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Cloning and function analysis of Zea mays adverse stress inducible promoter

A promoter sequence, corn technology, applied in the field of bioengineering, can solve the problem of few inducible promoters, and achieve the effect of strong comprehensive resistance

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, there are still few inducible promoters that can be used in transgenic research

Method used

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  • Cloning and function analysis of Zea mays adverse stress inducible promoter
  • Cloning and function analysis of Zea mays adverse stress inducible promoter
  • Cloning and function analysis of Zea mays adverse stress inducible promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]Embodiment 1: Cloning of the adversity stress inducible promoter of maize anion peroxidase gene (ZmapH)

[0036] The promoter of maize anion peroxidase gene (ZmapH) (promoter sequence comprises the DNA nucleotide sequence of -1bp to-1150bp region relative to the transcription initiation site of SEQ ID NO:1), in maize anion peroxidase The oxidase gene (ZmapH) was identified in the sequence of the 5' untranslated region.

[0037] The maize anion peroxidase gene (ZmapH) is registered in NCBI GenBank (accession number: AF037033.1), and the sequence listing shows the DNA sequence of the plant adversity stress-inducible promoter and 5' untranslated region of the above-mentioned gene of the present invention. exist figure 1 In , the start codon ATG for protein synthesis is underlined, and the base A of the transcription start site is shown with +1. And use the promoter analysis website to analyze the core elements of the promoter. Promoter analysis website http: / / www.dna.aff...

Embodiment 2

[0042] Embodiment 2: Construction of plant adversity stress inducible vector

[0043] The promoter of the maize anion peroxidase gene (ZmapH) cloned in Example 1 and the 5' untranslated region ZmapH Pro (see the sequence listing) of 543 bp were inserted into the vector to construct a plant stress-inducible vector.

[0044] Specifically, the plant expression vector pCAMBIA1301 and the recombinant plasmid pMD 18-T::ZmapH Pro were respectively digested with BamHI and NcoI, and then inserted into the BamHI and NcoI restriction sites of the vector pCAMBIA1301. The vector is called pCAMBIA 1301::ZmapH Pro, and it is used to drive the expression of GUS gene, which was identified by enzyme digestion ( Figure 5 ) obtained the promoter fragment, which was the same as the expected result.

[0045] exist Image 6 Among them, the gene GUS encoding β-glucuronidase was used as the reporter gene, and the selection marker was hygromycin resistance gene. In addition, 35s-pro represents the ...

Embodiment 3

[0046] Example 3: Identification of the activity of maize adversity stress-inducible promoters

[0047] By the heat shock transformation method, the vector pCAMBIA 1301::ZmapH Pro constructed in Example 2 is transferred to Agrobacterium tumefaciens EHA105, and the plasmid is extracted ( Figure 7 ) and PCR identification ( Figure 8 ).

[0048] In order to identify the stress-induced activity of the promoter, the method of Jefferson et al. (EMBO J, 1987) was used to test the activity of GUS after drought treatment, one of the stress treatments of mature embryos of maize.

[0049] More specifically, soak the corn seeds to accelerate germination, then cut the seeds in half longitudinally, induce culture with 20% PEG for 24 hours, put the corn seeds in the GUS detection solution at 37°C overnight, GUS detection solution: 1mg / 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 ferr...

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Abstract

The invention which belongs to the technical field of biological engineering provides a Zea mays adverse stress inducible promoter sequence. The adverse stress inducible promoter sequence is characterized in that: the promoter sequence comprises DNA nucleotide sequences relative to a zone from -1bp to -1150bp at the transcription start site of SEQ ID NO: 1. The invention also provides an adverse stress inducible plant expression vector for Zea mays transformation. The plant expression vector comprises the Zea mays adverse stress inducible promoter and a 5' untranslated region of ZmapH (Zea mays anionic peroxidase H); and PCR primers of SEQ ID NO:2 and SEQ ID NO:3 are suitable for the amplification of DNA segments containing the SEQ ID NO:1. The Zea mays adverse stress inducible promoter can be used to promote adverse stress resistant gene to efficiently express, and has a positive meaning to the research of the transgenic technology of the Zea mays stress tolerant species and the cultivation of the strong comprehensive resistance high quality high yield new transgenic species.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an inducible promoter sequence derived from a maize anion peroxidase gene (ZmapH) and capable of high-level expression in maize. Background technique [0002] The drought ecological crisis has seriously affected the sustainable development of human beings. Therefore, in agricultural production, there is an urgent need for food crops and vegetation that grow well under drought-tolerant and water-stressed environments. With the rapid development of modern genetic engineering technology, plant drought tolerance has shifted from traditional plant physiology research to molecular research. Crop molecular breeding technology makes it possible to finely adjust the effect of breeding by substituting the gene level for the original genome level. [0003] The regulation of gene expression in higher plants has become a hot spot in the field of molecular biology research, ...

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 潘洪玉陶冶张世宏刘金亮陈景源孙庚贾承国李桂华
Owner JILIN UNIV