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Low-temperature drought induced promoter of zeamays and activity analysis

A drought-inducible, promoter sequence technology, applied in the field of low temperature, drought-inducible promoter sequence, DNA sequence, 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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  • Low-temperature drought induced promoter of zeamays and activity analysis
  • Low-temperature drought induced promoter of zeamays and activity analysis
  • Low-temperature drought induced promoter of zeamays and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Example 1: Cloning of the low temperature and drought inducible promoter of the maize zinc finger protein gene ZmZFP

[0037] The promoter of the maize zinc finger protein gene ZmZFP (the promoter sequence comprises the DNA nucleotide sequence of the -1bp to -1645bp region relative to the transcription start site of SEQ ID NO: 1), in the 5' region of the maize ZmZFP gene identified in the sequence.

[0038] The maize zinc finger protein gene ZmZFP is registered in NCBI GenBank (accession number: EU963900.1), and the sequence listing shows the DNA sequence of the plant low temperature and drought inducible promoter and 5' untranslated region of the above gene of the present invention. In the list of promoter sequences in this document, the base G of the transcription start site is indicated by +1, and the base ATG is underlined in red. And use the promoter analysis website to analyze the core elements of the promoter. Promoter analysis website http: / / bioinformatics.psb....

Embodiment 2

[0043] Example 2: Construction of Plant Low Temperature and Drought Inducible Vector

[0044] The low temperature and drought inducible promoter of the maize zinc finger protein gene ZmZFP cloned in Example 1 and the 5' untranslated region ZmZFPPro (see sequence listing) of 230 bp were inserted into the vector to construct a plant low temperature and drought inducible carrier.

[0045] More specifically, the plant expression vector pCAMBIA1301 and the recombinant plasmid pMD18-T::ZmZFPPro were respectively digested with PstI and BglII, and then inserted into the PstI and BalII restriction sites of the vector pCAMBIA1301. This vector is called pCAMBIA 1301::ZmZFPPro, 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.

[0046] exist Figure 7 Among them, the gene GUS encoding β-glucuronidase was used as the reporter gene, and the s...

Embodiment 3

[0047] Example 3: Identification of the activity of a maize low temperature and drought inducible promoter

[0048] By electric shock transformation, the vector pCAMBIA1301::ZmZFPPro constructed in Example 2 was transferred to Agrobacterium tumefaciens EHA105, and the plasmid was extracted and identified by PCR ( Image 6 ).

[0049] In order to identify the low-temperature and drought-induced activity of the promoter, the method of Jefferson et al. (EMBO J, 1987) was used to treat maize embryos with low temperature (4°C) and drought and then detect the activity of GUS.

[0050] More specifically, the corn seeds are soaked to accelerate germination, and then the seeds are cut in half longitudinally, respectively induced and cultured at 4°C and 20% PEG for 24 hours, and the corn seeds are placed 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), 10m...

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Abstract

The invention relates to a low-temperature drought induced promoter of zeamays and activity analysis, and belongs to the technical field of biological engineering. The invention provides a low-temperature drought induced promoter sequence of the zeamays. The sequence comprises a deoxyribonucleic acid (DNA) nucleotide sequence relative to a -1 bp to -1,645 base pair (bp) region of the transcription starting point of SEQ ID No:1; a low-temperature drought induced plant expression vector for zeamays conversion is provided simultaneously, and comprises the low-temperature drought induced promotersequence of the zeamays and a 5' untranslated region of zeamays zinc finger protein (ZmZFP) gene; a transgenic zeamays mature embryo converted by the plant expression vector is provided; and polymerase chain reaction (PCR) primers of SEQ ID No: 2 and SEQ ID No: 3 are suitable for amplifying DNA fragments containing the sequence of SEQ ID No: 1. The low-temperature drought induced promoter can be used for promoting the high-efficiency expression of the low-temperature drought gene and is applied in drought transgenic plant which are not resistant to low temperature, so that the plants have thecharacters of low temperature and drought, and thus, the low-temperature drought induced promoter has a positive significance for solving the problem of food crisis in low-temperature and drought 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 gene expression under low temperature and drought stress. In particular, it relates to a low-temperature and drought-inducible promoter sequence derived from a maize zinc finger protein gene ZmZFP and highly expressed in plants. 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, drought and other adversities are the main abiotic stress factors affecting plant growth and development. In recent years, due to global climate change, extreme weather events such as droughts, floods, freezing, and heat waves have occurred frequently, which has adversely affected my country's agricultural production. For example, during the period from last winter to early sp...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/84A01H5/10C12N15/11
Inventor 潘洪玉陈宣明张世宏刘金亮余刚赵淑莉贾承国李桂华
Owner JILIN UNIV
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