesis of nicotine, multiple key genes need to be introduced and expressed in a coordinated manner. In the existing technology, heterologous expression of multiple genes faces two major challenges: (1) Low efficiency of multi-gene vector construction; traditional vector construction methods (such as enzyme digestion and ligation, homologous recombination) are difficult to achieve precise assembly of more than 6 genes, and gene redundancy or deletion is easy to occur, resulting in poor vector stability; (2) Insufficient heterologous host adaptability; most studies focus on microorganisms (such as Escherichia coli, yeast) or model plants (such as Nicotiana benthamiana), but microorganisms require in vitro culture equipment, model plants lack industrial application value, and effective heterologous synthesis of nicotine has not yet been achieved in edible Solanaceae plants with economic value, mainly because the transformation method is not optimized for its cell characteristics and the coordination of multi-gene expression is poor.

[0005] Furthermore, existing transformation technologies often suffer from low infection efficiency and gene silencing issues in Agrobacterium-mediated transformation, while the parameters (such as pressure and time) of the vacuum permeation method lack specific optimization for Solanaceae plants, resulting in low nicotine synthesis efficiency after transformation. Therefore, developing a vector system capable of efficiently assembling multiple genes and adapted to Solanaceae plants, along with a corresponding transformation method, is of great significance for achieving heterologous and efficient nicotine synthesis. Summary of the Invention

[0006] The purpose of this invention is to provide a method for heterologous synthesis of nicotine in Solanaceae plants mediated by Agrobacterium, which can significantly improve the infection efficiency of Agrobacterium and the nicotine synthesis efficiency.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A nicotine heterologous synthesis multi-gene aggregation vector includes eight key genes of the nicotine biosynthesis pathway: NtAO2, NtQS, and NtQPT2 genes of the NAD pathway; NtODC2, NtPMT1, and NtMPO1 genes of the polyamine pathway; and A622 and NtBBL genes of the two-ring condensation pathway. The vector is constructed using pYLTAC380GW as the backbone vector via the Transgene-Stacking II system, and the eight key genes are sequentially inserted into the backbone vector.

[0008] Furthermore, the NtBBL gene is either the NtBBLa gene or the NtBBLb gene.

[0009] Furthermore, all eight key genes were derived from tobacco root cDNA and obtained through PCR cloning.

[0010] Furthermore, pYL322-d1 and pYL322-d2 were used as donor vectors during the construction process, and the vector construction was completed through 7 rounds of genome assembly.

[0011] Furthermore, the multi-gene aggregation vector is prepared by the following method: (1) Gene cloning: NtAO2, NtQS, NtQPT2, NtODC2, NtPMT1, NtMPO1, A622 and NtBBL genes were obtained from tobacco root cDNA by PCR amplification. (2) Construction of donor vector: The gene obtained in step (1) is inserted into the pYL322-d1 or pYL322-d2 donor vectors respectively to obtain a recombinant donor vector containing a single gene; (3) Multigen assembly: Using pYLTAC380GW as the backbone vector, the recombinant donor vector was assembled in 7 rounds using the Transgene-StackingII system. After each round of assembly, the recombinant donor vector was digested with I-SceI and PI-SceI at 37℃ for 5 hours for verification. (4) Vector final construction: The NtBBL gene was integrated into the assembly product of step (3). The reaction conditions were 25℃ for 5 h, and Proteinase K was added to stop the reaction at 37℃ for 15 min. (5) Vector verification: The correctness of the final vector was verified by digesting NotI at 37℃ for 30 min, and the nicotine heterologous synthesis multi-gene polymerization vector was obtained.

[0012] A method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants includes the following steps: (1) Activation of Agrobacterium engineered strain and preparation of infection solution: Agrobacterium containing a recombinant vector of the key gene for nicotine synthesis was cultured as a seed culture and induced by acetylsyleugenol (AS) to expand the culture to the OD of the bacterial solution. 600 The value is 1.8-2.2; after centrifugation, wash at least once with a mixture containing MgCl2 and MES, then add AS to adjust the OD of the bacterial culture. 600 Adjust the concentration to 0.9-1.0, let stand at room temperature for 2-3 hours to obtain the vacuum inoculum; (2) Pretreatment of leaves of Solanaceae plants: Select functional leaves of healthy Solanaceae plants aged 3-4 weeks, wash them and make tiny wounds on the back of the leaves that do not penetrate the leaves. (3) Vacuum inoculation: The pretreated leaves are immersed in the inoculation solution and inoculated in a vacuum pump at a pressure of 0.04 MPa for 5 minutes; after inoculation, the leaves are taken out, washed and the residual bacterial solution on the surface is dried. (4) Co-culture: The infected Solanaceae plants were cultured under light conditions to complete the heterologous synthesis of nicotine; The key genes for nicotine synthesis include the NtAO2, NtQS, and NtQPT2 genes of the NAD pathway, the NtODC2, NtPMT1, and NtMPO1 genes of the polyamine pathway, and the A622 and NtBBL genes of the bicyclic condensation pathway, wherein the NtBBL gene is either the NtBBLa or NtBBLb gene.

