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Application of EIN2 protein in inhibiting gene expression

A technology for inhibiting genes and proteins, which is applied in the application field of EIN2 protein in inhibiting gene expression, and can solve the problem of not including protein functional domains.

Inactive Publication Date: 2015-12-23
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although evolutionary analysis shows that the C-terminus of EIN2 (EIN2CEND) is relatively conserved, bioinformatics analysis shows that this region does not contain any typical protein functional domains known so far (JiandGuo, 2013)

Method used

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  • Application of EIN2 protein in inhibiting gene expression
  • Application of EIN2 protein in inhibiting gene expression
  • Application of EIN2 protein in inhibiting gene expression

Examples

Experimental program
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Effect test

Embodiment 1EI

[0019] Example 1 EIN2 protein subcellular localization

[0020] The coding sequence of red fluorescent protein mRFP was fused with EIN2 gene, and the recombinant gene 35S::EIN2-mRFP was constructed by using 35S promoter. Then, the recombinant was transformed into the ethylene complete insensitivity mutant ein2-5 by using the method mediated by Agrobacterium to obtain the transgenic line 35S::EIN2-mRFP / ein2-5. Through resistance screening and phenotypic analysis, the 35S::EIN2-mRFP / ein2-5 strain with the same response to ethylene as wild-type Arabidopsis Col-0 was obtained, which indicates that the fusion protein EIN2-mRFP in this strain It has the same function as the plant endogenous protein EIN2. Using this transgenic material and by means of confocal laser fluorescence microscopy, the subcellular localization of the fusion protein EIN2-mRFP upon application of exogenous ethylene was analyzed. like figure 1 As shown in A, the transgenic line 35S::EIN2-mRFP / ein2-5 was grow...

Embodiment 2

[0023] Example 2 EIN2 inhibits the translation of EBF1 mRNA and activates ethylene response through 3' UTRT

[0024] Chinese patent ZL201110110101.0 has disclosed that EBF1mRNA 3'UTR, as a cis-acting element, can inhibit the translation process from mRNA to protein, and the 3'UTR plays a negative role in the translation process of EBF1mRNA. In terms of genetic relationship, EIN2 activates downstream ethylene signaling by negatively regulating the function of EBF1 / 2 (Anetal., 2010; Guo and Ecker, 2003). This example detects whether the EIN2 protein, which participates in the formation of the translation inhibition structure P-body, has the function of regulating translation.

[0025]Due to the lack of specific antibodies against endogenous EBF1 protein, this example recombines the MYC tag coding sequence with the EBF1 protein coding sequence (CodingSequence, referred to as CDS), so that commercial MYC tag antibodies can be used to detect MYC-EBF1 Protein level; construct a "lo...

Embodiment 3

[0028] Example 3 EIN2 interacts with EBF1mRNA3'UTR and EIN5 in vivo

[0029] Example 2 shows that EIN2 directly or indirectly acts on the 3'UTR of EBF1 mRNA to inhibit the translation of EBF1. This example detects whether EIN2 interacts with the 3'UTR of EBF1 mRNA in plants. First, RNAImmunoprecipitation (RNAIP) technology was used to detect whether EIN2 interacted with mRNA in M1C / EIN2ox and M1F / EIN2ox strains, image 3 A shows that RNA-IP detects the interaction between EIN2 and EBF1 mRNA. In the EIN2ox background, the transgenic lines overexpressing the recombinant genes M1C and M1F were grown on MS medium containing 10μMACC in the dark for 3 days; after that, the total nucleic acid-protein complexes of the cells were extracted and IP was performed using HA antibody reaction, followed by RNA extraction and RT-PCR detection of M1C and M1F. image 3 In A, EIN2ox expresses the recombinant protein fused with the full length of EIN2 and the HA tag, and TUB4 is the RT-PCR refer...

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Abstract

The invention discloses an application of EIN2 protein in inhibiting gene expression. Cytoplasm in EIN2 protein acts with 3'UTR of EBF1mRNA, and is positioned at P-body together with protein such as EIN5, so that the translation process of EBF1mRNA can be inhibited. Due to expression of EIN2 protein in plants, particularly high-class plants, the translation process of any gene with EBF1 3'UTR can be inhibited; only by combining a coding sequence of a target gene with EBF1 3'UTR, expression of the gene can be horizontally started or ended after transcription by expressing EIN2 under certain conditions.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of EIN2 protein in inhibiting gene expression. Background technique [0002] Loss of function of the EIN2 (Ethylene Insensitive2) gene (AT5G03280) in the model plant Arabidopsis leads to complete ethylene insensitivity. This is the only single gene deletion mutation found so far that results in complete ethylene insensitivity mutant, indicating that EIN2 protein is a positive regulatory component of ethylene response, and also indicates that EIN2 protein is a key component of ethylene signaling pathway (Alonso et al. , 1999; Jian and Guo, 2013). [0003] The Arabidopsis EIN2 protein has 1294 amino acids, and its N-terminal contains 12 transmembrane domains (1-461), which are responsible for localizing the protein to the endoplasmic reticulum membrane. At the same time, its C-terminus (462-1294) is a hydrophilic region. Although evolutionary analysis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/82A01H5/00
CPCC07K14/415C12N15/8216
Inventor 郭红卫李文阳
Owner PEKING UNIV
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