Method for inducing transgenic hairy roots of chinese toon
By infecting Toona sinensis explants with Agrobacterium rhizogenes K599, transgenic hairy roots of Toona sinensis were successfully induced, solving the problem of verifying the function of Toona sinensis genes and realizing efficient and low-cost transformation and gene function research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
- Filing Date
- 2024-04-18
- Publication Date
- 2026-06-23
Smart Images
Figure HDA0004798702250000011 
Figure HDA0004798702250000012 
Figure HDA0004798702250000013
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for inducing transgenic hairy roots of Toona sinensis. Background Technology
[0002] Chinese toon, a plant belonging to the genus Toona of the family Meliaceae, has attracted attention due to its unique aroma and rich nutritional secondary metabolites. Currently, key genes for the agronomic traits of Chinese toon have been identified, but a gene function system for Chinese toon is lacking. The application of the hairy root system can be used for rapid and effective verification of the gene function of Chinese toon. Summary of the Invention
[0003] The purpose of this invention is to provide a method for inducing transgenic hairy roots of Toona sinensis.
[0004] To achieve the objective of this invention, in a first aspect, this invention provides a method for inducing transgenic hairy roots of Toona sinensis, which involves infecting Toona sinensis explants with Agrobacterium rhizogenes K599 containing the target gene to induce the explants to generate hairy roots.
[0005] The explant is a wound formed by cutting off part of the hypocotyl or root of a Toona sinensis seedling, or a wound formed by pricking the seedling with a needle.
[0006] Furthermore, the method includes the following steps:
[0007] (1) Cultivation of Toona sinensis seedlings;
[0008] (2) The expression vector containing the target gene was introduced into Agrobacterium rhizogenes K599 and activated by shaking until OD. 600 The concentration is 0.5-1.5, then it is spread on a solid plate and incubated for 12-24 hours;
[0009] (3) Cut off part of the hypocotyl or root of the Chinese toon seedling directly to form a wound, or prick the seedling with a needle to form a wound, and apply Agrobacterium rhizogenes cultured on a solid plate to the wound.
[0010] (4) After application, transfer to nutrient soil for cultivation to induce the formation of hairy roots.
[0011] Step (1) includes: selecting fresh, plump seeds from the current year and removing the wing membranes, soaking the seeds overnight to facilitate full absorption of water, then spreading the seeds flat on a seedling tray with a damp towel or paper towel, and placing them in an environment of 20℃-25℃. Spraying with 1 / 4 MS salt solution daily for moisturizing and germination culture for 7-21 days.
[0012] Step (2) includes: transforming Agrobacterium rhizogenes K599 with the expression vector containing the target gene, inoculating it into solid TY medium with the corresponding resistance, incubating it at 28°C for 2-3 days, picking single clones and culturing them in liquid TY medium with the corresponding resistance until OD. 600The concentration is 0.5-1.5. Take about 300 μL and spread it on a solid TY medium plate. Continue to incubate for 12-24 hours until the Agrobacterium rhizogenes completely covers the plate.
[0013] The liquid TY medium was formulated as follows: 5 g / L peptone and 3 g / L yeast extract, pH adjusted to around 7.0, and after autoclaving, 10 mmol / L calcium chloride solution and 50 mg / L kanamycin were added.
[0014] Solid TY medium is liquid TY medium with 15 g / L agar powder added.
[0015] Step (3) includes: cutting off part of the hypocotyl or root of the Chinese toon seedling to form a wound, or pricking the seedling with a needle to form a wound, applying a colony of Agrobacterium rhizogenes cultured on a solid plate to the wound, inserting it into a culture tank or a seedling tray with a lid containing nutrient soil, and maintaining a humidity of more than 80%.
[0016] Step (4) includes: transferring the plants to a light culture room at a temperature of 23-26℃, and using Agrobacterium tumefaciens infection solution containing plasmids (OD) for the first week after inoculation. 600 =0.5) watering, and continue to cultivate under light for 21-30 days (preferably 21 days) to obtain transgenic hairy roots.
[0017] The formulation of the infiltration solution is: 10 mmol / L MgCl2, 10 mmol / L MES and 100 μmol / L AS.
[0018] The target gene includes, but is not limited to, fluorescent marker genes, such as RUBY, GUS, uvGFP, and other fluorescent marker genes.
[0019] Secondly, the present invention provides the application of the hairy roots of Toona sinensis prepared according to the method in the study of Toona sinensis gene function and the genetic improvement breeding of Toona sinensis.
