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Hybrid liriodendron efficient transient gene expression method using suspension cell protoplast as receptor

A technology for hybridizing Liriodendron tulipifera and protoplasts, which can be applied in the fields of biochemical equipment and methods, genetic engineering, introduction of foreign genetic material using vectors, etc., and can solve problems such as lack of research.

Pending Publication Date: 2017-03-15
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Polyethyleneimine (PEI) is a commonly used cationic polymer, and it is a non-viral gene carrier commonly used in vitro or in vivo in animal cell transgenesis, but less research has been carried out in plant gene transfection

Method used

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  • Hybrid liriodendron efficient transient gene expression method using suspension cell protoplast as receptor
  • Hybrid liriodendron efficient transient gene expression method using suspension cell protoplast as receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Plasmid preparation

[0039] The purity and concentration of the plasmid have an important impact on the transient transformation of protoplasts. The purity is low, and the conversion is difficult to succeed. If the concentration is too low, the transformation efficiency will be very low. Therefore, the purity and concentration of the plasmid is one of the key factors affecting the transformation efficiency. In this example, QIAGEN Plasmid Midi Kits were used to extract and purify the plasmid (PJIT166-GFP), which can conveniently and quickly obtain a high-purity plasmid. The specific process is:

[0040] 1) Pick a well-dispersed single colony on the plate, inoculate it in 2 mL of LB medium containing 100 mg / L ampicillin, and incubate with shaking at 37°C and 300 rpm for 8 h;

[0041] 2) Take the shaken bacterial liquid, add it to 50 mL LB medium containing 100 mg / L ampicillin at a ratio of 1:500, and culture with shaking at 37 °C and 300 rpm for 12-16 h;

...

Embodiment 2

[0055] Example 2 Separation and Purification of Hybrid Liriodendron Liriodendron Suspension Cell Protoplasts

[0056] Isolation of a large number of highly active protoplasts is the basis for the instantaneous transformation of protoplasts, and many factors such as the growth state of plant materials, the type and purity of enzymes, the time and temperature of enzymatic hydrolysis will affect the efficiency and quality of the isolated protoplasts.

[0057] In this example, using the suspension cells of Liriodendron tulipifera as materials, based on parameters such as special enzymatic hydrolysis solution, osmotic pressure, and enzymatic hydrolysis time, an effective method for preparing protoplasts of suspension cells of Liriodendron tulipifera was established. The specific steps are as follows:

[0058] 1) Preparation of enzymatic solution: Weigh 0.15 g of Cellulase R-10, 0.1 g of Macerozyme R10 and 0.01 g of PectolaseY-23 in 6.25 mL of 0.8 M mannitol, 2.5 mL of 80 mM KCl, 1 m...

Embodiment 3

[0068] Example 3 Transformation

[0069] The method for the instant transformation of protoplasts of hybrid Liriodendron chinensis suspension line cells mediated by PEI, the concrete steps are as follows:

[0070] 1) For a single reaction, add a total of 3 μg of plasmid DNA to a 2 mL centrifuge tube.

[0071] 2) Add 1 μg of PEI storage solution, add PBS until the system reaches 50 μL, mix thoroughly by ultrasonication for 2 minutes, and then let stand at room temperature for 30 minutes to prepare PEI / DNA gene carrier complex.

[0072] 3) Take 200uL of protoplasts from hybrid Liriodendron tulipifera suspension cells and mix them with PEI / DNA gene carrier complex, and gently tap with your fingers to mix the plasmid DNA and protoplast solution (or gently pipette with a wide-mouth pipette tip), Mix it well, and let it stand at room temperature for 5-10 min.

[0073] 4) Add 1mL of W5 solution and gently mix up and down to stop the transfection reaction, centrifuge at 1100rpm for ...

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Abstract

The invention discloses a hybrid liriodendron efficient transient gene expression method using suspension cell protoplast as the receptor. The method includes: preparing the hybrid liriodendron suspension cell protoplast, preparing plasmid, converting and observing. The method has the advantages that the hybrid liriodendron suspension cell protoplast is used as the receptor system for genetic transformation, a PEI mediated method is used, green fluorescent protein GFP genes are used as the reporter genes, and the efficient transient expression of the GFP genes is detected through a fluorescent microscope; the hybrid liriodendron suspension cells are used as the source of the protoplast, the hybrid liriodendron suspension cells are abundant, consistent in state, weak in background signal and convenient in material taking, and the built transient expression system can provide a fast and convenient operation platform for the researches of the gene functions of liriodendron and magnoliaceae plants.

Description

technical field [0001] The invention relates to a genetic transformation method of the hybrid tulip tree, in particular to a highly efficient instant gene expression method of the hybrid tulip tree using suspension cell protoplasts as receptors. Background technique [0002] Liriodendron belongs to the ancient Magnoliaceae plants, and from the phylogenetic point of view, it belongs to the original angiosperms. The study of Magnoliaceae genes and their functions will have an important impact on elucidating the phylogeny of angiosperms. At present, there may be problems such as false positives caused by heterologous systems using the transgenic systems of common model plants Arabidopsis and tobacco to carry out research on the gene function and signal transduction of Liriodendron. The research on gene function of palm tree and magnoliaceae is of great significance. Transgenic systems mainly include stable transfection transgenic systems and transient expression gene systems....

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC12N15/8206C12N15/8212
Inventor 陈金慧霍爱玲施季森陈桢雨杨立明成铁龙
Owner NANJING FORESTRY UNIV
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