A method for transient expression of genes in the leaves of a sensate motor plant, d. tentaculata
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING UNIV OF SCI & TECH
- Filing Date
- 2026-03-09
- Publication Date
- 2026-06-09
Smart Images
Figure CN122168666A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a method for transient gene expression in plants, particularly a method for transient gene expression in the leaves of the Venus flytrap, a plant with sensible motility. Background Technology
[0002] The Venus flytrap (Dionaea muscipula) is an insectivorous plant with a trapping mechanism, and its unique physiological and ecological characteristics have attracted widespread attention. For example, the Venus flytrap's rapid movement mechanism, high sensitivity to stimuli, and special metabolic pathways during insect trapping provide rich material for plant science research. However, the genetic transformation system of the Venus flytrap is still immature, severely restricting in-depth research on its gene function and molecular mechanisms. Currently, transgenic research on the Venus flytrap mainly relies on traditional stable genetic transformation methods. Whether through induced shoot regeneration or genetic modification via seed cultivation, these methods require lengthy tissue culture, plant regeneration, and screening processes, often lasting several months or even longer. This method is not only time-consuming and complex, but also has low transformation efficiency, making it difficult to meet the research needs for rapid verification of gene function.
[0003] Transient gene expression technology is a rapid and efficient method for gene function research, enabling the expression of target genes in a short time and avoiding the cumbersome steps of stable transformation. Currently, transient expression technology has been successfully applied in various plants, such as tobacco, Arabidopsis thaliana, and rice, facilitating gene function research. However, in Venus flytraps, existing transient expression methods (such as the immersion method and vacuum infiltration method) suffer from low efficiency, poor reproducibility, and difficulty in achieving effective delivery in living leaves; a highly efficient and reliable transient expression system has not yet been established.
[0004] Therefore, there is an urgent need to develop a rapid and efficient transient gene expression method suitable for live Venus flytraps to overcome the technical bottlenecks of long stable transformation cycles and low transient expression efficiency. Summary of the Invention
[0005] This invention provides a method to overcome the shortcomings of the prior art.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for transiently expressing a gene in the leaves of the Venus flytrap, a plant with sensitive motility, comprising the following steps: S1: Construct an Agrobacterium expression vector containing the target gene and prepare Agrobacterium infection solution; S2: Using a syringe needle, make a scratch on the midrib on the back of a non-detached Venus flytrap leaf, and inject the Agrobacterium infection solution into the Venus flytrap leaf tissue through the scratch. S3: After injection, the Venus flytrap is first cultured in the dark, and then placed under light conditions for further cultivation.
[0007] To optimize the above technical solution, the specific measures also include: Furthermore, in S1, the OD of the Agrobacterium infection solution... 600 The value is 0.8 to 1.0, preferably 1.0, to balance the infectivity and the damage to sensitive leaf tissues.
[0008] Furthermore, the target gene includes, but is not limited to, reporter genes, resistance genes (disease resistance, insect resistance, stress resistance, etc.), metabolic pathway-related genes, environmental response genes, signal transduction genes, and other functional genes.
[0009] Furthermore, in step S2, the method of injecting the Agrobacterium infection solution into the Venus flytrap leaf tissue through the scratch is as follows: the Agrobacterium infection solution is drawn into the syringe and injected into the Venus flytrap leaf tissue with the assistance of a flow pump.
[0010] Further, in S2, the syringe is a disposable sterile syringe with a specification of 1 mL and a needle specification of 30G (inner diameter of 0.15 mm and outer diameter of 0.31 mm); the specific injection method is as follows: after the syringe draws the Agrobacterium infection solution, it is fixed on the flow pump, and the flow rate of the flow pump is 0.5~1.0 mL / h.
