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Polypeptide fragment capable of activating saccharomycete and plant cell gene expression and verification method of polypeptide fragment

A technology of polypeptide fragments and plant cells, applied in the field of genetic engineering

Active Publication Date: 2015-12-30
甘肃中科博瑞生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no reports of polypeptide fragments in AtDPBF4 / EEL that can activate gene expression at home and abroad

Method used

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  • Polypeptide fragment capable of activating saccharomycete and plant cell gene expression and verification method of polypeptide fragment
  • Polypeptide fragment capable of activating saccharomycete and plant cell gene expression and verification method of polypeptide fragment
  • Polypeptide fragment capable of activating saccharomycete and plant cell gene expression and verification method of polypeptide fragment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Analysis of AtDPBF4 / EEL deletion mutation based on yeast one-hybrid system

[0064] (1) Construction of recombinant plasmids

[0065] According to the Arabidopsis AtDPBF4 / EEL gene information included in GenBank, a pair of specific primers were designed and synthesized for the cloning of D1:

[0066] D1 upstream primer: CGGAATTCCACTTAGGAAGTTCTGGAAAACCAC

[0067] D1 downstream primer: CGGGATCCAGGCGCTTGTGGTGTATCCGATAATC

[0068] According to the Arabidopsis AtDPBF4 / EEL gene information included in GenBank, a pair of specific primers were designed and synthesized for the cloning of D2:

[0069] D2 upstream primer: CGGAATTCCTTGTTCGTCAGGGAAGCTTGACGTTAC

[0070] D2 downstream primer: CGGGATCCAGGCGCTTGTGGTGTATCCGATAATC

[0071] According to the Arabidopsis AtDPBF4 / EEL gene information included in GenBank, a pair of specific primers were designed and synthesized for the cloning of D3:

[0072] D3 upstream primer: CGGAATTCACTACTACTACTCATAAGCAGCCTAC

[0073] D3 do...

Embodiment 2

[0106] Example 2 Transient expression analysis of Arabidopsis mesophyll protoplasts

[0107] (1) Recombinant plasmid construction

[0108] According to the results of the AtDPBF4 / EEL deletion mutation analysis in the yeast system, the DNA fragments covering each other at the 3' ends were synthesized, and the complete AD1 coding sequence was synthesized by chain extension, and the complete AD2 coding was amplified in vitro by PCR. The region sequence, extension product, amplification product and transient expression analysis effector vector pHQEff were digested by EcoRI and BamHI respectively, and the linearized vector, PCR product and extension product were ligated overnight for 16 with T4 DNA ligase, and then the ligation product was Transformed into Escherichia coli (Escherichiacoli) DH5α strain, cultured overnight at 37°C. The next day, positive colonies were selected for culture and recombinant plasmids were prepared. Using empty plasmid and recombinant plasmid as templa...

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Abstract

The invention provides a polypeptide fragment capable of activating saccharomycete and plant cell gene expression and a verification method of the polypeptide fragment. In a saccharomycete one-hybrid system, a bidirectional deletion mutantion analysis is performed on arabidopsis AtDPBF4 / EEL, and two polypeptide fragments AD1 and AD2 capable of activating genetic transcription in saccharomycete cells are discovered; a coding area sequence of AD1 and AD2 is synthesized in vitro and connected to an effect carrier pHQEff for transient expression analysis of an arabidopsis protoplast, and recombinant plasmids are respectively named as pHQEff-AD1 and pHQEff-AD2; the transient expression analysis result of the arabidopsis protoplast shows that AD1 and AD2 can both activate the expression of a reporter gene (GUS). The discovery of the polypeptide fragments AD1 and AD2 lays a foundation for further building drought-resistant and saline-alkaline tolerant transgenic plants, and has an important guidance meaning on cultivation and improvement of gene engineering plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a polypeptide fragment capable of activating gene expression of yeast and plant cells and a verification method. Background technique [0002] AtDPBF4 / EEL gene encodes a basic leucine zipper (b-ZIP) transcription factor, and its expression product plays a role in gene expression regulation in late embryonic development of Arabidopsis seeds, seed germination and abscisic acid (ABA) signal transduction play an important regulatory role. Like other typical transcription factors, AtDPBF4 / EEL also contains two important functional domains: DNA binding domain (DBD) and transcription activation domain (TAD), the former binds to the upstream activation sequence (UAS) of the gene promoter region, The latter binds to certain regions in a protein complex called Mediator, which also simultaneously interacts with the RNA polymerase II transcription machinery, these pr...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/82
CPCC07K14/415C12N15/8206
Inventor 马建忠肖成斌马燕林王永刚张伟杰
Owner 甘肃中科博瑞生物工程有限公司
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