A method for genetic transformation of eucommia ulmoides based on ruby reporter gene and agrobacterium rhizogenes mediation
By using the RUBY reporter gene and Agrobacterium rhizogenes-mediated method on young Eucommia ulmoides branches, the barrier of Eucommia gum was overcome, the genetic transformation efficiency was improved, a rapid gene function verification system was established, and the stable identification of Eucommia ulmoides gene function was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- RES INST OF NON TIMBER FORESTRY CHINESE ACAD OF FORESTRY
- Filing Date
- 2026-03-13
- Publication Date
- 2026-06-16
AI Technical Summary
Eucommia ulmoides has low genetic transformation efficiency. In existing technologies, the adhesiveness and hardness of Eucommia ulmoides gum form a barrier, resulting in low infection efficiency and making it difficult to establish a rapid and efficient gene function verification system.
Using the RUBY reporter gene and Agrobacterium rhizogenes-mediated method, young Eucommia ulmoides branches were selected as infection material. Plasmids were transformed into Agrobacterium rhizogenes K599 competent cells by liquid nitrogen freeze-thaw method. Agrobacterium was cultured in a specific culture medium with the addition of substances such as acetylsuccinone. Vacuum infection technology was used to improve infection efficiency.
A genetic transformation system for Eucommia ulmoides that can be observed with the naked eye without special equipment was successfully established, which improved the efficiency of genetic transformation, enabled rapid and stable identification of gene functions, and revealed the molecular regulatory mechanism of Eucommia ulmoides.
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Figure CN122214397A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of forest tree genetic engineering, specifically a method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes. Background Technology
[0002] As a traditional Chinese medicine, Eucommia ulmoides Oliv. has always been known for its bark used medicinally. In recent years, with the continuous development and upgrading of the Eucommia industry, the development and utilization of Eucommia seed oil, Eucommia rubber, and the active ingredients in Eucommia leaves have received increasing attention. Therefore, elucidating the formation mechanisms of certain special traits of Eucommia through biochemical and molecular biological methods has become a new hot topic and direction. However, due to the difficulty in establishing genetic transformation systems for forest trees, especially ancient species like Eucommia, constructing a rapid and efficient gene function verification system is particularly important.
[0003] In plant genetic transformation, the selection and application of reporter genes are crucial for transgenic efficiency. Currently, various reporter gene systems are widely used, such as green fluorescent protein (GFP), GUS staining, and luciferase LUC; however, these systems require specialized equipment or expensive substrates for observation. Betalains are produced using tyrosine as a substrate, through the CYP76AD1... + DODA + and GT + These are natural products catalyzed by three enzymes. Based on this principle, researchers designed a novel, visual reporter system, named RUBY, by coupling these three enzymes into an open reading frame. The intensity of the red color indicates the efficiency of transgenic processes and gene expression. This reporter system has already been successfully expressed in model plants such as Arabidopsis thaliana, rice, soybean, and maize. It is a natural, pollution-free, cost-effective, and easy-to-observe reporter system.
[0004] However, since Eucommia ulmoides is a woody plant that is difficult to culture, and it contains substances such as eucommia gum, which have high viscosity and hardness, the eucommia gum will form a barrier during the genetic transformation of Eucommia ulmoides, resulting in low infection efficiency. Therefore, there are few reports on the genetic transformation of Eucommia ulmoides in the existing technology. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to provide a genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes, thereby increasing the genetic transformation efficiency of Eucommia ulmoides.
[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:
[0007] A genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes includes the following steps:
[0008] Step 1: Take young Eucommia ulmoides branches as inoculation material;
[0009] Step 2: Transform the plasmid containing the RUBY reporter gene into Agrobacterium rhizogenes K599 competent cells to obtain Agrobacterium rhizogenes containing the RUBY reporter gene;
[0010] Step 3: After expanding and resuspending Agrobacterium rhizogenes containing the RUBY reporter gene, an infection solution was obtained; then, young Eucommia ulmoides branches were placed in the infection solution for vacuum infection to obtain infected young Eucommia ulmoides branches.
[0011] Step 4: Insert the infected young Eucommia branches into a nutrient pot containing vermiculite for cultivation; after callus formation, continue cultivation until red roots grow.
