Method for expressing proteins by using plant petal cell protoplast

A technology of protoplasts and plant petals, applied in the biological field, can solve the problems of no specificity of tissues and organs, not as good as the real state of petal cells, etc., and achieve the effect of improving efficiency

Inactive Publication Date: 2015-08-12
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The preparation of protoplasts has a variety of materials, mostly young leaves, germs, callus, etc. The existing protoplast preparation methods for ornamental plants mostly use callus as materials, callus cells are loose, cell walls are thin, and enzymolysis cell wall is easier
However, callus cells do not have tissue-organ specificity, so they are not as close to the real state of flowers as petal cells when they are used to study the functions of genes related to flower fragrance and flower color.

Method used

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  • Method for expressing proteins by using plant petal cell protoplast
  • Method for expressing proteins by using plant petal cell protoplast

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Embodiment 1

[0039] In the following, the petals of the plum blossom variety "Green Calyx with long stamens" are used as materials, and the vector pSuper1300-GFP encoding green fluorescent protein driven by the pSuper promoter is used as the exogenous target gene to be transformed, and the present invention will be further described in conjunction with the accompanying drawings.

[0040] Tissue processing: select six newly bloomed plum blossoms (such as figure 1 a), remove the petals (such as figure 1 b), stack 10-20 petals together (such as figure 1 c), use a blade to cut the petals into filaments (1-1.5cm in length and 0.3-0.8mm in width such as figure 1 d), the finer the cut, the more conducive to enzymatic hydrolysis;

[0041] Enzymolysis: Prepared enzymolysis solution 20mL (2% (m / v) Cellulase R-10, 2% (m / v) Resolase R-10, 0.4M Mannitol, 20mM KCl, 10mM CaCl 2, 20mM MES with a pH of 5.7, 0.1% (m / v) BSA and 5mM β-mercaptoethanol) were added to a glass petri dish with a radius of 90mm,...

Embodiment 2

[0051] This example is used to illustrate the preparation of green peach petal protoplasts and the expression of exogenous green fluorescent protein GFP. Method is identical with the preparation of the plum flower petal protoplast of embodiment 1 and exogenous gene transformation (see figure 2 ).

[0052] like figure 2 Shown in e is the morphology of peach petal protoplasts and the expression of green fluorescent protein GFP observed under a 20× laser confocal microscope, and the scale bar is 200 μm. From left to right: the images obtained under 484nm excitation light, under bright field, and after synthesis. According to statistics, the transformation efficiency is about 85%, so this method is also very suitable for the preparation of Peach petal protoplasts and the expression of exogenous target genes.

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Abstract

The present invention relates to a method for expressing proteins by using plant petal cell protoplast. The method comprises: (1) adopting plant petals as a material, and carrying out enzymolysis to obtain petal cell protoplast; (2) standing the petal cell protoplast for 20-40 min at a temperature of 2-8 DEG C; and (3) transforming plasmid into the protoplast by using a PEG and Ca<2+>-mediated method, and expressing the target protein. Compared with the method in the prior art, the method of the present invention has the following beneficial effects that: the target protein can be expressed in a short time, the protoplast protein expression system can be used for research on target protein subcellular positioning and protein molecule interaction, the activity of the protoplast is normal, and the target protein expression detection results show that the method can be used for research on gene transcription level, protein expression level and substance metabolism level change in living cells and is the rapid and convenient target gene function research method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for expressing protein with plant petal cell protoplasts and its application. Background technique [0002] Genes in organisms mostly perform their functions by encoding proteins. The study of plant functional genes ultimately comes down to the study of protein functions. At present, the research and functional verification of genes are mostly done by transgenic to model plants (such as Arabidopsis, tobacco, etc.). Due to the differences between woody plants and herbaceous plants, as well as the differences between species, the phenotypes exhibited by transgenic model plants may not necessarily reflect the true function of the target gene in other plants. Taking Meihua as an example, there is currently no mature genetic transformation system for plum blossoms, and it is impossible to verify the function of genes by obtaining transgenic plum blossom lines. Moreover, even if...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 包菲张启翔程堂仁潘会堂王佳孙丽丹马开峰杨炜茹周育真
Owner BEIJING FORESTRY UNIVERSITY
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