Application of ursolic acid in improving transient expression efficiency of tobacco leaves

Abstract

The invention provides application of ursolic acid in improving transient expression efficiency of tobacco leaves, and belongs to the technical field of bioengineering. The invention creatively finds that when ursolic acid is applied to a tobacco transient expression system, the transient expression efficiency of tobacco leaves can be remarkably improved. Experiments prove that after a proper amount of ursolic acid is added for treatment, the yield of exogenous protein in the tobacco leaves is greatly increased, so that powerful support is provided for large-scale production and scientific research of high-value biological products; ursolic acid can be used as a universal transient expression enhancement means, and is suitable for various different biotechnology application scenes. As a natural product existing in nature, ursolic acid has the characteristics of good biocompatibility and low toxicity, and does not cause harm to experimental subjects. Meanwhile, the synthesis cost is relatively low, the material is easy to obtain, and the principle of sustainable development is met. Therefore, ursolic acid can be predicted to have a wide application prospect in the field of transient expression of tobacco.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to the application of ursolic acid in improving the transient expression efficiency of tobacco leaves. Background Art

[0002] The transient expression system is an important tool in modern plant biotechnology. This technology can obtain the expression product of the target gene in a short time without going through the cumbersome and time-consuming stable transformation process. This fast and efficient characteristic makes transient expression widely used in fields such as gene function research, protein production, and metabolic engineering.

[0003] Tobacco (Nicotiana benthamiana), as a model plant, has become one of the most commonly used hosts in transient expression experiments due to its easy operation, clear genetic background, and high compatibility with a variety of foreign DNA fragments. However, despite the high praise for the convenience and flexibility of the tobacco transient expression system, its efficiency still has certain limitations. For example, in some cases, the transient expression level is relatively low, making it difficult to meet the needs of large-scale production; in addition, the time window of transient expression is relatively short, which is not conducive to long-term tracking or the study of complex biological processes. Therefore, finding methods and technologies that can effectively improve the transient expression efficiency of tobacco has become an important topic in this field.

[0004] To overcome the above problems, scientists have tried various strategies to optimize the transient expression system: Using strong promoters: By selecting promoters with high transcriptional activity (such as the CaMV 35S promoter), the initial expression level of the target gene can be increased to a certain extent. However, this method usually only provides limited improvement and may lead to non-specific expression or other side effects. Optimizing vector design: Modifying plasmid structures, including adjusting the replication origin, the position of the multiple cloning site, and introducing auxiliary elements, etc., helps to improve the transient expression efficiency. However, these modifications often require complex molecular biology operations, and the effects vary depending on specific experimental conditions.

[0005] Application of chemical inducers: Some small molecule compounds have been proven to promote the uptake and expression of foreign DNA by plant cells, such as salicylic acid, methyl jasmonate, etc. Although these substances can significantly enhance the transient expression level under certain conditions, their mechanism of action is not fully understood, and there may be problems such as toxicity and environmental risks. Optimization of Agrobacterium-mediated co-culture: Adjusting factors such as Agrobacterium concentration and infection time will also affect the results of transient expression. However, the range of changes in these parameters is limited and cannot fundamentally solve the problem of low efficiency.

[0006] As can be seen from the above, although existing optimization methods can improve transient expression performance to a certain extent, they still face many challenges in practical applications, especially in the pursuit of efficient, stable and harmless technical solutions, where there is still much room for improvement. Summary of the Invention

[0007] The present invention provides an application of ursolic acid in improving the transient expression efficiency of tobacco leaves. After treatment with an appropriate amount of ursolic acid, the yield of exogenous proteins in tobacco leaves increases significantly, and it can be used as a general transient expression enhancement means applicable to various different biotechnological application scenarios.

[0008] To achieve the above object, the present invention provides an application of ursolic acid in improving the transient expression efficiency of tobacco leaves.

[0009] Preferably, the tobacco leaves are selected from at least one of Nicotiana benthamiana, flue-cured tobacco, sun-cured tobacco, air-cured tobacco, oriental tobacco, cigar tobacco, chewing tobacco and snuff.

[0010] Preferably, during application, ursolic acid is added to the bacterial suspension and then introduced into the tobacco leaves. After 4-5 days, the fluorescence expression of the tobacco leaves can be observed using a protein hybridization imaging system.

[0011] Preferably, the bacterial suspension is a bacterial suspension added with the LUC reporter gene or GFP fluorescent protein, and the concentration OD 600 value is 0.5-0.8, and the pH value is 6-8.

[0012] Preferably, the concentration of ursolic acid is 0.010 mg / ml - 0.050 mg / ml.

[0013] The present invention also provides an application of ursolic acid in the preparation of a plant agent for improving the transient expression efficiency of tobacco leaves.

[0014] The present invention also provides a plant agent mainly composed of ursolic acid at a concentration of 0.010 mg / ml - 0.050 mg / ml.

[0015] The present invention also provides a method for increasing the yield of exogenous proteins in tobacco leaves, which can be achieved by introducing a plasmid containing a target gene into tobacco leaves pretreated with 0.010 mg / ml - 0.050 mg / ml ursolic acid.

