Efficient protein subcellular localization detection method based on citrus protoplasts
A protoplast and protein technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, biological testing, etc., can solve the problems of low success rate, high cost, time-consuming and laborious, etc., and achieve the goal of improving the success rate and saving costs Effect
Active Publication Date: 2020-01-17
HUAZHONG AGRI UNIV
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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.
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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 Labor...
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The invention discloses an efficient protein subcellular localization detection method based on citrus protoplasts. The method includes the steps: (1) converting organelle fluorescent protein labeledvectors into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens; (2) infecting citrus explants by the recombinant agrobacterium tumefaciens to obtain expression organelle fluorescent protein labeled citrus materials; (3) cloning genes to be detected to fluorescent labeled expression vectors with fluorescence different from that in the step (1) to obtain fluorescent labeled vectors for the genes to be detected; (4) performing enzymolysis on the citrus materials obtained in the step (2), and separating and purifying the citrus materials to obtain expression organelle fluorescent protein labeled citrus protoplasts; (5) culturing the citrus protoplasts obtained by the step (4) of fluorescent labeled vector conversion of the genes to be detected for 24-48 hours by PEG (polyethylene glycol) mediating, observing the citrus protoplasts under a laser scanning confocal microscope and indicating that expression proteins of the genes to be detected are localized in an organelle if two fluorescent protein labels are overlapped. The method is simple, convenient, efficient and low in cost, and success rate is greatly increased.
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...
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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