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Corn low-temperature inducible promoter and activity analysis

A low-temperature induction, promoter sequence technology, applied in the field of bioengineering, can solve the problem of few inducible promoters

Inactive Publication Date: 2012-04-04
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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  • Corn low-temperature inducible promoter and activity analysis
  • Corn low-temperature inducible promoter and activity analysis
  • Corn low-temperature inducible promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cloning of the low temperature inducible promoter of the maize DRE response element binding protein gene ZmDBP3

[0035] The promoter of the corn DRE response element binding protein gene ZmDBP3 (the promoter sequence comprises the DNA nucleotide sequence of the -1bp to -1151bp region relative to the transcription start site of SEQ ID NO: 1), at the 5th position of the corn ZmDBP3 gene ' region sequence was identified.

[0036]The maize DRE response element binding protein gene ZmDBP3 is registered in NCBI GenBank (accession number: FJ805751.1), and the sequence listing shows the DNA sequence of the plant low-temperature-inducible promoter and 5' untranslated region of the above-mentioned gene of the present invention. In the list of promoter sequences in this document, base C of the transcription initiation site is indicated with +1. And use the promoter analysis website to analyze the core elements of the promoter.

[0037] Promoter analysis website http:...

Embodiment 2

[0042] Embodiment 2: Construction of plant low temperature inducible vector

[0043] The low-temperature-inducible promoter of the maize DRE response element binding protein gene ZmDBP3 cloned in Example 1 and the 255 bp 5' untranslated region ZmDBP3Pro (see sequence listing) were inserted into the vector to construct a plant low-temperature-inducible vector.

[0044] More specifically, the plant expression vector pCAMBIA1301 and the recombinant plasmid pMD18-T::ZmDBP3Pro were respectively digested with EcoRI and NcoI, and then inserted into the EcoRI and NcoI restriction sites of the vector pCAMBIA1301. This vector is called pCAMBIA 1301::ZmDBP3Pro, and is used to drive the expression of GUS gene, identified by PCR ( Figure 4 ) and enzyme digestion identification ( Figure 5 ) to obtain the recombinant plasmid of the promoter and the vector.

[0045] exist Figure 7 Among them, the gene GUS encoding β-glucuronidase is used as the reporter gene, and the selection marker is...

Embodiment 3

[0046] Example 3: Identification of activity of maize low temperature inducible promoter

[0047] By electric transformation method, the carrier pCAMBIA1301::ZmDBP3Pro constructed in embodiment 2 is transferred in Agrobacterium tumefaciens EHA105, extracts plasmid and carries out PCR identification ( Figure 6 ).

[0048] To identify the low-temperature-induced activity of the promoter, maize embryos were treated at low temperature (4°C) by the method of Jefferson et al. (EMBO J, 1987), and then the activity of GUS was detected.

[0049] More specifically, the corn seeds are soaked to accelerate germination, and then the seeds are cut in half longitudinally, induced and cultured at 4°C for 24 hours, and 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 ferrocyanide Potassium, 0.1% Trit...

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Abstract

The invention discloses a corn low-temperature inducible promoter and activity analysis, belonging to the technical field of bioengineering, and provides an obtained corn low-temperature inducible promoter sequence, which comprises a DNA (Deoxyribonucleic Acid) nucleotide sequence ranging from a -1bp region to a -1151bp region relative to a transcription start site of SEQ ID NO:1. The invention further provides a low-temperature inducible plant expression vector for transforming corn, which comprises a corn low-temperature inducible promoter sequence and a 5' non-translational region of a corn dehydration-responsive element (DRE) binding protein gene ZmDBP3, as well as a genetically-modified corn mature embryo transformed by the plant expression vector. PCR primers shown as SEQ ID NO:2 and SEQ ID NO:3 are suitable for amplifying DNA fragments, including the sequence SEQ ID NO:1. The corn low-temperature inducible promoter can be used for starting efficient expression of a low-temperature-resistant gene, can be applied to low-temperature-intolerant plants to realize the low-temperature resistance of plants, and plays a positive role in solving the problem of food crisis in low-temperature areas.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a DNA sequence, which can be used as a promoter to regulate the expression of genes under low temperature stress, and specifically relates to a low temperature induction gene ZmDBP3 derived from corn DRE response element binding protein gene ZmDBP3 and highly expressed in plants type promoter sequence. Background technique [0002] China is a large agricultural country with a large population, and food safety production is related to the national economy, the people's livelihood and social stability. Low temperature injury (including chilling injury and freezing injury) is a serious meteorological disaster affecting most parts of my country, especially in the Northeast and the Huanghuaihai region. Both reduce grain production by more than 5 billion kilograms. Due to my country's vast territory and diverse terrain, low-temperature freezing damage occurs in a very wide range,...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/11A01H5/00
Inventor 潘洪玉陈宣明张世宏刘金亮赵淑莉余刚贾承国李桂华
Owner JILIN UNIV
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