Efficient transient gene expression method for hybrid liriodendron taking mesophyll protoplast as receptor

A technology for hybridizing Liriodendron tulipifera and protoplasts, applied in biochemical equipment and methods, genetic engineering, using vectors to introduce foreign genetic material, etc., to achieve the effect of fast and convenient operation platform

Inactive Publication Date: 2017-03-08
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, efficient PEG-mediated transient expression systems have only been established in plants such as Arabidopsis thaliana, rice, cotton, and poplar. Affected by species differences, Magnoliaceae plants have not yet been established in a short period of time due to the large amount of wax in their leaves. A method for rapidly isolating its mesophyll protoplasts and an effective transient expression system

Method used

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  • Efficient transient gene expression method for hybrid liriodendron taking mesophyll protoplast as receptor
  • Efficient transient gene expression method for hybrid liriodendron taking mesophyll protoplast as receptor

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Embodiment 1

[0039] Example 1 Plasmid preparation

[0040] Using QIAGEN Plasmid Midi Kits to extract and purify the plasmid (PJIT166-GFP), the plasmid with high purity can be obtained conveniently and quickly. The specific steps are as follows:

[0041] 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;

[0042] 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 to 16 h;

[0043] 3) Centrifuge at 6000×g for 15 minutes at 4°C to collect the bacterial pellet, and completely discard the supernatant;

[0044]4) Resuspend the bacteria with 4 mL Buffer P1;

[0045] 5) Add 4 mL Buffer P2, invert up and down 4-6 times to mix completely, and let stand at room temperature for 5 min for lysis;

[0046] 6) Add 4 mL of pre-cooled Buffer P3, immediately invert 4-6 ...

Embodiment 2

[0056] Example 2 Isolation and Purification of Hybrid Liriodendron Liriophyll Mesophyll Cell Protoplasts

[0057] The quality of protoplasts directly affects the transformation efficiency, 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 isolated protoplasts. Mesophyll cell protoplasts retain many physiological responses and cell activities of plants to a greater extent, so they have been widely selected as the material source of plant cell protoplasts. At present, the mesophyll cell protoplasts of many plants such as Arabidopsis thaliana, tobacco, poplar, rice, corn, and cotton can be efficiently isolated in a short period of time, while the leaves of Liriodendron genus are similar to Ginkgoceae and Magnolia magnolia. , the upper epidermis contains a homologous series of leaf waxes, so its epidermis is difficult to remove by tearing methods s...

Embodiment 3

[0067] Example 3 Transformation

[0068] So far, efficient protoplast transient expression systems have been established in many plants such as Arabidopsis thaliana, tobacco, poplar, Populus euphratica, grape, and corn. However, there are some differences in the parameters of the PEG-mediated transient transformation system of protoplasts from different sources. For example, in the recently established PEG-mediated transient transformation system of Populus euphratica suspension cell protoplasts, the optimal parameters are: in a 240 μL transformation system, 1×10 5 The protoplasts and 20 μg plasmids were transfected with 20% PEG-4000 for 20 minutes, and the transformation rate was more than 50%. The optimal parameters for the protoplasts of grape suspension cells were: transformation in 220 μL System, 4×10 5 The protoplasts were added to 20 μg of plasmid, under the mediation of 25% PEG-4000, statically transfected for 30 minutes, and the transformation rate of more than 60% ...

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Abstract

The invention discloses an efficient transient gene expression method for hybrid liriodendron taking mesophyll protoplast as a receptor. The efficient transient gene expression method for the hybrid liriodendron taking mesophyll protoplast as the receptor comprises the following steps: preparation of the mesophyll protoplast of the hybrid liriodendron, preparation of plasmids, conversion and observation. The protoplast of mesophyll cells of the hybrid liriodendron is used as a receptor system of genetic transformation, by a PEG mediated method, green fluorescent protein (GEP) genes are used as reporter genes, and efficient transient expression of GFP genes is detected through a fluorescence microscope. The leaf blade of the hybrid liriodendron is used as a source of the protoplast, materials are taken conveniently, vitality is high, and an established transient expression system can provide a rapid and convenient operation platform for research of gene functions of liriodendron tulipifera and magnoliaceae.

Description

technical field [0001] The invention relates to a genetic transformation method of a hybrid tulip tree, in particular to a highly efficient instantaneous genetic transformation method of a hybrid tulip tree. 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. Affected by the long growth cycle of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8206C12N15/8212
Inventor 施季森霍爱玲夏兵陈金慧陈桢雨杨立明
Owner NANJING FORESTRY UNIV
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