[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:
[0021] (I) This invention marks the first successful application of Agrobacterium rhizogenes to induce transgenic hairy roots in Toona sinensis. The efficiency of transgenic hairy roots was directly observed using the red RUBY gene and GUS reporter gene carried by the vector, with the calculated transformation efficiency exceeding 90% by line. Furthermore, the invention successfully transformed the uvGFP gene, which exhibits strong green fluorescence, and can be used for verification and screening of transgenic hairy roots.
[0022] (II) This invention obtains transgenic Toona sinensis tissue under non-tissue culture conditions. The operation process is simple, the technology is simple, the cost is low, and the efficiency is high. Furthermore, complete transgenic plants can be generated through transgenic hairy roots, overcoming the difficulty of verifying the function of Toona sinensis genes. Attached Figure Description
[0023] Figure 1 This is a preferred embodiment of the present invention showing the gene transformation of the RUBY system of Toona sinensis; wherein, A: induction of transgenic hairy roots of Toona sinensis seedlings in a culture tank; B: red indicates successfully transformed transgenic hairy roots of the RUBY system.
[0024] Figure 2 In a preferred embodiment of the present invention, the uvGFP fluorescent marker gene is irradiated with ultraviolet light. The red arrow points to the hairy roots that produce green transgenes when irradiated with a handheld fluorescent flashlight.
[0025] Figure 3 In a preferred embodiment of the present invention, the hairy roots transfected with the GUS gene were stained. The fact that the hairy roots stained blue with GUS indicated that the GUS gene had been successfully introduced into Toona sinensis. Detailed Implementation
[0026] This invention aims to provide a simple, efficient, and rapid method for inducing transgenic hairy roots in Toona sinensis. Transgenic hairy roots are produced by inserting T-DNA from a plasmid containing the target gene expression gene into plant tissues or organs using Agrobacterium rhizogenes. The induction culture of hairy roots can generate a large number of transgenic hairy roots, which can be directly used for research on the function of the target gene and as bioreactors to synthesize target metabolites. Furthermore, they can be regenerated to form transgenic plants.
[0027] The present invention adopts the following technical solution:
[0028] (1) Cultivation of Toona sinensis seedlings
[0029] Select fresh, plump seeds from the current year and remove the wing membranes. Soak the seeds overnight to facilitate full water absorption. Then, spread the seeds evenly on a damp towel or paper towel in a seedling tray and place them in an environment of 20℃-25℃. Spray with 1 / 4 MS salt solution daily for moisturizing and germination culture for 7-21 days.
[0030] (2) Preparation of Agrobacterium tumefaciens
[0031] The expression vector containing the target gene was transformed into Agrobacterium rhizogenes K599 and cultured at 28°C for about 2 days on a solid TY culture medium with appropriate resistance. Single colonies were then picked and cultured in liquid TY medium with appropriate resistance until Agrobacterium OD. 600 The pH value is approximately 0.5-1.5. Take about 300 μL and spread it onto a solid TY medium plate, then continue culturing for 12-24 hours until the Agrobacterium rhizogenes completely covers the plate. The liquid TY medium formula is: 5 g / L peptone and 3 g / L yeast extract, adjust the pH to approximately 7.0, autoclave, and then add 10 mmol / L calcium chloride solution and 50 mg / L kanamycin.
[0032] (3) Infection by Agrobacterium rhizogenes
[0033] Cut off part of the hypocotyl or root of the Chinese toon seedling directly, or make a wound by pricking it with a needle. Apply a cluster of Agrobacterium rhizogenes cultured on a solid plate to the wound, and insert it into a culture jar or a covered seedling tray filled with nutrient soil, keeping the humidity above 80%.
[0034] (4) Induction and culture of Agrobacterium rhizogenes
[0035] The plants were then transferred to a plant light culture room at a temperature of 23-26℃, and infected with Agrobacterium tumefaciens containing plasmids (OD200) for the first week after inoculation. 600 =0.5) watering, continued light culture for 21 days, and then used handheld fluorescence and other methods to screen transgenic hairy roots to verify the function of the target gene. Infection solution formula: 10 mmol / L MgCl2, 10 mmol / L MES and 100 μmol / L AS.
[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
[0037] Example 1: Transformation of Chinese toon seedling hypocotyl into RUBY gene expression system
[0038] (1) Cultivation of Toona sinensis seedlings
[0039] Select fresh, plump seeds from the current year and remove the wing membranes. Soak the seeds overnight to facilitate full water absorption. Then, spread the seeds evenly on a seedling tray lined with gauze and place them in a 23℃ environment. Spray with 1 / 4 MS salt solution daily for 10 days to maintain moisture and promote germination.
