Search

CN-121975864-A - Genetic transformation method suitable for Chinese medicine radix sileris

CN121975864ACN 121975864 ACN121975864 ACN 121975864ACN-121975864-A

Abstract

The invention discloses a genetic transformation method suitable for Chinese medicine divaricate saposhnikovia root, belonging to the technical field of biology. According to the genetic transformation method, the leaves are used as the infection objects, the infected leaves differentiate callus, then the regeneration seedlings are differentiated, the test period of genetic transformation is shortened, and the differentiation rate of the regeneration seedlings is improved. According to the invention, through the induction callus, bacteriostasis and resistance screening culture medium of specific mass concentration components and through limiting the infection time of agrobacterium, the bacteriostasis rate, differentiation rate and positive conversion rate are improved, a high-efficiency and stable windproof genetic transformation system is established, and the support is provided for precisely improving the characteristics of the windproof varieties and cultivating new windproof varieties with high quality, high yield and multiple resistance.

Inventors

  • CHEN YANG
  • YU HAORAN
  • JIN YIFENG
  • LIANG XIAOXUE
  • CHI XU
  • YOU XUE
  • WANG YUWEN
  • WANG PINSU

Assignees

  • 齐齐哈尔大学

Dates

Publication Date
20260505
Application Date
20260306

Claims (10)

  1. 1. A genetic transformation method applicable to Chinese herb divaricate saposhnikovia root, which is characterized by comprising the following steps: S1, preparing and culturing an explant, namely cutting divaricate saposhnikovia leaf, sterilizing, inoculating in an induced callus culture medium for preculture, wherein the induced callus culture medium comprises the following mass concentration components of 4.4-4.6 g/L of MS culture medium, 0.4-0.6 mg/L of 2,4-D, 1.2-1.4 mg/L, IBA 0.4.4-0.6 mg/L of 6-BA, 30-32 g/L of sucrose and 7.0-7.2 g/L of agar, and the pH=6.0; S2, preparing agrobacterium tumefaciens, namely transferring a plant expression vector into competent cells of agrobacterium tumefaciens GV3101, and screening and identifying to obtain positive transgenic agrobacterium tumefaciens; S3, preparing an infection liquid, namely placing positive agrobacterium tumefaciens in a liquid LB culture medium for culture, centrifuging, removing a supernatant, adding the culture liquid, fully mixing thalli, and preparing a transgenic infection liquid, wherein the infection liquid comprises the following components in mass concentration of 1/2 MS of culture medium 2.2-2.4 g/L and sucrose 30-32 g/L; S4, inducing the regenerated seedlings of the divaricate saposhnikovia root, namely immersing the pre-cultured leaves in an invasion solution containing positive root cancer agro-pole colonies, taking out the leaves after the invasion, wiping the bacterial solution on the surfaces by using sterile filter paper, transferring the bacterial solution to an induced callus culture medium for dark culture, transferring the bacterial solution to a bacteriostatic culture medium for dark culture, changing the bacteriostatic culture medium every 30d, transferring the regenerated seedlings differentiated for 2 times into a resistance screening culture medium for culture to obtain the successfully genetically transformed divaricate saposhnikovia root seedlings, and culturing the divaricate saposhnikovia root seedlings in the resistance screening culture medium; The bacteriostatic culture medium comprises the following mass concentration components of 4.4-4.6 g/L of MS culture medium, 0.4-0.6 mg/L of 2,4-D, 1.2-1.4 mg/L, IBA, 0.4-0.6 mg/L, timentin-400 mg/L, 30-32 g/L of sucrose and 7.0-7.2 g/L of agar, and pH=6.0; the resistance screening culture medium comprises the following mass concentration components of 4.4-4.6 g/L of MS culture medium, 0.4-0.6 mg/L of 2,4-D, 1.2-1.4 mg/L, IBA, 0.4-0.6 mg/L, timentin-400 mg/L of 6-BA, 25-35 mg/L of hygromycin, 30-32 g/L of sucrose and 7.0-7.2 g/L of agar, and pH=6.0; s5, identifying transgenic plants, namely extracting total DNA of the windproof seedlings with successful resistance screening, and carrying out PCR detection.
  2. 2. The genetic transformation method according to claim 1, wherein the sterilization method in S1 is that the windproof blades are cut in a beaker, soaked in a 75% ethanol solution 3min times, washed 3-5 times with sterile water, soaked in a 1% sodium hypochlorite solution 5-min times, and washed 5-6 times with sterile water after the soaking is completed.
  3. 3. The genetic transformation method according to claim 1, wherein the callus induction medium in S1 consists of MS medium 4.4 g/L,2, 4-D0.5 mg/L, 6-BA 1.2 mg/L, IBA 0.5 mg/L, sucrose 30 g/L and agar 7.2 g/L, pH=6.0.
  4. 4. The genetic transformation method according to claim 1, wherein the agrobacterium in S2 is agrobacterium tumefaciens.
  5. 5. The genetic transformation method according to claim 4, wherein the agrobacterium contains an overexpression vector capable of expressing the target gene.
  6. 6. The genetic transformation method according to claim 1, wherein the infection solution in S3 comprises 1/2MS medium 2.4 g/L and sucrose 30 g/L.
  7. 7. The genetic transformation method according to claim 1, wherein the infection time in S4 is 8-10 min and the dark culture time is 2-3 d.
  8. 8. The genetic transformation method according to claim 1, wherein the bacteriostatic medium in S4 consists of the following mass concentration components of MS medium 4.4 g/L,2, 4-D0.5 mg/L, 6-BA 1.2 mg/L, IBA 0.5 mg/L, timentin 300 mg/L, sucrose 30 g/L and agar 7.2 g/L.
  9. 9. The genetic transformation method according to claim 1, wherein the resistance selection medium in S4 consists of the following mass concentration components of MS medium 4.4g/L,2, 4-D0.5 mg/L, 6-BA 1.2 mg/L, IBA 0.5 mg/L, timentin 300 mg/L, hygromycin 30 mg/L, sucrose 30 g/L and agar 7.2 g/L.
  10. 10. The genetic transformation method according to claim 1, wherein the conditions of the cultivation are 25℃in daytime, 15℃in night, 60% relative humidity, 600. Mu. Mol.m -2 ·s -1 in illumination intensity, 16h in light cultivation in a cultivation period of one day, and 8h in dark cultivation.

