Application and method of bri1 in plant immune signal verification
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A verification method, plant technology, applied in the application field of plant immune signal verification, can solve the problems of limiting multi-antigen recombinant proteins, interference, and difficulty in accurately determining whether immune components have elicitor activity
Active Publication Date: 2022-02-15
SICHUAN AGRI UNIV +1
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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
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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.
[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 field of biotechnology, and relates to the application and method of BRI1 in the verification of plant immune signals. In the present invention, the plant development-related regulatory gene BRI1 is applied to the verification of plant immune signals for the first time. The recombinant protein was constructed by recombining the immune function receptor protein and the receptor protein BRI1; the recombinant protein can specifically bind to exogenous PAMPs molecules and activate the expression of Arabidopsis development-related reporter genes. To measure the immune stimulating activity of PAMPs molecules. The method of the invention breaks the boundaries of plant immunity and development research, and realizes the quantitative analysis of the activation ability of a single immune signal to its receptor protein under the condition of the presence of multiple elicitors by examining the brassinosteroid-related signals, which can effectively The verification of the effectiveness of different immune components in the multi-antigen recombinant immune protein provides a new technical means for the study of the mechanism of action of the recombinant immune protein and botany research.
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...
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