Construction method of agrobacterium tumefaciens-mediated transient transformation system of taxus chinensis var mairei
By constructing the Agrobacterium-mediated transient transformation system of southern yew, using callus and suspended cells, the problem of inefficient transient transformation of yew is solved, and efficient gene expression is achieved and the operation process is simplified.
Patent Information
- Application Number
- CN202510367758.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-07-08
AI Technical Summary
The existing transient transformation methods of yew are inefficient, the traditional stable transformation technology has a long cycle, and the existing transient transformation methods are difficult to achieve efficient gene expression due to problems such as high lignification of yew cell walls and expensive equipment.
By inducing yew to form callus and obtaining suspended cells, Agrobacterium infects yew cells to shorten the transformation time, and Agrobacterium mediated method is used to construct a transient transformation system.
It effectively shortens the gene expression time, improves the conversion efficiency, simplifies the operation process, and reduces equipment costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological gene transformation, and particularly relates to a method for constructing an Agrobacterium-mediated transient transformation system for Taxus chinensis var. mairei. Background Art
[0002] Taxus chinensis var. mairei is a rare and endangered species unique to China and has attracted much attention because its bark, branches, leaves and roots are rich in the anti-cancer active substance paclitaxel. However, the natural Taxus grows slowly and the paclitaxel content is extremely low. Although chemical synthesis and cell culture technologies provide alternative ways for paclitaxel production, the regulation of its complex metabolic pathway is still limited by the lag in the research on Taxus gene functions. Therefore, developing efficient gene function research technologies to directionally regulate genes related to paclitaxel synthesis or optimize the metabolic network is expected to become a breakthrough for increasing the yield.
[0003] Gene function research depends on an efficient genetic transformation system. Traditional stable transformation technologies need to go through long processes such as explant regeneration, resistance screening and plant cultivation, with a long cycle. Moreover, as a gymnosperm, Taxus has a dense cell wall structure and weak regeneration ability, resulting in extremely low transformation efficiency. In contrast, transient transformation technology can quickly verify the gene expression or metabolic regulation effect in a short time and provides an effective tool for related gene research. Existing Taxus transient transformation methods mostly rely on protoplasts or particle bombardment. For the former, due to the high degree of lignification of the Taxus cell wall, it is difficult to enzymatically isolate and the protoplast viability is low; for the latter, expensive equipment is required, and mechanical bombardment is likely to damage cells, resulting in unstable transformation efficiency. Although Agrobacterium-mediated method has been widely used in various plants, due to the influence of Taxus explant browning, most existing Taxus transient transformation methods are difficult to achieve high-efficiency expression. Summary of the Invention
[0004] In order to overcome the deficiencies of the prior art, the present invention provides a method for constructing an Agrobacterium-mediated transient transformation system for Taxus chinensis var. mairei. First, callus of Taxus is induced to form and sufficient suspension cells are provided as materials in a short time, and then Taxus cells are infected with Agrobacterium, effectively shortening the transient transformation time.
[0005] The above object of the present invention is achieved by the following technical solutions: A method for constructing an Agrobacterium-mediated transient transformation system for Taxus chinensis var. mairei, the steps are as follows:
[0006] 1. Rapidly prepare Taxus callus: Take young Taxus stem segments rinsed with sterile water, induce culture, and then through subculture to obtain Taxus callus;
[0007] 2. Obtain Taxus suspension cells: Select Taxus callus that is white, uniform in texture, and loose for suspension culture. After culturing, take samples and filter through multiple layers of gauze to obtain Taxus cell clusters, thus obtaining the infection materials.
[0008] 3. Prepare the infection bacterial solution: Inoculate the Agrobacterium strain overexpressing the GFP gene into LB liquid medium containing antibiotics for amplification culture. After centrifuging the bacterial solution, discard the supernatant, add the resuspension solution, and resuspend and shake the bacterial cells to obtain the infection bacterial solution.
[0009] 4. Infect Taxus cells with Agrobacterium: Add the infection materials obtained in step 2 to the infection bacterial solution obtained in step 3. After infecting by shaking on a shaker, perform a drying treatment on multiple layers of gauze, rinse and filter with 1 / 2 MS liquid medium and then dry again. Then conduct dark co-culture. After the co-culture ends, rinse, filter, and dry again with 1 / 2 MS liquid medium.
