An optimized RNA stabilization element and its use in enhancing transient expression in tobacco

Abstract

This invention, entitled "An Optimized RNA Stabilizing Element and Its Application in Enhancing Transient Expression in Tobacco," belongs to the field of plant genetic engineering technology. The technical problem to be solved is to provide an optimized RNA stabilizing element capable of enhancing transient expression in tobacco and its application in this process. The key technical solution is to provide a method for enhancing the transient expression level of tobacco, comprising: introducing an optimized RNA stabilizing element into the expression vector of the target gene, wherein the optimized RNA stabilizing element is a 5′UTR core motif containing the nucleotide sequence shown in SEQ ID NO:1. This method effectively overcomes the bottleneck of exogenous gene expression in plants, providing a highly efficient expression enhancement tool for plant bioreactors and molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to an optimized RNA stabilizing element and its application in enhancing transient expression in tobacco. Background Technology

[0002] For understanding the technical content of this invention:

[0003] In the field of plant genetic engineering, efficient expression of exogenous genes is crucial for the production of recombinant proteins (such as vaccines, antibodies, and enzymes) and the improvement of crop agronomic traits (such as stress resistance and nutritional value). Traditional strategies mainly rely on using strong promoters (such as the cauliflower mosaic virus 35S promoter) to enhance transcription levels. However, post-transcriptional regulation, especially mRNA stability and translation efficiency, has become a significant bottleneck limiting the final protein accumulation. The 5′ untranslated region of mRNA plays a central role in regulating translation initiation, mRNA stability, and subcellular localization. Specific cis-acting elements within this region can interact with translation initiation factors or RNA-binding proteins, thereby significantly affecting gene expression levels.

[0004] Relevant patent documents retrieved: This document, published in China (CN111826394A) on October 27, 2020, discloses a plant transient expression vector comprising a 5'UTR region, an MCS region, and a SEKDEL sequence. The invention utilizes artificially synthesized sequences to enhance vector transcription, while selectively adding amino acid sequences to improve the translation level and stability of the target protein, ultimately increasing the overall accumulation of the target protein in plant leaf cells.

[0005] Currently, the number of known universal translation enhancement elements for plants is limited, and their efficiency varies, such as the Ω sequence from tobacco mosaic virus. These elements are usually derived from viruses or model plants, and their effectiveness is inconsistent across different species or genetic backgrounds. Furthermore, there is a lack of rational design and optimization for specific expression systems (such as transient expression). In addition, existing technologies lack systematic methods for discovering and validating novel and highly efficient plant endogenous RNA stabilizing elements. Most studies still rely on empirical modification of the UTR of single genes or combinations of limited known elements, making it difficult to achieve breakthrough enhancements in expression levels.

[0006] Therefore, there is an urgent need in this field for a systematic approach that can discover universal and effective RNA stability regulatory elements from the highly expressed genes of plants themselves, and establish a complete system for functional verification and application, in order to solve the problems of low expression efficiency and poor stability of exogenous genes in plants, thereby promoting the development of plant bioreactor technology and precision molecular breeding. Summary of the Invention

[0007] The purpose of this invention is to provide: This invention discloses an optimized RNA stabilizing element and its application in enhancing transient expression in tobacco, along with related technologies, to address technical problems such as providing an optimized RNA stabilizing element or a combination thereof capable of enhancing transient expression in tobacco. The RNA stabilizing element provided by this invention can effectively overcome the bottleneck of exogenous gene expression levels in plants, offering an efficient and reliable new tool for the development of plant bioreactors and molecular breeding.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of standard chemical terms can be found in the references: *Genetic Engineering*, Higher Education Press, August 1, 2013, 2nd edition; and *Molecular Cloning: A Laboratory Manual (4th Edition)*, Science Press Co., Ltd., February 2017, 1st edition.

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as vector construction, preparation of chemocompetent cells, vector digestion, and SDS-PAGE gel preparation, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] As used herein, the term "promoter" includes all sequences capable of driving the transcription of coding sequences in cells (e.g., plant or animal cells). For example, promoters can be cis-acting control elements, including enhancers, promoters, transcription terminators, origins of replication, chromosome integration sequences, and 5' and 3' untranslated regions or intron sequences, all of which are involved in the regulation of transcription. These cis-acting sequences can interact with proteins or other biomolecules to perform transcription.