[0012] This invention selects young Eucommia ulmoides branches as the infection material. The young branches exhibit vigorous cell division, thinner cell walls, and relatively low eucommia gum content, making them more conducive to infection. Furthermore, by transforming a plasmid containing the RUBY reporter gene into Agrobacterium rhizogenes K599 competent cells using a liquid nitrogen freeze-thaw method, the plasmid is better transformed into Agrobacterium, thereby improving the subsequent infection efficiency on Eucommia ulmoides. In preparing the infection solution, Agrobacterium rhizogenes containing the RUBY reporter gene is cultured in a specific medium (LB liquid medium resistant to 50 mg / L spectinomycin and 50 mg / L kanamycin), the Agrobacterium concentration is adjusted, and substances such as acetylsyleugenone are added to the resuspension to enhance the infectivity of Agrobacterium. During subsequent infection, the young Eucommia ulmoides branches are placed in the infection solution for vacuum infection with specific parameters. By creating a negative pressure environment, the infection solution is promoted to penetrate the infection material more effectively. Through the synergistic effect of the above-mentioned technical means, the hindering effect of Eucommia gum can be overcome to a certain extent, thereby increasing the genetic transformation efficiency of Eucommia.
[0013] The above-mentioned genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes involves obtaining Eucommia ulmoides seedlings in step 1 as follows: Eucommia ulmoides seeds that have been sterilized and had their surface pericarps removed are placed in MS solid medium and cultured for 10-20 days under a light-dark cycle of 16 hours / 8 hours until the cotyledons are fully open and the true leaves emerge, thus obtaining Eucommia ulmoides seedlings. The seedlings are then grown in an environment where the volume ratio of nutrient soil to vermiculite is 2:1. The tender Eucommia ulmoides branches at the top of the seedlings are then cut off. These conditions are conducive to the growth of Eucommia ulmoides.
[0014] The above-mentioned method for genetic transformation of Eucommia ulmoides based on RUBY reporter gene and Agrobacterium rhizogenes involves MS solid medium containing 4.43 g / L MS powder, 30 g / L sucrose and 8 g / L agar. The pH of MS solid medium is 5.8-6.0. Too high or too low pH will result in low seed germination rate or poor plant growth.
[0015] The above-mentioned genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes involves the following sterilization method: take out plump and uniform Eucommia ulmoides kernels, wash them with a 75% ethanol solution, then disinfect them with a 10% sodium hypochlorite solution, and finally wash them several times with sterilized ddH2O to complete the sterilization of Eucommia ulmoides kernels.
[0016] The above-mentioned method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes is characterized in that, in step 2, a plasmid containing the RUBY reporter gene is transformed into Agrobacterium rhizogenes K599 competent cells using a liquid nitrogen freeze-thaw method. Using the liquid nitrogen freeze-thaw method allows for better transformation of the plasmid into Agrobacterium, which is beneficial for improving the subsequent infection efficiency of Eucommia ulmoides.
[0017] The liquid nitrogen freeze-thaw method includes the following steps:
[0018] Step 2.1: Add the plasmid containing the RUBY reporter gene to Agrobacterium K599 competent cells, mix with pipette, and then incubate on ice for 30 min, freeze in liquid nitrogen for 5 min, and heat shock in a 37°C water bath for 5 min in sequence.
[0019] Step 2.2: Add 1 mL of antibiotic-free LB liquid culture medium, revive at 28℃ and 180 rpm for 4 h, centrifuge at 4000 rpm for 5 min, discard the supernatant, resuspend the obtained bacterial body and spread it evenly on resistant LB solid medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin, and incubate at 28℃ for 40-50 hours.
[0020] Step 2.3: Pick white single-celled colonies and inoculate them into 2 mL of resistant LB liquid medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin. Incubate at 28°C and 220 rpm to obtain Agrobacterium rhizogenes containing the RUBY reporter gene.
[0021] In the above-mentioned method for genetic transformation of Eucommia ulmoides based on RUBY reporter gene and Agrobacterium rhizogenes, step 2.2 involves LB liquid culture medium containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.
[0022] The above-mentioned method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes is described, wherein the RUBY reporter gene is driven by the 35S promoter.