[0016] Preferably, the target gene is selected from at least one of the group consisting of:

[0017] Genes related to metabolic synthesis: Key genes for linalool: LIS, key genes for geraniol: GES, key genes for limonene: LIMS, key genes for β-ionone: CCD1, key genes for phenethyl alcohol: PAR, key genes for taxol: TS, key genes for artemisinin: ADS, key genes for caffeine: XMT, key genes for anthocyanin: DFR, key genes for carotenoid: PSY, key genes for lignin: PAL, key genes for flavonoids: CHS, key genes for alkaloids: STR, key genes for terpenoids: DXS, key genes for saponins: β-AS, key genes for glucosinolates: CYP79B2, key genes for salicylic acid: ICS, key genes for jasmonic acid: AOS, key genes for gibberellin: GA20ox, key genes for abscisic acid: NCED, key genes for vitamin C: GLOase, key genes for vitamin E: HPT, key genes for polyphenols: PAL, key genes for terpenes: GPPS, key genes for diterpenoids: CPS, key genes for triterpenoids: OSC, key genes for phenolic acids: C4H, key genes for sterols: SQS, key genes for polysaccharides: SUS, key genes for biodiesel precursors: DGAT;

[0018] Genes related to the production of health care, nutrition or traditional Chinese medicine ingredients: Key gene for taxadiene: TXS; Key gene for artemisinic acid: CYP71AV1; Key gene for ginsenoside: DS; Key gene for glycyrrhizic acid: CYP88D6; Key gene for ginkgolide: Ginkgolide cyclase; Key gene for flavonoids: CHS; Key gene for saponins: CYP72A; Key gene for alkaloids: Nicotine synthase; Key gene for polysaccharides: Polysaccharide synthase; Key gene for volatile oils: Monoterpene synthase; Key gene for vitamin A: β-Carotene hydroxylase; Key gene for vitamin C: L-Galactono-1,4-lactone dehydrogenase; Key gene for vitamin E: TocopHerol synthase; Key gene for lycopene: Lycopene synthase; Key gene for lutein: Lutein synthase; Key gene for resveratrol: Resveratrol synthase; Key gene for fructooligosaccharides: Fructosyltransferase; Key gene for dietary fiber: Cellulose synthase; Key gene for polyphenols: PolypHenol oxidase; Key gene for antioxidant components: SOD; Key gene for anti-inflammatory components: COX; Key gene for immunomodulatory components: Immunoglobulin synthase; Key gene for antiviral components: Interferon synthase; Key gene for antibacterial components: Antimicrobial peptidesynthase; Key gene for disease resistance: R gene; Key gene for insect resistance: Bt gene; Key gene for drought resistance: Dehydrin gene; Key gene for salt resistance: Salt tolerance gene; Key gene for cold resistance: Cold tolerance gene; Key gene for miRNA: miRNA precursor synthase; Key gene for secondary metabolism regulation: MYB; Key gene for anthocyanins: Anthocyanin synthase; Key gene for sapogenins: Saponin synthase; Key gene for dammarenediol: CYP716A47; Key gene for chalcone: FLS; Key gene for tocopherol: TocopHerol cyclase

[0019] Medicinal protein, vaccine synthesis-related genes: Key genes for hepatitis B surface antigen: HBsAg, key genes for norovirus vaccine: NVCP, key genes for rabies virus glycoprotein: RVG, key genes for human papillomavirus vaccine: HPV L1, key genes for influenza virus hemagglutinin: HA, key genes for cholera toxin B subunit: CTB, key genes for malaria vaccine candidate antigen: MSP1, key genes for HIV antigen: gp120, key genes for dengue virus antigen: DENVE, key genes for tuberculosis antigen: ESAT-6, key genes for anti-Ebola virus antibody: 6D8, key genes for anti-HIV antibody: 2G12, key genes for anti-rabies monoclonal antibody: SO57, key genes for anti-cancer antibody: Herceptin, key genes for anti-inflammatory antibody: Infliximab, key genes for interferon-α: IFN-α, key genes for interferon-γ: IFN-γ, key genes for interleukin-2: IL-2, key genes for granulocyte colony-stimulating factor: G-CSF, key genes for erythropoietin: EPO, key genes for glucocerebrosidase: GBA, key genes for α-galactosidase: GLA, key genes for lysozyme: LYZ, key genes for superoxide dismutase: SOD, key genes for insulin: INS, key genes for human serum albumin: HSA, key genes for collagen: COL1A1, key genes for growth hormone: GH, key genes for vascular endothelial growth factor: VEGF, and key genes for lactoferrin: LF;

[0020] Functional genes that improve leaf performance after overexpression in leaves, including: 1) Improving stress resistance: DREB1A, RD29A, AREB1, NAC6, LEA3, SOS1, NHX1, HKT1, ZAT10, AVP1, CBF1, COR15A, ICE1, LOS2, ZAT12; 2) Improving disease resistance: NPR1, PR1, RPS2, EDS1, PAD4, N gene; 3) Increasing yield: RBCS, FBPase, SBPase, PPDK, AGPase; 4) Promoting tillering and branching: TB1, IPA1, D10, D14, D27; 5) Shortening flowering time: FT, SOC1, LFY, AP1, CO; 6) Regulating photoperiod: GI, pHYA, pHYB, CRY1, CRY2; 7) Other functions: Gene editing-related gene CAS protein-coding gene, regulating hormone signals GA20ox, GA2ox, DELLA, ARF, PIN1, enhancing antioxidant capacity SOD, CAT, APX, GPX, GR;

[0021] Other marker genes: Green fluorescent protein GFP, red fluorescent protein RFP, yellow fluorescent protein YFP, betaine synthesis gene RUBY, luciferase gene LUC.

[0022] Preferably, the plasmid containing the target gene is obtained by the following method:

[0023] The target gene is obtained by a synthesis method, and the target gene is constructed into the pacI and NotI sites of the transient expression vector PJL-TRBO by restriction enzyme digestion and ligation.

