Method for realizing transient expression of exogenous target gene in rhododendron molle plant by using vacuum agrobacterium infection method
By combining vacuum Agrobacterium infection with ultrasonic pretreatment, the problem of low genetic transformation efficiency in rhododendrons was solved, enabling efficient and transient expression of exogenous genes on Rhododendron plants, simplifying the operation process and reducing costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING FORESTRY UNIV
- Filing Date
- 2025-12-31
- Publication Date
- 2026-05-08
AI Technical Summary
Existing genetic transformation methods for azaleas are inefficient and time-consuming, and traditional methods are difficult to achieve batch processing and efficient expression of exogenous genes.
The vacuum Agrobacterium infection method combined with ultrasonic pretreatment was adopted. The infection solution was uniformly penetrated into the Rhododendron molle plants through vacuum penetration, realizing the instantaneous expression of exogenous target genes, simplifying the operation process and improving efficiency.
This study achieved efficient, uniform, and high-intensity transient expression of exogenous genes in Rhododendron plants, shortening the experimental cycle and reducing operational difficulty and cost, thus providing a convenient tool for molecular biology research on Rhododendrons.
Smart Images
Figure CN121992014A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of forest tree genetic engineering technology, specifically to a method for achieving transient expression of exogenous target genes in Rhododendron molle plants via vacuum Agrobacterium infection. Background Technology
[0002] Rhododendron is one of China's ten most famous flowers, possessing high ornamental and cultural value. Rhododendron molle is a rare yellow-flowered species among rhododendrons, and its ornamental and medicinal value deserves further attention. Current research on rhododendrons mainly focuses on resource distribution, morphological classification, and stress physiology, with limited progress in molecular biology research. One reason for this limitation is the lack of convenient and effective genetic transformation methods. Current research on rhododendron genetic transformation primarily focuses on stable genetic transformation, i.e., the differentiation of callus from infected explants through tissue culture, while transient genetic transformation uses injection methods. However, both methods suffer from low efficiency; tissue culture requires a long culture period, and injection infection is time-consuming and labor-intensive, sometimes even risking failure. Therefore, a highly efficient genetic transformation method for Rhododendron molle is urgently needed in this field. This invention utilizes the principle of vacuum permeation to uniformly and efficiently penetrate the infection solution into the Rhododendron molle plant. It can process a large number of samples at the same time, saving time and effort, and providing a more effective tool for molecular biology research on Rhododendron. Summary of the Invention
[0003] This invention addresses the problems of low efficiency and long processing time in current rhododendron genetic transformation methods by providing a method for transient expression of exogenous target genes in *Rhododendron molle* plants using vacuum Agrobacterium infection. This method significantly reduces the difficulty of conducting genetic transformation in *Rhododendron molle* by directly using seedlings for vacuum infection, and dozens of seedlings can be transformed simultaneously in a single process, greatly reducing the time required. Furthermore, no further special cultivation is needed after infection, further reducing costs.
[0004] To achieve the above objectives, the present invention adopts the following technical solution.
[0005] A method for transient expression of exogenous target genes in Rhododendron molle plants using vacuum Agrobacterium infection includes the following steps:
[0006] (1) Sample treatment of Rhododendron simsii seedlings, the sample treatment including: stopping watering before infection, and immersing the whole seedling in water for ultrasonic treatment before infection.
[0007] (2) Immerse the seedlings of Rhododendron molle treated in step (1) in Agrobacterium tumefaciens infection solution containing the target gene and perform cyclic vacuum treatment. The cyclic vacuum treatment includes: evacuating to below -0.07 MPa, maintaining for a period of time and then restoring to normal pressure. This process is repeated at least twice.
[0008] (3) After the plants were vacuum-infected, they were cultured in the dark and then transferred to normal culture to achieve transient expression of the target gene.
[0009] In some embodiments, the specific conditions for the cyclic vacuum treatment are: a vacuum pressure of -0.07 MPa, maintained for 3-7 minutes and then restored to normal pressure, repeated 2-5 times.
[0010] In some embodiments, the specific conditions for the cyclic vacuum treatment are: a vacuum pressure of -0.07 MPa, maintained for 5 minutes and then restored to normal pressure, repeated 4 times.
[0011] In some embodiments, in step (1), the rhododendron seedlings are seedlings less than 1 year old.
