Efficient transient expression system based on spraying technology

Abstract

The invention provides an efficient transient expression system based on a spraying technology, and belongs to the technical field of bioengineering. According to the efficient transient expression system based on the spraying technology, agrobacterium tumefaciens containing target genes is suspended through suspension liquid and then directly sprayed to leaves through a sprayer, and transient expression of the target genes can be promoted / achieved. The main components in the suspension provided by the invention are cane sugar, organic silicon and the like which are low in cost, so that efficient transient expression based on a spraying technology is realized; particularly, the method has wide application prospects in the aspects of outdoor creation of high-added-value target metabolites, medicinal proteins, vaccines, functional gene characteristic agricultural products, health-care and nutritional ingredients and traditional Chinese medicine ingredients in the field or improvement of agronomic traits of crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to an efficient transient expression system based on spraying technology. Background Art

[0002] Plants as bioreactors for the production of metabolites show broad application prospects. Their unique advantages include high cost-effectiveness, good safety, environmental friendliness, strong ability for correct protein folding and modification, as well as high flexibility and versatility. These characteristics make the plant system not only favored in academic research but also have great potential in industrial applications.

[0003] Tobacco transient expression technology has been widely used in the fields of biotechnology and plant metabolic engineering in recent years as a rapid, efficient, and economical method. This technology uses Agrobacterium-mediated transient expression of foreign genes without integration into the host genome. This method can not only accelerate the research process but also provide a new way for industrial-scale production of valuable metabolites. Through transient expression technology, plants can rapidly and efficiently produce a variety of valuable compounds, such as recombinant proteins, vaccine components, antibodies, enzymes, secondary metabolites, etc. For example, in the medical field, plants have been successfully used to manufacture therapeutic antibodies and prophylactic vaccines, providing a new way to quickly respond to public health crises; in the agricultural field, metabolic engineering is used to transform plants to improve their resistance to pests and diseases or improve nutritional quality, providing new ideas for sustainable agricultural development. Therefore, the production of metabolites by plants not only helps to solve current technical problems but also promotes innovation and development in multiple industries, becoming one of the important platforms for future biofabrication.

[0004] However, although transient expression technology shows promising application potential in the field of biosynthesis, the current conventional method is still to introduce foreign DNA into plant cells such as tobacco through vacuum infiltration technology, which is time-consuming and laborious and is only suitable for laboratory conditions and difficult to operate in the field. Therefore, this greatly limits the application of transient expression technology in production. Agrobacterium spraying may be an effective way to solve the above problems. In the field, an infection solution containing the target gene is directly sprayed onto the leaves of plants such as tobacco using a spraying system. This method is highly operable and simple to promote. However, unfortunately, there has been no successful application case so far because the method is very inefficient, so that Agrobacterium cannot effectively enter the plant leaves and express. Therefore, how to improve the technical effect of spraying is the key problem to solve the field application of plant transient expression. Summary of the Invention

[0005] The present invention provides an efficient transient expression system based on spraying technology, achieving efficient transient expression based on spraying technology, and having broad application prospects especially in creating high-value target metabolites, medicinal proteins, vaccines, functional gene characteristic agricultural products, health care and nutritional components, traditional Chinese medicine components or improving crop agronomic traits.

[0006] To achieve the above object, the present invention provides an efficient transient expression system based on spraying technology. Agrobacterium containing the target gene is suspended by a suspension, and then directly sprayed onto the leaves with a sprayer, which can promote / achieve the transient expression of the target gene.

[0007] Preferably, the suspension consists of tap water and 1% silicone. It can be understood that silicone, as an adjuvant, helps the infection solution better enter the plant and play its role. The reasons are as follows: Silicone can enhance the wettability of the leaf surface, reduce the surface tension of water, make the liquid better spread on the leaf surface, increase the contact area, and thus improve the material penetration efficiency. Improving stomatal penetration helps to open the leaf stomata and promote the entry of substances into the plant through the stomata. Enhancing cutin penetration can soften the leaf cutin, making it easier for foreign substances to penetrate and improving the absorption efficiency. Improving adhesion and persistence can enhance the adhesion of the liquid on the leaves, reduce loss, extend the action time, and indirectly improve the efficiency of substances entering the leaves. Reducing evaporation, the formed film can reduce water evaporation, keep the leaf surface moist, and extend the material penetration time. It can be clearly understood from the principle that the following substances may be able to replace silicone or enhance its effect as a synergistic agent. Surfactants (saponins (such as tea saponin, soap tree extract), sophorolipids (microbial fermentation products)); natural oils and esters (such as orange oil, pine oil, coconut oil methyl ester, lignosulfonic acid); synthetic polymers (polyglycerol esters, polyaspartic acid); minerals and nanomaterials (nano-silica, silicates (such as potassium silicate), bentonite); co-surfactants (zwitterionic surfactants (such as betaines), fluorinated surfactants (such as perfluoropolyethers)); natural synergists (chitosan, humic acid); functional additives (urea, glycerol or propylene glycol); polysaccharides (such as xanthan gum, chitosan), proteins (such as hydrolyzed protein), using gene editing technology or other bioengineering technologies to promote the expression of plant stomatal regulatory proteins and indirectly enhance the penetration efficiency of silicone, etc. It can also be understood that based on general knowledge, adding surfactants such as Tween 20 and sodium benzenesulfonate to the suspension can also achieve similar effects, and the technologies using other surfactants to achieve the purpose of the present invention are also within the protection scope of the present invention.