[0040] (2) Preparation of Agrobacterium tumefaciens
[0041] The RUBY expression system vector was transformed into Agrobacterium rhizogenes K599 and cultured at 28°C for about 2 days on a solid TY culture medium with appropriate resistance. Single colonies were then picked and cultured in liquid TY medium with appropriate resistance until Agrobacterium OD. 600 The pH value is approximately 0.8. Take 300 μL and spread it onto a solid TY medium plate, then continue incubation for 18 hours until the Agrobacterium rhizogenes completely covers the plate. The liquid TY medium formula is: 5 g / L peptone and 3 g / L yeast extract, pH adjusted to approximately 7.0, autoclaved, and then supplemented with 10 mmol / L calcium chloride solution and 50 mg / L kanamycin. The solid TY medium is the liquid TY medium with 15 g / L agar powder added.
[0042] (3) Agrobacterium rhizogenes infects the hypocotyl of Toona sinensis seedlings
[0043] Cut the roots of the Chinese toon seedlings from the bottom of the hypocotyl to create a wound. Gather the Agrobacterium rhizogenes cultured on solid plates into a bacterial clump. Apply the clump to the cut of the seedling and insert it into a culture jar filled with nutrient soil, maintaining a humidity of over 80%.
[0044] (4) Induction and culture of Agrobacterium rhizogenes
[0045] The plants were then transferred to a plant light culture room at 23°C, and infected with Agrobacterium tumefaciens containing plasmids (OD200) at 2, 4, and 6 days post-infection. 600 =0.5) watering, water 1 ml from the base of each plant, continue light culture for 21 days and then observe whether red transgenic hairy roots are produced ( Figure 1 Infection solution formulation: 10 mmol / L MgCl2, 10 mmol / L LMES and 100 μmol / L AS.
[0046] Example 2: Transformation of Toona sinensis seedling hypocotyls into uvGFP reporter gene
[0047] (1) Cultivation of Toona sinensis seedlings
[0048] Select fresh, plump seeds from the current year and remove the wing membranes. Soak the seeds overnight to facilitate full water absorption. Then, spread the seeds evenly on a seedling tray lined with gauze and place them in a 23℃ environment. Spray with 1 / 4 MS salt solution daily for 15 days to maintain moisture and promote germination.
[0049] (2) Preparation of Agrobacterium tumefaciens
[0050] Vectors containing both the uvGFP reporter gene and the GUS gene were transformed into Agrobacterium rhizogenes K599 and cultured at 28°C for 2 days on solid TY culture medium with appropriate resistance. Single colonies were then picked and cultured in liquid TY with appropriate resistance until Agrobacterium OD. 600 The pH value is approximately 0.8. Take 300 μL and spread it onto a solid TY medium plate, then continue incubation for 18 hours until the Agrobacterium rhizogenes completely covers the plate. The liquid TY medium formula is: 5 g / L peptone and 3 g / L yeast extract, pH adjusted to approximately 7.0, autoclaved, and then supplemented with 10 mmol / L calcium chloride solution and 50 mg / L kanamycin. The solid TY medium is the liquid TY medium with 15 g / L agar powder added.
[0051] (3) Agrobacterium rhizogenes infects the roots of Toona sinensis seedlings
[0052] Cut the roots of the Chinese toon seedlings from the bottom of the hypocotyl to create a wound. Gather the Agrobacterium rhizogenes cultured on solid plates into a bacterial clump. Apply the clump to the cut of the seedling and insert it into a culture jar filled with nutrient soil, maintaining a humidity of over 80%.
[0053] (4) Induction and culture of Agrobacterium rhizogenes
[0054] The plants were then transferred to a plant light culture room at a temperature of 23°C, and the inoculum was treated with an inoculum solution (OD) on days 2, 4, and 6 after inoculation. 600 =0.5) watering, 1 ml water is poured from the base of each plant. After continuing light culture for 21 days, handheld fluorescence screening is used to select hairy roots that clearly produce green fluorescence ( Figure 2 Further, GUS staining was used to verify whether the green fluorescent hairy roots had been introduced with the GUS gene and turned blue. Figure 3 Infection solution formulation: 10 mmol / L MgCl2, 10 mmol / L MES and 100 μmol / L AS.
[0055] Example 3: Transformation of Toona sinensis seedlings by root infection
[0056] (1) Cultivation of Toona sinensis seedlings
[0057] Select fresh, plump seeds from the current year and remove the wing membranes. Soak the seeds overnight to facilitate full water absorption. Then, spread the seeds evenly on a seedling tray lined with gauze and place them in a 23℃ environment. Spray with 1 / 4 MS salt solution daily for 21 days to maintain moisture and promote germination.