[0023] In the above-mentioned genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes, step 3, the preparation method of the infection solution is as follows:
[0024] Step 3.1: Transfer Agrobacterium rhizogenes containing the RUBY reporter gene to resistant LB liquid medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin, and culture at 28°C and 220 rpm until the concentration of Agrobacterium rhizogenes containing the RUBY reporter gene reaches OD500. 600 The concentration was 0.6, resulting in the expanded culture of Agrobacterium.
[0025] Step 3.2: After centrifuging the Agrobacterium culture obtained in Step 3.1, discard the supernatant, resuspend the bacteria in resuspending solution, and adjust its final concentration to OD. 600 The concentration of Agrobacterium was 0.6. After standing for 3 hours in the dark at room temperature, an infection solution was obtained for infecting young Eucommia ulmoides branches. Adjusting the concentration of Agrobacterium in the infection solution and then standing it in the dark at room temperature for 3 hours helps to activate the infection solution, making it more active and facilitating successful infection.
[0026] In the aforementioned method for genetic transformation of Eucommia ulmoides mediated by the RUBY reporter gene and Agrobacterium rhizogenes, step 3.2 involves a resuspension containing 10 mM MES, 10 mM MgCl2, and 200 μM acetylsyringone. MES ensures an acidic system, allowing acetylsyringone to initiate gene expression in an acidic environment; MgCl2 provides divalent cations and regulates osmotic pressure.
[0027] The above-mentioned genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes, in step 3, the specific method of vacuum infection is as follows: A hole is punctured at the base of a young Eucommia ulmoides branch with a needle, and then the branch is placed in the infection solution. A vacuum pump is used for suction infection, reducing the pressure from 1 atmosphere to 0.1 atmospheres. Excessive pressure poses safety hazards, affecting the personal safety of experimental personnel and causing greater damage to the plant; insufficient pressure prevents gene transfer. Vacuuming is performed for 5 minutes, followed by slow venting for 20 minutes. After infection, the infected area is washed with sterilized distilled water. Excessive vacuuming and venting time may affect plant growth, while insufficient time may prevent gene transfer.
[0028] In the aforementioned genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes, step 4 involves the following culture conditions: the plants are first covered with a plastic bag and cultured in the dark at 23°C for two days, then removed and placed in a normally lit incubator. Dark culture is for seedling establishment; directly placing them in light can easily cause the plants to wilt and die.
[0029] The technical solution of the present invention achieves the following beneficial technical effects:
[0030] (1) This invention selects young Eucommia ulmoides branches as the infection material. The young Eucommia ulmoides branches have vigorous cell division, thin cell walls, and relatively low Eucommia gum content, which is more conducive to infection. Combined with the transformation of plasmids containing the RUBY reporter gene into Agrobacterium rhizogenes K599 competent cells by liquid nitrogen freeze-thaw method, the plasmid is better transformed into Agrobacterium, which is conducive to improving the subsequent infection efficiency of Eucommia ulmoides. In addition, when preparing the infection solution, Agrobacterium rhizogenes containing the RUBY reporter gene is cultured in a specific culture medium (LB liquid medium resistant to spectinomycin and kanamycin with a final concentration of 50 mg / L) and the concentration of Agrobacterium is adjusted. The infection ability of Agrobacterium is enhanced by adding substances such as acetylsuccinone to the resuspension. In the subsequent infection, the young Eucommia ulmoides branches are placed in the infection solution for vacuum infection with specific parameters. By creating a negative pressure environment, the infection solution is promoted to enter the infection material more effectively. Through the synergistic effect of the above-mentioned technical means, the hindering effect of Eucommia gum can be overcome to a certain extent, thereby increasing the genetic transformation efficiency of Eucommia.