[0008] Preferably, the suspension contains 1%, 2% or 5% sucrose or does not contain sucrose.

[0009] Preferably, the suspension does not contain MgCl2 and / or MES.

[0010] Preferably, the leaf is selected from at least one of Nicotiana benthamiana, flue-cured tobacco, sun-cured tobacco, air-cured tobacco, oriental tobacco, cigar tobacco leaf, chewing tobacco, and snuff.

[0011] Preferably, the target gene is selected from at least one gene in the group consisting of genes suitable for normal expression in tobacco leaves:

[0012] Metabolic synthesis-related genes: Key gene for linalool: LIS (Linalool Synthase), derived from lavender, citrus, etc., with the function of floral and citrus scents; Key gene for geraniol: GES (Geraniol Synthase), derived from rose, citronella, etc., with the function of rose scent; Key gene for limonene: LIMS (Limonene Synthase), derived from citrus fruits, with the function of citrus scent; Key gene for β-ionone: CCD1 (Carotenoid Cleavage Dioxygenase 1), derived from rose, tomato, etc., with the function of floral and fruity scents; Key gene for phenylethyl alcohol: PAR (Phenylacetaldehyde Reductase), derived from rose, jasmine, etc., with the function of floral scent; Key gene for taxol: TS (Taxadiene Synthase), derived from Taxus chinensis, with the function of anti-cancer drug; Key gene for artemisinin: ADS (Amorpha-4,11-diene Synthase), derived from Artemisia annua, with the function of anti-malaria drug; Key gene for caffeine: XMT (Xanthosine Methyltransferase), derived from coffee, tea, etc., with the function of central nervous stimulant; Key gene for anthocyanin: DFR (Dihydroflavonol 4-Reductase), derived from grape, blueberry, etc., with the function of antioxidant; Key gene for carotenoids: PSY (Phytoene Synthase), derived from carrot, tomato, etc., with the functions of antioxidant and pigment; Key gene for lignin: PAL (Phenylalanine Ammonia-Lyase), derived from wood, with the function of cell wall structure; Key gene for flavonoids: CHS (Chalcone Synthase), derived from soybean, apple, etc., with the function of antioxidant; Key gene for alkaloids: STR (Strictosidine Synthase), derived from Catharanthus roseus, with the function of anti-cancer drug; Key gene for terpenoids: DXS (1-Deoxy-D-Xylulose 5-Phosphate Synthase), derived from various plants, with the function of various biological activities; Key gene for saponins: β-AS (β-Amyrin Synthase), derived from Panax ginseng, Glycyrrhiza glabra, etc., with the function of immunomodulation;Key genes of glucosinolates: CYP79B2, derived from cruciferous plants, functions as a defense compound; key gene of salicylic acid: ICS (Isochorismate Synthase), derived from willow, functions as plant defense; key gene of jasmonic acid: AOS (Allene Oxide Synthase), derived from various plants, functions as a plant hormone; key gene of gibberellins: GA20ox (Gibberellin 20-Oxidase), derived from fungi and plants, functions as plant growth regulation; key gene of abscisic acid: NCED (9-cis-Epoxycarotenoid Dioxygenase), derived from various plants, functions as a plant hormone; key gene of ascorbic acid: GLOase (L-Galactono-1,4-lactone Dehydrogenase), derived from citrus and tomato, functions as an antioxidant; key gene of tocopherols: HPT (Homogentisate Phytyltransferase), derived from wheat and corn, functions as an antioxidant; key gene of polyphenols: PAL (Phenylalanine Ammonia-Lyase), derived from grape and tea, functions as an antioxidant; key gene of monoterpenes: GPPS (Geranyl Diphosphate Synthase), derived from pine and mint, functions as aroma and defense; key gene of diterpenes: CPS (Copalyl Diphosphate Synthase), derived from pine and rice, functions as defense; key gene of triterpenes: OSC (Oxidosqualene Cyclase), derived from ginseng and licorice, functions as biological activity; key gene of phenolic acids: C4H (Cinnamate 4-Hydroxylase), derived from various plants, functions as an antioxidant; key gene of sterols: SQS (Squalene Synthase), derived from soybean and corn, functions as a cell membrane component; key gene of polysaccharides: SUS (Sucrose Synthase), derived from various plants, functions as energy storage;Key genes of biodiesel precursors (Triacylglycerols): DGAT (Diacylglycerol Acyltransferase), derived from oil crops, acting as an energy source;

