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Corn stress-inducible promoter and activity analysis

An inducible, promoter sequence technology, applied in the field of bioengineering, can solve problems such as plant growth retardation

Inactive Publication Date: 2013-02-13
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, overexpression of the DREB1A gene also resulted in plant growth retardation under normal growth conditions; in contrast, gene expression driven by the stress-inducible promoter rd29A not only made plants more stress tolerant but also had minimal effects on their growth. negative impact

Method used

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  • Corn stress-inducible promoter and activity analysis
  • Corn stress-inducible promoter and activity analysis
  • Corn stress-inducible promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Cloning of drought-inducible promoter of maize DRE binding protein gene (Zm DBF3)

[0037] The promoter of the maize DRE binding protein gene (Zm DBF3) (the promoter sequence contains the DNA nucleotide sequence of the region from -1bp to -1299bp relative to the transcription start site of SEQ ID NO:1), in the maize DRE binding protein The 5'untranslated region sequence of the gene (ZmDBF3) was identified.

[0038] The maize DRE binding protein gene (Zm DBF3) is registered in NCBI GenBank (accession number: NM_001112181.1). The sequence table shows the DNA sequence of the plant drought-inducible promoter and 5'untranslated region of the above gene of the present invention. in figure 1 In the case, the initiation codon ATG of protein synthesis is underlined, and the base A of the transcription initiation site is shown as +1. And use the promoter analysis website to analyze the core elements of the promoter. The promoter analysis website is http: / / bioinformatics.p...

Embodiment 2

[0042] Example 2: Construction of plant drought-inducible vector

[0043] The promoter of the corn DRE binding protein gene (Zm DBF3) cloned in Example 1 and the 148 bp 5'untranslated region Zm DBF3Pro (see sequence table) were inserted into the vector to construct a plant drought-inducible vector.

[0044] More specifically, the plant expression vector pCAMBIA1301 and the recombinant plasmid pMD 18-T::Zm DBF3Pro were double digested with EcoRI and NcoI respectively, and then they were inserted into the EcoRI and NcoI digestion sites of the vector pCAMBIA1301. The vector is called pCAMBIA 1301::Zm DBF3Pro, which is used to drive the expression of the GUS gene and was identified by restriction enzyme digestion ( image 3 ) A promoter fragment was obtained, which was the same as expected.

[0045] in Figure 4 Among them, the gene GUS encoding β-glucuronidase is used as the reporter gene, and the selection marker is the hygromycin resistance gene. In addition, 35s-pro represents the H...

Embodiment 3

[0046] Example 3: Identification of the activity of maize drought-inducible promoter

[0047] The vector pCAMBIA 1301::Zm DBF3Pro constructed in Example 2 was transferred to Agrobacterium tumefaciens EHA105 by the heat shock transformation method, and the plasmid was extracted and identified by restriction enzyme digestion.

[0048] In order to identify the drought-inducing activity of the promoter, the method of Jefferson et al. (EMBO J, 1987) was used to drought-treated maize embryos infected by Agrobacterium and then tested the activity of GUS.

[0049] More specifically, the corn seeds are soaked to accelerate germination, then the seeds are cut longitudinally, cut in half, and cultured with 20% PEG for 24 hours. The corn seeds are placed in the GUS test solution at 37°C overnight, GUS test solution: 3mg / ml X-gluc (5-bromo-4-chloro-3-indole-β-D-glucuronide), 50mM sodium phosphate buffer solution (PH=7.0), 10mM EDTA, 0.5mM potassium ferricyanide, 0.5mM sub Potassium ferricyanide,...

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Abstract

A corn stress-inducible promoter and activity analysis belong to the technical field of bioengineering. The invention provides an obtained corn stress-inducible promoter sequence, a stress-inducible plant expression vector for corn transformation and genetically modified plants transformed by plant expression vector; the corn stress-inducible promoter sequence contains a DNA (deoxyribonucleic acid) nucleotide sequence from the -1bp region to the -1299bp region relative to the transcription start site of SEQ ID NO: 1; the stress-inducible expression vector contains the corn stress-inducible promoter sequence and a 5' untranslated region of a corn DRE-binding protein gene; the genetically modified plants transformed by the plant expression vector contain the corn stress-inducible promoter sequence and the 5' untranslated region of the corn DRE-binding protein gene; and PCR (polymerase chain reaction) primers of SEQ ID NO: 2 and SEQ ID NO: 3 are suitable for amplifying the sequence DNA segment containing the SEQ ID NO: 1. The corn stress-inducible promoter can be used for promoting the high-efficiency expression of drought-resistant genes and in drought-resistant genetically modified plants, and has a positive significance in solving grain crisis in arid regions and increasing grain yield.

Description

Technical field [0001] The present invention belongs to the technical field of bioengineering, and specifically relates to a drought-inducible promoter sequence derived from maize drought DRE-binding protein 3 gene and highly expressed in plants, and also relates to a plant drought-inducible expression vector containing the promoter sequence, And use the promoter to produce transgenic plants. Background technique [0002] Adversity stress is one of the most important factors and challenges affecting grain yield in agricultural production. Factors such as soil degradation, lack of fresh water, variability in climate (elephant) and other factors have led to the deduction of zero-yield barren, saline-alkali, and tidal flats (above 1.5 billion mu), and crops directly caused by adversity stresses such as salinity, drought and low temperature each year The reduction in production still exceeds about 20% of the total output. Among these stress factors that seriously affect crop produc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11A01H5/00
Inventor 潘洪玉王凤婷刘金亮张世宏贾承国李桂华
Owner JILIN UNIV
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