[0014] The term "nucleotide" as used in this article refers to a small molecule composed of a pentose sugar, a phosphate group, and a nitrogenous base. It is the basic unit of nucleic acids (DNA and RNA) and plays a crucial role in genetic information storage, energy metabolism, and cell signal transduction. In a nucleotide, the pentose sugar provides the backbone structure for RNA or DNA; DNA contains deoxyribose, and RNA contains ribose. The phosphate group is used to link sugar molecules (pentose sugars) to form phosphodiester bonds in the nucleic acid chain. The nitrogenous bases are purines (adenine, guanine) and pyrimidines (cytosine, thymine, uracil), which pair via hydrogen bonds (e.g., AT, CG). Different nucleotides are sometimes represented by their nitrogenous bases.

[0015] As used in this article, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid. Vectors can be, for example, plasmids, granules, viruses, or bacteriophages. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells.

[0016] As used in this article, the term "expression vector" refers to a vector that, when present in a suitable environment, can direct the expression of one or more transgene-encoded proteins carried by the vector.

[0017] In a first aspect, the present invention provides a method for enhancing the transient expression level of tobacco, comprising: introducing an optimized RNA stabilization element into an expression vector of a target gene, wherein the optimized RNA stabilization element is a 5′UTR core motif containing the nucleotide sequence shown in SEQ ID NO: 1.

[0018] This includes technical features such as the target gene and optimized RNA stabilizing elements.

[0019] Furthermore, the target gene is a gene encoding enhanced green fluorescent protein; Furthermore, the nucleotide sequence of the gene encoding enhanced green fluorescent protein is shown in SEQ ID NO:2.

[0020] According to some embodiments of the present invention, the optimized RNA stabilizing element is obtained through the following steps: (a) Screening for highly expressed candidate genes based on transcriptome data; (b) Through algorithmic analysis and prediction, core motifs (motif-P05) with enhanced expression potential are identified from the 5′UTR region of the candidate genes. (c) Introduce the core motif into a reporter vector for functional validation and screen for optimized sequences that can significantly improve the expression level of reporter genes.

[0021] The method for enhancing transient expression levels in tobacco includes the following steps: (1) Synthesize the 5′UTR core motif shown in SEQ ID NO: 1; (2) Construct a plant expression vector to fuse the 5′UTR core motif with the coding sequence of enhanced green fluorescent protein at its N-terminus for expression; (3) Transform the expression vector constructed in step (2) into Agrobacterium to obtain recombinant Agrobacterium; (4) Use the recombinant Agrobacterium from step (3) to infect tobacco leaves.

[0022] Further, the Agrobacterium mentioned in step (3) is selected from at least one of Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105, Agrobacterium EHA101, Agrobacterium AGL1, Agrobacterium C58C1, Agrobacterium A281, and Agrobacterium GV2260. Preferably, the Agrobacterium mentioned in step (3) is Agrobacterium GV3101.

[0023] Furthermore, the tobacco mentioned in step (4) includes, but is not limited to, tobacco Benzodiaceae, common tobacco, forest tobacco, velvety tobacco, yellow tobacco, and pink-blue tobacco.

[0024] Preferably, the tobacco mentioned in step (4) is Benzoic tobacco.

[0025] In a second aspect, the present invention provides: an optimized RNA stabilizing element for enhancing transient expression levels in tobacco, which is a 5′UTR core motif containing the nucleotide sequence shown in SEQ ID NO: 1.

[0026] The 5′UTR core motif is motif-P05.

[0027] Among them, the technical feature is the nucleotide sequence of the 5′UTR core motif.

[0028] The nucleotide sequence of the 5′UTR core motif is selected from: the nucleotide sequence shown in SEQ ID NO:1; or a sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO:1.