[0013] Genes related to the production of health care, nutrition or traditional Chinese medicine components: Key genes of taxadiene: TXS; Key genes of artemisic acid: CYP71AV1; Key genes of ginsenosides: DS; Key genes of glycyrrhizic acid: CYP88D6; Key genes of ginkgolides: Ginkgolide cyclase; Key genes of flavonoids: CHS; Key genes of saponins: CYP72A; Key genes of alkaloids: Nicotine synthase; Key genes of polysaccharides: Polysaccharide synthase; Key genes of volatile oils: Monoterpene synthase; Key genes of vitamin A: β-Carotene hydroxylase; Key genes of vitamin C: L-Galactono-1,4-lactone dehydrogenase; Key genes of vitamin E: Tocopherol synthase; Key genes of lycopene: Lycopene synthase; Key genes of lutein: Lutein synthase; Key genes of resveratrol: Resveratrol synthase; Key genes of fructooligosaccharides: Fructosyltransferase; Key genes of dietary fiber: Cellulose synthase; Key genes of polyphenols: Polyphenol oxidase; Key genes of antioxidant components: SOD; Key genes of anti-inflammatory components: COX; Key genes of immunomodulatory components: Immunoglobulin synthase; Key genes of antiviral components: Interferon synthase; Key genes of antibacterial components: Antimicrobial peptidesynthase; Key genes of disease resistance: R gene; Key genes of insect resistance: Bt gene; Key genes of drought resistance: Dehydrin gene; Key genes of salt resistance: Salt tolerance gene; Key genes of cold resistance: Cold tolerance gene; Key genes of miRNA: miRNA precursor synthase; Key genes of secondary metabolism regulation: MYB; Key genes of anthocyanins: Anthocyanin synthase; Key genes of sapogenins: Saponin synthase; Key genes of dammarenediol: CYP716A47; Key genes of chalcone: FLS; Key genes of tocopherol: Tocopherol cyclase;

[0014] Medicinal protein, vaccine synthesis-related genes: Key gene of hepatitis B surface antigen (HBsAg): HBsAg, applied as hepatitis B vaccine; Key gene of Norwalk virus vaccine (Norwalk Virus Capsid Protein): NVCP, applied as Norwalk virus vaccine; Key gene of rabies virus glycoprotein (Rabies Virus Glycoprotein): RVG, applied as rabies vaccine; Key gene of human papillomavirus vaccine (HPV L1 Protein): HPV L1, applied as cervical cancer vaccine; Key gene of influenza virus hemagglutinin (Influenza Hemagglutinin): HA, applied as influenza vaccine; Key gene of cholera toxin B subunit (Cholera Toxin B Subunit): CTB, applied as cholera vaccine; Key gene of malaria vaccine candidate antigen (MalariaAntigen MSP1): MSP1, applied as malaria vaccine; Key gene of human immunodeficiency virus antigen (HIV gp120): gp120, applied for research on AIDS vaccine; Key gene of dengue virus antigen (Dengue Virus E Protein): DENV E, applied as dengue vaccine; Key gene of tuberculosis antigen (TB Antigen ESAT-6): ESAT-6, applied as tuberculosis vaccine; Key gene of anti-Ebola virus antibody (Anti-Ebola Antibody): 6D8, applied for the treatment of Ebola virus; Key gene of anti-HIV antibody (Anti-HIV Antibody 2G12): 2G12, applied for research on AIDS treatment; Key gene of anti-rabies monoclonal antibody (Anti-Rabies Monoclonal Antibody): SO57, applied for the treatment of rabies; Key gene of anti-cancer antibody (Anti-Cancer Antibody Trastuzumab): Herceptin, applied for the treatment of breast cancer; Key gene of anti-inflammatory antibody (Anti-TNF Antibody): Infliximab, applied for the treatment of rheumatoid arthritis; Key gene of interferon-α (Interferon-alpha): IFN-α, applied for antiviral and anti-cancer; Key gene of interferon-γ (Interferon-gamma): IFN-γ, applied for immunomodulation; Key gene of interleukin-2 (Interleukin-2): IL-2, applied for cancer immunotherapy; Key gene of granulocyte colony-stimulating factor (G-CSF): G-CSF, applied for promoting leukocyte production; Key gene of erythropoietin (Erythropoietin, EPO): EPO, applied for the treatment of anemia;Key genes of Glucocerebrosidase: GBA, application: treatment of Gaucher's disease; key genes of Alpha-Galactosidase: GLA, application: treatment of Fabry disease; key genes of Lysozyme: LYZ, application: antibacterial agent; key genes of Superoxide Dismutase (SOD): SOD, application: antioxidant therapy; key genes of Insulin: INS, application: treatment of diabetes; key genes of Human Serum Albumin (HSA): HSA, application: plasma substitute; key genes of Collagen: COL1A1, application: tissue engineering; key genes of Growth Hormone: GH, application: treatment of growth disorders; key genes of Vascular Endothelial Growth Factor (VEGF): VEGF, application: angiogenesis therapy; and key genes of Lactoferrin: LF, application: antibacterial, immunomodulation;