[0024] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0025] The present invention creatively discovers that when ursolic acid is applied to the tobacco transient expression system, the transient expression efficiency of tobacco leaves can be significantly improved. Experiments have proved that after treatment with an appropriate amount of ursolic acid, the yield of foreign proteins in tobacco leaves has increased significantly, which provides strong support for the large-scale production and scientific research of high-value biological products; ursolic acid can be used as a general transient expression enhancement means and is applicable to various different biotechnological application scenarios. As a natural product existing in nature, ursolic acid itself has good biocompatibility and low toxicity characteristics and will not cause harm to experimental subjects. At the same time, its synthesis cost is relatively low, it is easy to obtain, and it conforms to the principle of sustainable development. Therefore, we can foresee that ursolic acid has broad application prospects in the field of tobacco transient expression. Description of the Drawings

[0026] Figure 1 Observation results four days after injecting tobacco with solutions containing different volumes of ursolic acid, where the solution concentrations are 0.010 mg / ml, 0.025 mg / ml, 0.050 mg / ml, 0.099 mg / ml, 0.148 mg / ml, and 0.196 mg / ml respectively;

[0027] Figure 2 Observation results one day after injecting tobacco with solutions containing different volumes of ursolic acid, where the solution concentrations are 0.010 mg / ml, 0.050 mg / ml, and 0.099 mg / ml respectively;

[0028] Figure 3 Observation results two days after injecting tobacco with solutions containing different volumes of ursolic acid, where the solution concentrations are 0.010 mg / ml, 0.050 mg / ml, and 0.099 mg / ml respectively;

[0029] Figure 4 Observation results four days after injecting tobacco with solutions containing different volumes of ursolic acid, where the solution concentrations are 0.010 mg / ml, 0.050 mg / ml, and 0.099 mg / ml respectively;

[0030] Figure 5Observation results after seven days of injecting tobacco with solutions containing different volumes of ursolic acid. Among them, the solution concentrations are 0.010 mg / ml, 0.050 mg / ml, and 0.099 mg / ml respectively;

[0031] Figure 6 Observation results after four days of injecting tobacco with solutions using LUC as the reporter gene. Among them, the solution concentrations are 0.010 mg / ml, 0.099 mg / ml, and 0.196 mg / ml respectively;

[0032] Figure 7 Observation results after four days of injecting tobacco with solutions using GFP as the reporter gene. Among them, the solution concentrations are 0.010 mg / ml, 0.099 mg / ml, and 0.196 mg / ml respectively;

[0033] Figure 8 Observation results after four days of injecting tobacco with solutions of different pH values. Among them, the pH values are 1, 7, 10, and 14 respectively;

[0034] Figure 9 Observation results after four days of injecting tobacco with solutions of different pH values. Among them, the pH values are 4, 6, 7, and 8 respectively;

[0035] Figure 10 Observation results after four days of injecting tobacco with solutions of different pH values. Among them, the pH values are 2, 5, 7, and 10 respectively;

[0036] Figure 11 For injecting tobacco with solutions of different OD 600 values. Observation results after four days. Among them, the solution concentration is 0.010 mg / ml, and the OD 600 values are 0.1, 0.5, 1, and 1.5 respectively;

[0037] Figure 12 For injecting tobacco with solutions of different OD600 values. Observation results after four days. Among them, the solution concentration is 0.010 mg / ml, and the OD 600 values are 0.2, 0.4, 0.6, and 0.8 respectively;

[0038] Figure 13 Observation results after one day of injecting tobacco seedlings with solutions containing ursolic acid. Among them, the solution concentration is 0.010 mg / ml;

[0039] Figure 14 Observation results after two days of injecting tobacco seedlings with solutions containing ursolic acid. Among them, the solution concentration is 0.010 mg / ml;

[0040] Figure 15Observation results four days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0041] Figure 16 Observation results seven days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0042] Figure 17 Observation results one day after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0043] Figure 18 Observation results two days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0044] Figure 19 Observation results three days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0045] Figure 20 Observation results four days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0046] Figure 21 Observation results five days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0047] Figure 22 Observation results six days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml;

[0048] Figure 23 Observation results seven days after injecting tobacco seedlings with a solution containing ursolic acid, where the solution concentration is 0.010 mg / ml. Detailed implementation method

[0049] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0050] Experimental materials

[0051] Nicotiana benthamiana, LUC bacterial solution, GFP bacterial solution, MES, MgCl 2 , acetosyringone (AS), ursolic acid

[0052] Example 1 Different Concentrations

[0053] To determine at which concentration ursolic acid can most effectively promote the transient expression efficiency, we set up multiple concentration gradients of ursolic acid for verification. At the same time, two experiments were conducted. In the first experiment, 6 different concentrations with dense concentration gradients were set to screen the concentration range where ursolic acid works efficiently. In the second experiment, based on the first experiment, the concentration range was narrowed down to 3 different concentrations to further verify the optimal working concentration for promoting the transient expression of foreign genes.

[0054] The first experiment:

[0055] 1) Add 1 ml of LUC bacterial solution to 100 ml of YEP medium (containing 100 μl of 50 mg·l -1 Rif and 100 μl of 50 mg·l -1 Kan), and culture it with shaking at 28 °C for 12 - 18 h.

[0056] 2) Transfer the LUC bacterial solution to a 50 - ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0057] 3) Discard the supernatant, add 200 ml of suspension (add 4 ml of MES, 2 ml of MgCl 2 , make up to 200 ml with ddH 2 O, and add 200 μl of AS before use) to resuspend the bacterial cells.

[0058] 4) Use a spectrophotometer to detect the OD 600 value of the bacterial suspension, and adjust the concentration of the bacterial suspension to OD600 = 0.672 with the suspension, then let the bacterial suspension stand in the dark at room temperature for 3 h.

