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Method for rapidly locating protein position in guard cell of plant leaf

A technology of guard cells and plant leaves, applied in the field of protein localization in plant cells, can solve the problems of complexity and long cycle, and achieve the effect of simple operation, good repeatability and good labeling effect.

Inactive Publication Date: 2019-01-25
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the period of protein subcellular localization using tobacco leaves is relatively long, and it can only be observed after 48 hours after the transformation is completed, and the growth status of the leaves is required, which is relatively complicated.

Method used

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  • Method for rapidly locating protein position in guard cell of plant leaf
  • Method for rapidly locating protein position in guard cell of plant leaf
  • Method for rapidly locating protein position in guard cell of plant leaf

Examples

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no. 1 example

[0032] see figure 1 , the embodiment of the present invention provides a method for rapidly locating the position of a protein in a guard cell of a plant leaf, and the method is carried out according to the following steps:

[0033] 1. Construction of plant expression vector: using high-fidelity enzyme pfu series, design primers for the target gene according to the primer blast in NCBI, and use the cDNA obtained by extracting plant material RNA and inversion as a template, and using PCR technology to clone and sequence the target gene; After the expression vector and the gene fragment are digested and recovered with the same endonuclease, the gene fragment is connected to the expression vector pBI121-EGFP by ligase, and then the target gene expression vector plasmid is prepared; the target gene expression vector plasmid is in pBI121- A vector obtained by excising the GUS gene and replacing it with the EGFP gene on the basis of GUS, using CaMV 35S as the promoter, marking the t...

no. 2 example

[0040] see image 3 , Figure 4 and Figure 5 , another method for rapidly locating the position of proteins in guard cells of plant leaves provided by the embodiment of the present invention is different from the first embodiment in that the components of the washing solution are 7.2%-7.3% of mannitol and MES (2-morpholinoethanesulfonic acid) 0.04%-0.05%, pH adjusted to 5.7-5.8 with 1M KOH.

no. 3 example

[0042] see image 3 , Figure 4 and Figure 5 , another method for rapidly locating the position of proteins in guard cells of plant leaves provided in the embodiment of the present invention is different from the first and second embodiments in that the composition of the suspension is 0.08% MES by mass fraction -0.1%, Mannitol 7.2%-7.5% and MgCl 2 ·6H 2 O 0.3%-0.4%, adjust pH to 5.7-5.8 with 1 M KOH.

[0043] Wherein, the enzymatic hydrolysis solution is filtered through a 100-120 mesh sieve.

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Abstract

The invention relates to a method for rapidly locating the position of a protein in a guard cell of a plant leaf, belonging to the field of a method for locating the protein in a plant cell, in particular to the field of a method for locating the protein in a plant guard cell. The invention aims at solving the problems of long cycle and complex operation of protein subcellular localization by thetraditional method. The method for quickly locating the position of the protein in the guard cell of the plant leaf comprises the following steps: 1, construction of a plant expression vector; 2, separation of the guard cell of the plant leaf; 3, transformation of the guard cell; 4, incubation and culture of the guard cell; and 5, localization of the protein in the guard cell. The method providedby the invention is used for localization of proteins in plant guard cells.

Description

technical field [0001] The invention belongs to the field of protein localization methods in plant cells, in particular to the field of protein localization methods in plant guard cells. Background technique [0002] Green fluorescent protein (GFP) derived from jellyfish, its endogenous fluorophore can efficiently emit clearly visible green fluorescence when excited by ultraviolet or blue light (395nm and 479nm), which is an important tool for monitoring living cells and tissues. Ideal probes for gene expression and protein localization, it has been widely used as a reporter gene for in vivo cytological experimental studies. Most of the previous studies on the localization of plant proteins in cells used the transient transformation method of tobacco leaves. However, the use of tobacco leaves for protein subcellular localization requires a long period of time, which can be observed 48 hours after the transformation is completed, and the growth conditions of leaves are requi...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/82C12N5/04G01N21/64
CPCC12N5/04C12N15/66C12N15/82G01N21/6486
Inventor 荆艳萍卜芋芬潘成隋鑫张越
Owner BEIJING FORESTRY UNIVERSITY
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