[0015] Functional genes that can significantly increase crop stress resistance, improve yield, enhance quality, and shorten flowering time after overexpression in leaves, including: 1) Improving stress resistance: DREB1A (Dehydration-Responsive Element Binding Protein 1A), RD29A (Responsive to Desiccation 29A), AREB1 (ABA-Responsive Element Binding Protein 1), NAC6 (NAM, ATAF, and CUC 6), LEA3 (Late Embryogenesis Abundant 3), SOS1 (Salt Overly Sensitive 1), NHX1 (Na+ / H+ Antiporter 1), HKT1 (High-Affinity K+ Transporter 1), ZAT10 (Zinc Finger of Arabidopsis thaliana 10), AVP1 (Vacuolar Pyrophosphatase 1), CBF1 (C-Repeat Binding Factor 1), COR15A (Cold-Regulated 15A), ICE1 (Inducer of CBF Expression 1), LOS2 (Low Expression of Osmotically Responsive Genes 2), ZAT12 (Zinc Finger of Arabidopsis thaliana 12); 2) Improving disease resistance: NPR1 (Non-Expressor of PR Genes 1), PR1 (Pathogenesis-Related Protein 1), RPS2 (Resistance to Pseudomonas syringae 2), EDS1 (Enhanced Disease Susceptibility 1), PAD4 (Phytoalexin Deficient 4), N gene (NtCN);3) Increase yield: RBCS (Ribulose-1,5-Bisphosphate Carboxylase Small Subunit), FBPase (Fructose-1,6-Bisphosphatase), SBPase (Sedoberptulose-1,7-Bisphosphatase), PPDK (Pyruvate Orthophosphate Dikinase), AGPase (ADP-Glucose Pyrophosphorylase); 4) Promote tillering and branching: TB1 (Teosinte Branched 1), IPA1 (Ideal Plant Architecture 1), D10 (Dwarf 10), D14 (Dwarf 14), D27 (Dwarf 27); 5) Shorten flowering time: FT (Flowering Locus T), SOC1 (Suppressor of Overexpression of Constans 1), LFY (Leafy), AP1 (Apetala 1), CO (Constans); 6) Regulate photoperiod: GI (Gigantea), PHYA (Phytochrome A), PHYB (Phytochrome B), CRY1 (Cryptochrome 1), CRY2 (Cryptochrome 2); 7) Other functions: Genes related to gene editing, CAS protein-coding genes, regulate hormone signals GA20ox (Gibberellin 20-Oxidase), GA2ox (Gibberellin 2-Oxidase), DELLA (DELLA Protein), ARF (Auxin Response Factor), PIN1 (Pin-Formed 1), enhance antioxidant capacity SOD (Superoxide Dismutase), CAT (Catalase), APX (Ascorbate Peroxidase), GPX (Glutathione Peroxidase), GR (Glutathione Reductase);

[0016] Marker genes: Green fluorescent protein (GFP), Red fluorescent protein (RFP), Yellow fluorescent protein (YFP), Betaine synthesis gene (RUBY), Luciferase gene (LUC).

[0017] Preferably, the Agrobacterium containing the target gene is obtained by the following method:

[0018] The target gene was obtained by a synthetic method, and the target gene was constructed into the pacI and NotI sites of the transient expression vector PJL-TRBO by restriction enzyme digestion and ligation methods;

[0019] By chemical transformation method, the target gene was introduced into the competent Agrobacterium tumefaciens GV3101, and then positive clones were selected by sequencing identification for propagation;

[0020] The Agrobacterium tumefaciens was cultured to 500 - 1000 mL, centrifuged to precipitate, the supernatant was removed, washed with water, and then centrifuged again to remove the supernatant, leaving the precipitate;

[0021] The precipitated Agrobacterium tumefaciens was transported to the outdoor field, resuspended with ordinary water, and then it was ready for use.

[0022] It can be understood that the synthetic method, chemical transformation method, sequencing identification method, etc. for obtaining the target gene involved in the above methods are all conventional methods well-known in the art, and can be selected and applied according to different target genes in the art.

[0023] The present invention also provides an application of the high-efficiency transient expression system according to any one of the above technical solutions in the transient expression of field plants.

[0024] Preferably, the field plants are selected from at least one of tobacco, lettuce, rice, wheat, flax, soybean, and cabbage.

[0025] The present invention also provides an application of the high-efficiency transient expression system according to any one of the above technical solutions in creating high-value-added target metabolites, pharmaceutical proteins, vaccines, functional gene characteristic agricultural products, or improving crop agronomic traits.

