Genetic transformation method of agrobacterium tumefaciens-mediated lonicera maackii embryos

Abstract

The invention provides a genetic transformation method of agrobacterium tumefaciens-mediated lonicera maackii embryos. Comprising the following steps: (1) obtaining lonicera maackii embryos; (2) putting the lonicera maackii embryos into infection liquid, and infecting by a vacuum infiltration method to obtain infected lonicera maackii embryos; the infection solution is obtained by the following method: culturing agrobacterium in an infection culture solution, and centrifuging the obtained culture to obtain a precipitate; resuspending the precipitate in an infection suspension to obtain an infection solution; the agrobacterium contains a target gene; and (3) carrying out first culture on the infected lonicera maackii embryo under a dark condition, and then carrying out second culture in gauze to obtain the lonicera maackii containing the target gene. Therefore, honeysuckle containing the target gene can be conveniently and quickly obtained, and the function of the target gene can be researched.

Description

Technical Field

[0001] The invention relates to the technical field of plant transgenic technology, and in particular to a method for genetic transformation of honeysuckle seed embryos mediated by Agrobacterium. Background Art

[0002] Honeysuckle is a plant of the genus Lonicera in the family Caprifoliaceae. It is a common tree species in my country and one of the four major ornamental tree species in landscaping. The whole plant of Honeysuckle can be used as medicine. The leaves have antipyretic, immune-enhancing and antibacterial effects, the roots have bactericidal and antimalarial effects, and the flowers can be used as a substitute for honeysuckle. The active ingredients of Honeysuckle include chlorogenic acid, terpenes, volatile oils and flavonoids.

[0003] Seeds are an important life stage of higher plants. They are the juveniles of plants and have many physiological and biochemical processes in plants. They are ideal materials for studying plant growth and development patterns. In addition, seeds are also important for plant system development and plant domestication research. So far, there is still a lack of genetic transformation methods for Lonicera japonica, which limits the exploration and utilization of its genetic resources. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. This study invented a method for genetic transformation using Lonicera japonica seed embryos, which provides an effective tool for future research on its gene function.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium, comprising:

[0007] (1) Obtaining honeysuckle seed embryos;

[0008] (2) placing the honeysuckle seed embryos into an infiltration solution and infecting them by vacuum infiltration to obtain infected honeysuckle seed embryos;

[0009] The infection solution is obtained by the following method:

[0010] Agrobacterium is cultured in an infection culture medium, and the obtained culture is centrifuged to obtain a precipitate;

[0011] The precipitate is resuspended in an infection suspension to obtain the infection solution;

[0012] The Agrobacterium contains the target gene.

[0013] (3) The infected Lonicera japonica embryos are first cultured in a dark condition, and then second cultured in gauze to obtain Lonicera japonica embryos containing the target gene.

[0014] According to an embodiment of the present invention, the above-mentioned method for genetic transformation of honeysuckle seed embryos mediated by Agrobacterium may further include the following technical features:

[0015] According to an embodiment of the present invention, step (2) further comprises:

[0016] (2-1) placing the honeysuckle seed embryos into an infiltration dye solution, and performing a first vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 30 minutes;

[0017] (2-2) treating the first vacuum infiltration treatment of the honeysuckle embryos at room temperature and pressure for 5 to 10 minutes;

[0018] (2-3) Continue to perform a second vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 5 minutes to obtain infected honeysuckle embryos.

[0019] According to an embodiment of the present invention, the infection culture solution comprises:

[0020] Yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 45-55 mg / L, rifampicin 45-55 mg / L, 1 mM 2-morpholineethanesulfonic acid, and 0.02 mM acetosyringone. All concentrations are working concentrations.

[0021] According to an embodiment of the present invention, the infection suspension comprises:

[0022] 2.033 g / L magnesium chloride hexahydrate, 10 mM 2-morpholineethanesulfonic acid, and 0.2 mM acetosyringone. All concentrations are working concentrations.

[0023] According to the embodiment of the present invention, the Agrobacterium is inoculated in the infection culture solution, cultured at 25-35° C. and 180-200 rpm for 15-18 hours, and then the infection culture solution is added continuously, cultured at 25-35° C. and 180-200 rpm to obtain a culture, wherein the OD600 of the culture is 0.5-0.8;

[0024] The culture was centrifuged at 8000-1000 rpm for 40-60 seconds, and the supernatant was discarded to obtain a precipitate;

[0025] The precipitate is resuspended in an infection suspension to obtain the infection solution.