[0013] Furthermore, the Agrobacterium is Agrobacterium EHA105 strain.

[0014] Furthermore, the solanaceous plant mentioned is tomato or eggplant.

[0015] Furthermore, the recombinant vector containing the key gene for nicotine synthesis mentioned in step (1) is pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-PMT1-AO2-BBLb, with a full length of 39592bp, containing the HygR selection marker and the CaMV35S enhancement promoter.

[0016] Further, in step (1), the seed culture is as follows: Agrobacterium strain stored at -80℃ is streaked into LB solid medium containing kanamycin, incubated upside down at 28℃ for 48h, and then single clones are picked and inoculated into LB liquid medium containing kanamycin, and shaken at 28℃ and 180-220rpm for 14-18h.

[0017] Further, in step (1), the AS-induced expansion culture is as follows: the seed culture is transferred to LB liquid medium containing kanamycin at a volume ratio of 1:80-120, AS is added to a final concentration of 150 μM, and the culture is shaken at 28°C and 180-220 rpm for 14-18 h.

[0018] Furthermore, in step (1), the centrifugation conditions are 4500-5500 rpm and 8-12 min at room temperature; in the mixed solution containing MgCl2 and MES, the concentration of MgCl2 is 15 mM, the concentration of MES is 15 mM, and the pH is 5.5-5.7.

[0019] Furthermore, the co-cultivation conditions in step (4) are: temperature 24-26℃, light duration 16h / dark duration 8h, relative humidity 60%-70%; tomatoes are co-cultivated for 3 days and eggplants for 4 days, and the leaves are kept dry during the cultivation period.

[0020] Furthermore, the procedure also includes a step for detecting nicotine content: after co-culturing, the leaves are dried, ground, and ultrasonically extracted with a 75% methanol solution containing 0.1M NaOH, and then detected using a plant nicotine enzyme-linked immunosorbent assay (ELISA) kit.

[0021] The technical solution of this application achieves the following technical effects through the synergistic application of multi-gene polymerization vectors, supporting preparation methods, and transformation technology of Solanaceae plants: 1. In terms of vector construction, the ordered assembly and stable expression of key genes required for nicotine synthesis were achieved. Through the combination of the Transgene-Stacking II system and Gateway BP cloning technology, and after multiple rounds of enzyme digestion verification, the integrity and accuracy of the genome assembly were significantly improved, solving the problems of easy deletion and disordered sequence in multi-genome assembly using traditional methods. The vector exhibits good passage stability in Agrobacterium and Solanaceae plant cells, and can maintain co-expression of genes for a long period, providing a stable genetic basis for nicotine synthesis.

[0022] 2. The vector preparation method is designed with standardized steps (gene cloning → donor construction → multiple rounds of assembly → final verification), which has strong reproducibility. Vectors prepared in different batches show high consistency after enzyme digestion detection, which significantly shortens the vector construction cycle and improves preparation efficiency.

[0023] 3. After optimization of the transformation technology for Solanaceae plants, the infection effect was significantly improved. Using a 0.04 MPa vacuum permeation parameter for 5 minutes, the cell damage rate of eggplant and tomato leaves was low, and the target gene was effectively introduced. 3-4 days after transformation, ELISA detection showed nicotine accumulation in both eggplant and red water chestnut tomato, with significantly higher levels than the control group. Moreover, no additional precursor substances were required, as the synthesis could be achieved using the Solanaceae plants' own metabolic system.