[0026] Preferably, the target metabolite is selected from at least one of the group consisting of: Artemisinin, Taxol, Caffeine, Anthocyanins, Carotenoids, Flavonoids, Alkaloids, Terpenoids, Saponins, Glucosinolates, Salicylic Acid, Jasmonic Acid, Gibberellins, Abscisic Acid, Ascorbic Acid, Tocopherols, Polyphenols, Lignin, Aromatic Compounds, Polysaccharides, Phytosterols, Phenolic Acids, Tannins, Coumarins, Phytohormones, Essential Oils, Plant Pigments, Plant Antioxidants, Plant Antimicrobial Peptides, Plant Fibers, Quercetin, Rutin, Apigenin, Soy Isoflavones, Anthocyanins, Ginsenosides, Glycyrrhizin, Ginkgolides, Berberine, Morphine, Nicotine, Quinine, Chlorogenic Acid, Gallic Acid, Ferulic Acid, Ellagic Acid, Rosmarinic Acid, Ganoderma Lucidum Polysaccharides, Lentinan, Lycium BarbarumPolysaccharides), Astragalus Polysaccharides, Tremella Polysaccharides, Menthol, Eucalyptol, Geraniol, Linalool, Limonene, Resveratrol, Curcumin, Lycopene, Lutein, β-Carotene, Coenzyme Q10, D-Limonene, GABA, Glutathione, Melatonin;

[0027] Hepatitis B surface antigen (HBsAg), Interferon-alpha, Interferon-gamma

[0028] (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, Alpha-Galactosidase, Lysozyme, Superoxide Dismutase (SOD), Anti-Ebola Antibody, Anti-HIV Antibody 2G12, Anti-Rabies Monoclonal Antibody, Anti-Cancer Antibody Trastuzumab, Hepatitis B Vaccine, Norwalk Virus Vaccine, Rabies Vaccine, Human Papillomavirus Vaccine (HPV Vaccine), Influenza Vaccine, Cholera Vaccine, Malaria Vaccine, HIV Vaccine, Dengue Vaccine, TB Vaccine, Rotavirus Vaccine, Pneumococcal Vaccine, Pertussis Vaccine, Tetanus Vaccine, Diphtheria Vaccine, Measles Vaccine, Rubella Vaccine, Varicella Vaccine, Ebola Vaccine, and Zika Vaccine.

[0029] Preferably, the spraying amount of the high-efficiency transient expression system is 2 - 5 mL / leaf, and it is sufficient to completely spray the entire leaf.

[0030] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0031] 1. In the prior art, the Agrobacterium infiltration solution in a conventional laboratory contains different chemical substances such as MgCl 2 , MES (2-(N-morpholino)ethanesulfonic acid), etc. For the convenience of field operation, the present invention directly replaces the infiltration solution with ordinary tap water, without adding the above-mentioned chemical drugs, and there is no need to sterilize the infiltration solution, greatly reducing the cost.

[0032] 2. In the prior art, conventional transient infiltration techniques require vacuum or injection operations, while the present invention only needs to directly spray the suspension of Agrobacterium containing the target gene onto the leaves with a sprayer to achieve the transient expression of the target gene, without the need for special expensive equipment, and the operation is very convenient.

[0033] 3. The main components in the suspension provided by the present invention are low-cost sucrose, silicone, etc., realizing efficient transient expression based on spraying technology, and having broad application prospects especially in creating tobacco leaves with high target metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shows the phenotypic changes after spray treatment of GFP provided by the embodiment of the present invention. Among them, CK represents ddH 2 O, A represents tap water + sucrose + 1% silicone, B represents tap water + sucrose + acetosyringone;

[0035] Figure 2 Shows the phenotypic changes after spray treatment of RUBY (betaine, a secondary metabolite) provided by the embodiment of the present invention. Among them, CK represents ddH 2 O, A represents tap water + sucrose + acetosyringone, B represents tap water + sucrose + 1% silicone, C represents tap water + acetosyringone + Silwet L-77;

[0036] Figure 3 Shows the transient expression of GFP in Nicotiana benthamiana at different times provided by the embodiment of the present invention. Among them, CK represents ddH 2 O, A represents tap water + sucrose + acetosyringone + 1% silicone, B represents tap water + sucrose + acetosyringone;

[0037] Figure 4 Shows the transient expression of GFP in hydroponic Nicotiana benthamiana under different treatment conditions provided by the embodiment of the present invention. Among them, CK represents ddH 2 O, A represents tap water + acetosyringone, B represents tap water + sucrose + acetosyringone, C represents tap water + sucrose + 1% silicone, D represents tap water + 1% silicone;

[0038] Figure 5 For the transient expression of RUBY (betaine, a secondary metabolite) in hydroponic Nicotiana benthamiana under different condition treatments provided by the embodiments of the present invention, where CK represents ddH 2 O, A represents tap water + acetosyringone, B represents tap water + sucrose + acetosyringone, C represents tap water + sucrose + 1% silicone, D represents tap water + 1% silicone;

[0039] Figure 6 For the transient expression of GFP in Nicotiana benthamiana under different silicone concentration treatments provided by the embodiments of the present invention;

[0040] Figure 7 For the transient expression of GFP in cultivated tobacco provided by the embodiments of the present invention. Specific embodiments

[0041] 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 embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1 Plant test materials

[0043] The plant materials used in the experiment include: Nicotiana benthamiana, and the cultivated tobacco (Nicotiana tabacum) variety is Havana. The growth conditions of hydroponic Nicotiana benthamiana and cultivated tobacco are at a temperature of 22 - 25°C, 16 h light / 8 h dark, and a humidity of 70 - 80%. Six-week-old plants were selected for spray inoculation. For cultivated tobacco, slightly larger seedlings about 7 - 8 weeks old were selected.