[0026] According to an embodiment of the present invention, the Agrobacterium contains a vector, the vector contains a target gene, and the vector is a plasmid.

[0027] According to an embodiment of the present invention, the first culture time is 20 to 30 hours.

[0028] According to an embodiment of the present invention, the second culture is carried out at 20-25° C., with 16 hours of light and 8 hours of darkness, and the culture time is 5-10 days.

[0029] According to an embodiment of the present invention, the target gene is from at least one of plants, animals and microorganisms, such as honeysuckle, sugar beet or other plants.

[0030] According to an embodiment of the present invention, it includes: a reporter gene, wherein the reporter gene is a Ruby gene.

[0031] The second aspect of the present invention provides a transgenic plant, wherein the transgenic plant is Lonicera japonica, and the transgenic plant is obtained by using the Agrobacterium-mediated genetic transformation method of Lonicera japonica embryos described in the first aspect.

[0032] The beneficial effects achieved by the present invention are at least:

[0033] The present invention provides a method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium. The method can be used to conveniently and quickly study the genes of Lonicera japonica. The method provided by the present invention provides a convenient method for the gene regulation mechanism, gene function verification, protein interaction, etc. of Lonicera japonica, provides a theoretical basis for the genetic improvement and improved variety breeding of Lonicera japonica, especially plays an important role in the functional research of forest seeds, and has high practical application value.

[0034] It has many advantages, including:

[0035] 1. Shorten the period of obtaining transgenic honeysuckle: there is no need to wait for the transgenic plants to mature and produce seeds to obtain stably transformed plants, thus overcoming the limitation of the long growth period of honeysuckle seeds in traditional methods.

[0036] 2. Simple and fast operation: seeds do not need to be pre-cultured, and the tissue culture step can be selectively omitted after transformation.

[0037] 3. Avoid tissue regeneration: Seed embryos have the ability to construct morphology, providing a new way of transgenic manipulation for plants with difficult tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a flow chart of the transformation of honeysuckle embryos according to Example 1 of the present invention.

[0039] Figure 2 This is a diagram showing the transformation results of Lonicera japonica embryos provided in Example 1 of the present invention (scale bar = 1 mm, A1-A3 are wild-type embryos; B1-B3 are embryos transformed with 35S::Ruby vector).

[0040] Figure 3 The electrophoretic diagrams are obtained by PCR identification of Ruby gene expression in embryos according to Example 1 of the present invention (1-6 are embryos showing red color after infection, 7-10 are wild-type embryos). DETAILED DESCRIPTION

[0041] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0042] At present, there is still a lack of genetic transformation methods for Honeysuckle, which limits the exploration and utilization of its genetic resources. The present invention provides an Agrobacterium-mediated genetic transformation method for Honeysuckle embryos, which provides an effective tool for studying the gene functions of Honeysuckle.

[0043] To this end, the present invention provides a method for genetic transformation of honeysuckle embryos mediated by Agrobacterium, comprising:

[0044] (1) Obtaining honeysuckle seed embryos;

[0045] (2) placing the honeysuckle seed embryos into an infiltration solution and infecting them by vacuum infiltration to obtain infected honeysuckle seed embryos;

[0046] The infection solution is obtained by the following method:

[0047] Agrobacterium is cultured in an infection culture medium, and the obtained culture is centrifuged to obtain a precipitate;

[0048] The precipitate is resuspended in an infection suspension to obtain the infection solution;

[0049] The Agrobacterium contains the target gene.

[0050] (3) The infected Lonicera japonica embryos are first cultured in a dark condition, and then second cultured in gauze to obtain Lonicera japonica embryos containing the target gene.

[0051] The honeysuckle seed embryos mentioned are seed embryos with complete structures and good activity.

[0052] According to a specific implementation, step (2) further comprises:

[0053] (2-1) placing the honeysuckle seed embryos into an infiltration dye solution, and performing a first vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 30 minutes;

[0054] (2-2) treating the first vacuum infiltration treatment of the honeysuckle embryos at room temperature and pressure for 5 to 10 minutes;

[0055] (2-3) Continue to perform a second vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 5 minutes to obtain infected honeysuckle embryos.

