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A highly efficient method for detecting protein subcellular localization based on citrus protoplasts

A protoplast, protein technology, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., can solve the problems of low success rate, high cost, complicated process, etc. Effect

Active Publication Date: 2021-08-31
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the traditional method for detecting the subcellular localization of citrus proteins is complicated, has a low success rate, is time-consuming and laborious, and is expensive.

Method used

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  • A highly efficient method for detecting protein subcellular localization based on citrus protoplasts
  • A highly efficient method for detecting protein subcellular localization based on citrus protoplasts
  • A highly efficient method for detecting protein subcellular localization based on citrus protoplasts

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Embodiment 1

[0032] This example relates to the stable genetic transformation of citrus callus and the protoplast transformation of citrus callus. The callus stable genetic transformation vector is a fusion vector carrying mitochondrial-localized red fluorescent protein RFP (Red fluorescent protein) (the mitochondrial localization tag is fused with red fluorescent protein, and the expression is driven by the same promoter), and the vector map is detailed in figure 1, the carrier resistance is herbicide Basta resistance, denoted as mt-rb (Nelson B K, Cai X, Andreas Nebenführ. A multi-color set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants[J]. The Plant Journal , 2007, 51(6):1126-1136.). The cloned coxII expression product of the gene to be tested is located on the mitochondria. The green fluorescent marker GFP (Green fluorescent protein) expression subcellular localization vector pM999-GFP used comes from the rice research team of the State Key Labo...

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Abstract

The invention discloses a method for efficiently detecting protein subcellular localization based on citrus protoplasts, which comprises the following steps: (1) transforming the organelle fluorescent protein marker carrier into Agrobacterium to obtain recombinant Agrobacterium; Implant, obtain the citrus material that expresses organelle fluorescent protein marker; (3) clone the gene to be tested into the fluorescent marker expression vector whose fluorescence is different from step (1), obtain the fluorescent marker carrier of the gene to be tested; (4) enzymolysis step ( 2) The obtained citrus material is separated and purified to obtain citrus protoplasts expressing organelle fluorescent protein markers; (5) mediated by PEG, the citrus protoplasts obtained in step (4) are transformed into fluorescent marker vectors of the genes to be tested, and cultured for 24- After 48 hours, observed under a laser confocal microscope, the coincidence of the two fluorescent protein markers indicated that the expression protein of the gene to be tested was located in the organelle. The method of the invention is simple, convenient, efficient, low in cost, and the success rate is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently detecting protein subcellular localization based on citrus protoplasts. Background technique [0002] Citrus is the largest fruit tree in the world and the most important fruit tree in southern my country. Due to the high heterozygosity of the citrus genome, the complex genetic background, and the characteristics of long childhood and multiple embryos, the progress of citrus gene function research has been hindered. With the development of genomics and the advancement of sequencing technology, multiple citrus genomes have been sequenced, and more and more citrus genes have been identified. Protein is the product of gene expression. The structure and function of protein are closely related to its subcellular location. Protein must be in a specific position to exert gene function. Therefore, detection of protein subcellular localization is an essential step i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02G01N33/68G01N21/64C12N15/84
CPCC12N15/8212G01N21/6486G01N33/5097G01N33/68
Inventor 伍小萌杨雯惠刘丹王鹏蔚郭文武
Owner HUAZHONG AGRI UNIV
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