[0058] (2) Preparation of Agrobacterium tumefaciens
[0059] Vectors containing both the uvGFP reporter gene and the GUS gene were transformed into Agrobacterium rhizogenes K599 and cultured at 28°C for 2 days on solid TY culture medium with appropriate resistance. Single colonies were then picked and cultured in liquid TY with appropriate resistance until Agrobacterium OD. 600 The pH value is approximately 0.8. Take 300 μL and spread it onto a solid TY medium plate, then continue incubation for 18 hours until the Agrobacterium rhizogenes completely covers the plate. The liquid TY medium formula is: 5 g / L peptone and 3 g / L yeast extract, pH adjusted to approximately 7.0, autoclaved, and then supplemented with 10 mmol / L calcium chloride solution and 50 mg / L kanamycin. The solid TY medium is the liquid TY medium with 15 g / L agar powder added.
[0060] (3) Infection by Agrobacterium rhizogenes
[0061] Cut off part of the hypocotyl or root of the Chinese toon seedling directly, or make a wound by pricking it with a needle. Apply a cluster of Agrobacterium rhizogenes cultured on a solid plate to the wound, insert it into a seedling tray filled with nutrient soil, and cover it to maintain a humidity of more than 80%.
[0062] (4) Induction and culture of Agrobacterium rhizogenes
[0063] The plants were then transferred to a plant light culture room at a temperature of 23-26℃, and infected with Agrobacterium tumefaciens containing plasmids (OD200) for the first week after inoculation. 600=0.5) Irrigate, continue light cultivation and observe whether transgenic hairy roots are produced. Infection solution formula: 10 mmol / L MgCl2, 10 mmol / L MES and 100 μmol / L AS.
[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for inducing transgenic hairy roots of Toona sinensis, characterized in that, The explants of Toona sinensis were infected with Agrobacterium rhizogenes K599 containing the target gene to induce the formation of hairy roots. The explant is formed by removing part of the hypocotyl or root of a Toona sinensis seedling, or by pricking the seedling with a needle; The method includes the following steps: (1) Cultivation of Toona sinensis seedlings; (2) The expression vector containing the target gene was introduced into Agrobacterium rhizogenes K599 and activated by shaking until OD. 600 The concentration is 0.5-1.5, then it is spread on a solid plate and incubated for 12-24 hours; (3) Cut off part of the hypocotyl or root of the Chinese toon seedling to form a wound, or prick the seedling with a needle to form a wound, and apply Agrobacterium rhizogenes cultured on a solid plate to the wound; (4) After application, transfer to nutrient soil for cultivation to induce the formation of hairy roots; Step (3) includes: cutting off part of the hypocotyl or root of the Chinese toon seedling to form a wound, or pricking the seedling with a needle to form a wound, applying a cluster of Agrobacterium rhizogenes cultured on a solid plate to the wound, inserting it into a culture tank or a seedling tray with a lid containing nutrient soil, and maintaining a humidity of more than 80%. Step (4) includes: transferring the plants to a light culture room at a temperature of 23-26℃, irrigating them with Agrobacterium tumefaciens infection solution containing the target gene for the first week after inoculation, and continuing light culture for 21-30 days to obtain transgenic hairy roots; The formulation of the infiltration solution is: 10 mmol / L MgCl2, 10 mmol / L MES and 100 μmol / L AS; The OD of the Agrobacterium infection solution containing the target gene 600 =0.
5.
2. The method according to claim 1, characterized in that, Step (1) includes: selecting fresh, plump seeds from the current year and removing the wing membranes, soaking the seeds overnight to facilitate full absorption of water, then spreading the seeds flat on a seedling tray with a damp towel or paper towel, and placing them in an environment of 20℃-25℃. Spraying with 1 / 4 MS salt solution daily for moisturizing and germination culture for 7-21 days.
3. The method according to claim 1, characterized in that, Step (2) includes: transforming Agrobacterium rhizogenes K599 with the expression vector containing the target gene, inoculating it into solid TY medium with the corresponding resistance, incubating it at 28°C for 2 days, and picking single clones to culture in liquid TY medium with the corresponding resistance until OD. 600 The concentration is 0.5-1.
5. Take 300 μL and spread it on a solid TY medium plate. Continue to incubate for 12-24 hours until the Agrobacterium rhizogenes completely covers the plate. The liquid TY medium was formulated as follows: 5 g / L peptone and 3 g / L yeast extract, pH adjusted to 7.0, autoclaved, and then 10 mmol / L calcium chloride solution and 50 mg / L kanamycin were added. Solid TY medium is liquid TY medium with 15 g / L agar powder added.
4. The method according to claim 1, characterized in that, The target gene includes a fluorescently labeled gene.
5. The method according to claim 4, characterized in that, The fluorescently labeled genes were selected from RUBY, GUS, and uvGFP genes.
6. The application of the hairy roots of Toona sinensis prepared according to any one of claims 1-5 in the study of Toona sinensis gene function and genetic improvement breeding of Toona sinensis.