[0010] 5. Detect the expression of foreign genes: After the co-culture ends, use a confocal microscope to observe the fluorescence expression intensity of the GFP protein in Taxus cells. Extract total RNA from the remaining samples and reverse transcribe it into cDNA, and detect the infection effect of Agrobacterium pBI121GD-GFP by real-time fluorescence quantitative PCR technology.
[0011] Further, in step 1, the induction culture duration is 2 weeks. The induction medium used for induction culture is 3.21 g / L B5 medium, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 4 g / L agar, 30 g / L sucrose, pH 5.5; the induction culture conditions are 25 ± 1°C, dark culture.
[0012] Further, in step 2, samples are taken after 15 days of suspension culture. The suspension medium used for suspension culture is 3.21 g / L B5 medium, 0.4 mg / L 2,4-D, 0.3 mg / L NAA, 1.2 mg / L 6-BA, 30 g / L sucrose, pH 5.8; the suspension culture conditions are 25 ± 1°C, dark culture.
[0013] Further, in step 3, add the resuspension solution to 50 mL of the bacterial solution. The added resuspension solution is 5 mL of 1 / 2 MS resuspension solution. Resuspend and shake the bacterial cells for three hours to obtain the infection bacterial solution; the composition of the 1 / 2 MS resuspension solution is: weigh 0.1025 g of MgCl2, 0.218 g of MES, and 1.96 mg of ACE and dissolve them in 50 mL of 1 / 2 MS.
[0014] Further, in step 3, the Agrobacterium strain overexpressing the GFP gene is the GV3103 strain containing the recombinant plasmid; the recombinant plasmid is plasmid pBI121GD-GFP.
[0015] Further, in the preparation of the 1 / 2MS liquid medium in Step 3: Weigh 2.37 g / L of MS powder + 15 g / L of sucrose, and adjust the pH to 5.8 with KOH.
[0016] Further, in Step 4, the drying time is 10 minutes, and the co-cultivation time is one day.
[0017] Further, in Step 5, the primer sequences of the GFP gene in the fluorescence quantitative PCR experiment design are respectively: GFP-F 'AAGCAGAAGAACGGCATCA, GFP-R 'TCCAGCAGGACCATGTGAT.
[0018] The beneficial effects of the present invention compared with the prior art are as follows: The present invention saves the time required for gene expression through the transformation method. The present invention first induces Taxus chinensis to form callus and provides sufficient suspension cells as materials in a short time, and then uses Agrobacterium to infect Taxus chinensis cells, effectively shortening the transient transformation time. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The present invention will be further described below in conjunction with the drawings and specific embodiments
[0020] Figure 1 is a schematic diagram of the callus induced by the induction medium;
[0021] Figure 2 is a schematic diagram of Taxus chinensis suspension cells in the exponential growth phase under a confocal microscope;
[0022] Figure 3 is a schematic diagram of the confocal microscope detection results of Taxus chinensis suspension cells successfully transformed with GFP under different Agrobacterium infection times;
[0023] Figure 4 is a schematic diagram of the real-time fluorescence quantitative PCR detection results under different Agrobacterium infection times;
[0024] Figure 5 is a schematic diagram of the confocal microscope detection results of Taxus chinensis suspension cells successfully transformed with GFP under different Agrobacterium infection concentrations;
[0025] Figure 6 is a schematic diagram of the real-time fluorescence quantitative PCR detection results under different Agrobacterium infection concentrations. DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.
[0027] Example 1 Obtaining Taxus callus and suspension cell materials
[0028] (1) Induction of Taxus callus
[0029] Cut the young stem segments of Taxus chinensis var. mairei, rinse them with sterile water until there is no dust, cut them into stem segments about 1 cm long in a laminar flow hood, shake them in 75% anhydrous ethanol for 30 seconds, then rinse them twice with sterile water, detoxify them in 0.2% HgCl2 solution for 15 minutes, and then rinse them three times with sterile water. After drying on multiple layers of gauze, inoculate them into the induction medium for tissue culture. The callus state is as shown in Figure 1 shown, and almost all Taxus stem segments have generated callus, with an induction rate exceeding 95%.