[0031] (2) This invention successfully established an Agrobacterium rhizogenes-mediated gene transformation system in Eucommia ulmoides using RUBY as a reporter gene. The transgenic roots can be observed with the naked eye without the need for special equipment. This system can rapidly and stably identify the function of Eucommia ulmoides genes, further revealing their intrinsic molecular regulatory mechanisms, and has profound significance for the study of Eucommia ulmoides gene function. Attached Figure Description
[0032] Figure 1 (a) is a phenotypic diagram of wild-type callus; Figure 1 (b) is a phenotypic diagram of transgenic Eucommia ulmoides red callus tissue;
[0033] Figure 2 (a) is a phenotypic diagram of the root system of the wild-type plant; Figure 2 (b) is a phenotypic diagram of the red root system of the transgenic Eucommia ulmoides regenerated plant. Detailed Implementation
[0034] A genetic transformation method for Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes includes the following steps:
[0035] Step 1: Cultivation of young Eucommia ulmoides branches as infection material
[0036] Take out the whole kernels of Huazhong No. 8, which are plump and uniform in texture, wash them with 75% ethanol solution for 1 min, then disinfect them with sodium hypochlorite solution with 10% available chlorine for 10 min, and finally wash them 5 times with sterilized ddH2O for 1 min each time to obtain sterilized Eucommia ulmoides kernels. Place the sterilized Eucommia ulmoides kernels in MS solid medium (4.43 g / L MS powder, 30 g / L sucrose, 8 g / L agar, pH: 5.8-6.0) and culture them in a culture room under 16 h light / 8 h dark conditions for 15 days until the cotyledons are fully opened and the true leaves emerge to obtain Eucommia ulmoides seedlings. After 2 days of acclimatization, place the Eucommia ulmoides seedlings in an environment with a volume ratio of 2:1 of nutrient soil and vermiculite. After the plants have fully adapted to the external environment, cut off the tender Eucommia ulmoides branches at the top of the seedlings for infection treatment.
[0037] Step 2: Plasmid transformation containing the RUBY reporter gene
[0038] Agrobacterium rhizogenes competent cells K599 (AC1080) from Weidi Company were used for Agrobacterium rhizogenes transformation. The specific steps are as follows:
[0039] ① Add 100 ng of plasmid containing the RUBY reporter gene to 100 μL of Agrobacterium K599 competent cells, mix well by pipetting, and incubate on ice for 30 min;
[0040] ② Quickly place the centrifuge tubes in liquid nitrogen and freeze for 5 min. Then take them out and heat shock them in a 37°C water bath for 5 min. Then add 1 mL of antibiotic-free LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) and revive them in a shaker at 28°C and 180 rpm for 4 h.
[0041] ③ After centrifuging at 4000 rpm for 5 min, discard the supernatant, resuspend the bacterial cells and spread them evenly on solid LB medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin resistance. Incubate at 28℃ for about 48 hours.
[0042] ④ Pick white single-celled colonies and inoculate them into 2 mL of liquid LB medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin resistance. Incubate at 28°C and 220 rpm.
[0043] Step 3: Prepare the inoculum solution. Place the young Eucommia ulmoides branches in the inoculum solution for vacuum inoculum treatment to obtain inoculum-inoides branches; specifically:
[0044] ① Transfer 1 mL of Agrobacterium rhizogenes containing the RUBY reporter gene to 10 mL of LB liquid medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin resistance. Incubate at 28°C and 220 rpm until the concentration of Agrobacterium rhizogenes containing the RUBY reporter gene reaches OD. 600 The concentration was 0.6, resulting in the expanded culture of Agrobacterium.
[0045] ② After centrifuging the expanded Agrobacterium culture at 4000 rpm for 20 min, discard the supernatant and resuspend the bacteria in resuspending buffer (10 mM MME, 10 mM MgCl2, 200 μM acetylsuccine). Adjust the final Agrobacterium concentration to OD. 600 The concentration was 0.6. After standing for 3 hours at room temperature in the dark, an inoculum solution was obtained for infecting young Eucommia ulmoides branches.
[0046] ③ Cut off the tender young Eucommia ulmoides branches growing in the incubator and poke several small holes at the base with a syringe needle to facilitate Agrobacterium infection. Then place them in the infection solution and use a vacuum pump to suction and infect them, reducing the pressure from 1 atmosphere to 0.1 atmospheres (the reading on the vacuum gauge is 0.09). Vacuum for 5 minutes, then slowly release the gas for 20 minutes. After infection, wash the infected parts with sterilized distilled water to obtain the infected young Eucommia ulmoides branches.