[0011] Furthermore, in S2, robust, fully expanded, but not yet triggered closure mature Venus flytrap leaves are selected. To maximize targeting of sensory motility-related cells and avoid severe damage, the injection target is precisely selected approximately 1-2 mm to either side of the midrib (main vein) on the abaxial surface of the leaf, avoiding the main trigger hairs, but located in the area below the epidermis rich in parenchyma cells and vascular bundle sheath extensions. This area has high cell activity and facilitates the diffusion of the infection solution along the leaf vein microenvironment to key parts such as the pulvinus (motor organ).
[0012] Furthermore, in S3, the dark culture time is 12-24 h, preferably 12 h, and the culture time under light conditions is 12-14 h, preferably 12 h.
[0013] Furthermore, in S3, during dark cultivation, the Venus flytrap leaves are wrapped with tin foil; during cultivation under light conditions, the temperature is 20~25 ℃, preferably 22 ℃, and the relative humidity is 65~75%, preferably 70%.
[0014] Further, in step S1, the method for culturing the Agrobacterium infection solution includes the following steps: S11. Streaking: Agrobacterium carrying the exogenous target gene plasmid vector is streaked on LB solid medium containing the corresponding antibiotic to activate the bacterial strain. S12, Small shake: Select an LB plate with single colonies, pick a round and smooth single colony into a centrifuge tube containing LB liquid medium with the corresponding antibiotic, mix well, and then incubate in a shaker at 26~30 ℃ and 180~240 rpm for 12~36 h until the bacterial solution becomes turbid and turns orange-yellow. S13. Expanding culture: Take the bacterial solution obtained in S12 at a volume ratio of 1:20~50, transfer it to LB liquid medium containing the corresponding antibiotic, mix well, and culture in a shaker at 26~30 ℃ and 180~240 rpm for 12~36 h until the bacterial solution becomes turbid and turns orange-yellow. S14. Cell treatment: Centrifuge the bacterial solution obtained in S13 at room temperature and 5000 rpm, discard the supernatant, and collect the bacterial cells; S15. Preparation of Agrobacterium infection solution: Resuspend the bacterial cells obtained in S14 using infection buffer and adjust the OD. 600 The value is 0.8~1.0, and the Agrobacterium infection solution is obtained.
[0015] Furthermore, in step S12, the culture is carried out at 28 ℃ and 220 rpm for 24 h; in step S13, the culture is carried out at 28 ℃ and 220 rpm for 16 h.
[0016] Further, in step S15, the infection buffer comprises MES, MgCl2, and acetylsylenone, wherein the molar ratio of MES, MgCl2, and acetylsylenone is 5-15 mmol: 5-15 mmol: 100-200 μmol, preferably the infection buffer contains 10 mM MES, 10 mM MgCl2, and 150 μM acetylsylenone; the pH of the infection buffer is 5-6, preferably 5.6.
[0017] Furthermore, in step S15, the Agrobacterium infection solution is mixed and then wrapped in tin foil and cultured in the dark for 2-3 hours, preferably 3 hours.
[0018] The beneficial effects of this invention are as follows: This invention provides a method for transient gene expression in the leaves of the Venus flytrap plant via injection delivery. By optimizing Agrobacterium-mediated transient expression technology and combining it with specific operational steps, a highly efficient and reliable method for transient gene expression in Venus flytraps is provided. This method can significantly shorten the verification cycle and rapidly assess the possibility of exogenous gene expression in Venus flytraps, solving the problems of long cycle and low efficiency of traditional stable transformation methods. This method has the advantages of simple operation, high efficiency, and good reproducibility, thus providing strong technical support and important technical means for gene function research, molecular mechanism analysis, and related biotechnology applications in Venus flytraps. It can be widely applied in plant science research and related biotechnology fields.