[0030] (2) Culture of Taxus suspension cells
[0031] Select white, uniform and loose Taxus callus and inoculate it into the suspension medium. The inoculation amount is 0.09 g / mL, and the initial pH is 5.8; sample when the suspension culture reaches 15 days. At this time, the cells of Taxus chinensis var. mairei are in the exponential growth phase and have good cell viability. The results are as shown in Figure 2 shown, and the Taxus cells are plump and in good condition under the confocal microscope.
[0032] Example 2 Preparation of infection bacterial solution
[0033] After constructing the recombinant plasmid pBI121GD-GFP, transfer it into Agrobacterium tumefaciens GV3103. Inoculate the recombinant Agrobacterium strain into 5 mL of LB liquid medium for culture. The formula of the LB liquid medium is: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, adjust pH = 7.0. Pipette 200 μL of the bacterial solution into 50 mL of LB liquid medium containing antibiotics (0.625 mg rifampicin; 2 mg gentamicin; 2.5 mg kanamycin; 0.392 mg acetosyringone and 0.1 g MES) for enlarged culture. The temperature of the shaker incubator is 28 °C, the rotation speed is 180 rpm, and culture is carried out under dark conditions for 16 - 18 h. Take the bacterial solution and centrifuge it at 4500 rpm in a 4 °C centrifuge for 10 min to remove the supernatant; add 5 mL of 1 / 2MS resuspension solution to the obtained precipitate. The composition of the 1 / 2MS resuspension solution is: 0.1025 g MgCl2, 0.218 g MES and 1.96 mg ACE dissolved in 50 mL of 1 / 2MS. Resuspend and shake the bacterial cells at 28 °C and 180 rpm for 3 hours to obtain the infection bacterial solution for standby.
[0034] Example 3 Infection of Taxus cells by Agrobacterium under different infection conditions
[0035] (1) Different Agrobacterium infection times:
[0036] Detect the OD of the bacterial solution using an ultraviolet spectrophotometer 600 Absorbance value. When the OD of the bacterial solution 600 reaches 0.6, centrifuge at 4500 rpm for 10 min, discard the supernatant, add the precipitate to 5 mL of pre-prepared 1 / 2MS resuspension to prepare an infection solution. Place Taxus chinensis suspension cells on multiple layers of gauze for filtration, and add the filtered cells to the infection solution. Infect for 10, 20, and 30 minutes respectively by shaking on a shaker at a temperature of 28 °C and a rotational speed of 180 r / min; after infection, dry on multiple layers of gauze for 10 minutes. After drying, rinse and filter twice with 1 / 2MS liquid medium and dry again for 10 minutes. Then, co-culture the infected cells in the dark at 25 ± 1 °C for 1 day. After the co-culture, rinse, filter, and dry twice again with 1 / 2MS liquid medium. Observe the fluorescence expression intensity of GFP protein in Taxus chinensis suspension cells using a laser confocal microscope, extract total RNA from the remaining samples and reverse transcribe it into cDNA, and detect the effects of Agrobacterium at different infection times by real-time fluorescence quantitative PCR technology. It is found that when the infection time is 20 minutes, both the protein fluorescence intensity and gene expression level are the highest.
[0037] (2) Different Agrobacterium infection concentrations:
[0038] Repeat the Agrobacterium amplification culture experiment in Example 3(1). When the OD of the bacterial solution reaches 600 values of 0.6, 0.8, and 1.0 respectively, centrifuge at 4500 rpm for 10 min, discard the supernatant, add the precipitate to 5 mL of pre-prepared 1 / 2MS resuspension to prepare an infection solution. Place Taxus chinensis suspension cells on multiple layers of gauze for filtration, and add the filtered cells to the infection solution. Infect by shaking on a shaker for 20 minutes at a temperature of 28 °C and a rotational speed of 180 r / min; after infection, dry on multiple layers of gauze for 10 minutes. After drying, rinse and filter twice with 1 / 2MS liquid medium and dry again for 10 minutes. Then, co-culture the infected cells in the dark at 25 ± 1 °C for 1 day. After the co-culture, rinse, filter, and dry twice again with 1 / 2MS liquid medium. After the co-culture, observe the fluorescence expression intensity of GFP protein in Taxus chinensis suspension cells using a confocal microscope, extract total RNA from the remaining samples and reverse transcribe it into cDNA, and detect the infection effect of Agrobacterium tumefaciens GV3103 by real-time fluorescence quantitative PCR technology. It is found that the optimal infection concentration is OD 600 value of 0.8.