[0012] In some embodiments, in step (1), the frequency of the ultrasonic treatment is 35-45 KHz, the power is 150-250 W, and the treatment time is 20-60 seconds.
[0013] In some embodiments, the ultrasonic treatment has a frequency of 40 kHz, a power of 200 W, and a processing time of 30 seconds.
[0014] In some embodiments, the Agrobacterium infection solution in step (2) is prepared by resuspending Agrobacterium cells in MMA infection solution, wherein the MMA infection solution contains: 5-15 mM magnesium chloride, 5-15 mM 2-morpholine ethanesulfonic acid and 100-300 μM acetylsuccinone.
[0015] In some embodiments, the MMA-assisted infiltration solution comprises: 10 mM magnesium chloride, 10 mM 2-morpholinoethanesulfonic acid, and 200 μM acetylsuccinone, and the OD of the infiltration solution after resuspension is [not specified]. 600 The value is 0.8-1.2.
[0016] In some embodiments, in step (3), the time for shading culture is 1-2 days, and the expression of the target gene is detected after normal culture for 5-9 days following the shading culture.
[0017] In some embodiments, the light-shielded culture time is 1 day, and the expression of the target gene is detected 7 days after normal culture.
[0018] Compared with the prior art, the beneficial effects of this application are as follows:
[0019] A novel transient expression system for Rhododendron molle (a woody ornamental plant) was developed by creatively combining ultrasonic pretreatment with circulating vacuum Agrobacterium infection technology. Compared to traditional injection methods, this approach significantly improves the delivery efficiency of exogenous genes through physical assistance, achieving uniform and high-intensity transient expression at the whole seedling level. This effectively overcomes the limitations of traditional methods, such as limited infection range, cumbersome operation, and difficulty in large-scale processing. Furthermore, compared to existing stable transformation systems relying on tissue culture, this method significantly shortens the experimental cycle, lowers the technical threshold and cost, and provides a powerful technical tool for rapid gene function verification, molecular breeding, and metabolic engineering research in Rhododendron molle and its close relatives. Experimental results demonstrate that the target gene is efficiently and specifically expressed in Rhododendron molle plants with good reproducibility after treatment using this method, opening up a new technical pathway for molecular biology research in the Rhododendron genus. Attached Figure Description
[0020] Figure 1 The image shows the experimental results of the sheep rhododendron with the green fluorescent protein gene exhibiting green fluorescence under a 488 nm excitation light source;
[0021] Figure 2 This is an electrophoresis image of transgenic plants identified by PCR using specific primers for the green fluorescent protein gene. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0023] Example 1
[0024] The plasmid pCAMBIA1300-GFP was selected and transformed into competent Escherichia coli DH5α using the freeze-thaw method. The transformed E. coli DH5α were evenly spread on LB solid agar plates containing 50 mg / L kanamycin for antibiotic screening. After incubation at 37°C overnight with the plates inverted, colony PCR was performed using universal primers contained in pCAMBIA1300-GFP and backups were made. Colonies showing the correct length band were sent to a biotechnology company for sequencing of the GFP base sequence.
[0025] Correctly sequenced *E. coli* bacteria were mixed into LB liquid medium containing kanamycin and incubated overnight in a shaker at 37°C and 200 rpm. Plasmids were extracted from the *E. coli* bacteria to obtain the target gene expression vector.
[0026] The obtained expression vector was transformed into competent Agrobacterium LBA4404 using the freeze-thaw method. The transformed Agrobacterium was then evenly spread on YEP solid agar plates containing 50 mg / L kanamycin and 25 mg / L rifampin for antibiotic screening. After inverting the plates and culturing at 28°C for 2 days, single colonies of Agrobacterium LBA4404 containing the target gene expression vector were obtained.
[0027] A single colony of Agrobacterium LBA4404 containing the target gene expression vector was picked and mixed with 30 ml of YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin. The mixture was then incubated in the dark for 12 h at 28°C and 200 rpm. Subsequently, 5 ml of the bacterial culture was mixed with 150 ml of YEP liquid medium containing 25 mg / L kanamycin, 20 mg / L rifampin and 20 μM acetylsyl syringone. The mixture was then incubated in the dark for 14 h at 28°C and 200 rpm.
