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Transcriptional repression domain as well as encoding genes and application thereof

A technology that encodes genes and structural domains, applied in the field of bioengineering, can solve the problems of inability to survive and low activity of transgenic plants

Active Publication Date: 2018-11-02
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the transcriptional repressive activity of EAR is too strong, it may lead to the inability of some transgenic plants fused with SRDX. Therefore, it is urgent to discover other transcriptional repressive domains with similar functions but slightly lower activity.

Method used

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  • Transcriptional repression domain as well as encoding genes and application thereof
  • Transcriptional repression domain as well as encoding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Construction of a recombinant expression vector based on pCIMBIA1300-35S.

[0022] Extract the RNA of Arabidopsis Col-0, reverse transcribe it into cDNA as a template, and perform PCR amplification with the following primers:

[0023] TCP7F: 5'GCTCTAGAaccATGTCTATTAACAACAAC 3' (SEQ ID NO: 4);

[0024] TCP7-3R: 5'CGGGATCCTTAATCCGGAAGACGTGGATCTTCCTCTCTTC 3' (SEQ ID NO: 5);

[0025] TCP7-6R: 5'CGGGATCCTTACAAATTAAGATCCGGAAGACGTGGATCTTCCTCTCTTC 3' (SEQ ID NO: 6);

[0026] TCP7-9R: 5'CGGGATCCTTAAGAAGCAAGCAAATTAAGATCCGGAAGACGTGGATCTTCCTCTCTTC 3' (SEQ ID NO: 7);

[0027] TCP7-12R: 5'CGGGATCCTTAACCGGATAAAGAAGCAAGCAAATTAAGATCCGGAAGACGTGGATCTTCCTCTCTTC 3' (SEQ ID NO: 8);

[0028] TCP7-13R: 5'CGGGATCCTTAACCGGATAAAGAAGCAAGCAAATTAAGATGACCCGGAAGACGTGGATCTTCCTCTCTTC 3' (SEQ ID NO: 9);

[0029] Insert the fragments into the pCIMBIA1300-35S vector Xba I and Bam H I restriction site 35S:TCP7-3 / 6 / 9 / 12 / 13AA Six sets of recombinant expression vectors.

Embodiment 2

[0030] Example 2. Construction of transformants.

[0031] After the obtained six sets of recombinant expression vectors were sequenced correctly, the constructed plasmids were respectively transformed into Agrobacterium GV3101 to obtain transformants.

Embodiment 3

[0032] Embodiment 3, acquisition of transgenic plants.

[0033] Transgenes were introduced into Arabidopsis thaliana by flower dipping method (Clough and Bent, 1998). The specific steps of the soaking method are as follows:

[0034] Pick a single colony and inoculate it in 5 ml YEB (Rif 50 mg / L, Kana 100 mg / L) and culture overnight at 30°C. Inoculate at 1:100 in 300 ml YEB (Rif 50 mg / L, Kana 100 mg / L) and expand until OD600=1.0-1.2.

[0035] To collect the bacteria, collect the bacteria into a 500 ml sterilized centrifuge bottle, centrifuge at 4000 rpm at room temperature for 20 min.

[0036] Discard the supernatant. Resuspend the bacteria in the transformation medium (1 / 2MS, 5% sucrose, 0.5 g / L MES, 10 μg 6-BA, adjust the pH to 5.7 with KOH, ready-to-use, without sterilization), add an appropriate amount of transformation medium to OD600=0.8-0.85. Add 0.03% silwet L-77.

[0037] Remove the siliques and flowers from the plants. Compact the soil with 4-6 pots of plants r...

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Abstract

The invention discloses a transcriptional repression domain as well as encoding genes and application thereof in regulating stress tolerance of plants. The transcriptional repression domain comprisesamino acid sequences as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3. Since the transcriptional inhibitory activity of the existing SRDX is too strong, transgenic plants in which certain transcription factors are fused with the SRDX may not survive. By contrast, inhibitory sequences provided by the invention have transcriptional inhibitory activity, but the transcriptional inhibitory activity is lower than that of the SRDX; and the transcriptional repression domain is of great scientific research and practical value to explore the function of transcription factors and gene expression regulation.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a transcriptional repression domain sEAR and its coding gene and its application in regulating the expression of downstream target genes by transcription factors. Background technique [0002] Plants develop from a seed to a complete plant, and each growth stage is always accompanied by gene expression and silencing. The dicot Arabidopsis thaliana is an important model plant that can be used to explore the regulation of gene expression. The theoretical research on gene expression regulation provides an important theoretical basis for improving the yield, quality and stress resistance of crops. [0003] Transcription factors play an important role in the regulation of gene expression. Transcription factors are divided into DNA-binding domains and transcriptional activation or repression domains. At present, there are mainly four types of inhibitory domains known in Arabi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC12N15/8216C12N15/8261C07K14/415
Inventor 崔素娟赵红桃张国芳
Owner HEBEI NORMAL UNIV
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