[0059] 5) Add 1 ml of the bacterial suspension to 6 2 - ml centrifuge tubes respectively, and then add 1 μl, 2.5 μl, 5 μl, 10 μl, 15 μl, 20 μl of 10 mg / ml ursolic acid in sequence and mix well.

[0060] 6) Use the bacterial suspension as a control. Select Nicotiana benthamiana plants with basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1 - ml syringe to slowly inject the solution into the leaves (try to ensure the same injection range on one leaf).

[0061] 7) On the fourth day after injection, observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system and take pictures.

[0062] The results are as Figure 1As shown in the figure, the experiment was divided into two groups. In Group 1, four sample points were injected on tobacco leaves, namely the control, ursolic acid at 0.010 mg / ml, 0.025 mg / ml, and 0.050 mg / ml. The above was repeated three independent experiments. In Group 2, four sample points were injected on tobacco leaves, namely the control, ursolic acid at 0.099 mg / ml, 0.148 mg / ml, and 0.196 mg / ml. The above was repeated three independent experiments. The results showed that ursolic acid showed a strong effect of promoting transient expression at a concentration of 0.01 - 0.05 mg / ml, and ursolic acid at 0.01 mg / ml had the best effect. In order to further verify the above conclusion, we conducted a second experiment.

[0063] Second experiment:

[0064] 1) Add 200 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture it with shaking at 28 °C for 12 - 18 h.

[0065] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0066] 3) Discard the supernatant, add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , make it up to 100 ml with ddH 2 O, and add 100 μl of AS before use) to resuspend the bacteria.

[0067] 4) Let the bacterial suspension stand in the dark at room temperature for 3 h.

[0068] 5) Add 1 ml of the bacterial suspension to three 2 ml centrifuge tubes respectively, and then add 1 μl, 5 μl, 10 μl of 10 mg / ml ursolic acid in sequence and mix well.

[0069] 6) Use the bacterial suspension as a control. Take Nicotiana benthamiana with basically the same size, gently prick the lower epidermis of the tobacco leaf with a needle (try not to pierce it), and use a 1 ml syringe to slowly suck the solution and inject it into the leaf (try to ensure the same injection range on one leaf).

[0070] 7) Observe the fluorescence expression of the tobacco leaves and take pictures using a protein hybridization imaging system on the first, second, fourth, and seventh days after injection.

[0071] The results are as Figures 2 - 5As shown, after 2 days of infection, differential phenotypes began to appear at the sample spots. Ursolic acid at 0.010 mg / ml could promote the expression of LUC, but ursolic acid at 0.050 mg / ml and 0.099 mg / ml did not show an obvious effect on promoting transient expression. After 4 days of treatment, the LUC expression reached the highest value, and ursolic acid at 0.010 mg / ml, 0.050 mg / ml, and 0.099 mg / ml all had an effect on promoting transient expression, among which ursolic acid at 0.010 mg / ml had a more obvious effect on promoting the expression of LUC.

[0072] Based on Experiments 1 and 2, the conclusion can be drawn that as a general conventional protein, LUC luciferase protein has general significance and can represent general proteins. When ursolic acid is added to the infection solution compared with the control, the expression of LUC is significantly enhanced, and the effect of ursolic acid on enhancing the transient expression of LUC depends on the concentration of ursolic acid. Bacterial suspensions with ursolic acid concentrations of 0.010 mg / ml, 0.025 mg / ml, 0.050 mg / ml, 0.099 mg / ml, 0.148 mg / ml, and 0.196 mg / ml all have a promoting effect on improving the transient expression efficiency of tobacco, but there are differences in the promotion time and the promotion effect.

[0073] Example 2 Different Bacterial Strains

[0074] In the concentration gradient experiment, it has been confirmed that ursolic acid at 0.01 mg / ml has the strongest promoting effect on the transient expression of LUC. Then, does the LUC protein affect the experimental results? Only the data of LUC may not be sufficient to show the promoting effect of ursolic acid on transient expression. To further clarify the effect of ursolic acid, we added green fluorescent protein GFP as a new verification protein, expecting to prove that ursolic acid also has a promoting effect on the transient expression of GFP, and at the same time, we set up bacterial suspensions containing two different expressed proteins, LUC and GFP, to infect tobacco leaves and observed the results with a control.

[0075] 1) Take 200 μl each of the LUC bacterial suspension and the GFP bacterial suspension and add them to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture with shaking at 28 °C for 12 - 18 h.

[0076] 2) Transfer the LUC bacterial suspension and the GFP bacterial suspension to 50 ml centrifuge tubes respectively, and centrifuge at 4000 r·min -1 for 10 min.

[0077] 3) Discard the supernatant, and add 100 ml of suspension to the LUC bacterial cells and the GFP bacterial cells respectively (add 2 ml of MES, 1 ml of MgCl 2, make up to 100 ml with ddH 2 O, and resuspend the bacteria with an additional 100 μl of AS) before use.

[0078] 4) Let the bacterial suspension stand in the dark at room temperature for 3 h.

[0079] 5) Add 1 ml of the LUC bacterial suspension to each of several 2-ml centrifuge tubes, and then sequentially add 1 μl, 10 μl, and 20 μl of 10 mg / ml ursolic acid, and mix well; perform the same treatment on the GFP bacterial suspension.

[0080] 6) Use the LUC bacterial suspension and the GFP bacterial suspension as controls respectively. Select Nicotiana benthamiana plants of basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1-ml syringe to slowly inject the solution into the leaves (try to ensure that the injection range on one leaf is consistent).

[0081] 7) Observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system on the fourth day after injecting the LUC bacterial solution, and take pictures; observe the tobacco leaves and take pictures on the fourth day after injecting the GFP bacterial solution.