[0047] ④ After inoculation, insert the infected young Eucommia ulmoides branches into nutrient pots containing vermiculite, cover with a plastic bag, and incubate in the dark at 23℃ for two days. Then, remove them and place them in a normal light incubator for growth. After callus formation, observe the efficiency of red callus formation and continue cultivation until red roots grow.
[0048] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.
Claims
1. A method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes, characterized in that, Includes the following steps: Step 1: Take young Eucommia ulmoides branches as inoculation material; Step 2: Transform the plasmid containing the RUBY reporter gene into Agrobacterium rhizogenes K599 competent cells to obtain Agrobacterium rhizogenes containing the RUBY reporter gene; Step 3: After expanding and resuspending Agrobacterium rhizogenes containing the RUBY reporter gene, an infection solution was obtained; then, young Eucommia ulmoides branches were placed in the infection solution for vacuum infection to obtain infected young Eucommia ulmoides branches. Step 4: Insert the infected young Eucommia branches into a nutrient pot containing vermiculite for cultivation; after callus formation, continue cultivation until red roots grow.
2. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1, characterized in that, In step 1, the method for obtaining young Eucommia ulmoides branches is as follows: place sterilized Eucommia ulmoides kernels with the surface pericarp removed in MS solid medium and culture them for 10-20 days under conditions of 16 hours of light / 8 hours of darkness until the cotyledons are fully opened and the true leaves emerge, thus obtaining Eucommia ulmoides seedlings; then place the Eucommia ulmoides seedlings in an environment with a volume ratio of 2:1 of nutrient soil and vermiculite, and cut off the young Eucommia ulmoides branches at the top of the seedlings.
3. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 2, characterized in that, The MS solid medium contains 4.43 g / L MS powder, 30 g / L sucrose and 8 g / L agar, and the pH value of the MS solid medium is 5.8-6.
0.
4. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 2, characterized in that, The sterilization method is as follows: take out plump and uniform Eucommia ulmoides kernels, wash them with 75% ethanol solution, then disinfect them with 10% sodium hypochlorite solution, and finally wash them several times with sterilized ddH2O to complete the sterilization of Eucommia ulmoides kernels.
5. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1, characterized in that, In step 2, the plasmid containing the RUBY reporter gene is transformed into Agrobacterium rhizogenes K599 competent cells using the liquid nitrogen freeze-thaw method.
6. A method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1 or 5, characterized in that, The RUBY reporter gene is driven by a 35S promoter.
7. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1, characterized in that, In step 3, the preparation method of the infiltration solution is as follows: Step 3.1: Transfer Agrobacterium rhizogenes containing the RUBY reporter gene to resistant LB liquid medium containing 50 mg / L spectinomycin and 50 mg / L kanamycin, and culture at 28°C and 220 rpm until the concentration of Agrobacterium rhizogenes containing the RUBY reporter gene reaches OD500. 600 The concentration was 0.6, resulting in the expanded culture of Agrobacterium. Step 3.2: After centrifuging the Agrobacterium culture obtained in Step 3.1, discard the supernatant, resuspend the bacteria in resuspending solution, and adjust the final concentration of Agrobacterium to OD. 600 The concentration was 0.
6. After standing for 3 hours at room temperature in the dark, an inoculum solution was obtained for infecting young Eucommia ulmoides branches.
8. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 7, characterized in that, In step 3.2, the resuspension contains 10 mM MES, 10 mM MgCl2 and 200 μM acetylsuccinone.
9. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1, characterized in that, In step 3, the specific method of vacuum inoculation is as follows: use a needle to puncture the bottom of the young Eucommia ulmoides branches, then place the young Eucommia ulmoides branches into the inoculation solution, use a vacuum pump to perform suction inoculation, reduce the pressure from 1 atmosphere to 0.1 atmosphere, pump for 5 minutes, release for 20 minutes, and after the inoculation is completed, wash the inoculated part with sterilized distilled water.
10. The method for genetic transformation of Eucommia ulmoides based on the RUBY reporter gene and Agrobacterium rhizogenes mediated according to claim 1, characterized in that, In step 4, the cultivation conditions are as follows: first, cover with a plastic bag and incubate in the dark in an incubator at 23°C for two days, then take it out and place it in a normal light incubator for growth.