[0029] Furthermore, the nucleotide sequence of the 5′UTR core motif is selected from sequences that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:1. Furthermore, the nucleotide sequence of the 5′UTR core motif is selected from a sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO:1; Furthermore, the nucleotide sequence of the 5′UTR core motif is selected from a sequence that has at least 95% sequence identity with the sequence shown in SEQ ID NO:1; Furthermore, the nucleotide sequence of the 5′UTR core motif is selected from a sequence that has at least 99% sequence identity with the sequence shown in SEQ ID NO:1.

[0030] Thirdly, the present invention provides a recombinant plant expression vector, wherein the recombinant plant expression vector contains the above-mentioned optimized RNA stability element.

[0031] The element is operatively connected upstream of the target gene coding sequence.

[0032] Fourthly, the present invention provides a recombinant Agrobacterium, wherein the recombinant Agrobacterium contains the above-mentioned recombinant plant expression vector.

[0033] Fifthly, the present invention provides the application of the above-mentioned optimized RNA stabilizing element, the above-mentioned recombinant plant expression vector, or the above-mentioned recombinant Agrobacterium in improving the transient expression level of exogenous proteins in plants.

[0034] The exogenous protein mentioned above includes GFP.

[0035] The plant is selected from any one of tobacco, Arabidopsis thaliana, tomato, potato, soybean, and cotton. Preferably, the plant is tobacco.

[0036] The tobacco mentioned includes, but is not limited to, Nicotiana benthamiana, common tobacco, forest tobacco, velvety tobacco, yellow-flowered tobacco, and pink-blue tobacco.

[0037] Furthermore, the tobacco mentioned is Benzoic tobacco.

[0038] The present invention has at least the following beneficial effects: This invention provides an optimized RNA stability element and its application system, specifically relating to a method for enhancing transient expression levels in tobacco using a specific 5'UTR core motif (motif-P05). The method includes integrating the core motif into a target gene expression vector, constructing an optimized plant expression vector, and transiently expressing the gene in *Nicotiana benthamiana* using an Agrobacterium GV3101-mediated transformation system.

[0039] This invention realizes a complete technical system from element screening and vector construction to expression verification. By regulating the dual mechanisms of mRNA stability and translation efficiency, it effectively breaks through the bottleneck of plant exogenous gene expression and provides an efficient expression enhancement tool for plant bioreactors and molecular breeding. Attached Figure Description

[0040] Figure 1 Strategies for building carriers.

[0041] Figure 2 For vector enzyme digestion verification; Lane 1: Plasmid; Lane 2: Plasmid Digested with PstI; Lane M: DNA Marker.

[0042] Figure 3 This is a fluorescence detection image.

[0043] Figure 4 To analyze the quantitative results of fluorescence imaging in ImageJ.

[0044] Figure 5 The above image shows the results of Western blot detection of eGFP expression, while the bottom image shows the quantitative results of WB band grayscale values ​​analyzed by ImageJ. Detailed Implementation

[0045] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0047] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0048] Example 1: Screening of OptRSE Component Core Motion 1. Multi-omics data integration for screening candidate genes Data source: Integrates omics data from three levels: mRNA transcriptomics: measures the transcriptional level of genes.

[0049] Quantitative proteomics: measuring the abundance of corresponding proteins.

[0050] mRNA degradome: provides clues about mRNA stability or post-translational regulation.

[0051] Screening criteria: 131 genes showing high expression at both the mRNA and protein levels (high mRNA level + high protein abundance) were successfully screened. These genes may be key genes for core cellular functions or high-abundance proteins, and their expression is strictly and efficiently regulated.

[0052] 2. Regulatory sequence extraction For these 131 candidate genes, three key regulatory region sequences were extracted: Promoter region: The 2 kb region upstream of the gene, which mainly contains regulatory elements for transcription initiation.

[0053] 5' Untranslated region: Affects the efficiency and stability of mRNA translation initiation.

[0054] 3' Untranslated region: Affects mRNA stability, localization, and translation efficiency.

[0055] 3. Prediction of cis-acting elements Tools: Unbiased de novo prediction of the 5' untranslated region was performed using MEME software.