[0082] The results are as Figures 6 - 7As shown, 4 days after injecting the tobacco leaves with the LUC bacterial solution, the infiltration solution supplemented with ursolic acid showed significantly stronger LUC protein expression compared to the control; there was a similar promoting effect. When infecting tobacco leaves with GFP for 4 days, the infiltration solution containing ursolic acid showed stronger GFP signals compared to the control, indicating that ursolic acid also promoted the transient expression of GFP. This shows that ursolic acid has universality in promoting the transient expression of recombinant proteins. It is understandable that when other recombinant proteins are used, including but not limited to: hepatitis B surface antigen (HBsAg), interferon-alpha, interferon-gamma, interleukin-2, granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), insulin, human serum albumin (HSA), collagen, growth hormone, vascular endothelial growth factor (VEGF), lactoferrin, glucocerebrosidase, artemisinin, taxol, caffeine, anthocyanins, carotenoids, flavonoids, alkaloids, terpenoids, saponins, glucosinolates, salicylic acid, jasmonic acid, gibberellins, abscisic acid, ascorbic acid, tocopherols, etc., ursolic acid will have a significant promoting effect on their transient expression in tobacco leaves.

[0083] Example 3 Different pH values

[0084] pH value is an important factor affecting transient expression in tobacco. The change in pH value can affect promoter activity, and thus affect the transient expression of foreign genes. More importantly, pH value has a great impact on protein stability, including affecting enzyme activity, and thus affecting protein synthesis and degradation. Improper pH value may lead to protein misfolding and reduce the expression efficiency. At the same time, the change in pH value will affect cell metabolism, and thus affect the supply of energy and raw materials required for gene expression; extreme pH values will damage cells and reduce the transient expression efficiency. Therefore, it is very necessary to explore the effect of ursolic acid on transient expression efficiency under different pH conditions in the process of promoting tobacco transient expression. We set up three experiments. The first experiment was to explore the tolerance of tobacco leaves to the infection solution during transient expression under different pH conditions, and initially screen out the available pH values. The second experiment was to screen different pH values, hoping to obtain the pH range in which ursolic acid has a promoting effect on transient expression. The third experiment was a partial repetition of the second experiment, and at the same time, the pH value range was narrowed to obtain the pH value condition with the best effect of ursolic acid.

[0085] The first experiment:

[0086] 1) Add 500 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture with shaking at 28 °C for 12 - 18 h.

[0087] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0088] 3) Discard the supernatant, add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , make up to 100 ml with ddH 2 O, and add 100 μl of AS before use) to resuspend the bacterial cells.

[0089] 4) Use a pH meter to detect the pH value of the bacterial suspension, and adjust the pH value of the resuspended solution to 1, 7, 10, and 14 with HCl and KOH respectively, and let it stand in the dark at room temperature for 3 h.

[0090] 5) Take two Nicotiana benthamiana plants of basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce), and use a 1 ml syringe to slowly inject the solution into the leaves (try to ensure the same injection range on one leaf).

[0091] 6) Observe the tobacco leaves and take pictures on the fourth day after injection.

[0092] The results are as Figure 8As shown, when the pH is 1, 10, or 14 and transient expression lasts for 4 days, the tobacco leaves show severe wilting. Therefore, the infection solution under this pH concentration range causes greater damage to tobacco leaves. When the pH is 7, the tobacco leaves show a relatively normal state. Therefore, it can be judged that when the pH is strongly acidic or alkaline, it is not conducive to the transient expression of tobacco, providing a basis for pH selection in our second and third experiments.

[0093] The second experiment:

[0094] 1) Add 500 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture it with shaking at 28 °C for 12 - 18 h.

[0095] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0096] 3) Discard the supernatant, add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , and make up to 100 ml with ddH 2 O. Add 100 μl of AS before use) to resuspend the bacterial cells.

[0097] 4) Use a pH meter to detect the pH value of the bacterial suspension, and adjust the pH value of the resuspended solution to 1, 7, 10, and 14 with HCl and KOH respectively, and let it stand in the dark at room temperature for 3 h.

[0098] 5) Take two Nicotiana benthamiana plants of basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1 ml syringe to slowly inject the solution into the leaves (try to ensure the same injection range on one leaf).

[0099] 6) Four days after injection, use a protein hybridization imaging system to observe the fluorescence expression of the tobacco leaves and take pictures.

[0100] The results are as Figure 9 shown. In the same leaf, inject the infection solutions containing 0.01 mg / ml ursolic acid and expressing LUC Agrobacterium at different pH values. Four days after infection, the infection sites develop normally. By observing the LUC expression, it is found that the promoting effect of ursolic acid on LUC expression shows differences under different pH conditions, and the promoting effect of ursolic acid on LUC expression is more significant when the pH is 6 - 8.

[0101] The third experiment:

[0102] 1) Add 500 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l-1 Rif and 50 μl of 50 mg·l -1 Kan), and culture it with shaking at 28 °C for 12 - 18 h.

[0103] 2) Transfer the LUC bacterial solution to a 50 - ml centrifuge tube and centrifuge it at 4000 r·min -1 for 10 min.

[0104] 3) Discard the supernatant, add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , and make it up to 100 ml with ddH 2 O, and add 100 μl of AS before use) to resuspend the bacterial cells.

[0105] 4) Detect the pH value of the bacterial suspension with a pH meter, and adjust the pH value of the resuspended solution to 2, 5, 7, and 10 with HCl and KOH respectively, and let it stand in the dark at room temperature for 3 h.

[0106] 5) Take two Nicotiana benthamiana plants of basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce it), and use a 1 - ml syringe to slowly inject the solution into the leaves (try to ensure the same injection range on one leaf).