[0019] Specifically, this invention utilizes an Agrobacterium-mediated transient expression system to establish a method for transient gene expression in the leaves of the Venus flytrap, a plant with sensitive motility. This overcomes the limitation of existing transient expression technologies that cannot be directly applied to Venus flytraps in other plants. The method is simple to operate, requiring no complex tissue culture and plant regeneration processes, and can directly achieve gene expression in the mature leaves of living Venus flytraps. By optimizing the infection solution concentration and injection method, this invention significantly improves gene delivery and expression efficiency, exhibits good reproducibility, and has a wide range of applications, making it applicable to other carnivorous plants or plant species that are difficult to stably transform. Attached Figure Description
[0020] Figure 1 The photo shows Agrobacterium-infected solution being injected into the Venus flytrap leaf using a sterile syringe. Figure 2 This is a fluorescence detection image of the EGFP reporter gene expressed on Venus flytrap leaves using the Agrobacterium transient expression system. Detailed Implementation
[0021] The present invention will be further described below with reference to specific embodiments.
[0022] This embodiment provides a method for transient gene expression in the leaves of the Venus flytrap, a plant with sensitive movement.
[0023] I. Preparation of Agrobacterium infection solution: The GV3101 Agrobacterium carrying the EGFP reporter gene was stored in our laboratory for our own use. The specific preparation steps of the Agrobacterium infection solution are as follows: (1) Plate streaking: The constructed GV3101 Agrobacterium containing the EGFP gene was taken out of the -80 ℃ freezer and operated on a clean bench. After the bacterial culture was thawed, GV3101 was spread onto LB solid plates containing 50 μg / mL Kan and 25 μg / mL Rif and streaked. The culture medium was sealed with sealing film and incubated upside down at 28 ℃ for 48 h. The plates were removed when good colony growth was observed and single colonies were cultured.
[0024] (2) Small shake: Take out the LB plate with a single colony. On the clean bench, select a 2 mL centrifuge tube and add 1 mL of LB liquid medium containing 50 μg / mL Kan and 25 μg / mL Rif. Pick a round and smooth single colony into a 2 mL centrifuge tube, mix well, and incubate at 28 ℃ and 220 rpm on a shaker for 24 h until the bacterial solution becomes turbid and turns orange-yellow; (3) Expanding culture: Take the bacterial solution after small shaking at a volume ratio of 1:25, transfer it to LB liquid medium containing the corresponding antibiotic, mix well, and culture in a shaker at 28 ℃ and 220 rpm for 16 h until the bacterial solution becomes turbid and turns orange-yellow. (4) Cell treatment: The OD of the above Agrobacterium bacterial solution was measured using an ultraviolet spectrophotometer. 600 The value was 0.8 ~ 1.0. Centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, and collect the bacterial cells. (5) Preparation of Agrobacterium infection solution: Resuspend the bacterial cells in infection buffer and adjust OD. 600 The pH value was 1.0. The infection buffer contained 10 mM MES, 10 mM MgCl2, and 150 μM acetylsylgenone, with a pH of 5.6. After mixing, the solution was wrapped in aluminum foil and incubated in the dark at room temperature for 3 h.
[0025] II. Agrobacterium infection: such as Figure 1 As shown, healthy, vigorous, and disease-free Venus flytraps with good growth were selected as the experimental subjects. A 1 mL disposable sterile syringe was used, with the needle replaced with a 30G needle (inner diameter 0.15 mm, outer diameter 0.31 mm). For infection, the Agrobacterium infection solution was first drawn into the syringe and fixed onto a flow pump, with the flow rate set at 0.5 mL / h. A scald was made on the midrib of the underside of a non-external Venus flytrap leaf, and the Agrobacterium infection solution was injected into the leaf tissue with the assistance of the flow pump. The infected Venus flytrap leaf was then wrapped in aluminum foil and cultured in the dark for 12 h, followed by 12 h of light culture at 22 ℃ and 70% relative humidity to promote transient expression of the target gene.
[0026] Fluorescence imaging was used to detect transiently expressed EGFP reporter genes in transgenic leaves within 1 to 7 days after treatment.
[0027] Fluorescence detection analysis, with continuous collection for one week, showed that compared with the negative control, the Venus flytrap leaves injected with Agrobacterium containing the EGFP reporter gene exhibited a significant green fluorescent signal, such as... Figure 2 As shown, this phenomenon indicates that active exogenous EGFP protein was successfully expressed in the leaves of the sensitive motility plant Venus flytrap using the Agrobacterium transient expression system.
