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Borage promoter and application thereof

A technology of promoter and borage, applied in the field of genetic engineering

Active Publication Date: 2020-03-17
INST OF AGRO FOOD SCI & TECH SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the current lack of effect of borage promoter on gene regulation, the present invention provides a borage promoter and its application

Method used

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  • Borage promoter and application thereof
  • Borage promoter and application thereof
  • Borage promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Promoter and cloning and element analysis

[0020] Healthy borage leaves were cut, ground in liquid nitrogen, and genomic DNA was extracted by CTAB method. A pair of primers for PCR amplification were designed and synthesized according to the sequence of the known Δ-6 fatty acid dehydrogenase gene promoter:

[0021] Forward primer: 5'-AACCGTCAATAATGATGAAAATTTTG-3';

[0022] Reverse primer: 5'-TGAATAAAATATAAGAGGGTAGACTTTAAG-3'.

[0023] The PCR reaction system was as follows: 4.0 μL of cDNA, 5.0 μL of 10×Pyrobest Buffer, 2 μL of 2 mmol / L dNTP, 0.5 μL of Pyrobest enzyme, 2.0 μL of 10 μmol upstream and downstream primers, and the final volume was 50 μL.

[0024] The PCR reaction program was: pre-denaturation at 94 °C for 7 min, followed by 30 cycles: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, after the cycle was completed, an extension reaction at 72 °C for 10 min, followed by incubation at 4 °C.

[0025] 1% agarose gel electrophoresis to detect PCR produ...

Embodiment 2

[0030] Example 2 Regulation of genes by promoters

[0031] 1. Vector Construction

[0032] Synthetic promoter-GUS gene, with an EcoRI restriction site added upstream and a Bg1II restriction site added downstream. The above gene was transformed into the pCAMBIA-1303 vector which also removed the 35S promoter by double enzyme digestion to obtain the pCAMBIA1303-proBoD6D vector. Subsequently, the pCAMBIA1303-proBoD6D positive vector was transfected into Agrobacterium tumefaciens EHA105. The following sequences are used as primers to detect positive transformants:

[0033] proBoD6D-FP (EcoRI):

[0034] 5'-GAATTCAACCGTCAATAATGATGAAAATTTTG-3';

[0035] proBoD6D-RP (Bg1II):

[0036] 5'-AGATCTTGAATAAAATATAAGAGGGTAGACTTTAAG-3'.

[0037] The PCR reaction system was as follows: 0.2 μL of plasmid, 5 μL of 10×Pyrobest Buffer, 2 μL of 2 mmol / LdNTP, 0.5 μL of Pyrobest enzyme, 2.0 μL of 10 μmol upstream and downstream primers, and the final volume was 50 μL.

[0038] The PCR reaction p...

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PUM

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Abstract

The invention belongs to the technical field of biology, provides a borage promoter, and provides a gene expression cassette, a vector, a receptor and a transgenic plant containing the borage promoter. The borage promoter can be used for transfecting target genes into a corresponding receptor and / or plant through the vector. The borage promoter provided by the invention can express various proteingenes such as enzymes and structural proteins in host plants.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a borage promoter and its application. Background technique [0002] A promoter is a DNA sequence for the positioning of RNA polymerase, usually located upstream of a gene. Once the RNA polymerase locates and binds to the promoter, the transcription process can be initiated. Therefore, the promoter is an important cis element for the regulation of gene expression. The essence of the promoter regulation mode is the trans element such as RNA polymerase and other protein cofactors. Interaction. Today, with the vigorous development of genetic engineering technology, to construct an expression vector capable of expressing heterologous proteins at a high level, in addition to the general requirements of cloning vectors, the most important thing is to carry out DNA fragments that can control foreign insertion. A DNA sequence for efficient transcription and translation, known as a promo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC07K14/415C12N15/8222
Inventor 孙金月郭溆王新坤刘超王青
Owner INST OF AGRO FOOD SCI & TECH SHANDONG ACAD OF AGRI SCI
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