Method for constructing double-hormone response promoter on basis of interaction relationship between transcription factors

A technology of transcription factors and promoters, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of unable to meet the requirements of pathogen-induced promoters, single signal, unable to change the mutual antagonism between hormones, etc. problem, to achieve the effect of changing the pathogenic induction characteristics, high activity and fast response

Inactive Publication Date: 2019-08-09
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing a dual-hormone-responsive promoter based on the interaction between transcription factors, so as to solve the problem that the existing promoter responds to a single induction signal and cannot change the mutual antagonism between hormones. However, the problem of being unable to meet the requirements of pathogen-inducible promoters for plant disease-resistant genetic engineering

Method used

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  • Method for constructing double-hormone response promoter on basis of interaction relationship between transcription factors
  • Method for constructing double-hormone response promoter on basis of interaction relationship between transcription factors
  • Method for constructing double-hormone response promoter on basis of interaction relationship between transcription factors

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Embodiment 1

[0044] Utilize pattern chart to further analyze the core content of the present invention

[0045] A method for constructing dual hormone-responsive promoters based on the interaction between transcription factors, such as figure 1 shown, including the following steps:

[0046] 1) Select a natural promoter pA as the basic backbone, which includes multiple cis-acting elements CS1, CS2, and CS3 induced in response to hormone A; selection includes cis-acting elements CS4 and CS4 induced in response to hormone B CS5 assembles into response element cassettes; the hormone A is not the same as the hormone B.

[0047] 2) According to the literature or database, find that the transcription factors bound by CS1, CS2, CS3, CS4 and CS5 are TF1, TF2, TF3, TF4 and TF5 respectively.

[0048] 3) Using Bimolecular Fluorescent Complementation (BiFC) and other technologies, respectively detect whether there is an interaction between transcription factors TF4 and TF5 and transcription factors ...

Embodiment 2

[0052] Interaction analysis between transcription factors

[0053] 2.1 Extraction of RNA from Arabidopsis plants

[0054] Use a scalpel to injure the roots of Colombian wild-type Arabidopsis plants, and then use the root irrigation inoculation method to water for 10 8 spores / mL of Verticillium dahliae V991 spore suspension, 20mL per plant, inoculated with Verticillium dahliae for 24 hours, using the leaves of Arabidopsis plants as materials, using the plant RNA rapid extraction kit (Aidlab), and operating the process according to the instructions Extraction of RNA from Arabidopsis plants. Then, using RNA as a template, a strand of cDNA was synthesized using TaKaRa's reverse transcription kit according to the operating instructions, and the coding sequence used for the synthesis of transcription factors was stored at -20°C.

[0055] 2.2 Acquisition of transcription factor coding sequences

[0056] PR-1a activation using the Plant Promoter Database via http: / / www.dna.affrc.go...

Embodiment 3

[0062] Example 3 Construction of pathogen-inducible promoter

[0063] 3.1 Construction of response component box

[0064] Typical cis-acting elements that respond to JA induction mainly include G-box and GCC-box. Combining cis-acting elements in the form of double copies, and there needs to be a certain distance between different cis-acting elements, so that the strength of the promoter can be improved without excessive expansion such as high background activity negative impact. To increase the strength of the JA-induced response, GCC-box and G-box were combined in double copies, respectively. due to tobacco NTPMT The G-box element in the promoter and the intermediate junction pair of the GCC motif NTPMT The induction of the promoter response to JA plays an important role, therefore, the JA response module is designed as follows: with a segment from tobacco NTPMT The middle junction sequence of the promoter G-box element and the GCC motif connects two doubled JA response...

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Abstract

The invention discloses a method for constructing a double-hormone response promoter on the basis of the interaction relationship between transcription factors. The method comprises the steps of selecting a natural promoter comprising a plurality of cis-acting elements induced by a response hormone A as a basic framework; selecting cis-acting elements induced by a response hormone B to form a response element box; determining the position of the response element box inserted into the natural promoter according to the interaction relationship between the transcription factor combined with the cis-acting elements in the response element box and the transcription factor combined with adjacent cis-acting elements inserted into the natural promoter in order to construct the double-hormone response promoter. The promoter obtained by means of the method can obtain signals from a plurality of signal paths or different transcription factor types, eliminate or weaken the influence of mutually antagonistic signals during regulation and control of gene expression, and synergistically improve the expression level of genes. During application in disease-resistant genetic engineering, the resistance of transgenic plants to pathogenic bacteria of different ecological types can be more effectively improved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for constructing a dual hormone-responsive promoter based on the interaction relationship between transcription factors. Background technique [0002] The promoter is an integral part of the gene (gene), which controls the initiation time of gene transcription and the degree of expression. Promoters are like "switches" that determine the activity of genes. The promoter itself does not control gene activity, but controls gene activity by combining with transcription factor (TF). Promoters commonly used in genetic engineering include three types: constitutive promoters, tissue-specific promoters, and inducible promoters. Genes regulated by constitutive promoters can be expressed in most tissues of transgenic plants, and the expression of genes regulated by them is continuous, the expression of RNA and protein is relatively constant, there is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/113C12N15/82A01H5/00A01H6/82A01H6/60
CPCC12N15/10C12N15/113C12N15/8222C12N15/8282C12N2310/10
Inventor 李先碧裴炎范艳华牛国清肖月华侯磊金丹童胜林道尧
Owner SOUTHWEST UNIVERSITY
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