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Method for regulating cellular pathway by using plant hormone ABA (Abscisic Acid) and small molecular substance PYR (Pyrabactin)

A technology of small molecular substances and plant hormones, applied in the field of biological method inventions, can solve problems such as inappropriateness and immune system damage, and achieve the effect of reducing damage and low cost

Inactive Publication Date: 2018-08-17
JILIN UNIV
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Problems solved by technology

However, both this method and optogenetics have certain limitations
Rapamycin is an immunosuppressor, which has certain damage to the immune system; the optogenetics system is not suitable for the regulation of intracellular light signaling pathways

Method used

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  • Method for regulating cellular pathway by using plant hormone ABA (Abscisic Acid) and small molecular substance PYR (Pyrabactin)
  • Method for regulating cellular pathway by using plant hormone ABA (Abscisic Acid) and small molecular substance PYR (Pyrabactin)
  • Method for regulating cellular pathway by using plant hormone ABA (Abscisic Acid) and small molecular substance PYR (Pyrabactin)

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Embodiment 1

[0040] A method for regulating the Wnt signaling pathway in human HEK293T cells by using the plant hormone ABA and the small molecular substance PYR, comprising the following methods:

[0041] (1) Carrier design and construction:

[0042] First, using the primers described in claim 1, using Arabidopsis thaliana cDNA and HEK293T cell cDNA as templates, using pcr technology to amplify each gene fragment, and then using the corresponding primers, using two fragments as templates, carry out overlap PCR; At this time, ABI1 and PYR1 were fused together with the C-terminus of LRP6, a key protein in the Wnt signaling pathway, and PCR products such as ABI1, PYR1, ABI1-LRP6C, and PYL1-LRP6C were recovered using agarose gel, and the endonuclease XbaI XmaI double Digest linearized pCIi 4xmyc, pCI egfp, and pCI mcherry vectors, use Infusion method to connect, transform DH5a, colony PCR verification, pick clones and send them for sequencing, and ensure the correct sequencing; using the ab...

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Abstract

The invention provides a method for regulating a cellular pathway by using plant hormone ABA (Abscisic Acid) and a small molecular substance PYR (Pyrabactin). The method comprises the following steps:constructing ABA receptor proteins ABI1 and PYR1 in Arabidopsis thaliana on a human cell expression vector, then transferring into HEK293T cells respectively, adding ABA in order that the two receptor proteins undergo induced interaction, and adding a PYR inhibitor in order that the interaction degree is weakened; fusing other human cell signal pathway key proteins with the two receptor proteins,and making the two receptor proteins undergo interaction under the induction of the ABA, so that a fused signal pathway also undergoes ABA induced aggregation. According to the method provided by theinvention, a key protein LRP6 on a human Wnt pathway is selected. After fusion expression of an ABA receptor and LRP6, LRP6 aggregates under the induction of ABA, thereby promoting the production ofbeta-catenin and opening up an intracellular signal pathway. After the PYR inhibitor is added, the production amount of the beta-catenin is reduced, and the cellular pathway is closed.

Description

technical field [0001] The invention relates to the field of biological method inventions, specifically, a method for regulating cell pathways by using plant hormone ABA and small molecular substance PYR. Background technique [0002] Optogenetics (Optogenetics) mainly uses photoreceptors that can undergo multimerization after receiving light induction or photoreceptors that can undergo photospecific interactions with their interacting proteins, and then couple related signal transduction proteins to Regulates cell signal transduction. So far, many photoreceptors have been improved to fuse key proteins in signaling pathways to regulate and control many pathways in organisms, such as receptor tyrosine kinase Ras-MAPK signaling pathway, PI3K-Akt signaling pathway and Wnt signaling pathway. At the gene level, the researchers used elements such as CRY2-CIB1, FKF1-GI, UVR8-COP1, and LOV proteins to regulate the transcription and expression of specific genes in mammalian cells. ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C12N2840/002
Inventor 左泽乘张力莫伟亮张峻川李苏迪石翔肖勇张文礼张宝昌
Owner JILIN UNIV
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