[0028] The cultured Agrobacterium was harvested by centrifugation at 6000 rpm for 10 min. The harvested bacteria were resuspended in an MMA-co-infection buffer containing 10 mM magnesium chloride, 10 mM 2-morpholine ethanesulfonic acid, and 200 μM acetylsuccinone. The OD of the resuspended buffer was measured. 600 =1.0, then let stand for 2-3 hours.
[0029] Stop watering 1-year-old Rhododendron seedlings two days before infection. Before infection, immerse the whole seedling in water for ultrasonic treatment at 40 kHz frequency, 200 W power, and 30 s.
[0030] The seedlings were placed in the invasive staining solution, a portable vacuum pump was connected and evacuated, the pump was turned on, and the pressure was set to -0.07 MPa. This pressure was maintained for 5 minutes, then restored to normal pressure. This cycle was repeated 4 times, for a total of 20 minutes. The right root was then immersed in water and kept in the dark for 1 day. After 7 days of normal culture, the seedlings were irradiated with a 488 nm excitation light source to observe GFP expression. Figure 1 As shown, the seedling leaves emitted a distinct green fluorescence. RNA was sampled and extracted, then reverse transcribed to obtain cDNA, and PCR was performed using GFP-specific primers, yielding bands.
[0031] Plants emitting fluorescence were subjected to PCR amplification of the target gene. WT represented uninfected plants, and OE1 represented plants emitting significant fluorescence. WT and OE1 were subjected to PCR with three different concentrations of cDNA, from low to high concentration (left to right). Plants OE2 to OE6 were used for verification, and many of these plants showed bands, demonstrating the reliability of the fluorescence detection method. Figure 2).
[0032] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.
Claims
1. A method for transient expression of exogenous target genes in *Rhododendron molle* plants using vacuum Agrobacterium infection, characterized in that... Includes the following steps: (1) Sample treatment of Rhododendron simsii seedlings, the sample treatment including: stopping watering before infection, and immersing the whole seedling in water for ultrasonic treatment before infection. (2) Immerse the seedlings of Rhododendron molle treated in step (1) in Agrobacterium tumefaciens infection solution containing the target gene and perform cyclic vacuum treatment. The cyclic vacuum treatment includes: evacuating to below -0.07 MPa, maintaining for a period of time and then restoring to normal pressure. This process is repeated at least twice. (3) After the plants were vacuum-infected, they were cultured in the dark and then transferred to normal culture to achieve transient expression of the target gene.
2. The method according to claim 1, characterized in that, The specific conditions for the cyclic vacuum treatment are: vacuum pressure of -0.07 MPa, maintained for 3-7 minutes and then restored to normal pressure, repeated 2-5 times.
3. The method according to claim 2, characterized in that, The specific conditions for the cyclic vacuum treatment are: vacuum pressure of -0.07 MPa, maintained for 5 minutes and then restored to normal pressure, repeated 4 times.
4. The method according to claim 1, characterized in that, In step (1), the Rhododendron seedlings are seedlings less than 1 year old.
5. The method according to claim 1 or 4, characterized in that, In step (1), the frequency of the ultrasonic treatment is 35-45 KHz, the power is 150-250 W, and the treatment time is 20-60 seconds.
6. The method according to claim 5, characterized in that, The ultrasonic treatment has a frequency of 40 kHz, a power of 200 W, and a processing time of 30 seconds.
7. The method according to claim 1, characterized in that, The Agrobacterium infection solution in step (2) is prepared by resuspending Agrobacterium cells in MMA infection solution. The MMA infection solution contains: 5-15 mM magnesium chloride, 5-15 mM 2-morpholine ethanesulfonic acid and 100-300 μM acetylsuccinone.
8. The method according to claim 7, characterized in that, The MMA-assisted infiltration solution contains: 10 mM magnesium chloride, 10 mM 2-morpholinoethanesulfonic acid, and 200 μM acetylsuccinone, and the OD of the infiltration solution after resuspension is... 600 The value is 0.8-1.
2.
9. The method according to claim 1, characterized in that, In step (3), the light-shielding culture time is 1-2 days, and the expression of the target gene is detected after normal culture for 5-9 days after light-shielding culture.
10. The method according to claim 9, characterized in that, The light-shielded culture time was 1 day, and the expression of the target gene was detected 7 days after normal culture.