[0044] Example 2 Selection of suspension formulation

[0045] The following will observe the transient expression effect of the target gene obtained by the simplest spray method by treating the above tobacco with suspensions of different formulations.

[0046] The experimental group was 2 hydroponic Nicotiana benthamiana plants treated by spraying, and the control group CK, hydroponic tobacco, was treated with ddH 2 O.

[0047] The first group of suspension formulation

[0048] Suspension formulation: Comparison between tap water + 2% sucrose + 1% silicone (A) and tap water + 2% sucrose + 0.2% acetosyringone (B).

[0049] The PJL-TRBO-GFP (green fluorescent protein) and PEAQ-RUBY (betaine, a secondary metabolite) strains were cultured separately in YEP (Kan+Rif) medium and placed in a shaker at 28°C with a shaking speed of 200 rpm for 18 - 20 h to make the bacterial solution as concentrated as possible. The shaken bacterial solutions were poured into 50 ml centrifuge tubes respectively, centrifuged at 4000 rpm for 10 min to collect the bacterial cells, and the bacterial cells were suspended with the above-mentioned Suspensions A and B respectively, and then poured into a clean and sterile 50 ml small sprayer, and directly sprayed on the front and back leaves of tobacco plants without standing. When spraying, it should be uniform. After the spraying treatment, cover the whole plant with a transparent cover for a period of time to prevent the rapid evaporation of the bacterial solution and observe the expression of the plant. Among them, PJL-TRBO-GFP was obtained by ligating the GFP gene between pacI and NotI through general molecular biology techniques of restriction enzyme digestion and ligation using the vector PJL-TRBO as the backbone; PEAQ-RUBY was obtained by ligating the gene expressing RUBY protein between AgeI and XhoI through general molecular biology techniques of restriction enzyme digestion and ligation using the vector PEAQ-HT as the backbone.

[0050] The results are as Figure 1 shown. Figure 1 In the experiment, it can be seen that the green fluorescent protein in the leaves began to be expressed on the 5th day after spraying P. benthamiana with the GFP suspension. Under ultraviolet light, scattered green fluorescent protein could be seen, but the expression effect was very weak. However, by the 7th day, the protein expression level increased significantly, and the expression level after treatment with 1% silicone solution was significantly higher than that after treatment with acetosyringone solution. By the 10th day, the expression level of fluorescent protein after treatment with 1% silicone solution was significantly higher than that of the control and the treatment with acetosyringone solution. The results indicate that 1% silicone solution can promote the expression of GFP protein.

[0051] Figure 2 It can be seen from

[0052] that the gene expression of RUBY in different suspensions on the 7th day. Obvious red spots appeared in the suspension with 1% silicone added, and the expression effect of RUBY was more significant after adding Silwet L-77, and the expression was more significant on the 10th day. This result is similar to the expression trend of GFP fluorescent protein.

[0053] The second group of suspension formula

[0054] To further verify the effect of the silicone solution, a first repeated experiment was conducted based on the above corresponding experiments by preparing a second set of suspension formulations.

[0055] Suspension formulation: Comparison between tap water + sucrose + acetosyringone + 1% silicone (A) and tap water + sucrose + acetosyringone (B).

[0056] Photos were taken at different times. As shown in Figure 3 shown, Figure 3 in which the effect of the suspension with 1% silicone added was significant. It can be seen that green fluorescent protein had started to be expressed in the leaves of Nicotiana benthamiana on the 5th day after spraying treatment, and the protein expression level increased significantly on the 8th day. The expression level after adding the silicone solution was significantly higher than that after adding the acetosyringone solution. On the 10th day, the expression level of fluorescent protein after adding the silicone solution was significantly higher than that of the control and the suspension with acetosyringone added. The results showed that the 1% silicone solution could significantly promote the efficient transient expression of foreign genes by direct spraying. This was the same as the trend of the first result.

[0057] The third set of suspension formulations

[0058] For the second repeated experiment, two bacterial suspensions of GFP and RUBY were still used for simultaneous treatment. Since the ultimate goal of this experiment was to apply it to agricultural production, the selected suspension components were relatively simple.

[0059] The third set of suspension formulations: Comparison between tap water + acetosyringone (A), tap water + sucrose + acetosyringone (B), tap water + sucrose + 1% silicone (C), and tap water + 1% silicone (D).

[0060] Figure 4 and 5 showed the phenotypic changes of the leaves observed at different time points after direct spraying on the leaves. Green fluorescence had started to appear in the leaves treated with the silicone solution added to the suspension on the 3rd day after spraying. There were no significant phenotypic changes in the leaves treated with only acetosyringone without surfactant. As time passed, the effect became more significant. From the local photos of the leaves on the 10th day, it could be seen that the expression of GFP in the leaves treated with silicone was more significant compared to the control and the suspension with only acetosyringone added.

