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Preparation of phyllostachys pubescens protoplast and establishment of transient conversion system

A protoplast and instantaneous transformation technology, applied in the field of genetic engineering, can solve the problems of limited genetic transformation system of forest trees and the inability to progress in research

Active Publication Date: 2020-10-30
ZHEJIANG FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on forest trees is imminent, whether it is the verification of gene function or the regulation of gene expression. Due to the limitation of the forest tree genetic transformation system, these studies cannot continue

Method used

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  • Preparation of phyllostachys pubescens protoplast and establishment of transient conversion system
  • Preparation of phyllostachys pubescens protoplast and establishment of transient conversion system
  • Preparation of phyllostachys pubescens protoplast and establishment of transient conversion system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: the preparation method of moso bamboo protoplast

[0032] Step (1). The cultivation of bamboo seedlings:

[0033] First, the seeds of Phyllostachys edulis were sterilized with 75% alcohol for 5 min, ddH 2 O rinse 3 times, rinse off the alcohol on the surface of Phyllostachys pubescens as much as possible. with ddH 2 Spread the O-infiltrated filter paper in a petri dish, spread the sterilized moso bamboo seeds inside, rinse with warm water in the morning and evening, keep the moso bamboo seeds moist, and culture them at 25°C in the dark. After a week or so, the bamboo seeds will turn white, and then transfer the white bamboo seeds to a place filled with ddH 2 O in a hydroponic box, placed in a light incubator for cultivation, and the cultivation temperature was controlled at 25°C. First, light with a light intensity of 10,000lx was used for 14 hours, and then placed in the dark for 10 hours. Watered once every 2 days, about 25 days. Moso bamboo seedling...

Embodiment 2

[0046] Example 2: PEG-Ca 2+ Transient Transformation of Phyllostachys pubescens Protoplasts

[0047] Step (1). Prepare a PEG solution with a concentration of 40% (ready to use, it can be prepared 1 hour before conversion), and bathe in a 50°C-60°C water bath until the PEG solid is completely dissolved.

[0048] Step (2). Add 10 μl of 3 μg / μl GFP DNA to be transformed into a 2 ml round bottom centrifuge tube, add 100 μl of protoplast suspension, and mix gently.

[0049] Step (3). Slowly add 110ul of PEG solution to the mixture, control the injection time of each sample at 1min, mix gently, and incubate the transformation mixture at 25°C for 10-25 minutes in the dark.

[0050] Step (4). The mixture was added to 440 μl of W5 solution to stop the transformation process, then mixed upside down, and centrifuged at 150×g for 1 minute at room temperature (increasing speed 9 decelerating 2).

[0051] Step (5). Remove the supernatant, add 1ml of W5 solution, and incubate in the dark a...

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Abstract

The invention discloses preparation of phyllostachys pubescens protoplast and establishment of a transient conversion system. According to the invention, 4.5-6% (wt / vol) cellulase R10 and 1-1.6% (wt / vol) segregating enzyme R10 are used for dissociating phyllostachys pubescens leaf sheaths to obtain protoplasts; after the obtained protoplast is separated, purified and pretreated, the exogenous geneis efficiently transferred into the phyllostachys pubescens protoplast by utilizing PEG-Ca <2+> for transient expression.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a preparation method of moso bamboo protoplasts and a method for transferring exogenous genes into moso bamboo protoplasts through chemical induction and performing transient expression. Background technique [0002] Plant protoplasts are protoplast masses without cell walls obtained through enzymatic hydrolysis, but have the biological activity of living cells. It can be used as an ideal material for molecular biology, cell biology and other research. [0003] Transient transformation uses protoplasts as recipients, and introduces the transient expression vector with the target gene into plant protoplasts through a specific method, and does not integrate into the plant genome, and only uses the original vector itself for expression. The key step of the protoplast transient transformation system is to introduce foreign genes into protoplasts for expression. There are m...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206
Inventor 沈锦波张梦弟李岩
Owner ZHEJIANG FORESTRY UNIVERSITY
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