[0107] 6) On the fourth day after injection, observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system and take pictures.

[0108] The results are as Figure 10 shown. In the same leaf, the infection solutions containing 0.01 mg / ml of ursolic acid and expressing LUC Agrobacterium with different pH values were injected respectively. After 4 days of infection, the infection sites developed normally. By observing the LUC expression, it was found that the promoting effect of ursolic acid on LUC expression showed differences under different pH conditions. Among them, the promoting effect of ursolic acid on LUC expression was more significant when the pH was 7.

[0109] Combining the two experiments, it can be inferred that when the pH value of the bacterial suspension is between 6 - 8, ursolic acid has a more significant promoting effect on the high transient expression efficiency of tobacco. More specifically, when the pH is 7, the promoting effect of carinol on the transient expression efficiency of tobacco in our experiment is the best. When the pH value is too low or too high, it is not conducive to the transient expression of tobacco, and when it is 14, it will cause the necrosis of the tobacco leaves.

[0110] Example 4 Different OD 600 values

[0111] The OD value at 600 nm is used to measure the density of Agrobacterium in the infection solution. An OD value that is too high or too low will affect the expression efficiency. A too high bacterial solution concentration may cause damage to plant cells and inhibit the expression of foreign genes. A too low bacterial solution concentration will reduce the infection efficiency and result in insufficient expression levels of foreign genes. An appropriate OD value can ensure the effective infection of plant cells by Agrobacterium and promote the efficient expression of foreign genes. A too high OD value may trigger the defense response of plant cells, such as oxidative stress or cell death, affecting transient expression. An appropriate OD value can maintain the viability of plant cells and ensure the efficient expression of foreign genes. Therefore, it is very necessary to explore the effect of OD600 on the transient expression efficiency of ursolic acid in tobacco. In order to determine the optimal OD value, we conducted two experiments. The second experiment was to obtain a more accurate OD value based on the first experiment.

[0112] The first experiment:

[0113] 1) Add 1 ml of LUC bacterial solution to 100 ml of YEP medium (containing 100 μl of 50 mg·l -1 Rif and 100 μl of 50 mg·l -1 Kan), and culture with shaking at 28 °C for 12 - 18 h.

[0114] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0115] 3) Discard the supernatant, add 200 ml of suspension (add 4 ml of MES, 2 ml of MgCl 2 , make up to 200 ml with ddH 2 O, and add 200 μl of AS and 0.01 mg / ml of ursolic acid before use) to resuspend the bacterial cells.

[0116] 4) Detect the OD 600 value of the bacterial suspension with a spectrophotometer, and adjust the concentration of the bacterial suspension to OD600 = 0.1, 0.5, 1, 1.5 with the suspension, then let the bacterial suspension stand in the dark at room temperature for 3 h.

[0117] 5) Add 1 ml of the bacterial suspension and 1 μl of 10 mg / ml ursolic acid to 4 separate 2 ml centrifuge tubes respectively, and mix well.

[0118] 6) Select Nicotiana benthamiana plants with basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1 ml syringe to slowly inject the solution into the leaves (try to ensure the same injection range on one leaf).

[0119] 7) On the fourth day after injection, observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system and take pictures.

[0120] The results are as follows Figure 11 As shown, different OD value (0.1, 0.5, 1, 1.5) infection solutions containing 0.01 mg / ml ursolic acid and Agrobacterium expressing LUC were respectively injected into the same leaf. Four days after infection, the infection sites developed normally. By observing the LUC expression, it was found that the promoting effect of ursolic acid on LUC expression showed differences under different OD values. When the OD value was 0.5, the promoting effect of ursolic acid was more obvious.

[0121] The second experiment:

[0122] 1) Add 1 ml of LUC bacterial solution to 100 ml of YEP medium (containing 100 μl of 50 mg·l -1 Rif and 100 μl of 50 mg·l -1 Kan), and culture with shaking at 28 °C for 12 - 18 h.

[0123] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0124] 3) Discard the supernatant, add 200 ml of suspension (add 4 ml of MES, 2 ml of MgCl 2 , make up to 200 ml with ddH 2 O, and add 200 μl of AS and 0.01 mg / ml of ursolic acid before use) to resuspend the bacterial cells.

[0125] 4) Detect the OD 600 value of the bacterial suspension with a spectrophotometer, and adjust the concentration of the bacterial suspension to OD600 = 0.2, 0.4, 0.6, 0.8 with the suspension, then let the bacterial suspension stand in the dark at room temperature for 3 h.

[0126] 5) Add 1 ml of the bacterial suspension and 1 μl of 10 mg / ml ursolic acid to each of the 4 2 ml centrifuge tubes and mix well.

[0127] 6) Select Nicotiana benthamiana plants with basically the same size, gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1 ml syringe to slowly inject the solution into the leaves (try to ensure that the injection range on one leaf is the same).

[0128] 7) Four days after injection, observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system and take pictures.

[0129] The results are as follows Figure 12As shown, based on the first experiment, we set the OD value range around 0.5. In the same leaf, we injected infection solutions with different OD values (0.2, 0.4, 0.6, 0.8), all containing 0.01 mg / ml ursolic acid and Agrobacterium expressing LUC. After 4 days of infection, the infection sites developed normally. By observing the LUC expression, it was found that under different OD value conditions, the promoting effect of ursolic acid on LUC expression showed differences. Among the OD values of 0.4, 0.6, and 0.8, the promoting effect of ursolic acid was more obvious. Further comparison showed that the transient expression efficiency of tobacco was the highest when the OD600 value of the bacterial suspension was 0.8.