[0056] "Unbiased" means that it does not rely on known motif databases, but instead directly seeks significantly enriched, conservative sequence patterns based on the sequence itself.

[0057] Objective: To identify 200 potential DNA or RNA core motifs that may co-regulate this group of highly expressed genes within this regulatory region, and to select the motif sequence 5 (motif-P05) with the highest overall ranking for plant expression vector construction. (The nucleotide sequence of motif-P05 is shown in SEQ ID NO.1: ATACCGAGTGATTCTATCGACTTTCCCAAAGAATCAAA.) Example 2: Construction and transformation of pUC18-eGFP vector (empty vector) 1. Homologous recombination cloning of the eGFP gene based on SalI / PstI restriction sites 1.1 Materials Linearized vector: pUC18 backbone digested with SalI / PstI; (Enzymes used were all from NEB, SalI catalog number: R0138; PstI catalog number: R0140).

[0058] Insert fragment: an eGFP fragment with 15 bp homologous arms at both ends to the pUC18 multiple cloning site (in this invention, the amplification primers for the insert fragment are all 41 bp, containing 15 bp of sequence homologous to the vector + 6 bp restriction site + 20 bp target sequence amplification primers).

[0059] PCR amplification enzyme: 2× PCR Master Mix (Yeasen, 2× Hieff® PCR Master Mix (With Dye), catalog number 10102ES).

[0060] Recombinase: Exnase® II homologous recombinase (using Exnase® II homologous recombinase from TransGen Biotech, corresponding to the ClonExpress® II One-Step Cloning Kit. The kit's catalog number is C112).

[0061] Host: Escherichia coli DH5α competent cells (purchased from Weidi Biotechnology, catalog number DL1001).

[0062] 1.2 Methods 1.2.1 Carrier linearization pUC18 was double-digested with SalI and PstI. The reaction conditions were: after gentle mixing and brief centrifugation, the mixture was incubated in a 37°C water bath or metal bath for 1-2 hours. After nucleic acid electrophoresis, the linearized vector fragment was purified by gel recovery.

[0063] Table 1

[0064] 1.2.2 Homologous recombination primer design Homologous recombination primers (41 bp) were designed at both ends of the eGFP fragment: 5' amplification primer: cggggatcctctagaGTCGACATGGTGAGCAAGGGCGAGGA (SEQ ID NO:3); 3' amplification primer: atacgaacgaaagctCTGCAGTTACTTGTACAGCTCGTCCA (SEQ ID NO:4).

[0065] 1.2.3 PCR amplification of the eGFP insert fragment Table 2

[0066] 1.2.4 Recombination reaction Mix the linearized vector and eGFP fragment according to the proportions in the table below, add Exnase® II recombinase, and react at 37°C for 30 min.

[0067] Table 3

[0068] 1.2.4 Transformation and Screening The recombinant product was transformed into DH5α competent cells, plated on LB plates containing Kana (50 µg / mL), and incubated overnight at 37°C.

[0069] 1.2.5 Identification Single colonies were picked, plasmids were extracted and sequenced to confirm the plasmid pUC18-eGFP.

[0070] The eGFP coding sequence is shown in SEQ ID NO.2: .

[0071] 2. pUC18-eGFP vector was transformed into Agrobacterium GV3101 Competent cells: GV3101 chemocompetent cells, purchased from: Weidi Biotechnology, catalog number AC1001.

[0072] (1) Freeze-thaw transformation: Take 100 μL of competent cells and add 2 μL of the constructed pUC18-eGFP vector plasmid DNA (about 300 ng); place on ice for 5 min, in liquid nitrogen for 5 min, and heat shock in a water bath at 37℃ for 5 min; add 700 μL of antibiotic-free LB liquid medium and shake at 28℃ for 2-3 hours to recover.

[0073] (2) Plate screening: Take the revived bacterial culture and spread it on LB solid plates containing rifampicin (Rif, 50 µg / mL), gentamicin (Gent, 50 µg / mL) and bacterial resistance markers (kanamycin 50 µg / mL) on the carrier. Incubate upside down at 28°C for 2-3 days until single colonies appear.