[0056] The vacuum infiltration treatment mentioned is a technology that uses a vacuum environment to infiltrate liquid or gas into the interior of a solid material. Through vacuum infiltration treatment, Agrobacterium can be better adsorbed and infiltrated into the infected solid material through pressure difference to improve the infection efficiency; and the pressure of the vacuum infiltration treatment is less than -0.08MPa, which can improve the infection efficiency. Specifically in the present invention, two vacuum infiltration treatments are set to avoid damage to the honeysuckle embryos caused by long-term vacuum treatment, and at the same time further improve the infection efficiency of the honeysuckle embryos. The vacuum infiltration treatment time mentioned is counted from when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08MPa, and the treatment under normal temperature and pressure conditions mentioned is counted from when the pressure shown on the vacuum gauge of the vacuum tank is atmospheric pressure.

[0057] The infection culture medium mentioned includes: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 45-55 mg / L spectinomycin, 45-55 mg / L rifampicin, 1 mM 2-morpholineethanesulfonic acid, and 0.02 mM acetosyringone. The infection culture medium composed of these substances can better promote the infection effect of Agrobacterium.

[0058] According to an embodiment of the present invention, the infection suspension comprises:

[0059] 2.033 g / L magnesium chloride hexahydrate, 10 mM 2-morpholineethanesulfonic acid, and 0.2 mM acetosyringone.

[0060] During the experiment, the Agrobacterium can be first inoculated into the infection culture solution, cultured at 25-35°C and 180-200rpm for 15-18 hours, and then the infection culture solution is continuously added, cultured at 25-35°C and 180-200rpm to obtain a culture, wherein the OD600 of the culture is 0.5-0.8 (for example, 0.6);

[0061] The culture was centrifuged at 8000-1000 rpm for 40-60 seconds, and the supernatant was discarded to obtain a precipitate;

[0062] The precipitate is resuspended in an infection suspension to obtain the infection solution.

[0063] Wherein, described Agrobacterium contains carrier, described carrier contains target gene, and described carrier is plasmid.Agrobacterium-mediated genetic transformation is a kind of technology widely used in plant genetic engineering, is to utilize the Ti plasmid of Agrobacterium that foreign gene (target gene) is imported into plant gene.Herein, the Agrobacterium that can be used can be Agrobacterium tumefaciens (Agrobacterium tumefaciens), such as strains such as GV3101, LBA4404 or EHA105, and these strains can carry and transmit foreign gene.Then first target gene is inserted into the carrier that is suitable for Agrobacterium-mediated transformation, such as pCAMBIA1301 or other carriers that are suitable for plant gene transformation.These carriers usually contain necessary marker genes (such as GUS or GFP) and necessary promoter and terminator.

[0064] According to a specific embodiment, the first culture time is 20 to 30 hours.

[0065] According to a specific implementation manner, the second culture is carried out at 20-25° C., with 16 hours of light and 8 hours of darkness, and the culture time is 5-10 days.

[0066] According to a specific embodiment, a reporter gene is also included, and the reporter gene is a RUBY gene. These reporter genes can be used as screening markers to help screen out successfully transformed plants, and use PCR, Southern hybridization and other methods to verify whether the exogenous gene is successfully integrated into the plant genome. Then the selected transgenic plants can be regenerated on an appropriate culture medium, and after obtaining the regenerated plants, the stability and expression of the transgenic plants can be further verified by molecular biological methods.

[0067] The technical scheme of the present invention is described below by specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples can be obtained commercially.

[0068] Example 1

[0069] References Figure 1 As shown, Example 1 provides a method for transforming Lonicera japonica seed embryos, which specifically includes:

[0070] (1) Streak the Agrobacterium strain containing the 35S::Ruby (Ruby is a reporter gene) plasmid on LB resistance activation medium, suspend and culture the single clone colonies in the infection medium, collect the Agrobacterium, and obtain an infection medium with an OD600 of 0.6-0.8 after resuspending the infection suspension.

[0071] 1) Streak the Agrobacterium strain containing the 35S::Ruby plasmid on LB resistance activation medium and culture it in a 28°C incubator for 2 to 3 days.

[0072] 2) Pick a single colony from step 1) and inoculate it into 1 mL of infection culture medium. Shake the culture medium at 28°C and 180-200 rpm for 16 hours to activate it.