[0130] Example 5 Different times

[0131] Foreign genes usually start to express within 24 - 48 hours after Agrobacterium infection. After a certain period of time, the peak of transient expression usually appears, and then it weakens due to the degradation or silencing of foreign genes. Observing before the gene expression reaches the peak may lead to a low detected expression level. Observing after the expression peak may underestimate the expression level due to gene silencing or protein degradation. Therefore, does ursolic acid affect the time point when the peak of foreign protein appears during transient expression? Or under the condition of adding ursolic acid, how long after invading tobacco leaves can the recombinant protein achieve the highest expression efficiency? These problems will all affect the experimental effect of ursolic acid in practical applications. The experiment was carried out in two times. In the first experiment, the samples were observed discontinuously; in order to make the results more accurate, the second experiment with continuous observation for 7 days was carried out.

[0132] The first experiment:

[0133] 1) Add 200 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture it with shaking at 28 °C for 12 - 18 h.

[0134] 2) Transfer the LUC bacterial solution to a 50 ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0135] 3) Discard the supernatant, and add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , and make it up to 100 ml with ddH 2 O, and add 100 μl of AS before use) to resuspend the bacterial cells.

[0136] 4) Let the bacterial suspension stand in the dark at room temperature for 3 h.

[0137] 5) Add 10 ml of LUC bacterial suspension to a 15-ml centrifuge tube, and then add 10 μl of 10 mg / ml ursolic acid and mix well.

[0138] 6) Use the LUC bacterial suspension as a control. Select 8 Nicotiana benthamiana plants of basically the same size. Gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1-ml syringe to slowly draw up the solution and inject it into the leaves (try to ensure that the injection area on one leaf is consistent).

[0139] 7) After injecting the LUC bacterial solution, take two tobacco plants on the first, second, fourth, and seventh days respectively, observe the fluorescence expression of the tobacco leaves using a protein hybridization imaging system, and take pictures.

[0140] The results are as Figures 13 - 16 shown. After injecting the tobacco with the infection solution expressing LUC containing ursolic acid, obvious differences were observed at different days. The expression of LUC protein could be observed starting from the second day, but the expression level was not high. As the injection time increased, the transient expression efficiency of LUC in tobacco reached an observable peak on the fourth day.

[0141] Second experiment:

[0142] 1) Add 200 μl of LUC bacterial solution to 50 ml of YEP medium (containing 50 μl of 50 mg·l -1 Rif and 50 μl of 50 mg·l -1 Kan), and culture with shaking at 28 °C for 12 - 18 h.

[0143] 2) Transfer the LUC bacterial solution to a 50-ml centrifuge tube and centrifuge at 4000 r·min -1 for 10 min.

[0144] 3) Discard the supernatant, and add 100 ml of suspension (add 2 ml of MES, 1 ml of MgCl 2 , make up to 100 ml with ddH 2 O, and add 100 μl of AS before use) to resuspend the bacterial cells.

[0145] 4) Let the bacterial suspension stand in the dark at room temperature for 3 h.

[0146] 5) Add 10 ml of LUC bacterial suspension to a 15-ml centrifuge tube, and then add 10 μl of 10 mg / ml ursolic acid and mix well.

[0147] 6) Use the LUC bacterial suspension as a control. Select 14 Nicotiana benthamiana plants of basically the same size. Gently prick the lower epidermis of the tobacco leaves with a needle (try not to pierce through), and use a 1-ml syringe to slowly draw up the solution and inject it into the leaves (try to ensure that the injection area on one leaf is consistent).

[0148] 7) After injecting the LUC bacterial solution, two strains of tobacco were taken on the first to seventh days respectively, and the fluorescence expression of the tobacco leaves was observed using a protein hybridization imaging system and photographed.

[0149] The results are as Figures 17 - 23 shown. After injecting the tobacco with the infection solution expressing LUC containing ursolic acid, obvious differences were observed at different days. The expression of LUC protein could be observed starting from the second day, but the expression level was not high. Starting from the third day, the expression of LUC began to increase significantly. As the injection time increased, the transient expression efficiency of LUC in tobacco reached an observable peak on the fourth day, and the peak could last until the seventh day, still maintaining a relatively high expression level. Therefore, in actual operation, we can start observing or collecting the expression of the recombinant protein on the fourth day, when the expression level is the highest.

Claims

1. Application of ursolic acid in improving the efficiency of transient expression in tobacco leaves.

2. The use according to claim 1, characterized in that: The tobacco leaf is selected from at least one of Nicotiana benthamiana, flue-cured tobacco, air-cured tobacco, sun-cured tobacco, oriental tobacco, cigar tobacco, chewing tobacco and snuff.

3. The use according to claim 1 or 2, characterized in that: When used, ursolic acid is added to the bacterial suspension and then introduced into tobacco leaves. After 4-5 days, the fluorescence expression of the tobacco leaves can be observed using a protein hybridization imaging system.

4. The use according to claim 3, characterized in that: The bacterial suspension was added with LUC reporter gene or GFP fluorescent protein. The concentration of the bacterial suspension was OD 600 The value is 0.5-0.8, and the pH value is 6-8.

5. The use according to claim 3, characterized in that: The concentration of ursolic acid is 0.010mg / ml-0.050mg / ml.

6. Application of ursolic acid in the preparation of a botanical agent for improving the efficiency of transient expression in tobacco leaves.

7. A botanical agent, characterized in that The main component is ursolic acid with a concentration of 0.010mg / ml-0.050mg / ml.

8. A method for increasing transient expression of exogenous genes in tobacco leaves, characterized in that: This can be achieved by introducing a plasmid containing the target gene into tobacco leaves pretreated with 0.010 mg / ml-0.050 mg / ml ursolic acid.

