Ginseng non-tissue culture genetic transformation method and application

Abstract

The invention discloses a ginseng non-tissue culture genetic transformation method and application. According to the method, a ginseng seedling with a wound surface at a main root or a main side root is used as a transformation receptor, and agrobacterium is used for genetic transformation. Ginseng transgenic operation can be carried out under the non-tissue culture condition, and ginseng hairy roots with transformation results can be accurately identified through the red marker of RUBY in experiments. The whole period of the method is about 60-90 days, rapid and efficient genetic transformation of the ginseng can be achieved, and a solid foundation is provided for molecular breeding of the ginseng. The method provides a basis for realizing efficient research of ginseng gene functions, provides a feasible scheme for improving the ginseng breeding speed, and has good research potential and wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and in particular relates to a method for non-tissue culture genetic transformation of ginseng and its application. Background Art

[0002] Ginseng ( Panax ginseng CA Mey.) is a perennial Panax ginseng plant of the Araliaceae family. It has the functions of replenishing vital energy, restoring pulse and strengthening deficiency, nourishing spleen and lung, producing essence and nourishing blood, and is known as the "king of herbs". However, ginseng has a long cultivation cycle (the growth cycle of garden ginseng is about 4 to 5 years, while the growth cycle of forest ginseng is about 15 to 25 years), serious diseases, and common continuous cropping obstacles. Therefore, the cultivation of high-quality, high-yield, and highly resistant new ginseng varieties has always been a focus of attention in this field.

[0003] Traditional breeding methods have problems of long cycle and low efficiency. Molecular breeding methods can effectively solve the difficulties of traditional breeding. However, the bottleneck of ginseng molecular breeding is a simple and efficient genetic transformation system. The existing ginseng genetic transformation system mainly adopts tissue culture method, which has a long cycle (more than one year), large workload, low efficiency and easy pollution. How to find a simpler and more efficient genetic transformation method is an urgent problem to be solved in this industry. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to carry out efficient genetic transformation of ginseng and / or how to reduce the genetic transformation cycle of ginseng and / or how to breed ginseng quickly.

[0005] In order to solve the above technical problems, the present invention first provides a method for genetic transformation of ginseng, which may include genetic transformation of ginseng seedlings with wounds on the roots as transformation receptors. In the above method, the ginseng seedlings may be ginseng seedlings with all fibrous roots and part of lateral roots removed. The part of lateral roots may be 2nd to 3rd level lateral roots. The ginseng seedlings may be two-year-old seedlings. The diameter of the taproot and / or retained lateral roots of the ginseng seedlings may be 25-400 mm.

[0006] In the above method, the fibrous roots can be tiny branch roots of ginseng, that is, 3rd to 4th level branch roots and / or absorbing roots.

[0007] In the above method, the ginseng seedling with wounds on the root can be prepared according to a method comprising the following steps: removing all fibrous roots of the ginseng seedling and removing part of the lateral roots so that the incision area of ​​the main root or the retained lateral root can be 25-400 mm 2 , and obtained ginseng seedlings with wounds on the roots.

[0008] In the above method, the main root can be a root developed from the radicle, that is, a primary root; and the lateral root can be a relatively strong branch root of ginseng, that is, a primary branch root.

[0009] In the above method, the genetic transformation may include infiltrating the wound of the ginseng seedling with the wound on the root into an Agrobacterium infection solution carrying the target gene for infection treatment.

[0010] The above method may include culturing the infected ginseng seedlings to obtain transgenic ginseng hairy roots, thereby completing the genetic transformation of ginseng.

[0011] In the above method, the infection time may be greater than or equal to 12 hours.

[0012] In the above method, the OD of the Agrobacterium infection solution is 600nm The value can be 0.8-1.0.

[0013] The application of the method described above in ginseng breeding also falls within the protection scope of the present invention.

[0014] The application of the above-mentioned transgenic ginseng hairy roots in ginseng breeding also falls within the protection scope of the present invention.

[0015] The transgenic ginseng hairy roots described above also fall within the protection scope of the present invention.

[0016] The invention discloses a ginseng genetic transformation system based on root regeneration, which can carry out transgenic operation of ginseng under non-tissue culture conditions, and can accurately identify ginseng hairy roots of transformation results through the red mark of RUBY.