[0073] 3) Add fresh infection culture medium to the infection culture medium in step 2) to make up to 5 mL, and shake the culture at 28°C and 180-200 rpm for 3 hours until the OD600 of the infection culture medium is about 0.6.

[0074] 4) Take 1 mL of the infection culture medium in step 3) into a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 45 seconds at room temperature, discard the supernatant, add 1 mL of infection suspension to resuspend the precipitate, and let it stand for 3 hours after resuspension.

[0075] Among them, the formula of LB resistance activation medium is: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar 5 g / L, spectinomycin 50 mg / L, and rifampicin 50 mg / L.

[0076] The infection culture medium formula is: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 50 mg / L, rifampicin 50 mg / L, 1 mM 2-morpholineethanesulfonic acid, and 0.02 mM acetosyringone.

[0077] The formula of the infection suspension is: 2.033 g / L magnesium chloride hexahydrate, 10 mM 2-morpholineethanesulfonic acid, 0.2 mM acetosyringone. The infection suspension is prepared before use.

[0078] (2) Obtain honeysuckle seed embryos through the following steps.

[0079] 1) Soak the honeysuckle seeds in distilled water for 24 hours.

[0080] 2) Under a stereo microscope, use a scalpel soaked in the infection solution to carefully cut the seed coat along the hilum, then use the back of the scalpel to squeeze the seed cotyledons to slightly expose the embryo, and carefully pick out the embryo with intact structure and good activity. The peeled embryo should be immediately immersed in the infection solution.

[0081] (3) The Lonicera japonica embryos obtained in step (2) are infected by vacuum infiltration using the infection solution obtained in step (1).

[0082] 1) Open the centrifuge tube containing the infection solution and the embryos soaked in the infection solution and place it in a vacuum tank. Use a vacuum pump to evacuate the vacuum. Start timing when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08 MPa. Perform vacuum infiltration treatment for 30 minutes at room temperature.

[0083] 2) After slowly releasing the air to restore the pressure of the vacuum tank to atmospheric pressure, open the vacuum tank and take out the centrifuge tube. When the pressure indicated by the vacuum gauge of the vacuum tank reaches atmospheric pressure, start timing. Treat at room temperature and pressure for 5 minutes. During this period, gently shake the centrifuge tube to suspend the embryo.

[0084] 3) Open the centrifuge tube containing the infection solution and the embryos soaked in the infection solution and place it in a vacuum tank again. Use a vacuum pump to evacuate the tank. Start timing when the pressure shown on the vacuum gauge of the vacuum tank is less than -0.08 MPa. Perform vacuum penetration treatment for 5 minutes at room temperature.

[0085] 4) Wrap the centrifuge tube containing a small amount of resuspension solution and vacuum-infiltrated seed embryos with tin foil and incubate at 28°C in the dark for 24 hours.

[0086] (4) The Lonicera japonica embryos infected in step (3) are further cultured in moist gauze, and the transformation is verified by PCR.

[0087] 1) Cut the gauze into 8cm×8cm size, place 5 layers of gauze flatly in a square culture dish, and soak them completely with sterile distilled water.

[0088] 2) Use a plastic pipette or a pipette (with the tip cut off) to suck up the embryos after light-proof culture and transfer them evenly to the moist gauze in the culture dish.

[0089] 3) Cover the culture dish and place it in a light incubator at 22°C, 16 hours of light and 8 hours of darkness. After about 2 to 7 days, some embryos will show a red phenotype.

[0090] 4) Six red embryos and four wild-type embryos were randomly selected, and DNA was extracted using Edwards reagent and PCR was performed. Ruby gene bands were amplified from all six red embryos. Figure 2 The Ruby gene expression results are shown in Figure 3 The results of Example 1 show that the target gene Ruby can be successfully transferred into the embryos of Lonicera japonica through the method provided by the present invention, and the genetic transformation system of Lonicera japonica embryos is successfully constructed.

[0091] Comparative Example 1

[0092] Comparative Example 1 is different from Example 1 in that in step (3), the centrifuge tube containing the infection solution and the seed embryos immersed in the infection solution is opened and placed in a vacuum tank, and a vacuum pump is used to evacuate the vacuum tank. When the pressure indicated by the vacuum gauge of the vacuum tank is less than -0.08 MPa, the timing is started, and the vacuum penetration treatment is carried out at room temperature for 20 to 30 minutes. The results show that the treated honeysuckle seed embryos do not show red.