[0061] The expression trends of RUBY and GFP processed together are the same. The results more precisely demonstrate that the silicone solution can significantly promote the transient expression of the target gene, and the effect of silicone is better than that of acetosyringone. Additionally, some studies have shown that the suspension affects the transient infection efficiency of Agrobacterium in plants, and sucrose is one of the important components. However, based on the experimental results of groups C and D, there is no obvious difference in the gene expression effects between the suspension with added sucrose and the suspension without added sucrose. Thus, it can be seen that adding 1% silicone solution to the suspension can indeed promote the transient expression of the target gene.

[0062] Example 3 Optimization of the Suspension Formula

[0063] To further optimize the optimal conditions of the suspension and screen the optimal concentration of silicone, the suspension only contains water + silicone solution, and four different gradients of the concentration of the added silicone solution are set: 0.2%, 0.5%, 1%, and 2%. The expression effects of GFP are observed on the 3rd day, 7th day, and 10th day respectively.

[0064] Figure 6 It can be seen that the phenotype of dry leaves appeared on the leaves on the 3rd day when the suspension sprayed with 2% silicone was used. Obvious phenotypes appeared on the 7th day. The expression effects of GFP treated with different concentrations of silicone are different. Among them, the expression effect of 1% is the best compared with other concentrations. For example, if the concentration of the 2% silicone adjuvant is too high, it may cause the decomposition of the cutin layer of the leaf epidermal cells, resulting in a decrease in the water retention ability of the leaves, cell damage, and ultimately leaf chlorosis and withering, showing the situation of leaf burning. While the concentrations of 0.2% and 0.5% of the silicone adjuvant are too low. For example, when the treated material is under the condition of 0.2% silicone, only weak GFP dots can be observed after 7 days of treatment, and the effect of 0.5% silicone is better than that of 0.2% silicone, and slightly stronger GFP green fluorescence can be observed, but it is still significantly lower than the expression under the condition of 1% silicone. Therefore, it can be concluded that whether the silicone is less than 1% or higher than 1%, the transient expression effect mediated by the spraying technique is not good, and the most ideal concentration of silicone is 1%.

[0065] Further observation shows that when the cultivated tobacco is treated with 1% silicone suspension and photographed at different times as Figure 7 , the results show that with the passage of time, the expression effect of GFP in the cultivated tobacco is significantly enhanced, and the expression effect of the cultivated tobacco is similar to that of Nicotiana benthamiana. On the 10th day, GFP almost covers the entire leaf in the sprayed leaves.

[0066] In the above embodiments, the treatment cases of two strains, GFP and RUBY, are given. This is because the ultimate goal of this experiment is to apply it to agricultural production, and the observation of the above two during the experiment is relatively simple and convenient. However, the present invention is not limited thereto. The core point of the present invention is to provide an efficient transient expression based on spraying technology applicable to agricultural production. When spraying, it is only necessary to suspend the Agrobacterium containing the target gene with a specific suspension. For example, at least the following scenarios can be achieved by using the novel spraying technology described in the present invention:

[0067] 1) Efficient transient expression of important aroma-producing products in field tobacco can directly obtain tobacco leaves containing the target aroma-producing products, reduce the subsequent use of tobacco industrial flavoring agents, greatly save costs, and the obtained novel tobacco leaves are natural aroma components, and the green cut is closer to nature. For example, citronellol is the main component of rose fragrance. Expressing the key genes ObGES, RrGER01, and RrCAD04 for citronellol synthesis can achieve the production of natural rose fragrance in tobacco leaves; ionone and linalool are important fragrance compounds. High-ionone tobacco leaves can be achieved by expressing PSY, GGPP, PDS, ZDS, LCY, and CCD; high-linalool tobacco leaves can be achieved by expressing DXS, DXR, MCT, HDS, and LIS.

[0068] 2) Efficient transient expression of important stress-resistant and disease-resistant genes in field tobacco can directly obtain non-transgenic tobacco with improved resistance and be directly applied to tobacco leaf production. For example, expressing the N gene (NtCN) against tobacco mosaic virus (TMV) can achieve the direct and efficient transient expression of this gene in the field, and the tobacco leaves produce the NtCN gene, achieving the acquisition of TMV-resistant tobacco leaves in a short time and at low cost. By expressing key genes to achieve high expression of quercetin, naringenin, phytoalexin, caffeic acid, and jasmonic acid, tobacco with improved disease resistance can be directly obtained.

[0069] 3) Efficient transient expression of important high-value-added metabolites in field tobacco can directly obtain tobacco leaves containing the target metabolites, realizing the development of novel tobacco leaves with real health attributes. For example, chlorogenic acid has important application potential in aspects such as anti-inflammatory, antibacterial, and blood sugar lowering. By expressing the key genes for chlorogenic acid synthesis NtHQT , Nt4CL , AtC3H and other genes to achieve high-chlorogenic acid tobacco leaves. Galantamine, a drug approved by the FDA for the treatment of Alzheimer's disease, can be expressed by simultaneously expressing tobacco NtCYP96T6, NtNMT1, and NtAKR1, and tobacco with a therapeutic effect on Alzheimer's disease can be obtained.

