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Kit and detection method for indicating subcellular localization of plant nucleolus and cajal body

A technology for subcellular localization and indication of plants, which is applied in the field of kits for indicating the subcellular localization of plant nucleoli and Kehao bodies. Achieve the effects of strong specificity, simple result judgment, and damage reduction

Inactive Publication Date: 2017-05-31
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, researchers often use DAPI, namely 4',6-diamidino-2-phenylindole as a fluorescent fuel to locate cell nuclei. Under a fluorescent microscope, this fuel will make plant cell nuclei appear purple fluorescent, but it The nucleolus and other subcellular organelles of the nucleus cannot be specifically indicated. The dye is a toxic carcinogen, and there is also a certain degree of instability during the staining process

Method used

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  • Kit and detection method for indicating subcellular localization of plant nucleolus and cajal body
  • Kit and detection method for indicating subcellular localization of plant nucleolus and cajal body
  • Kit and detection method for indicating subcellular localization of plant nucleolus and cajal body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Equipment of plant nucleolus and Kehao body subcellular localization kit

[0026] 1) Bacterial solution preparation: take YFP-NbFib2, CFP-NbFib2 (according to the sequence of NbFib2 reported in GenBank (GenBank accession number: AM269909.1)), YFP-P1 (self-prepared), CFP-P2 (self-prepared) 10 μL in 3 mL LB solution;

[0027] 2) Preparation of suspension: Take 2.5 mL of solution 1 and solution 2, 5 μL of inducer into a 25 mL sterile tube, add solution 3 to 25 mL;

Embodiment 2

[0028] Example 2: Detection method of the kit for subcellular localization of plant nucleolus and Kehao body

[0029] 1) Resuscitate the LB bacteria solution containing YFP-NbFib2 or CFP-NbFib2 in a shaker (28°C, 200 rpm) to OD 600 =0.6-0.8, get resuscitation fluid;

[0030] 2) Take 1 mL each of the resuscitated resuscitation fluids into 1.5 mL centrifuge tubes, centrifuge at 10,000 rpm for 1 min, add 1 mL of suspension to suspend the sediment, centrifuge at 10,000 rpm for 1 min, and repeat once;

[0031] 3) Suspend the precipitate with 1 mL of suspension, and let it stand at room temperature for 2-3 hours at 20°C-25°C;

[0032] 4) Prick 1-2 pinholes on the back of Nicotiana benthamiana leaves with a disposable syringe needle, draw an appropriate amount of the sediment suspension in 3) with a syringe, and slowly inject along the needle holes on the back of the leaves until the liquid soaks into the leaves;

[0033] 5) After culturing Nicotiana benthamiana for 2-3 days in a ...

Embodiment 3

[0036] Example 3: The detection method of the kit for detecting whether other proteins are localized in nucleolus and Kehao body subcellular localization

[0037] 1) Resuscitate the LB bacteria solution containing YFP-NbFib2 and CFP-P2 in a shaker (28°C, 200 rpm) to OD 600 =0.6-0.8;

[0038] 2) Take 1 mL each of the resuscitated bacterial solution into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm for 1 min, add 1 mL of suspension to suspend the sediment, centrifuge at 10,000 rpm for 1 min, and repeat once;

[0039] 3) Suspend the precipitate with 1 mL of suspension and let it stand at room temperature (20°C-25°C) for 2-3h;

[0040] 4) Prick 1-2 pinholes on the back of Nicotiana benthamiana leaves with a disposable syringe needle, and mix YFP-NbFib2 and CFP-P2 1:1 (v / v) The needle holes on the back of the leaves are injected into the leaves slowly until the liquid soaks the leaves;

[0041] 5) After culturing Nicotiana benthamiana for 2-3 days in a dark room, observe und...

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Abstract

The invention provides a kit and a detection method for indicating subcellular localization of plant nucleolus and a cajal body. The kit comprises two fusion protein tobacco fiber proteins, which are fusion protein YFP-NbFib2 with yellow fluorescence and fusion protein CFP-NbFib2 with cyan fluorescence. The specificity of the kit adopted in the invention is higher, and the nucleolus and cajal body subcellular organelle on the cell nucleus can be specifically indicated; the operation is simpler; pre-observed protein can be injected simultaneously, so that the damage for plants is reduced; the operation is safer; cancerogenic substances such as poisonous substances are prevented from contacting in the process of using DAPI; the result judgment is simple; and the color of the fluorescence is observed just in a confocal state.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and a detection method for indicating the subcellular location of plant nucleolus and Kehao body. Background technique [0002] The occurrence of plant diseases is often caused by pathogens, and the interaction between these pathogens, whether they are viruses, bacteria or fungi and plants, has become the focus of research for many years. Plant organelles play a very important role in the growth and development of plant cells. Therefore, how the proteins encoded by pathogens use the proteins in plant organelles to inhibit plant growth has become a research hotspot in recent years. [0003] Among plant organelles, the nucleus contains most of the cell's genetic material and serves to maintain the integrity of genes and thereby influence cellular activity. The nucleolus is one of the important organelles in the nucleus. It is the control center of the system and regu...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/64C12Q1/02
CPCG01N33/68G01N21/6486G01N33/5097
Inventor 郑璐平张冬梅丁作美张成龙吴祖建
Owner FUJIAN AGRI & FORESTRY UNIV
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