[0074] (3) Vector enzyme digestion verification: The successful transformation of the pUC18-eGFP vector plasmid into Agrobacterium GV3101 was quickly confirmed by vector enzyme digestion and named GV3101-pUC18-eGFP.

[0075] Example 3 Construction and transformation of the core motif fusion expression vector pUC18-eGFP (pUC18-motif-P05-eGFP) 1. Procedure for constructing the pUC18-eGFP-motif-P05 fusion expression vector using homologous recombination at SacI and SalI sites. 1.1 Carrier linearization The pUC18-eGFP vector was directly digested with SacI and SalI to remove the original small fragment in the MCS region upstream of eGFP, and the large backbone vector was recovered. (The enzymes used were all from NEB, SacI-HF catalog number: R3156; SalI catalog number: R0138). The enzyme digestion system is shown in the table below: Table 4

[0076] 1.2 Primer Design Design primers to amplify motif-P05, adding 15bp sequences homologous to the ends of the linearized vector at both ends: Upstream primer: gagaacacgggggacGAGCTCATACCGAGTGATTCTATCGA (SEQ ID NO:5); Downstream primer: gcccttgctcaccatGTCGACTTTGATTCTTTGGGAAAGTC (SEQ ID NO:6).

[0077] 1.3 PCR Amplification and Recombination (1) PCR amplification of motif-P05, the product has vector homologous arms at both ends.

[0078] Table 4

[0079] (2) Mix the linearized vector, the motif-P05 fragment with arms, and the homologous recombinase. (This reaction uses Exnase® II homologous recombinase from TransGen Biotech, corresponding to the ClonExpress® II One-Step Cloning Kit. The specific product number of this kit is C112.) Table 5

[0080] (3) Incubate at 37℃ for 15-30 minutes to complete seamless directional cloning.

[0081] See the build process Figure 1 .

[0082] 1.4 Transformation and Screening The bacteria were transformed into E. coli, screened on Kana plates, and their reading frames were verified by colony PCR and sequencing. The plasmid was named pUC18-motif-P05-eGFP.

[0083] 2. Agrobacterium GV3101 expressed using the pUC18-motif-P05-eGFP expression vector. Competent cells: GV3101 chemocompetent cells, purchased from: Weidi Biotechnology, catalog number AC1001.

[0084] (1) Freeze-thaw transformation: Take 100 μL of competent cells and add 2 μL of the constructed pUC18-motif-P05-eGFP vector plasmid DNA (about 300 ng); place on ice for 5 min, in liquid nitrogen for 5 min, and heat shock in a water bath at 37℃ for 5 min; add 700 μL of antibiotic-free LB liquid medium and shake at 28℃ for 2-3 hours to recover.

[0085] (2) Plate screening: Take the revived bacterial culture and spread it on LB solid plates containing rifampicin (Rif, 50 µg / mL), gentamicin (Gent, 50 µg / mL) and bacterial resistance markers (kanamycin 50 µg / mL) on the carrier. Incubate upside down at 28°C for 2-3 days until single colonies appear.

[0086] (3) Vector enzyme digestion verification: The successful transformation of the pUC18-motif-P05-eGFP plasmid into Agrobacterium GV3101 was quickly confirmed by vector enzyme digestion. See [link to vector enzyme digestion verification]. Figure 2 The results showed that the plasmid was successfully transformed into Agrobacterium GV3101 and named GV3101-pUC18-motif-P05-eGFP.

[0087] Example 4: Transient expression of tobacco 1. Preparation of Agrobacterium infection solution (1) Activation of bacterial strains: Single clones of GV3101-pUC18-eGFP and GV3101-pUC18-motif-P05-eGFP strains preserved in glycerol at -80℃ were scraped onto LB plates containing the corresponding antibiotics and cultured at 28℃ for 2 days.

[0088] (2) Preparation of MMA osmotic buffer: Prepare an MMA buffer with a final concentration of 10 mM MES, 10 mM MgCl2 and 100 μM acetosyringone by making up to volume with water.