[0017] The present invention reports for the first time a method for non-tissue culture genetic transformation of ginseng for root regeneration, the whole cycle of which is about 60 to 90 days, achieving rapid and efficient genetic transformation of ginseng, and providing a solid foundation for ginseng molecular breeding. The present invention fills the research gap of non-tissue culture genetic transformation of ginseng, provides a basis for efficient research on ginseng gene function, and provides a feasible solution for improving ginseng breeding speed, and has great research potential and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a ginseng seedling with good growth condition and cultivation environment.

[0019] Figure 2 This is the construction of 35S-RUBY plasmid for transgenic ginseng hairy roots.

[0020] Figure 3 The transgenic hairy root material of ginseng (the white arrows indicate the successfully transgenic hairy root material).

[0021] Figure 4 PCR verification of transgenic hairy roots of ginseng (NC, negative control; PC, vector pPositive control for PCR of CAMBIA3301-35S-RUBY; Sp1 and Sp2: transgenic ginseng hairy roots to be verified; M: molecular weight marker). DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0024] The ginseng in the following examples is the Dalmatian variety of ginseng (kindly provided by Mr. Jin Shiyuan, related literature: Jin Shiyuan. Jin Shiyuan's Traditional Identification Experience of Chinese Medicinal Materials [S]. Beijing: China Traditional Chinese Medicine Press, 2012: 100). The public can obtain this biological material from the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences for only use in repeating the present invention and not for other purposes.

[0025] Example 1. Method for non-tissue culture genetic transformation of ginseng and its application 1. Pre-cultivation of ginseng seedlings Two-year-old ginseng seedlings (variety: Dama Ya; cultivation area: Fusong, Jilin) ​​with good growth status (latent buds intact, main roots cylindrical or spindle-shaped, smooth epidermis, yellow-white or light yellow in color, no signs of rot, frostbite softening, mildew or pest infestation) were selected as the infective materials ( Figure 1 Select a suitable size culture pot, plant the ginseng seedlings in a soil mixture of humus and vermiculite in a ratio of 3:1 and add a small amount of compound fertilizer. After adding water, wrap it with plastic wrap and place it in a greenhouse environment at around 22 ºC for a week to adapt it to the greenhouse culture environment ( Figure 1 center right).

[0026] 2. Construction of transgenic vector and transformation of Agrobacterium rhizogenes right p The CAMBIA3301 plasmid was modified to change the GUS gene position in the plasmid to the RUBY gene, and a 35S promoter was used to construct a p CAMBIA3301-35S-RUBY vector (wherein the RUBY gene sequence is sequence 1 in the sequence list, which is available to the public from the applicant and is only used to repeat the present invention and cannot be used for other purposes), the map is as follows Figure 2 .

[0027] K599 competent Agrobacterium rhizogenes were prepared according to the instructions of the Agrobacterium Competence Preparation Kit (Biyuntian Biotechnology, Shanghai), and Agrobacterium transformation was performed according to the following steps: 0.1-1 μg of p CAMBIA3301-35S-RUBY plasmid DNA; let stand on ice for 15-20 min; freeze in liquid nitrogen for 1-3 min, and immediately put in a 37°C water bath for 5 min; add 500 μL of LB liquid culture medium, and culture in a shaking incubator at 30°C and 200 rpm for 4 h; draw 100 μL of the cultured bacterial liquid and spread it evenly on a solid LB culture medium containing 50 μg / mL of streptomycin and kanamycin; culture in a 30°C incubator for 2 days, pick a single colony, and obtain the recombinant Agrobacterium K599 / p CAMBIA3301-35S-RUBY.