[0039] The above-described embodiments are only the preferred embodiments of the present invention, rather than all the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious changes made without departing from the principle and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.
Claims
1. A method for constructing an Agrobacterium-mediated transient transformation system for Taxus chinensis var. mairei, characterized in that, The steps are as follows: S1. Rapidly prepare Taxus callus: Take the young stem segments of Taxus washed with sterile water, conduct induction culture, and then through subculture to obtain Taxus callus; S2. Obtain Taxus suspension cells: Select the white and evenly textured and loose Taxus callus for suspension culture, take samples after culture, filter through multiple layers of gauze to obtain Taxus cell clusters, and obtain the infection material; S3. Prepare the infection bacterial solution: Inoculate the Agrobacterium strain overexpressing the GFP gene into the LB liquid medium containing antibiotics for enlarged culture, discard the supernatant after centrifuging the bacterial solution, add the resuspension solution, and resuspend and shake the bacteria to obtain the infection bacterial solution; S4. Infect Taxus cells with Agrobacterium: Add the infection material obtained in step S2 to the infection bacterial solution obtained in step S3, after infecting by shaking on a shaker, conduct drying treatment on multiple layers of gauze, rinse and filter with 1 / 2MS liquid medium and then dry again, and then conduct dark co-culture. After the co-culture ends, rinse, filter and dry again with 1 / 2MS liquid medium; S5. Detect the expression of exogenous genes: After the co-culture ends, use a confocal microscope to observe the fluorescence expression intensity of the GFP protein in Taxus cells, extract total RNA from the remaining samples and reverse transcribe it into cDNA, and detect the infection effect of Agrobacterium pBI121GD-GFP by real-time fluorescence quantitative PCR technology.
2. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, characterized in that, In step 1, the induction culture duration is 2 weeks, and the induction medium used for induction culture is 3.21 g / L B5 medium, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 4 g / L agar, 30 g / L sucrose, pH 5.5; the induction culture conditions are 25±1°C, dark culture.
3. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, characterized in that, In step 2, samples are taken after 15 days of suspension culture. The suspension medium used for suspension culture is 3.21 g / L B5 medium, 0.4 mg / L 2,4-D, 0.3 mg / L NAA, 1.2 mg / L 6-BA, 30 g / L sucrose, pH 5.8: the suspension culture conditions are 25±1°C, dark culture.
4. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, characterized in that, In step 3, add the resuspension solution to 50 ml of the bacterial solution. The added resuspension solution is 5 mL of 1 / 2MS resuspension solution, and resuspend and shake the bacteria for three hours to obtain the infection bacterial solution; the composition of the 1 / 2MS resuspension solution is: weigh 0.1025 g of MgCl2, 0.218 g of MES and 1.96 mg of ACE and dissolve them in 50 mL of 1 / 2MS.
5. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, wherein In step 3, the Agrobacterium strain overexpressing the GFP gene is the GV3103 strain containing the recombinant plasmid; the recombinant plasmid is plasmid pBI121GD-GFP.
6. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, wherein The preparation of the 1 / 2MS liquid medium in step 3: Weigh 2.37 g / L of MS powder + 15 g / L of sucrose, and adjust the PH = 5.8 with KOH.
7. The construction method of the Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, characterized in that, In step 4, the drying time is 10 minutes, and the co-culture time is one day.
8. The method for constructing an Agrobacterium-mediated transient transformation system of Taxus chinensis var. mairei according to claim 1, wherein In step 5, the primer sequences of the GFP gene in the fluorescence quantitative PCR experimental design are: GFP-F 'AAGCAGAAGAACGGCATCA, GFP-R' TCCAGCAGGACCATGTGAT.