[0089] (3) Resuspension of bacterial cells: GV3101-pUC18-eGFP and GV3101-pUC18-motif-P05-eGFP colonies grown in the plate were eluted using osmosis buffer, and the final OD of the bacterial culture was adjusted. 600 To 0.1. The resuspended bacterial solution was left to stand at room temperature in the dark for 2-3 hours to allow the Vir gene to be fully induced, resulting in pUC18-eGFP empty vector control infection solution and pUC18-motif-P05-eGFP infection solution, respectively.

[0090] 2. Injection infection of tobacco leaves (1) Leaf selection: Select healthy, fully expanded leaves from the upper part of the plant. Usually, 2-3 leaves of a tobacco plant can be infected. Inject pUC18-motif-P05-eGFP infection solution on the left side of the leaf and pUC18-eGFP empty control infection solution on the right side.

[0091] (2) Injection procedure: Use a 1 mL sterile syringe to draw up the prepared pUC18-eGFP empty vector control infection solution and pUC18-motif-P05-eGFP infection solution. On the underside of the leaf (where there are more stomata, making it easier to penetrate), gently hold the leaf with your index finger, press the tip of the syringe (without the needle) against the underside of the leaf, and slowly and steadily inject the bacterial solution until a circular, moist infiltration spot (about 1-2 cm in diameter) appears on the upper side of the leaf.

[0092] (3) Culture conditions: Place the injected plants under normal light and temperature (22-25℃) for 72 hours.

[0093] Example 5: Fluorescence detection of differences in eGFP expression levels A GFP fluorescence detection system was constructed using a LUYOR-3415RG dual-wavelength fluorescent protein observation lamp to detect and screen tobacco plants transfected with green fluorescent protein (GFP) and red fluorescent protein (DsRed) genes. When the excitation light source emits blue fluorescence, observation using a yellow filter allows for the detection of tobacco plants containing green fluorescent protein (GFP). During image capture, the camera is held through the yellow filter, the phone's resolution is adjusted, and the image is taken at a fixed height to ensure consistent shooting angle and image quality for each photo.

[0094] 1. Fluorescence detection method: The fluorescence intensity difference between pUC18-motif-P05 (motif-P05-eGFP infection solution) on the left side of the leaf and the blank control pUC18-eGFP (eGFP empty control infection solution) on the right side was detected using a LUYOR-3415RG dual-wavelength fluorescent protein observation lamp. (See attached image) Figure 3 .

[0095] 2. Shooting requirements: When taking photos, the camera uses a yellow filter to adjust the phone's pixels and takes photos at a fixed height to ensure that the shooting angle and shooting quality of each photo are consistent.

[0096] 3. Fluorescent area statistics: The fluorescence imaging results were standardized and quantitatively analyzed using ImageJ image analysis software. The quantitative results are as follows: Figure 4 As shown.

[0097] Example 6: Western Blotting Detection of eGFP Expression Level Experimental methods: 1. Prepare the sample for loading (all steps are performed on ice or at 4°C) The frozen leaf tissue was thoroughly ground into a fine powder in liquid nitrogen, and 200 μL of 2× loading buffer (purchased from Beyotime; catalog number: P0288-5ml) was added and vortexed to mix. The mixture was then lysed on ice for 30 min, boiled at 95-100°C for 10 min, and centrifuged at 12000-14000 g for 5 min at 4°C. The supernatant (the total protein solution) was carefully aspirated and transferred to a new pre-chilled centrifuge tube, which was then placed on ice for later use.

[0098] 2. SDS-PAGE electrophoresis Install the precast gel or homemade SDS-PAGE gel into the electrophoresis tank and add 1× electrophoresis buffer (purchased from GenScript; catalog number: C31382410).

[0099] Load samples in sequence: leave one lane for pre-stained protein markers.

[0100] Constant voltage electrophoresis: 90V, 30 minutes (stacking gel), then 140V, 60-90 minutes (separating gel), until the bromophenol blue band reaches the bottom of the gel.