9. The method according to claim 8, characterized in that The target gene is selected from at least one of the group consisting of: Metabolic synthesis related genes: Linalool key gene: LIS, geraniol key gene: GES, limonene key gene: LIMS, β-ionone key gene: CCD1, phenylethanol key gene: PAR, paclitaxel key gene: TS, artemisinin key gene: ADS, caffeine key gene: XMT, anthocyanin key gene: DFR, carotenoid key gene: PSY, lignin key gene: PAL, flavonoid key gene: CHS, alkaloid key gene: STR, terpenoid key gene: DXS, saponin key gene: β-AS, Key genes for glucosinolates: CYP79B2, key genes for salicylic acid: ICS, key genes for jasmonic acid: AOS, key genes for gibberellins: GA20ox, key genes for abscisic acid: NCED, key genes for vitamin C: GLOase, key genes for vitamin E: HPT, key genes for polyphenols: PAL, key genes for terpenes: GPPS, key genes for diterpenes: CPS, key genes for triterpenes: OSC, key genes for phenolic acid: C4H, key genes for sterols: SQS, key genes for polysaccharides: SUS, key genes for biodiesel precursors: DGAT; Genes related to health care and nutrition or production of traditional Chinese medicine ingredients: Taxene Key gene: TXS; Artemisinic acid key gene: CYP71AV1; ginsenoside key gene: DS; Glycyrrhizic acid key gene: CYP88D6; Ginkgolide key gene: Ginkgolide cyclase; Flavonoid key gene: CHS; Saponin key gene: CYP72A; alkaloid key gene: Nicotine synthase; polysaccharide key gene: Polysaccharide synthase; volatile oil key gene: Monoterpene synthase; vitamin A key gene: β-Carotene hydroxylase; vitamin C key gene: L-Galactono-1,4-lactone dehydrogenase; vitamin E key gene: TocopHerol synthase; lycopene key gene: Lycopene synthase; lutein key gene: Lutein synthase; resveratrol key gene: Resveratrol synthase; oligofructose key gene: Fructosyltransferase; dietary fiber key gene: Cellulose synthase; polyphenol key gene: PolypHenol oxidase; antioxidant component key gene: SOD; anti-inflammatory component key gene: COX; immunomodulatory component key gene: Immunoglobulin synthase; antiviral component key gene: Interferon synthase; antibacterial component key gene: Antimicrobial peptidesynthase; disease resistance key gene: R gene; insect resistance key gene: Bt gene; drought resistance key gene: Dehydrin gene; salt tolerance key gene: Salt tolerance gene; cold tolerance key gene: Cold tolerance gene; miRNA key gene: miRNA precursor synthase; secondary metabolism regulation key gene: MYB; anthocyanin key gene: Anthocyanin synthase; sapogenin key gene: Saponin synthase; dammarenediol key gene: CYP716A47; chalcone key gene: FLS; tocopherol key gene: TocopHerol cyclase; Genes related to pharmaceutical proteins and vaccine synthesis: key genes for hepatitis B surface antigen: HBsAg, key genes for norovirus vaccine: NVCP, key genes for rabies virus glycoprotein: RVG, key genes for human papillomavirus vaccine: HPV L1, key genes for influenza virus hemagglutinin: HA, key genes for cholera toxin B subunit: CTB, key genes for malaria vaccine candidate antigens: MSP1, key genes for HIV antigens: gp120, key genes for dengue virus antigens: DENVE, key genes for tuberculosis antigens: ESAT-6, key genes for anti-Ebola virus antibodies: 6D8, key genes for anti-HIV antibodies: 2G12, key genes for anti-rabies monoclonal antibodies: SO57, key genes for anti-cancer antibodies: Herceptin, key genes for anti-inflammatory antibodies: Infliximab, key genes for interferon-α: IFN-α, Interferon-γ key gene: IFN-γ, interleukin-2 key gene: IL-2, granulocyte colony stimulating factor key gene: G-CSF, erythropoietin key gene: EPO, glucocerebrosidase key gene: GBA, α-galactosidase key gene: GLA, lysozyme key gene: LYZ, superoxide dismutase key gene: SOD, insulin key gene: INS, human serum albumin key gene: HSA, collagen key gene: COL1A1, growth hormone key gene: GH, vascular endothelial growth factor key gene: VEGF and lactoferrin key gene: LF; Functional genes that improve leaf performance after overexpression in leaves include: 1) Improve stress resistance: DREB1A, RD29A, AREB1, NAC6, LEA3, SOS1, NHX1, HKT1, ZAT10, AVP1, CBF1, COR15A, ICE1, LOS2, ZAT12; 2) Improve disease resistance: NPR1, PR1, RPS2, EDS1, PAD4, N gene; 3) Improve yield: RBCS, FBPase, SBPase, PPDK, AGPase; 4) Promote tillering and branching: TB1, IPA1, D10, D14, D27; 5) Shorten flowering time: FT, SOC1, LFY, AP1, CO; 6) Regulate photoperiod: GI, pHYA, pHYB, CRY1, CRY2; 7) Other functions: gene editing related genes CAS protein coding genes, regulate hormone signals GA20ox, GA2ox, DELLA, ARF, PIN1, enhance antioxidant capacity SOD, CAT, APX, GPX, GR; Other marker genes: green fluorescent protein GFP, red fluorescent protein RFP, yellow fluorescent protein YFP, betaine synthesis gene RUBY, luciferase gene LUC.

10. The method according to claim 8, characterized in that The plasmid containing the target gene is obtained by the following method: The target gene is obtained by a synthetic method, and then constructed into the pacI and NotI sites of the transient expression vector PJL-TRBO by enzyme ligation.

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