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Root specific promoter and recombinant expression vector thereof

An expression vector and promoter technology, applied in the field of plant genetic engineering, can solve the problems of plant growth inhibition, unfavorable transformation of plants, plant dwarfing, etc., and achieve the effect of enhancing expression activity and increasing inducible expression ability

Inactive Publication Date: 2008-10-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some cases, this may have some adverse effects on the growth and development of plants, which is not conducive to the realization of people's purpose of transforming plants
For example, after using the CaMV 35S promoter to drive the DREB1A gene to transform Arabidopsis plants, the drought resistance and salt tolerance of the transgenic plants were greatly improved, but due to the excessive ectopic expression of the gene, the growth of the plants under normal conditions was severely inhibited. dwarf plants, short leaves

Method used

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  • Root specific promoter and recombinant expression vector thereof
  • Root specific promoter and recombinant expression vector thereof
  • Root specific promoter and recombinant expression vector thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, the acquisition of SP promoter sequence

[0026] Using 1-week-old Colombian ecotype wild-type Arabidopsis plants as experimental materials, the genomic DNA was extracted and used as a template, primers were designed from the published Arabidopsis genome database, and the SP promoter sequence was amplified by PCR. Introduce BamHI and EcoRI restriction sites at both ends of the primer respectively, and the primer sequence is as follows: Upstream: 5′-ACGGGATCCACCATTCACGGTAGGTTCTTTTAT-3′, BamH I restriction site is introduced; Downstream: 5′-ACGGAATTCTCTTGCGTTTACTGTTTTTGC-3′, EcoR I enzyme is introduced cut site.

[0027] Perform agarose gel electrophoresis detection on the PCR amplification product, cut off the target band to purify and recover, connect the digested product to the pGEM-T Easy vector and transform Escherichia coli DH10β competent cells, and pick it on the Amp resistance plate From the grown single clone, the plasmid was extracted, identified ...

Embodiment 2

[0028] Embodiment 2, the construction of the recombinant expression vector carrying SP promoter and GUS gene

[0029] Plasmid p-SP and plasmid pCAMBIA1391 (purchased from pCAMBIA Company) were digested with BamH I and EcoR I for 3 hours at 37°C, respectively, and the digested products were analyzed by 1% agarose gel electrophoresis, and the 845bp SP promoter was recovered The sequence and the 12kb plasmid pCAMBIA1391 digested large fragment were ligated overnight at 4°C with T4 DNA ligase. The ligation product was transformed into Escherichia coli DH10β competent cells, and the single clone grown on the Kan resistance plate was picked, the plasmid was extracted, and identified by PCR and enzyme digestion. The obtained recombinant expression vector is named pCAMBIA1391-SP, and the physical map of the recombinant expression vector pCAMBIA1391-SP is as follows figure 1 shown.

Embodiment 3

[0030] Embodiment 3, SP promoter drives the specific expression of GUS gene in Arabidopsis root

[0031] 1. Transformation of wild-type Arabidopsis with recombinant expression vector pCAMBIA1391-SP

[0032] The recombinant expression vector pCAMBIA1391-SP constructed in Example 2 was transformed into Agrobacterium tumefaciens GV3101 competent cells, and single colonies growing on Rif and Kanamycin (Kan) resistant plates were picked for PCR identification.

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Abstract

The invention discloses a specific promoter of a root and a recombinant expression vector. The promoter adopts DNA molecular: a of following a) or b) or c) DNA molecular: a) is a DNA molecular which is composed of the deoxyribonucleotide shown in the sequence 1 in the sequence table; b) is a DNA molecular hybridized with a) defined DNA sequence under the strict condition; and c) is a specific expression DNA molecular in the root of the plant and has over 90 percents of homology and the gene with a adjusting and controlling purpose with a) defined DNA sequence. The invention also discloses a recombinant expression vector with the promoter, and the recombinant expression vector can be applied for cultivating the transgenic plant with a specific expressing target gene in the plant root.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a root-specific promoter and a recombinant expression vector thereof. Background technique [0002] The expression of eukaryotic genes is regulated by pre-transcriptional, transcriptional, post-transcriptional processing, transport, translation, and protein activity. The regulation of transcriptional level is the most basic link in gene expression, and the promoter is necessary for gene expression. It determines the space, time and intensity of exogenous gene expression, and is an important limiting factor for people to use genetic engineering and transgenic technology to transform plants. [0003] The coding sequence in the genome of most eukaryotes only accounts for 1%-10% of its genomic DNA, while a large part of the non-coding sequence plays a role in gene expression regulation, and the promoter sequence occupies a large proportion of the non-coding sequence . Promo...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H1/00A01H5/00C12N15/11C12N15/113
Inventor 王学臣张秀清魏鹏程陈其军陈乃芝任飞
Owner CHINA AGRI UNIV
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