[0101] 3. Transfer (wet transfer method) Key points: The PVDF membrane needs to be activated by soaking in methanol for 15 seconds beforehand, and then equilibrated in the anolyte. The filter paper needs to be soaked in both the catholyte and anolyte for 15 minutes. All components must be precisely aligned to avoid air bubbles. Prepare the transfer "sandwich": from negative to positive electrode, the sequence is: filter paper - gel - PVDF membrane - filter paper. Place the "sandwich" in the transfer tank, and perform the transfer at 26V, 1.5A for 10 minutes.

[0102] 4. Immunological testing Blocking: After the transfer is complete, place the membrane in a buffer solution containing 5% skim milk and block it on a shaker at room temperature for 3 hours.

[0103] Primary antibody incubation: Dilute the primary antibody (Mouses anti GFP-TagmAb) with 1xTBST at the recommended ratio of 1:10000 and incubate on a shaker at room temperature for 2 hours. (Primary antibody purchased from: ABclonal, catalog number: AE012) Wash the membrane: Wash the membrane three times rapidly with 1xTBST buffer on a shaker at room temperature, each time for 5-10 minutes.

[0104] Secondary antibody incubation: Dilute the corresponding HRP-labeled secondary antibody HRP Goat Anti-Mouse IgG (H+L) with 1xTBST at the recommended ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour. (Secondary antibody purchased from: ABclonal, catalog number: AS003) Wash the membrane: Wash the membrane three times rapidly with 1xTBST buffer on a shaker at room temperature, each time for 5-10 minutes.

[0105] 5. Chemiluminescence imaging and data analysis ECL development: Blot off excess TBST from the membrane, add 600 μL of ECL luminescent substrate evenly, and react for 1-2 minutes.

[0106] Imaging and Acquisition: Exposure and acquisition of images in a chemiluminescence imager or darkroom ( Figure 5 ).

[0107] Western blotting showed that motif-P05 significantly increased the expression level of the reporter protein eGFP (≥5-fold increase compared to the control).

[0108] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for enhancing the transient expression level of tobacco, characterized in that, include: An optimized RNA stabilizing element is introduced into the expression vector of the target gene. The optimized RNA stabilizing element is a 5′UTR core motif containing the nucleotide sequence shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that, The target gene is the gene encoding enhanced green fluorescent protein; the nucleotide sequence of the gene encoding enhanced green fluorescent protein is shown in SEQ ID NO:

2.

3. The method according to any one of claims 1-2, characterized in that, The method for enhancing transient expression levels in tobacco includes the following steps: (1) Synthesize the 5′UTR core motif shown in SEQ ID NO: 1; (2) Construct a plant expression vector to fuse the 5′UTR core motif with the coding sequence of enhanced green fluorescent protein at its N-terminus for expression; (3) Transform the expression vector constructed in step (2) into Agrobacterium to obtain recombinant Agrobacterium; (4) Use the recombinant Agrobacterium from step (3) to infect tobacco leaves.

4. The method according to claim 3, characterized in that, The Agrobacterium mentioned in step (3) is selected from at least one of Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105, Agrobacterium EHA101, Agrobacterium AGL1, Agrobacterium C58C1, Agrobacterium A281, and Agrobacterium GV2260.

5. The method according to claim 3, characterized in that, The tobacco mentioned in step (4) includes Nicotiana benthamiana, common tobacco, forest tobacco, velvety tobacco, yellow tobacco, and pink blue tobacco.

6. An optimized RNA stabilizing element for enhancing transient expression levels in tobacco, characterized in that, It is a 5′UTR core motif containing the nucleotide sequence shown in SEQ IDNO:

1.

7. A recombinant plant expression vector, characterized in that, The recombinant plant expression vector contains the optimized RNA stability element as described in claim 6.

8. A recombinant Agrobacterium, characterized in that, The recombinant Agrobacterium contains the recombinant plant expression vector of claim 7.

9. The application of the optimized RNA stabilizing element of claim 6, the recombinant plant expression vector of claim 7, or the recombinant Agrobacterium of claim 8 in improving the transient expression level of exogenous proteins in plants.

10. The application according to claim 9, characterized in that, The exogenous protein mentioned includes GFP.

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