[0028] 3. Non-tissue culture transformation of ginseng mediated by Agrobacterium rhizogenes containing RUBY reporter gene The genetic transformation of ginseng was carried out using Agrobacterium rhizogenes under non-tissue culture conditions. The specific steps are as follows: 3.1 Agrobacterium pre-culture Take out the recombinant Agrobacterium K599 / p CAMBIA3301-35S-RUBY was thawed on ice, activated by streaking in a clean bench, and incubated in a 28 ºC incubator to activate the bacteria until plaques grow. After picking a single clone, inoculate it into LB liquid medium containing antibiotics (kanamycin, 50 mg·L -1 ; Streptomycin, 50 mg·L -1 ) and shake-cultured K599 Agrobacterium rhizogenes until the bacterial solution OD 600nm The cell suspension was centrifuged at 5000 rpm at a concentration of 0.8, and the supernatant was removed. The bacteria were resuspended in infection solution (10 mM 2-morpholineethanesulfonic acid, 10 mM MgCl2, 200 μM acetosyringone, pH = 5.2), and induced in the dark for 3-5 h to obtain the recombinant Agrobacterium infection solution.

[0029] 3.2 Ginseng seedling preparation After the above Agrobacterium pre-culture is completed, the ginseng seedlings are taken out of the pot, and all the fibrous roots and part of the lateral roots are cut off with a blade, and a relatively flat wound surface is cut on the main root or the main lateral root. In this embodiment, a wound surface (about 56.7 mm in diameter) is cut on the main root (about 8.5 mm in diameter). 2 ).

[0030] 3.3 Infection and transformation Place the wound of the ginseng seedling obtained in step 3.2 in the recombinant Agrobacterium infection solution obtained in step 3.1, ensure that the wound is completely infiltrated by the infection solution, and infect overnight (greater than or equal to 12 hours). Then wash it three times with sterile water containing 400 mg / L Timentin, and then wash it five times with pure water for later use.

[0031] 4. Detection and identification of non-tissue culture transgenic hairy roots of ginseng Replant the ginseng seedlings infected in step 3 into the soil, regularly observe the appearance of hairy roots at the cut, and water them 1-2 times a week. In about 60-90 days, hairy roots will grow near the broken roots. p If the CAMBIA3301-35S-RUBY plasmid is successfully transgenic, it can produce red betalain, which is helpful for directly observing whether the transgenic success is successful. Figure 3 As indicated by the white arrows, the non-tissue culture transgenic hairy roots of ginseng are red, indicating that the transgenic process was successful.

[0032] The red hairy roots were cut off and DNA was extracted. The RUBY fragment was used for primer design. The primers were RUBY-F: 5'-CCATCCTCATCATCTCCGGC-3', RUBY-R: 5'-AAGTGTGCCATGATCCCAGG-3'. PCR amplification was performed according to the following system (94 ºC, 10 min; 35 cycles, each cycle was (94 ºC, 30 s; 55 ºC, 30 s; 72 ºC, 2 min); 72 ºC, 10 min; 4 ºC end), and then agarose gel electrophoresis was used to determine that the positive seedlings had the same fragment as the positive control ( Figure 4 ), further verifying the success of transgenic.

[0033] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for genetic transformation of ginseng, characterized in that: The method comprises the steps of using ginseng seedlings with wounds on their roots as transformation recipients for genetic transformation.

2. The method according to claim 1, characterized in that: the ginseng seedlings are ginseng seedlings with all fibrous roots and part of lateral roots removed.

3. The method according to claim 1 or 2, characterized in that: The ginseng seedling with wounds on the root is prepared according to a method comprising the following steps: removing all fibrous roots of the ginseng seedling and removing part of the lateral roots so that the incision area of ​​the main root or the retained lateral root is 25-400 mm 2 , and obtained ginseng seedlings with wounds on the roots.

4. The method according to claim 1, 2 or 3, characterized in that: The genetic transformation comprises infiltrating the wound of the ginseng seedling with the wound on the root into an Agrobacterium infection solution carrying the target gene for infection treatment.

5. The method according to any one of claims 1 to 4, characterized in that: The method comprises culturing the infected ginseng seedlings to obtain transgenic ginseng hairy roots, thereby completing the genetic transformation of ginseng.

6. The method according to any one of claims 1 to 5, characterized in that: The infection time is greater than or equal to 12 hours.

7. The method according to any one of claims 1 to 6, characterized in that: The OD of the Agrobacterium infection solution 600nm The value is 0.8-1.

0.

8. Use of the method according to any one of claims 1 to 7 in ginseng breeding.

9. Use of the transgenic ginseng hairy roots according to any one of claims 1 to 7 in ginseng breeding.

10. The transgenic ginseng hairy roots according to any one of claims 1 to 7.

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