[0028] Active exogenous EGFP protein was successfully expressed in the leaves of the Venus flytrap, a plant with sensual motility, demonstrating its feasibility for genetic engineering and confirming the possibility of modifying its physiological functions.
[0029] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the reagents, materials, and procedures used herein are all widely used in the relevant fields.
[0030] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensitive movement, characterized in that: Includes the following steps: S1: Construct an Agrobacterium expression vector containing the target gene and prepare Agrobacterium infection solution; S2: Using a syringe needle, make a scratch on the midrib on the back of a non-detached Venus flytrap leaf, and inject the Agrobacterium infection solution into the Venus flytrap leaf tissue through the scratch. S3: After injection, the Venus flytrap is first cultured in the dark, and then placed under light conditions for further cultivation.
2. The method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 1, is characterized in that: In S1, the OD of the Agrobacterium infection solution 600 The value is 0.8 to 1.
0.
3. The method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 1, is characterized in that: In step S2, the method of injecting the Agrobacterium infection solution into the Venus flytrap leaf tissue through the scratch is as follows: the Agrobacterium infection solution is drawn into the syringe and injected into the Venus flytrap leaf tissue with the assistance of a flow pump.
4. The method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 3, is characterized in that: In S2, the syringe is a disposable sterile syringe with a capacity of 1 mL and a needle size of 30G. The specific injection method is as follows: the syringe draws up the Agrobacterium infection solution and then fixes it on the flow pump, and the flow rate of the flow pump is 0.5~1.0 mL / h.
5. The method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 1, is characterized in that: In S3, the dark culture time is 12-24 h, and the culture time under light conditions is 12-14 h.
6. The method for transiently expressing genes in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 1, is characterized in that: In S3, during dark cultivation, the Venus flytrap leaves are wrapped with aluminum foil; When culturing under light, the temperature should be 20-25 ℃ and the relative humidity should be 65-75%.
7. The method for transiently expressing a gene in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 1, is characterized in that: In step S1, the method for culturing the Agrobacterium-infected solution includes the following steps: S11: Agrobacterium carrying the exogenous target gene plasmid vector is streaked on LB solid medium containing the corresponding antibiotic to activate the bacterial strain; S12: Select an LB plate with single colonies, pick a round and smooth single colony into a centrifuge tube containing LB liquid medium with the corresponding antibiotic, mix well, and incubate at 26~30 ℃ and 180~240 rpm for 12~36 h until the bacterial solution becomes turbid and turns orange-yellow. S13: Take the bacterial solution obtained in S12 at a volume ratio of 1:20~50, transfer it to LB liquid medium containing the corresponding antibiotic, mix well, and culture in a shaker at 26~30 ℃ and 180~240 rpm for 12~36 h until the bacterial solution becomes turbid and turns orange-yellow. S14: Centrifuge the bacterial culture obtained in S13 at room temperature and 5000 rpm, discard the supernatant, and collect the bacterial cells; S15: Resuspend the bacterial cells obtained in S14 using infection buffer and adjust OD. 600 The value is 0.8~1.0, and the Agrobacterium infection solution is obtained.
8. The method for transiently expressing a gene in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 7, characterized in that: In S12, the culture was carried out at 28 ℃ and 220 rpm in a shaker for 24 h. In S13, the culture was carried out at 28 ℃ and 220 rpm for 16 h.
9. The method for transiently expressing a gene in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 7, characterized in that: In step S15, the infection buffer comprises MES, MgCl2, and acetylsyringone, wherein the molar ratio of MES, MgCl2, and acetylsyringone is 5-15 mmol: 5-15 mmol: 100-200 μmol; and the pH of the infection buffer is 5-6.
10. The method for transiently expressing a gene in the leaves of the Venus flytrap, a plant with sensual motility, according to claim 7, characterized in that: In step S15, the Agrobacterium infection solution is mixed and then wrapped in tin foil and cultured in the dark for 2-3 hours.