[0093] The results showed that only one vacuum infiltration treatment without recovery and repeated vacuum infiltration treatment could not effectively transfer the target gene into the embryos of Lonicera japonica.

[0094] Comparative Example 2

[0095] Comparative Example 2 is different from Example 1 in that in step (3), the centrifuge tube containing the infection solution and the seed embryos immersed in the infection solution is opened and placed in a vacuum tank, and a vacuum pump is used to evacuate the vacuum tank. When the pressure indicated by the vacuum gauge of the vacuum tank is less than -0.08 MPa, the timing is started, and the vacuum penetration treatment is carried out for 35 minutes at room temperature. The results show that the seed embryos of Honeysuckle die.

[0096] The results showed that if only one vacuum infiltration treatment was performed without recovery or repeated vacuum infiltration treatment, even if the vacuum infiltration treatment time was extended, the target gene could not be successfully introduced into the honeysuckle embryos, and it would also lead to the death of the honeysuckle embryos, which may be related to the fragility of the embryos.

[0097] Comparative Example 3

[0098] Comparative Example 3 is different from Example 1 in that in step (4), the vacuum infiltration treated embryos are directly cultured in wet gauze. The results show that the treated Lonicera japonica embryos do not show red color. The results show that the target gene cannot be effectively introduced into the Lonicera japonica embryos by culturing in gauze without the first culture.

[0099] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations of the present invention. Those of ordinary skill in the art can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium, characterized in that: include: (1) Obtaining honeysuckle seed embryos; (2) placing the honeysuckle seed embryos into an infiltration solution and infecting them by vacuum infiltration to obtain infected honeysuckle seed embryos; The infection solution is obtained by the following method: Agrobacterium is cultured in an infection culture medium, and the obtained culture is centrifuged to obtain a precipitate; The precipitate is resuspended in an infection suspension to obtain the infection solution; The Agrobacterium contains a target gene; (3) The infected Lonicera japonica embryos are first cultured in a dark condition, and then second cultured in gauze to obtain Lonicera japonica embryos containing the target gene.

2. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: Step (2) further comprises: (2-1) placing the honeysuckle seed embryos into an infiltration dye solution, and performing a first vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 30 minutes; (2-2) treating the first vacuum infiltration treatment of the honeysuckle embryos at room temperature and pressure for 5 to 10 minutes; (2-3) Continue to perform a second vacuum infiltration treatment at a vacuum pressure of less than -0.08 MPa and room temperature for 5 minutes to obtain infected honeysuckle embryos.

3. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The infection culture solution comprises: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, spectinomycin 50 mg / L, rifampicin 50 mg / L, 1 mM 2-morpholineethanesulfonic acid, and 0.02 mM acetosyringone; The infective suspension comprises: 2.033 g / L magnesium chloride hexahydrate, 10 mM 2-morpholineethanesulfonic acid, and 0.2 mM acetosyringone.

4. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The Agrobacterium is inoculated into the infection culture solution, and cultured at 25-35° C. and 180-200 rpm for 15-18 hours, and then the infection culture solution is continuously added, and cultured at 25-35° C. and 180-200 rpm to obtain a culture, wherein the OD600 of the culture is 0.5-0.8; The culture was centrifuged at 8000-1000 rpm for 40-60 seconds, and the supernatant was discarded to obtain a precipitate; The precipitate is resuspended in an infection suspension to obtain the infection solution.

5. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The Agrobacterium contains a vector, the vector contains a target gene, and the vector is a plasmid.

6. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The first culture time is 20 to 30 hours.

7. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The second culture is carried out at 20-25° C., with 16 hours of light and 8 hours of darkness, and the culture time is 5-10 days.

8. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: The target gene is from at least one of plants, animals and microorganisms.

9. The method for genetic transformation of Lonicera japonica seed embryos mediated by Agrobacterium according to claim 1, characterized in that: include: The reporter gene is a Ruby gene.

10. A transgenic plant, wherein the transgenic plant is Lonicera japonica, and the transgenic plant is obtained by using the Agrobacterium-mediated genetic transformation method of Lonicera japonica seed embryos according to any one of claims 1 to 9.

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