Cloning and function analysis of Zea mays adverse stress inducible promoter

An adversity stress and inducible technology, applied in the field of inducible promoter sequences, can solve the problem of few inducible promoters and achieve the effect of strong comprehensive resistance

Inactive Publication Date: 2012-03-28
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] Example 1: Cloning of the stress inducible promoter of corn anion peroxidase gene (ZmapH)

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

[0037] The maize anion peroxidase gene (ZmapH) is registered in NCBI GenBank (accession number: AF037033.1). The sequence listing shows the DNA sequence of the plant stress-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. Promoter analysis website http: / / www.dna.affrc.go.jp / PLACE / signalscan.html.

[0038] Take mai...

Embodiment 2

[0042] Example 2: Construction of Plant Stress Inducible Vector

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

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

[0045] in Image 6 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 promoter of HPTII, 3...

Embodiment 3

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

[0047] The vector pCAMBIA 1301::ZmapH Pro constructed in Example 2 was transferred to Agrobacterium tumefaciens EHA105 by the heat shock transformation method, and the plasmid ( Figure 7 ) And perform PCR identification ( Picture 8 ).

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

[0049] More specifically, the corn seeds are soaked to accelerate germination, then the seeds are cut into two halves longitudinally, and incubated with 20% PEG for 24 hours. The corn seeds are placed in the GUS test solution at 37°C overnight, GUS test 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 fe...

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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 the corn anion peroxidase gene (ZmapH) and capable of being expressed at a high level in corn. Background technique [0002] The drought ecological crisis has seriously affected the sustainable development of mankind. 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 replace the original chromosome level at the gene level to finely regulate the effect of breeding. [0003] The regulation of gene expression in higher plants has become a hot spot in the field of molecular biology, and promote...

Claims

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Application Information

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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
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