[0070] 4) High-efficiency transient expression in field tobacco for the production of health care and nutritional components, and traditional Chinese medicine components. Using the method of the present invention, it is possible to produce various health care and nutritional components, and traditional Chinese medicine components such as Quercetin, Rutin, Apigenin, SoyIsoflavones, Anthocyanins, Ginsenosides, Glycyrrhizin, Ginkgolides, Berberine, Morphine, Nicotine, Quinine, Chlorogenic Acid, Gallic Acid, Ferulic Acid, Ellagic Acid, Rosmarinic Acid, Ganoderma Lucidum Polysaccharides, Lentinan, LyciumBarbarum Polysaccharides, Astragalus Polysaccharides, Tremella Polysaccharides, Menthol, Eucalyptol, Geraniol, Linalool, Limonene, Resveratrol, Curcumin, Lycopene, Lutein, β-Carotene, Coenzyme Q10, D-Limonene, GABA, Glutathione, Melatonin, etc.

[0071] 5) High-efficiency transient expression of medicinal, medical aesthetic proteins, vaccines, etc. in field tobacco can achieve large-scale production of medicinal proteins in the field. Currently, Nicotiana benthamiana can be used as a vector to transiently express and produce medicinal proteins. However, the biomass of Nicotiana benthamiana is much lower than that of cultivated tobacco. Due to the large size of cultivated tobacco plants, they are not suitable for hydroponic management. Due to limitations in conditions, conventional transient expression techniques can only be carried out under indoor conditions and cannot achieve low-cost, large-scale high-efficiency transient expression of field tobacco. However, the present invention can achieve high-efficiency transient expression of tobacco in the field, so it has great application potential in the production of medicinal proteins or vaccines. For example, by expressing genes encoding single-chain antibody fragments (scFv) or full-length antibodies for the treatment of cancers, autoimmune diseases, etc.; by expressing cytokines such as interferons and interleukins for immune regulation; by expressing genes encoding IFN-α, IL-2, etc. for X; by expressing genes encoding type I or type III collagen for skin repair and anti-aging; by expressing genes encoding EGF, etc. for skin regeneration; by expressing genes encoding HPV L1 protein to obtain vaccines such as HPV vaccines; by expressing genes encoding influenza virus hemagglutinin (HA) to obtain subunit vaccines such as influenza vaccines, etc.

[0072] 6) Realize the combined application of spraying transient expression technology and CRISPR / Cas gene editing. Currently, the transient expression for gene editing reported in the literature is basically carried out in the laboratory. Transient expression, as a technical means, can achieve a non-transgenic gene editing strategy quickly, but it cannot be used to quickly and efficiently carry out non-gene target operations using gene editing on a large scale in the field. As a way to deliver genes, the high-efficiency spraying transient expression technology achieved by the present invention can quickly and efficiently transfer Agrobacterium containing the CAS protein-encoding gene (such as cas9, cas12, cas13, etc.) and the target gene target sequence to tobacco leaf cells in the field, and express them efficiently, thereby editing the leaf genes, achieving gene function loss, and presenting a gene function loss phenotype in the current generation background of the tobacco.

Claims

1. An efficient instantaneous expression system based on spray technology, characterized in that: The Agrobacterium containing the target gene is suspended in a suspension and then sprayed directly onto the leaves with a sprayer to promote / achieve transient expression of the target gene.

2. The efficient transient expression system according to claim 1, characterized in that: The suspension consisted of tap water and 1% silicone.

3. The efficient transient expression system according to claim 2, characterized in that: The suspension contained 1%, 2% or 5% sucrose or no sucrose.

4. The efficient transient expression system according to claim 2 or 3, characterized in that: The suspension did not contain MgCl2 and / or MES.

5. The efficient transient expression system according to claim 1, characterized in that: The 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.

6. The efficient transient expression system according to claim 1, characterized in that: The target gene is selected from at least one gene in the group consisting of the following suitable for normal expression in tobacco leaves: 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.

7. The efficient transient expression system according to claim 6, characterized in that: Agrobacterium containing the target gene is obtained by the following method: The target gene was obtained by synthesis and constructed into the pacI and NotI sites of the transient expression vector PJL-TRBO by restriction ligation; The target gene was introduced into Agrobacterium competent GV3101 by chemical transformation, and then positive clones were selected for expansion by sequencing identification; Expand the culture of Agrobacterium to 500-1000 mL, centrifuge and remove the supernatant, wash with water and repeat centrifugation to remove the supernatant and keep the precipitate; Transport the precipitated Agrobacterium to the outdoor field, resuspend it with ordinary water and set aside.

8. Use of the high-efficiency transient expression system according to any one of claims 1 to 7 in transient expression of plants in the field.

9. Use of the efficient transient expression system according to any one of claims 1 to 7 in creating high value-added target metabolites, medicinal proteins, vaccines, functional gene specialty agricultural products, health and nutritional ingredients, traditional Chinese medicine ingredients, or improving crop agronomic traits.

10. The use according to claim 8 or 9, characterized in that: The spraying volume of the high-efficiency transient expression system is 2-5 mL / leaf, which is sufficient to completely spray the entire leaf.

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