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Application and method of BRI1 in plant immune signal verification

A verification method and plant technology, applied in the field of plant immune signal verification, can solve the problems of restricting multi-antigen recombinant proteins, interference, and difficulty in accurately determining whether an immune element has elicitor activity.

Active Publication Date: 2020-12-15
SICHUAN AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that in the process of multi-antigen recombinant protein verification, interference between different immune components often occurs, and it is difficult to accurately determine whether a certain immune component has elicitor activity
This greatly limits the in-depth study of the mechanism of multi-antigen recombinant proteins in plant immunity

Method used

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  • Application and method of BRI1 in plant immune signal verification
  • Application and method of BRI1 in plant immune signal verification
  • Application and method of BRI1 in plant immune signal verification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 molecular cloning experiment

[0042] 1. PCR amplification of BRI1KD

[0043] Using the Arabidopsis genomic DNA preserved in our laboratory as a template, according to the characteristics of the carrier HBT-GFP, the restriction endonuclease KpnI / SpeI was selected, and the primers for BRI1KD cloning were designed as shown in Table 1, and the reaction system was shown in Table 2. The PCR amplification reaction conditions are shown in Table 3, and the final product was stored at 4°C.

[0044] Table 1 PCR reaction primer information

[0045]

[0046] Table 2 PCR reaction system

[0047]

[0048]

[0049] Table 3 PCR amplification reaction conditions

[0050]

[0051] The PCR product was purified, and the PCR product was purified using a kit from Beijing Tiangen Co., Ltd.

[0052] 2.T 4 DNA ligase ligation reaction

[0053] The connection system is shown in Table 4, and the reaction steps are as follows:

[0054] (1) Add the reaction reagents ...

Embodiment 2

[0091] Embodiment 2 protoplast transformation experiment

[0092] 1. Preparation of Protoplasts from Arabidopsis Mesophyll Cells

[0093] The specific operation of Arabidopsis mesophyll cell protoplast preparation is as follows:

[0094] (1) Configure the protoplast enzymolysis solution, filter it with a 2.2 μm filter head and set it aside;

[0095] (2) Using col-0 wild-type Arabidopsis leaves cultivated for 4 weeks in an artificial climate as material, after removing the head and tail of the leaves, cut the leaves into 0.5-1mm filaments;

[0096] (3) rapidly immerse the blades of the cut filaments in the enzymolysis solution;

[0097] (4) Under the culture condition of 23°C, enzymatically hydrolyze the leaves for 4 hours in the dark;

[0098] (5) After the leaves are completely lysed, add an equal volume of W5 to the lysate, filter the enzymatic solution added with W5 into a 50 mL centrifuge tube with gauze, and centrifuge horizontally at 80 rcf / min for 2 min;

[0099] (6...

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Abstract

The invention belongs to the technical field of biology, and relates to application of BRI1 in plant immune signal verification and a method. The plant development related regulatory gene BRI1 is applied to plant immune signal verification for the first time. The immune function receptor protein and the receptor protein BRI1 are recombined to construct a recombinant protein; the recombinant protein can be specifically combined with exogenous PAMPs molecules and activate the expression of an arabidopsis thaliana development related reporter gene, and the immunostimulatory activity of the PAMPsmolecules to be detected is determined through the expression quantity of the reporter gene. According to the method, the boundary of plant immunity and development research is broken through, quantitative analysis of the activation capacity of a single immune signal on receptor protein of brassinolide is achieved by detecting brassinolide related signals under the condition that multiple excitonsexist, effectiveness of different immune elements in the multi-antigen recombinant immune protein can be effectively verified. And a new technical means is provided for action mechanism research andbotanical research of recombinant immune protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application and method of BRI1 in the verification of plant immune signals. Background technique [0002] Plants do not have the acquired immune system and immune cells that animals have, but use their own innate immunity to resist the invasion of pathogenic bacteria. After the plant perceives the pathogenic bacteria invading the body, it will activate two immune defense systems: the first is pathogen-associated molecular pattern-triggered immunity (PAMP-triggered immunity, PTI), which mainly relies on molecular pattern recognition on the surface of the cell membrane Receptors (Pattern recognition receptors, PRRs) recognize pathogenic bacteria associated molecular patterns (Pathogen associated molecμLar pattern, PAMPs), after PTI is triggered, plants will strengthen the cell wall through callose deposition in order to prevent pathogen invasion; the second For effector-triggered im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C07K19/00A01H5/00A01H6/20C12N1/21C12N5/10C12R1/19
CPCC12N15/8214C07K14/415C12N9/12C07K2319/03C07K2319/60
Inventor 蔡易李琦郭晋雅李君浩黄燕张怀渝唐自忠王浩锋
Owner SICHUAN AGRI UNIV
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