[0024] Overall, compared with traditional tobacco extraction methods, this technical solution produces fewer harmful impurities and reduces purification difficulty; compared with heterologous synthesis by microorganisms, it does not require complex cultivation equipment and is easier to achieve large-scale production based on existing crop planting systems, providing a practical technical path for the efficient synthesis of nicotine. Attached Figure Description

[0025] Figure 1 Schematic diagram of tobacco alkaloid metabolism; Figure 2 Electrophoresis diagram of the target gene's CDS region clone; Figure 3 Electrophoresis image of positive colonies selected from recombinant plasmids; Figure 4 Electrophoresis image of NotI restriction enzyme digestion; Figure 5 : pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-AO2-PMT1-BBLa / -BBLb bacterial culture PCR positive identification result; Figure 6 PCR detection results of BY-2 positive cell lines; Figure 7 Transient transformation experiment of tomato leaves; Figure 8 Experiment on instantaneous transformation of eggplant leaves; Figure 9 Nicotine content of different eggplant varieties before and after conversion; Figure 10 Nicotine content before and after conversion in different tomato varieties; Figure 11 Image of Agrobacterium strain EHA105 vector. Detailed Implementation

[0026] The present application will be further described below through specific embodiments.

[0027] Example 1: Construction of a multi-gene polymerase vector for heterologous nicotine synthesis 1.1 Material Preparation Gene source: cDNA from tobacco (Nicotiana acuminata) roots, extracted and preserved in our laboratory, with extraction method referring to the third edition of Molecular Cloning: A Laboratory Manual. Vector backbone: pYLTAC380GW vector (purchased from Addgene); Donor vectors: pYL322-d1, pYL322-d2 (purchased from South China Agricultural University); Enzymes: I-SceI, PI-SceI, NotI (purchased from NEB), Taq DNA polymerase (purchased from TaKaRa); Primers: PCR primers designed for the CDS region of key genes (sequences are shown in Table 1).

[0028] Table 1: PCR primer sequence list for key genes

[0029] 1.2 PCR amplification of key genes Using tobacco root cDNA as a template, PCR amplification was performed using the primers in Table 1. The reaction system (50 μL) consisted of: 10×PCR Buffer 5 μL, dNTPs (2.5 mM) 4 μL, forward and reverse primers (10 μM) 1 μL each, cDNA template 2 μL, Taq enzyme 0.5 μL, and ddH2O added to 50 μL. Reaction program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension (NtAO2 extension for 1 min 20 s, NtQS extension for 1 min 10 s, other genes adjusted at 1 kb / min), 35 cycles; 72℃ final extension for 10 min. The amplification products were verified by 1% agarose gel electrophoresis, see [link to relevant documentation]. Figure 2 The target gene CDS region clone electrophoresis results showed that NtAO2 (1256bp), NtQS (1142bp), NtQPT2 (1538bp), NtODC2 (1467bp), NtPMT1 (1845bp), NtMPO1 (1689bp), A622 (1053bp), NtBBLa (1326bp), and NtBBLb (1331bp) all obtained single target bands, consistent with the expected size.

[0030] 1.3 Donor Carrier Construction The PCR-recovered products were ligated into either the pYL322-d1 or pYL322-d2 vectors (NtAO2, NtQPT2, NtPMT1, and A622 were inserted into pYL322-d1, and the rest were inserted into pYL322-d2). The ligation system (10 μL) consisted of: 2 μL vector fragment, 5 μL target gene fragment, 1 μL T4 DNA ligase, 1 μL 10× Buffer, and 1 μL ddH2O. The ligation was carried out overnight at 16°C. Transformed *E. coli* DH5α competent cells, plated on LB agar plates containing 50 μg / mL spectinomycin, and incubated at 37°C for 16 h. Single clones were picked for PCR verification. Results are shown below. Figure 3 Electrophoresis image of positive colonies selected by recombinant plasmid; sequencing of positive clones confirms correct gene sequence, and recombinant donor vector is obtained.

[0031] 1.4 Multi-genome assembly Using pYLTAC380GW as the backbone vector, seven rounds of assembly were performed using the Transgene-Stacking II system: Round 1: Take plasmid pYLTAC380GW 300ng and pYL322d1-ODC2 500ng, mix them, and transform them together into Escherichia coli NS3529 competent cells. Spread the bacterial culture on LB solid medium containing kanamycin (Kan, 25 mg / L) and ChI (15 mg / L) and incubate at 37°C for 72 h. Collect the colonies with sterile ddH2O and extract the plasmid.

[0032] Add 50 ng of plasmid, 1 µL of I-SceI enzyme, and 1 µL of buffer to a 200 µL PCR tube, and bring the volume to 10 µL with ddH2O. Incubate at 37 °C (controlled by the PCR instrument) for 5 h. Transform into Escherichia coli NEB-10β, spread the bacterial culture on LB solid medium containing Kan (25 mg / L) antibiotic, and incubate overnight at 37 °C. Screen positive colonies by PCR, and proliferate and preserve strains containing a single positive band for plasmid extraction.

