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A method for the isolation, purification and efficient transformation of mesophyll protoplasts of Zhongshan fir variety 406

A technology of protoplasts and varieties, applied in the field of plant genetic engineering, can solve the problems such as the limitation of molecular biological function research of Zhongshan fir, and achieve the effect of simple operation

Active Publication Date: 2021-01-05
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the physiological and morphological studies of Zhongshan fir have achieved certain results, but like most conifer species, due to the immature genetic transformation system, the research on the molecular biology of Zhongshan fir is limited.

Method used

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  • A method for the isolation, purification and efficient transformation of mesophyll protoplasts of Zhongshan fir variety 406
  • A method for the isolation, purification and efficient transformation of mesophyll protoplasts of Zhongshan fir variety 406
  • A method for the isolation, purification and efficient transformation of mesophyll protoplasts of Zhongshan fir variety 406

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Protoplast Isolation and Purification

[0056] 1. Material selection and pretreatment

[0057] Generally speaking, if you want to obtain high-yield, highly active, and complete protoplasts, you generally choose young tissues with low keratinization and fibrosis, and materials that are easy to obtain. The present invention selects the young and tender leaf material of the tissue cultured seedlings of Zhongshan fir growing well in 30 days (such as figure 1 shown), in order to improve the efficiency and activity of protoplast isolation, the material needs to be pretreated in the dark for 30 hours (place the tissue culture seedlings in a constant temperature incubator and grow for 30 hours at 25°C in shade).

[0058] 2. Isolation and purification of the mesophyll protoplasts of Zhongshan fir

[0059] 1) Use a blade to quickly separate the Zhongshan fir slices from the petioles, and gently cut the leaves into 0.5-1mm wide leaf strips.

[0060] 2) Prepare enzyme ...

Embodiment 2

[0069] 1. Plasmid extraction

[0070] The vector p2FGW7.0 used for transformation in this example is a commercial vector, and the vector diagram is as follows figure 2 As shown, the vector contains a constitutive strong expression promoter P35S, which can be highly expressed in protoplasts, contains an Egfp tag, and can observe green fluorescent signals under ultraviolet light. Use the mini-extraction kit produced by Tiangen Company to extract the plasmid containing the vector. Generally, 15-20mL of overnight culture solution can extract about 50-80μg of the plasmid.

[0071] 1) Column equilibration step: Add 500 μL of equilibrium solution BL to the adsorption column CP4, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0072] 2) Take 5~15mL of overnight cultured bacterial solution containing p2FGW7.0 as the carrier (the concentration of the bacterial solution is controlled at ...

Embodiment 3

[0095] The present invention uses the leaves of Zhongshan Shanshan 406 tissue culture seedlings as materials, and based on the Arabidopsis thaliana mesophyll protoplast transformation system published in Nature Protocol in 2007 by the shen J laboratory of Harvard University, the enzymatic hydrolysis system of Zhongshan Shanshan fir mesophyll protoplasts is carried out. Optimization, cellulase set a gradient from 1% to 4% per 1%, pectinase set a gradient from 0.5% to 2% per 0.5%, a total of 16 sets of enzyme solution ratios, and observe the enzymatic hydrolysis after 12 hours The situation is counted, and each count is repeated three times. The results are shown in Table 1, showing: when the cellulase is 3%, and the pectinase is 1.5%, the enzymolysis effect is the best.

[0096] Table 1 The effect of different enzyme combinations and concentrations on the protoplast yield of Zhongshan fir seedlings (×10 6 / gFW)

[0097]

[0098] The effect of enzymatic hydrolysis time on th...

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Abstract

The invention discloses a Taxodium Zhongshanha 406 mesophyll protoplast separation, purification and high-efficiency conversion method. The method comprises the following steps: 1, carrying out dark pretreatment on a material tender leaves of 30 days-old good-growing tissue culture seedlings of Taxodium Zhongshanha; 2, separating and purifying mesophyll protoplasts; 3, extracting 50-80 [mu]g of a plasmid, which is a vector p2FGW7.0, from 15-20 mL of an overnight cultured bacterium liquid; and 4, converting the protoplasts through PEG mediation. The Taxodium Zhongshanha 406 mesophyll protoplast separation, purification and high-efficiency conversion method has the advantages of simplicity and easiness in operation, obtaining of massive, complete and pure Taxodium Zhongshanha mesophyll protoplasts, and high plasmid conversion rate reaching 80%. The method solves a problem that the gene functions of the Taxodium Zhongshanha cannot be deeply researched, provides a certain reference for the establishment of other coniferous species protoplast instantaneous expression systems, and has great application values in the field of forest tree gene engineering.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a method for isolating, purifying and efficiently transforming mesophyll protoplasts of Taxodium hybrids 'zhongshansha406'. Background technique [0002] Plant protoplast refers to "the part of the cell material that can be separated from the cell wall through plasmolysis". In recent years, with the development of genomics and proteomics, the transient expression of plant protoplasts has been widely used by scientists for high-throughput analysis of gene functions. Due to the metabolic process of protoplasts, the response to plant hormones, and the environmental Factors respond exactly the same as intact plant cells or tissue cells, so protoplasts provide us with a convenient and powerful tool for analyzing signal transduction pathways in plants. This technique has been widely used in the analysis of promoter activity, gene subcellular localization and protein-pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N5/04
CPCC12N5/04C12N15/8206C12N2509/00
Inventor 宣磊华建峰王莦丰王芝权杨颖於朝广范文才殷云龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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