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Method for inducing protein interaction and gene expression by pesticide

A technology for inducing protein and gene expression, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., to achieve the effect of increasing usage

Pending Publication Date: 2021-08-10
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The invention provides a method for pesticides to induce protein interaction and induce gene expression, which solves the limitations of existing compounds that induce protein interaction as gene expression inducers, and uses mandipropamid to control protein interaction. Another space Positional variants, breaking through the limitations of existing methods on the relative position of proteins in space

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  • Method for inducing protein interaction and gene expression by pesticide
  • Method for inducing protein interaction and gene expression by pesticide
  • Method for inducing protein interaction and gene expression by pesticide

Examples

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Embodiment 1

[0047] A method for pesticide-induced protein interaction and gene expression, in which mandipropamid induces complementation of three fragments of green fluorescent protein, the steps are:

[0048] Step 1: Construct plasmid 2 based on plasmid 1, ABI-CP N-terminal fusion GFP10, PYR1 MANDI Carboxy-terminally fused to GFP11.

[0049] Step 2: Plasmid 3 was constructed to induce expression of GFP1-9 fragments in Escherichia coli controlled by T7 promoter_lacO to match the IPTG induction system of Escherichia coli BL21 (DE3) strain.

[0050] Step 3: Transform plasmids 2 and 3 into Escherichia coli BL21(DE3) strain.

[0051] Step 4, detection of the expression products of plasmids 2 and 3 induced by mandipropamid in Escherichia coli: take the positive clones of Escherichia coli BL21 (DE3) strain background and shake the bacteria, wait for OD 600 When it reaches 0.40-0.60, add IPTG (isopropylthiogalactopyranoside) with a final concentration of 1mM to the bacterial solution to induc...

Embodiment 2

[0054] Mandipropamid induces the expression of the gene encoding green fluorescent protein:

[0055] Step 1: Construction of Plasmid 4 to Combine Split T7 RNA Polymerase with PYR1 in E. coli MANDI Fusion expression with ABI-CP respectively;

[0056] Step 2: construct plasmid 5, use the T7 promoter to drive the expression of the GFP-encoded gene, and detect the activity of mandipropamid-induced T7 RNA polymerase in Escherichia coli;

[0057] Step 3: transform plasmids 4 and 5 into E. coli Top10 strain. Note that strains with endogenous T7 RNA polymerase cannot be used, such as BL21(DE3) strains;

[0058] Step 4: Detect that the expression products of plasmids 4 and 5 are induced by mandipropamid in E. coli: Take the positive clones of the background of E. coli Top10 strains and shake them until OD 600 When it reaches 0.40-0.60, add mandipropamid with a final concentration gradient of 0.1μM, 1μM, 10μM, 100μM, 200μM, 400μM to the bacterial solution, and use the following param...

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Abstract

The invention discloses a method for inducing protein interaction and gene expression by a pesticide, the pesticide is mandipropamid, the protein interaction is induced as green fluorescent protein three-fragment complementation, and the gene expression is induced as green fluorescent protein coding gene expression; the provided ABI-CP is a topological structure variant using mandipropamid to control protein interaction, and the limitation of the existing method on the relative position of the fusion protein space is broken through; according to the method for regulating and controlling the gene expression by using mandipropamid, the limitation of the existing compound for inducing the gene expression is broken through due to the characteristics of the pesticide compound. The limitations include high cost, incapability of being massively used in the environment, non-physiologically neutral (capable of being rapidly metabolized), difficulty in entering cells to take effect and the like.

Description

technical field [0001] The invention relates to the technical field of gene expression regulation, in particular to a method for inducing protein interaction and gene expression induced by pesticides. Background technique [0002] Protein-molecular interactions are the basis of various biological processes. Detecting and manipulating protein-molecular interactions is a common means to detect and control biological functions such as signal transduction in organisms. For example, if a compound molecule is used to induce the interaction of two protein molecules, the spatial position of the fluorescent protein fused with the two proteins will be drawn closer, and the presence or concentration of the compound can be detected by fluorescence resonance transfer. For another example, if two proteins are induced to interact by light, these two proteins can be constructed as fusion proteins with a pair of effector proteins, and light can be used to control the interaction and functio...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N15/65C12R1/19
CPCC12N15/70C12N15/65C07K14/415C07K2319/60Y02A50/30
Inventor 袁圆苗靳
Owner NANTONG UNIVERSITY
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