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Dendrobium officinale instantaneous conversion method

A technology for instantaneous transformation of Dendrobium candidum, applied in biochemical equipment and methods, botany equipment and methods, fermentation, etc., can solve the problems of low conversion efficiency and long genetic transformation cycle of the transformation method of Dendrobium candidum

Pending Publication Date: 2022-07-05
SHAOGUAN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention aims to solve the problem of low transformation efficiency of the existing Dendrobium officinale transformation method caused by the long genetic transformation cycle of the existing Dendrobium officinale, and provides an efficient and rapid instant transformation method of Dendrobium officinale

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  • Dendrobium officinale instantaneous conversion method
  • Dendrobium officinale instantaneous conversion method
  • Dendrobium officinale instantaneous conversion method

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specific Embodiment approach 1

[0024] Embodiment 1: The instant transformation method of Dendrobium officinale in the present embodiment is carried out according to the following steps:

[0025] 1. After the surface of the Dendrobium candidum capsule is sterilized, the seeds of the Dendrobium candidum capsule are sown in the culture medium, and cultured aseptically for 50-60 days under the conditions of room temperature and illumination time of 16h / d to obtain the protocorm of Dendrobium candidum, with a diameter of 0.2 -0.5cm protocorm of Dendrobium officinale is soaked in high permeability treatment solution for 10-15min, and shakes every 1-2min during soaking;

[0026] 2. Transfer the Dendrobium officinale protocorm treated with the high osmotic treatment solution in step 1 to the infection solution containing Agrobacterium tumefaciens containing the recombinant target gene. The infection solution needs to be prepared in advance and kept at 4°C for 0.5- 1.0h; infection solution OD 600 0.9-1.0, and then ...

specific Embodiment approach 2

[0030] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the high osmotic treatment solution in step 1 is 1 containing 25% (w / v) sucrose + 0.01% (v / v) Triton X-100. / 4MS medium. It should be noted that Triton X-100 cannot be sterilized by high temperature, and should be added after filter sterilization;

specific Embodiment approach 3

[0031] Embodiment 3: This embodiment is different from Embodiment 1 or 2 in that: the Agrobacterium tumefaciens containing the recombinant target gene described in step 2 is an Agrobacterium tumefaciens transformed with an expression vector containing the recombinant target gene, wherein the recombinant target gene The gene is Dendrobium officinale gene. After the expression vector containing the target gene is transformed into Agrobacterium tumefaciens by the freeze-thaw method, the thalline used for the instant transformation of the present application is obtained, and the specific process is as follows:

[0032] 1. Take 0.5 μg of the expression vector plasmid containing the target gene and add it to 200 μL of Agrobacterium tumefaciens EHA105 competent cells, mix gently, and place on ice for 30 minutes;

[0033] 2. Freeze in liquid nitrogen for about 5 minutes, take out quickly, and heat shock in a water bath at 37°C for 5 minutes;

[0034] 3. Add 800 μL of antibiotic-free ...

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Abstract

The invention relates to an instantaneous transformation method for dendrobium officinale. In order to solve the problem of low transformation efficiency of a dendrobium officinale transformation method caused by long genetic transformation period of the existing dendrobium officinale, the method comprises the following steps: culturing a dendrobium officinale protocorm, soaking the dendrobium officinale protocorm in a high-permeability treatment solution, transferring the dendrobium officinale protocorm into an infection solution added with agrobacterium tumefaciens containing a recombinant target gene for infection, maintaining the pressure at 1.5 * 10 < 5 > Pa for 10-15 minutes after each 1.5 hours of infection in the process, and carrying out high-permeability treatment on the dendrobium officinale protocorm; and washing with medium permeation treatment liquid, culturing in dark, and culturing at room temperature. According to the method, the dendrobium officinale protocorm in a suitable physiological state, the improved high-permeability treatment liquid and the infection liquid are selected, and medium-permeability treatment is combined, so that the instant conversion efficiency of the dendrobium officinale is greatly improved.

Description

technical field [0001] The invention relates to a method for instantaneous transformation of Dendrobium officinale. Background technique [0002] Dendrobium officianle (Dendrobium officianle) is an endangered medicinal plant of the genus Dendrobium (Dendrobium Sw.), a perennial epiphytic herb of Orchidaceae (Orchidaceae). Valuable traditional Chinese medicine, known as "life-saving fairy grass" among the people. Analysis of modern pharmacology and chemical components shows that the main active components in Dendrobium officinale are polysaccharide compounds, which have the functions of improving immunity, lowering blood sugar, anti-tumor, anti-oxidation, protecting liver and stomach, and anti-cataract. Its physicochemical and structural characteristics closely related to medicinal activity. At the same time, since wild Dendrobium officinale is an epiphytic plant, it grows on the deep bark or trunk with high looseness and strong air permeability, and is also more common in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/65C12N15/56A01H5/00A01H6/62
CPCC12N15/8205C12N15/8209C12N9/2402C12Y302/01026
Inventor 刘羽佳刘博婷于白音韩伟理雅罗伟红赖思慧陈堰珊麦阮琳李学诗
Owner SHAOGUAN COLLEGE
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