[0033] Add 300 ng plasmid, 0.3 µL NotI enzyme, and 2 µL buffer sequentially to 200 µL PCR tubes, and bring the volume up to 20 µL with ddH2O. Incubate at 37 °C for 30 min. After the NotI enzyme digestion reaction is complete, perform agarose gel electrophoresis at 150 V for 2-3 h on ice, changing the electrophoresis buffer every 30 min.

[0034] Round 2: 300 ng of plasmid pYLTAC380GW-ODC2 and 500 ng of plasmid pYL322d2-QS were co-transformed into competent Escherichia coli NS3529 cells. The bacterial culture was spread on LB solid medium containing two antibiotics, Kan (25 mg / L) and Amp (15 mg / L) and incubated at 37°C for 48 h. Colonies were collected and plasmids were extracted.

[0035] Add 50 ng of plasmid, 1 µL of PI-SceI enzyme, and 1 µL of buffer to a 200 µL PCR tube, and bring the volume to 10 µL with ddH2O. Incubate at 37 °C for 5 h. Transform into Escherichia coli NEB-10β, spread the bacterial culture on LB solid medium containing Kan (25 mg / L) antibiotic, and incubate overnight at 37 °C. PCR screening of positive colonies was performed, and strains containing a single positive band were propagated, cultured, preserved, and had their plasmids extracted. Finally, NotI restriction enzyme digestion was performed for verification.

[0036] After multiple rounds of assembly, the expression plasmid pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-AO2-PMT1 was finally obtained.

[0037] After each round of assembly, 5 μL of product was taken and verified using I-SceI and PI-SceI (digestion at 37℃ for 5 h). The results are shown in the figure. Figure 4 NotI restriction enzyme digestion electrophoresis image. The digestion product electrophoresis shows that the fragment size is as expected and there are no impurities.

[0038] 1.5 Vector final construction and verification The assembly product from round 7 was used to perform Gateway BP cloning with a donor vector containing NtBBLa (or NtBBLb): The reaction system (20 μL) consisted of 8 μL of assembly product, 4 μL of donor vector, 2 μL of BP Clonase II enzyme mixture, and 6 μL of TE Buffer. After reacting at 25°C for 5 h, 1 μL of Proteinase K (20 mg / mL) was added and the reaction was terminated by treating at 37°C for 15 min. Transformed *E. coli* DH10B competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, single colonies were picked and cultured, plasmids were extracted and verified using NotI (digestion at 37℃ for 30 min). Results are shown in [Figure number missing]. Figure 5 The positive PCR results of pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-AO2-PMT1-BBLa / -BBLb bacterial culture showed that the size of the enzyme digestion fragment was consistent with the theoretical value, and the final vectors pYLTAC380GW-8Gene (containing NtBBLa) and pYLTAC380GW-8Gene-b (containing NtBBLb) were obtained.

[0039] Example 2: Functional validation of multi-gene aggregation vector in tobacco BY-2 cells 2.1 Cell Transformation Tobacco BY-2 suspension cells in the logarithmic growth phase were taken and transformed into Agrobacterium EHA105 using the vector constructed in Example 1 to obtain engineered bacteria; The engineered bacteria were added to the BY-2 cell suspension (containing 100 μM AS) at a ratio of 1:10 and co-cultured at 25°C and 130 rpm for 48 h. The cells were screened using MS solid medium containing 50 mg / L hygromycin and 200 mg / L termethin. The cells were subcultured every 14 days for a total of 3 screenings to obtain positive cell lines.

[0040] 2.2 Verification Results PCR detection: DNA was extracted from positive cell lines and amplified using specific primers for 8 genes. Figure 6 PCR detection results of BY-2 positive cell lines all yielded the target band, proving that the vector had been integrated into the cell genome.

