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Method for improving genetic transformation transient expression efficiency of neolamarckia cadamba

A genetic transformation and transient expression technology, applied in the field of improving the transient expression efficiency of tuanhua genetic transformation, can solve the problems of low transient expression rate and low transformation efficiency, and achieve simple equipment and materials, good operability and reproducibility Effect

Active Publication Date: 2015-05-20
GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at the problem of low transient expression rate and low transformation efficiency of Tuanhua in Agrobacterium-mediated genetic transformation, and provides a method for improving the transient expression rate of genetic transformation

Method used

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  • Method for improving genetic transformation transient expression efficiency of neolamarckia cadamba

Examples

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Embodiment 1

[0035] An example of the method for improving the transient expression efficiency of group flower genetic transformation described in the present invention, comprises the steps:

[0036] 1. Material selection:

[0037] Binary expression vector pBI 121, Agrobacterium C58, sterilized petri dish with filter paper, explants are hypocotyls and cotyledons of 20-25d seedlings of aseptic seedlings of Tuanhua seeds.

[0038] 2. Bacterial solution preparation:

[0039] The expression vector pBI 121 carrying the GUS gene was passed through CaCl 2 into Agrobacterium C58. After the PCR identification of the bacteria solution is correct, pick a single colony of the Agrobacterium transformant, and in the liquid LB medium with a mass volume concentration ratio of 50mg / L kanamycin, 50mg / L rifampicin and 25mg / L streptomycin Incubate overnight at 28°C in 2 mL with shaking. On the second day, transfer 50 μL of the bacterial solution into 100 mL of YEB liquid medium, and culture at 22°C and 130 ...

Embodiment 2

[0050] Another example of the method for improving the transient expression efficiency of group flower genetic transformation described in the present invention comprises the following steps:

[0051] 1. Material selection:

[0052] Binary expression vector pBI121, Agrobacterium LBA4404, sterilized Petri dish with filter paper, explants are sterile seedling hypocotyls and petiolate cotyledons of 20-25d seedling-aged Tuanhua seeds;

[0053] 2. Bacterial liquid preparation:

[0054] The expression vector pBI121 carrying the GUS gene was passed through CaCl 2 into Agrobacterium tumefaciens LBA4404 by the method; after the PCR identification of the bacterial solution was correct, pick a single colony of the Agrobacterium transformant, and in the mass volume concentration ratio of 50mg / L kanamycin (Kan), 50mg / L rifampicin Level (Rif) and 25mg / L streptomycin (Str) liquid LB medium 2mL 28 ℃ shaking culture overnight. On the second day, transfer 50 μL of bacterial liquid into 100 m...

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Abstract

The invention discloses a method for improving genetic transformation transient expression efficiency of neolamarckia cadamba. The method comprises six steps, including material selection, bacterium liquid preparation, explant dip dyeing, co-culturing, positive screening and transient expression detection. By adopting the method disclosed by the invention, the genetic transformation transient expression efficiency of the neolamarckia cadamba can be improved by 863.6% to 908.9%; the problems that the dip dyeing efficiency is low and transgenic plants of the neolamarckia cadamba are hard to obtain when an agrobacterium tumefaciens mediated method is adopted can be effectively solved; the growth of agrobacterium spots can be effectively inhibited; the co-culturing time of agrobacterium and explant can be prolonged without damaging the explant; the transient expression efficiency for transgenosis can be improved.

Description

technical field [0001] The invention belongs to the field of plant transgenic technology. In particular, it relates to a method for improving the transient expression efficiency of tuanhua genetic transformation. Background technique [0002] Tuanhua (Neolamarckia cadamba), also known as Huangliangmu, is a native broad-leaved tree species in southern my country. Because of its rapid growth and straight trunk, it is known as a "miracle tree". It has attracted widespread attention at home and abroad as early as the 1970s. Its wood property is equivalent to that of Chinese fir, and its fast-growing property is similar to that of eucalyptus and poplar. Bin is the active ingredient of the antihypertensive drug "Uncaria total alkaloids", which has a strong and long-lasting antihypertensive effect, and its potency is close to that of reserpine (Endo K, Oshima Y, Kikuchi H, et al.Hypotensive principles of Uncaria hooks.Planta medica.1983,49(3):188-190), these two alkaloids only ex...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/56
Inventor 黄浩白隆华翟勇进
Owner GUANGXI BOTANICAL GARDEN OF MEDICINAL PLANTS