Subcellular localization kit for infecting tobacco and application of subcellular localization kit

A subcellular localization and kit technology, applied in the field of agricultural biology, can solve the problems of low infection efficiency, obscure observation, and low expression of target genes, and achieve the effects of improving infection efficiency, easy observation, and enhancing expression.

Inactive Publication Date: 2018-03-13
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved: the present invention aims at the subcellular localization of tobacco, using Agrobacterium to infect tobacco, the host immune response is strong, resulting in low infection efficiency, and the expression of the target gene after infection is small, which is not obvious under confocal laser microscope observation Provide a subcellular localization kit for efficiently infecting tobacco, which can significantly improve the infection efficiency, enhance the expression of target genes, have strong fluorescence brightness, and the experimental operation is simple and safe

Method used

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  • Subcellular localization kit for infecting tobacco and application of subcellular localization kit
  • Subcellular localization kit for infecting tobacco and application of subcellular localization kit
  • Subcellular localization kit for infecting tobacco and application of subcellular localization kit

Examples

Experimental program
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Effect test

Embodiment 1

[0048] 1. Under ultra-clean environmental conditions, absorb 100 μL of bacterial solution A4 transfected with the target gene ICE1, and add it to 6 mL of LB liquid medium (containing 50 μg / mL kanamycin). Aspirate 100 μL of the nuclear-localized A3 bacterial solution and 50 μL of the A2 bacterial solution that reduces the plant immune response, and add them to 6 mL and 3 mL LB liquid medium (containing 50 μg / mL rifampicin) respectively. 3 tubes of bacterial solution were shaken overnight at 28°C and 250rpm (after overnight shaking, measure the OD value of the bacterial solution under ultra-clean environment conditions, at this time the OD value should be greater than 1.0, if the OD value of the bacterial solution is less than 1.0, continue to shake bacteria until the OD value reaches above 1.0).

[0049] 2. Centrifuge the overnight cultured bacterial solution at 5,000rpm (~5,580×g) for 15min, remove the supernatant, collect the precipitate, add the same volume of medium A1 dilu...

Embodiment 2

[0057] In this example, a subcellular localization experiment was carried out using conventional experimental methods.

[0058] 1. Shake the successfully detected Agrobacterium solution overnight at 28°C and 200rpm;

[0059] 2. Take 1-1.5mL bacterial liquid and add it to a sterilized 1.5mL centrifuge tube;

[0060] 3. 1000g / 10min, precipitate the bacteria (at room temperature), remove the supernatant, add 1mL permeate, and suspend the bacteria;

[0061] 4. Repeat step 3 to further remove a small amount of antibiotics;

[0062] 5. Take a small amount of suspended bacteria and dilute 10 times, measure the OD600 value, and multiply by 10, as the OD600 value of the suspended bacteria;

[0063] Note: If the OD600 value of the suspension is between 1.5-2.0, it means that there are a large number of dead cells, which will reduce the efficiency of transformation

[0064] 6. Determine the titer of the suspended bacterial liquid to the permeate, and calculate the dilution factor so t...

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Abstract

The invention provides a subcellular localization kit for infecting tobacco and application of the subcellular localization kit. The subcellular localization kit comprises a buffer reagent A1, a reaction bacterial liquid A2 for reducing plant immunity, nuclear localization agrobacterium liquid A3 and agrobacterium competence A4. According to the kit, autoimmune response of plants is suppressed byusing plant resistance response profilin, the infection efficiency of agrobacterium is improved, and the expression of an exogenous gene is enhanced, therefore, a fluorescence signal is relatively strong and is observed more easily. Moreover, the kit contains nuclear localization control components, so that contact with toxic and cancerogenic substances in the process of using DAPI (Diamidino-Phenyl-Indole) nuclear staining is avoided; meanwhile, the test cost is reduced, and environmental pollution is reduced.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology inventions, and specifically relates to a subcellular localization kit for infecting tobacco and its application. Background technique [0002] In the long-term struggle with exogenous microorganisms, plants have evolved different defense mechanisms to resist the attack of exogenous microorganisms. Plants rely on receptor kinases including FLS2 and EFR to sense bacterial invasion and activate immune responses, including oxygen burst, MAPK cascade, callose deposition, defense response gene expression, etc., to resist pathogen invasion. [0003] When Agrobacterium infects, it will cause the host's own pathogen resistance response, and this pathogen resistance response will be an important factor affecting the transformation of Agrobacterium. Once the endogenous immune response is too strong, the transformation efficiency will be greatly reduced, and the external The expression level of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/64
CPCG01N21/6486G01N33/5097
Inventor 刘同坤任海波陈丽李林张昌伟李英侯喜林
Owner NANJING AGRICULTURAL UNIVERSITY
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