[0041] Example 3: Agrobacterium-mediated vacuum infection and transformation of Solanaceae plants 3.1 Material Preparation Host plants: Tomato (Red Horseshoe and Black Horseshoe varieties, commercially available) and eggplant (Round Eggplant, Long Eggplant, and Thread Eggplant varieties, commercially available). Both were cultivated in a greenhouse (25℃, 16h light / 8h darkness, relative humidity 60%-70%) for 3-4 weeks. Healthy plants with unfolded leaves and no pests or diseases were selected for use. Agrobacterium and vector: Agrobacterium strain EHA105 (containing the recombinant vector pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-PMT1-AO2-BBLb, vector map see) Figure 11 It contains HygR selection markers, CaMV35S enhancer promoters, and Tnos terminators for each target gene (ODC2, QS, etc.), with a total length of 39592bp. Reagents and culture media: LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0, sterilized at 121℃ for 20min); Infection solution components: 15mM MgCl2, 15mM MES (pH 5.6), 150μM AS (acetylsylgenone); ELISA kit (plant nicotine specific, purchased from Beijing Solarbio Science & Technology Co., Ltd.); Tools: vacuum pump (ultimate vacuum ≤ 0.098 MPa), 1 mL syringe (with needle removed), sterile petri dish, filter paper.

[0042] 3.2 Activation of Agrobacterium engineered strains and preparation of infection solution (1) Seed culture: Agrobacterium EHA105 strain containing the target vector stored at -80℃ was streaked into LB solid medium containing 50mg / L kanamycin, and incubated upside down at 28℃ for 48h. Single clones (regular morphology and no contamination by other bacteria) were picked. (2) Expanded culture: Inoculate a single clone into 5 mL of LB liquid medium containing 50 mg / L kanamycin, and shake at 28°C and 200 rpm for 16 h to obtain seed culture; (3) AS induction and activation: Transfer the seed culture to 50 mL of fresh LB liquid medium (containing 50 mg / L kanamycin) at a ratio of 1:100, add AS to a final concentration of 150 μM, and continue to shake at 28 °C and 200 rpm for 16 h until the bacterial culture reaches OD. 600 The value reached 2.0; (3) Preparation of infection solution: Centrifuge the above bacterial solution at 5000 rpm and room temperature for 10 min, and discard the supernatant; gently resuspend the bacterial cells in a mixture of 15 mM MgCl2 and 15 mM ME S (pH 5.6), and wash twice; finally, add 150 μM AS to adjust the OD of the bacterial solution. 600 The solution is prepared by adjusting the pH to 0.95 and allowing it to stand at room temperature for 2-3 hours.

[0043] 3.3 Plant leaf pretreatment and vacuum inoculation (1) Leaf pretreatment: Select functional leaves from the middle of the plant, rinse them with sterile water, and dry the surface moisture with filter paper; use a 1mL syringe (with the needle removed) to lightly scratch the back of the leaf to create tiny wounds that do not penetrate the leaf (the spacing is about 0.5cm to avoid damaging the leaf veins). (2) Vacuum infiltration operation: Tomato: Invert the pretreated tomato leaves along with the plant so that the leaves are completely immersed in a sterile container containing 150mL of infection solution. Place the container in a vacuum pump, close the valve and slowly evacuate to 0.04MPa, maintaining this pressure for 5 minutes. Slowly release the vacuum (avoid damaging the leaves), remove the plant, gently rinse the leaf surface with sterile water to remove any residual bacterial solution, and blot dry with filter paper. Eggplant: The procedure is the same as for tomatoes, except that the plants are replaced with eggplant plants, and all other conditions are the same; (3) Co-cultivation: The infected tomato plants were placed in a greenhouse under standard conditions for 3 days, and the eggplant plants were cultivated for 4 days (during which the leaves were kept dry to prevent contamination by other bacteria).

[0044] 3.4 Conversion Effect Detection 3.4.1 Observation of phenotype and infection efficiency After cultivation, observe the leaf condition: the leaves of both tomatoes and eggplants remained bright green, without wilting or browning. Figure 7 Photographs of a tomato leaf instantaneous transformation experiment; Figure 8(Photographs of transient transformation experiments on eggplant leaves) show that the infection parameters are adapted to the characteristics of Solanaceae leaves, and the cell damage rate is <15%; One hundred tomato leaves (Red Horseshoe variety) were randomly selected and infected. The positive rate of PCR detection using the HygR marker on the vector reached 58%, proving that Agrobacterium was successfully introduced into the leaf cells.

[0045] 3.4.2 Nicotine content detection (ELISA method) (1) Sample preparation: Take 0.5g of infected leaves, dry them at 72℃ to constant weight, grind them into powder; add 5mL of 75% methanol solution containing 0.1M NaOH, extract by ultrasonication at 300W for 30min, centrifuge at 4℃ and 12000rpm for 15min, and take the supernatant as the test sample. (2) Detection and Results: Follow the instructions in the ELISA kit manual and plot a standard curve (R) using nicotine standards. 2 =0.997), measure the absorbance of the sample and calculate the content: Tomatoes: After conversion, the nicotine content of the Red Horseshoe variety reached 0.145 mg / g, and that of the Black Horseshoe variety reached 0.102 mg / g, both significantly higher than the unconverted control (nicotine was not detected in the CK group). Figure 10 Nicotine content before and after conversion in different tomato varieties.

