Method for separating and converting rape protoplast

A technology of protoplasts and rapeseed, applied in the field of plant genetic engineering, can solve problems affecting the progress of rapeseed scientific research, achieve the effects of improving scientific research work efficiency, reducing experimental costs, and shortening the experimental cycle

Inactive Publication Date: 2017-12-19
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, before the application of the present invention, there was no report on the use of rapeseed protoplast genetic transformation to carry out the functional verification of the target gene, which affected the progress of scientific research on rapeseed, an important oil crop.

Method used

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  • Method for separating and converting rape protoplast
  • Method for separating and converting rape protoplast
  • Method for separating and converting rape protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Effect of Enzymolysis Solution and Enzymolysis Time on Rapeseed Protoplast Isolation

[0026] The key to the successful separation of protoplasts is the enzyme used to enzymatically hydrolyze the cell wall. We set up two enzymatic hydrolysis solutions to compare the yield and quality of protoplasts under different enzyme concentrations. The specific methods are as follows:

[0027] The first group: enzymatic hydrolysis solution is prepared at the enzyme concentration commonly used for plant protoplast separation, and the enzymolysis solution contains 1.5wt% cellulase, 0.4wt% isolated enzyme, 0.5M mannitol, 20mM KCl, 20mM MES (pH, 5.7), 10mM CaCl 2 , 0.1wt% BSA;

[0028] The second group: 1wt% cellulase, 0.3wt% resolase, 0.5M mannitol, 20mM KCl, 20mM MES (pH, 5.7), 10mM CaCl 2 , 0.1wt% BSA;

[0029] The results showed that after 3 hours of enzymolysis, the protoplast output of the first group of treatments was (4 ± 0.2) × 10 5 per gram of leaves, the second...

Embodiment 2

[0036] Example 2 Transient expression of yellow fluorescent protein in rapeseed protoplasts

[0037] The solution used consisted of the following:

[0038] Enzyme solution: 1wt% cellulase, 0.3wt% isolated enzyme, 0.5M mannitol, 20mM KCl, 20mM MES (pH5.7), 10mM CaCl 2 , 0.1%wtBSA, ready to use;

[0039] W5 solution: 4mM MES, 154mM NaCl, 125mM CaCl 2 , 5mM KCl, 2mM MES (pH 5.7);

[0040] MMG solution: 0.4M mannitol, 15mM MgCl 2 and 4mM MES (pH 5.7).

[0041] 1. Separation and purification method of protoplasts

[0042] (1) Select rapeseed true leaves or 3-5 pieces of rapeseed cotyledons that have grown for about 2 weeks (the cotyledons can be obtained after 7 days of germination, the cultivation environment and technology are simple, easy to obtain, and greatly shorten the experimental cycle), and fix both ends of a piece of tape On the table, with the sticky side up, stick the leaves or cotyledons on the tape with the lower cuticle facing up, then tape the lower cuticle, ...

Embodiment 3

[0055] Transient expression of embodiment three reporter genes LUC and Ren in rape protoplasts

[0056] 1. Separation and purification of protoplasts

[0057] The same as the separation and purification method of protoplasts in Example 2.

[0058] 2. The method for transient transformation of rapeseed protoplasts mediated by PEG

[0059] Expression vector plasmid preparation: the vector used is pGreenII 0800-35S-LUC, and the vector map is as follows: Figure 6 As shown, the plasmid was extracted using the Tiangen Endotoxin Free Plasmid Extraction Kit, operated according to the instructions, and the concentration of the extracted plasmid was adjusted to about 1 μg / μL for later use.

[0060] (1) Add 10 μg plasmid DNA (1 μg / μL) to 200 μL protoplast-MMG suspension, flick the bottom of the tube to mix;

[0061] (2) Add 220 μL 40% PEG-4000, flick the bottom of the tube to mix, and place at room temperature for 15 minutes;

[0062] (3) Add 880 μL W5 solution to re-suspend, flick ...

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Abstract

The invention discloses a method for separating and converting rape protoplast. The method comprises the following steps: 1, selecting rape cotyledons or true leaves, and removing lower epidermis; 2, putting into enzymolysis liquid for carrying out enzymolysis, wherein the enzymolysis liquid is prepared from the following formula components: 1wt% of cellulase, 0.3wt% of macerozyme, 0.5M of mannitol, 20mM of KCl, 20nM of MES with a pH value of 5.7, 10mM of CaCl2 and 0.1wt% of BSA; 3, adding an isovolumetric W5 solution into a mixed solution obtained by means of enzymolysis, carrying out low speed centrifugation for removing supernatant, adding W5 for washing and resuspending, adding resuspended liquid into 20wt% of a sucrose solution for purifying, horizontally centrifuging, and taking supernatant green protoplast liquid; 4, centrifuging for removing the W5 solution, and resuspending protoplast precipitate by using an MMG solution; 5, converting the protoplast. The protoplast separating and converting method is established in rape for the first time; the rape protoplast is used as a receptor for converting, so that yellow fluorescent protein (YFP), luciferase (Luc) and renilla reniformis luciferase (Ren) are successfully expressed.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for isolating, cultivating and instantaneously transforming high-quality rapeseed protoplasts. A method for transferring exogenous genes into protoplasts of rape and performing transient expression through the mediation of polyethylene glycol (PEG). Background technique [0002] The production of oil crops not only plays an important role in national economic and social development, but also is one of the main factors to promote the sustainable development of China's agriculture. At present, China's vegetable oil production can only meet 1 / 2-2 / 3 of domestic consumption demand, and relies heavily on imports. It is the world's largest oil importer. Rapeseed is one of the most important oil crops in China, and rapeseed oil accounts for about 40% of China's edible oil. The annual planting area of ​​rapeseed in China is 7.5 million hectares, and the total output of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/04
CPCC12N15/8206C12N5/04
Inventor 华玮郑明杨红丽李晓康刘婧琳
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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