Method for regulating cellular pathway by using plant hormone GA (Gibberellic Acid) and small molecular substance PAC (Paclobutrazol)

A technology of small molecular substances and plant hormones, applied in the field of biological method invention, can solve the problems of immune system damage and incompatibility

Inactive Publication Date: 2018-08-17
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 GA (Gibberellic Acid) and small molecular substance PAC (Paclobutrazol)
  • Method for regulating cellular pathway by using plant hormone GA (Gibberellic Acid) and small molecular substance PAC (Paclobutrazol)
  • Method for regulating cellular pathway by using plant hormone GA (Gibberellic Acid) and small molecular substance PAC (Paclobutrazol)

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Embodiment 1

[0037]A method for regulating cell pathways by utilizing the plant hormone GA and the small molecular substance PAC, comprising the following methods:

[0038]

[0039] (1) Carrier design and construction:

[0040] First, using the primers described above, using the Arabidopsis thaliana cDNA and the HEK293T cell cDNA as templates, use pcr technology to amplify each gene fragment, and then use the corresponding primers to perform overlap PCR with two fragments as templates; The Arabidopsis DELLA protein GAI and the GA receptor GID1 were fused with the C-terminus of the key protein LRP6 in the Wnt signaling pathway, and the PCR products such as GAI, GID1, GAI-LRP6C, GID1-LRP6C were recovered by using agarose gel, and used in Dicer XbaI XmaI double-digest linearized pCI 4xmyc, pCI egfp and pCI mcherry vectors, use the Infusion method to connect, transform DH5a, colony PCR verification, pick clones and send them for sequencing, and ensure correct sequencing; use the above opera...

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Abstract

The invention provides a method for regulating a cellular pathway by using plant hormone GA (Gibberellic Acid) and a small molecular substance PAC (Paclobutrazol), and aims to realize controlled opening and closing of a signal pathway in human cells. The method comprises the following steps: constructing GA receptor proteins GID1 and GAI in Arabidopsis thaliana on a human cell expression vector; then transferring into HEK293T cells together; adding GA to induce interaction of the two proteins; adding an inhibitor PAC, so that the interaction degree is weakened. In the way, key proteins on a human cell signal pathway and the two receptor proteins are subjected to fusion expression, and the two receptor proteins undergo interaction under the induction of the GA, so that the fused signal pathway is also induced by the GA. A key protein LRP6 on a human Wnt pathway is selected. After fusion expression of GA receptor and LRP6, LRP6 aggregates under the induction of the GA, thereby promotingthe production of beta-catenin and opening up an intracellular signal pathway. After the inhibitor PAC 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 GA and small molecular substance PAC. Background technique [0002] Optogenetics is a new technology that combines light control technology with genetics for cell biology research, that is, expresses light-sensitive ion channel proteins on excitable target cells or organs, and activates them with light of corresponding wavelengths. Light-sensitive channels are used to fine-tune the physiological functions of cells, tissues, organs and animals. So far, many photoreceptors have been improved and used 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C12N2840/002
Inventor 左泽乘张峻川莫伟亮张力肖勇石翔张宝昌张文礼李苏迪
Owner JILIN UNIV
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