[0046] Eggplant: After conversion, the nicotine content of the long eggplant variety reached 0.093 mg / g, while that of the round eggplant varieties reached 0.068 mg / g and 0.055 mg / g, respectively, all higher than the unconverted control (CK group nicotine content <0.001 mg / g). Furthermore, the nicotine content of the long eggplant was higher than the 0.071 mg / g reported by the South China Botanical Garden for red eggplant. Figure 9 Nicotine content in different eggplant varieties before and after conversion.

[0047] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Equivalent changes or modifications made to the features and principles described in the claims of this invention should be included within the scope of this invention.

Claims

1. A method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants, characterized in that, Includes the following steps: (1) Activation of Agrobacterium engineered strain and preparation of infection solution: Agrobacterium containing a recombinant vector of the key gene for nicotine synthesis was cultured as a seed culture and induced by acetylsyleugenol (AS) to expand the culture to the OD of the bacterial solution. 600 The value is 1.8-2.2; after centrifugation, wash at least once with a mixture containing MgCl2 and MES, then add AS to adjust the OD of the bacterial culture. 600 Adjust the concentration to 0.9-1.0, let stand at room temperature for 2-3 hours to obtain the vacuum inoculum; (2) Pretreatment of leaves of Solanaceae plants: Select functional leaves of healthy Solanaceae plants aged 3-4 weeks, wash them and make tiny wounds on the back of the leaves that do not penetrate the leaves. (3) Vacuum inoculation: The pretreated leaves are immersed in the inoculation solution and inoculated in a vacuum pump at a pressure of 0.04 MPa for 5 minutes; after inoculation, the leaves are taken out, washed and the residual bacterial solution on the surface is dried. (4) Co-culture: The infected Solanaceae plants were cultured under light conditions to complete the heterologous synthesis of nicotine; The key genes for nicotine synthesis include the NtAO2, NtQS, and NtQPT2 genes of the NAD pathway, the NtODC2, NtPMT1, and NtMPO1 genes of the polyamine pathway, and the A622 and NtBBL genes of the bicyclic condensation pathway, wherein the NtBBL gene is either the NtBBLa or NtBBLb gene.

2. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that... The Agrobacterium mentioned is Agrobacterium strain EHA105.

3. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, The solanaceae plant mentioned is either tomato or eggplant.

4. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, The recombinant vector containing the key gene for nicotine synthesis mentioned in step (1) is pYLTAC380GW-ODC2-QS-QPT2-A622-MPO1-PMT1-AO2-BBLb, with a full length of 39592bp, containing the HygR selection marker and the CaMV35S enhancement promoter.

5. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, In step (1), the seed culture is as follows: Agrobacterium strain stored at -80℃ is streaked into LB solid medium containing kanamycin, incubated upside down at 28℃ for 48h, and then single clones are picked and inoculated into LB liquid medium containing kanamycin, and shaken at 28℃ and 180-220rpm for 14-18h.

6. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, In step (1), the AS induction and expansion culture is carried out by transferring the seed culture to LB liquid medium containing kanamycin at a volume ratio of 1:80-120, adding AS to a final concentration of 150 μM, and shaking the culture at 28℃ and 180-220 rpm for 14-18 h.

7. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, In step (1), the centrifugation conditions are 4500-5500 rpm and 8-12 min at room temperature; the concentration of MgCl2 in the mixed solution containing MgCl2 and MES is 15 mM, the concentration of MES is 15 mM, and the pH is 5.5-5.

7.

8. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, The co-cultivation conditions in step (4) are: temperature 24-26℃, light duration 16h / dark duration 8h, relative humidity 60%-70%; tomatoes are co-cultivated for 3 days and eggplants for 4 days. During the cultivation period, avoid getting water on the leaves.

9. The method for Agrobacterium-mediated heterologous synthesis of nicotine in Solanaceae plants according to claim 1, characterized in that, It also includes a nicotine content detection step: take the leaves after co-culture, dry them, grind them, extract them with ultrasonically in a 75% methanol solution containing 0.1M NaOH, and then detect them using a plant nicotine enzyme-linked immunosorbent assay (ELISA) kit.

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