Description

Genetic transformation method suitable for Chinese medicine radix sileris Technical Field The invention belongs to the technical field of biology, and particularly relates to a genetic transformation method suitable for Chinese medicine divaricate saposhnikovia root. Background Radix Saposhnikoviae Saposhnikovia divaricata (Trucz) is an important medicinal plant in China, and the active ingredients of the root have the effects of dispelling wind, relieving exterior syndrome, eliminating dampness, relieving pain, relieving spasm and the like. The extract of the root is commonly used for treating common cold, headache, rheumatalgia, tetanus and the like, and has higher medicinal and economic values. In recent years, people find that the limited natural resources are difficult to meet the increasing demands of medicine and research and development, and the long-term excavation of the wild resources inevitably breaks ecological balance, so that the natural resources are exhausted. The characteristics can be improved to a certain extent by means of gene transformation, the genetic transformation technology can edit key genes, the variety characteristics can be accurately improved, and a new variety with high quality, high yield and multiple resistance can be cultivated, however, in the prior art, the research on a windproof genetic transformation system is relatively less, and the research is mainly focused on the regeneration induction aspect of explants, the inventor of the invention discloses a windproof rapid propagation culture medium, a method and application thereof in the patent with publication number CN118489567A, and in addition, the patent with publication number CN115820717A discloses a windproof transgenic hairy root culture method, but the research on the windproof genetic transformation system is not reported yet. Therefore, the development of a high-efficiency and stable genetic transformation system suitable for the Chinese medicinal herb divaricate saposhnikovia root has very important significance. Disclosure of Invention The invention aims to provide a genetic transformation method suitable for Chinese medicine divaricate saposhnikovia root. In order to achieve the above object, the technical scheme of the present invention is as follows: A genetic transformation method applicable to the traditional Chinese medicine divaricate saposhnikovia root, which comprises the following steps: S1, preparing and culturing the explants, namely sowing the divaricate saposhnikovia seeds in nutrient soil, covering the soil, spraying the soil surface layer to be wet, culturing in a seedling culture room, cutting leaves after adult plants, sterilizing, and inoculating in an induced callus culture medium for preculture. S2, preparing agrobacterium tumefaciens, namely transferring a plant expression vector into competent cells of agrobacterium tumefaciens GV3101, and screening and identifying to obtain the agrobacterium tumefaciens containing the target genes. S3, preparing infection liquid, namely placing the positive agrobacterium tumefaciens of S2 into a liquid LB culture medium for culture, centrifuging, removing supernatant, adding the culture liquid, fully mixing thalli, and preparing the transgenic infection liquid. S4, inducing the divaricate saposhnikovia root regenerated seedlings, namely immersing the pre-cultured leaves in an invasion solution containing positive root cancer agro-rod colonies for infection, taking out the infected leaves after the infection, wiping off bacterial liquid on the surfaces by using sterile filter paper, transferring the bacterial liquid to an induced callus culture medium for dark culture for 2 d, transferring the bacterial culture medium to an antibacterial culture medium for dark culture, and replacing the culture medium every 30 d. Transferring the regenerated seedlings obtained by bacteriostasis culture differentiation to a resistance screening culture medium for culture to obtain the wind-proof seedlings successfully transformed by genetic engineering, and placing the wind-proof seedlings in the resistance screening culture medium for culture. S5, identifying transgenic plants, namely extracting total DNA of the windproof seedlings with successful resistance screening, and carrying out PCR detection. In a preferred embodiment of the present invention, the preparation method of the sterile material in S1 comprises the steps of cutting the wind-proof blades in a beaker, soaking in a 75% ethanol solution 3 min, washing 3-5 times with sterile water, adding a 1% sodium hypochlorite solution to soak 5-min, and washing 5-6 times with sterile water after the soaking is finished. The induced callus culture medium in the S1 comprises the following mass concentration components of 4.4-4.6 g/L of MS culture medium, 0.4-0.6 mg/L of 2,4-D, 1.2-1.4 mg/L, IBA 0.4.4-0.6 mg/L of 6-BA, 30-32 g/L of sucrose and 7.0-7.2 g/L of agar, and pH=6.0. In a preferred embodiment of the invention