Cloning and activity analysis of corn adversity inducing promoter

An inducible promoter sequence technology, applied in the field of plant genetic engineering, can solve the problem of few inducible promoters

Inactive Publication Date: 2012-12-05
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 activity analysis of corn adversity inducing promoter
  • Cloning and activity analysis of corn adversity inducing promoter
  • Cloning and activity analysis of corn adversity inducing promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the cloning of the promoter of maize lipoxygenase gene (ZmLOX10)

[0037] The promoter of maize lipoxygenase gene (ZmLOX10) (promoter sequence comprises the DNA nucleotide sequence of -1bp to-1273bp region relative to the transcription initiation site of SEQ ID NO:1), in maize lipid oxygenation The enzyme gene (ZmLOX10) was identified in the 5' untranslated region sequence.

[0038] ZmLOX10 is registered in NCBI GenBank (accession number: NM_001112510), and the sequence listing shows the DNA sequence of the maize stress-inducible promoter and 5' untranslated region of the above-mentioned gene of the present invention. In SEQ ID NO.1, the translation initiation codon ATG is underlined, using http: / / www.fruitfly.org / seq_tools / promoter.html The website predicts the transcription start site, which is marked with +1. And analyze the site with the promoter ( http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) analyzed the core elements of the prom...

Embodiment 2

[0043] Embodiment 2: Construction of maize lipoxygenase gene (ZmLOX10) promoter expression vector

[0044] The maize lipoxygenase gene (ZmLOX10) promoter cloned in Example 1 and the 5' untranslated region ZmLOX10Pro (see sequence listing) of 170bp were inserted into the vector, thereby constructing the maize lipoxygenase gene (ZmLOX10) Promoter expression vector.

[0045] More specifically, the recombinant plasmid pMD18-T::ZmLOX10Pro and the plant expression vector pCAMBIA1301( Figure 4 ), recover the target fragment and pCAMBIA1301 respectively, and connect them with T4 ligase to obtain the pCAMBIA1301::ZmLOX10Pro expression vector, which is used to drive the expression of the GUS gene. identified by PCR ( Figure 5 ) The obtained promoter fragment was the same as the expected result. Agrobacterium EHA105 was transformed by electric shock method, and PCR detection of bacteria liquid was carried out ( Image 6 ).

[0046] exist Figure 7 Among them, the gene GUS encodin...

Embodiment 3

[0047] Example 3: Identification of the activity of the maize stress-inducible promoter of the present invention

[0048] In order to identify the stress-induced activity of the promoter, the embryos of maize were drought-treated by the method of Jefferson et al. (EMBO J, 1987), and then the activity of GUS was detected.

[0049] 3.1 Preparation of acceptor material

[0050] Soak corn seeds in water for 1 day, let them fully absorb water, put them in an environment of 24°C for 1 day to accelerate germination, and then cut the seeds in half longitudinally for later use.

[0051] 3.2 Preparation of Agrobacterium bacteria solution

[0052] Agrobacterium EHA105 was inoculated on YEP (50mg / L rif, 50mg / L kan) solid medium, and cultured in the dark at 28°C until a single colony grew. Pick a single colony, inoculate it in 5 mL of YEP liquid medium containing the corresponding antibiotic, and cultivate it at 28°C with shaking at 200r / min. When the bacterial solution grows to OD 600...

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Abstract

The invention discloses cloning and activity analysis of a corn adversity inducing promoter, which belong to the technical field of plant gene engineering. The invention provides an obtained corn adversity inducing promoter sequence which comprises a DNA (Deoxyribonucleic Acid) sequence ranging from a -1bp region to a -1273bp region relative to the transcription initiation site of SEQ ID NO:1, and provides an adversity inducing plant expression vector for transforming corn, which comprises a corn adversity inducing promoter sequence and a 5' non-translational region of a corn lipoxygenase gene, and PCR (Polymerase Chain Reaction) primers shown as SEQ ID NO:2 and SEQ ID NO 3. The primers are characterized by being suitable for amplifying DNA fragments comprising the SEQ ID NO:1. The inducing promoter can be used for promoting high-efficiency expression of an adversity-relevant gene, is applied to cold-resistant, drought-resistant and salt-resistant transgenic plants, and plays a positive role in solving the problems of coldness, drought, crisis in food in salt damage regions, ecological degeneration and the like.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a DNA sequence, which can be used as a promoter to regulate the expression of genes under the stress of low temperature, drought and salt damage. Specifically, the invention relates to a lipoxygenase derived from corn (lipoxygenase) gene (ZmLOX10), and multiple stress-inducible promoter sequences highly expressed in plants. Background technique [0002] Against the backdrop of intensified global climate change, increased pressure on resources and the environment, the impact of the financial crisis, and the deepening downward trend of food supply, ensuring my country's food security has always been an urgent and important strategic task. Adversity stress is one of the most important factors and challenges affecting food production in agricultural production. Every year, crop yield reduction directly caused by adversity stresses such as salinity, drought, and low t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/84C12N15/11C12Q1/68
Inventor 贾承国潘洪玉张世宏刘金亮胡瑞学李桂华
Owner JILIN UNIV
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