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Corn fungus inducing promoter and activity analysis

An inducible and promoter technology, applied in the field of bioengineering, can solve the problem of less inducible promoters, and achieve the effect of solving the problem of food crisis

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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  • Corn fungus inducing promoter and activity analysis
  • Corn fungus inducing promoter and activity analysis
  • Corn fungus inducing promoter and activity analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cloning of the fungal-inducible promoter of maize endochitinase A gene ZmECA

[0035] The promoter of maize endochitinase A gene ZmECA (the promoter sequence comprises the DNA nucleotide sequence of the -1bp to -1729bp region relative to the transcription initiation site of SEQ ID NO: 1), at the 5' non-translation of the maize ZmECA gene identified in the region sequence.

[0036] The maize endochitinase A gene ZmECA is registered in NCBI GenBank (accession number: NM_001165432.1), and the sequence listing shows the DNA sequence of the plant fungal-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: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / .

[0038] Maize geno...

Embodiment 2

[0042] Embodiment 2: Construction of plant fungus inducible vector

[0043] The fungus-inducible promoter of the maize endochitinase A gene ZmECA cloned in Example 1 and the 5' untranslated region ZmECAPro of 99 bp (see sequence listing) were inserted into the vector, thereby constructing the plant fungus-inducible vector.

[0044] More specifically, the plant expression vector pCAMBIA1301 and the recombinant plasmid pMD18-T::ZmECAPro were respectively digested with HindIII and NcoI, and then inserted into the HindIII and NcoI restriction sites of the vector pCAMBIA1301. This vector is called pCAMBIA 1301::ZmECAPro, 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 the hygromycin resistance gene. In additi...

Embodiment 3

[0046] Example 3: Identification of the activity of the corn fungus-inducible promoter of the present invention

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

[0048] In order to identify the fungal inducible activity of the promoter, the corn embryo was treated with salicylic acid 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, then the seeds are cut in half longitudinally, induced by salicylic acid for 24 hours, and the corn seeds are placed in the GUS test solution at 37°C overnight, GUS test solution: 3mg / 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 Potas...

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Abstract

The invention discloses a corn fungus inducing promoter and active analysis, which belong to the technical field of bioengineering. The invention provides an obtained corn fungus inducing promoter sequence which comprises a DNA (Deoxyribonucleic Acid) sequence ranging from a -1bp region to a -1729bp region relative to the transcription initiation site of SEQ ID NO:1, and provides a fungus inducing plant expression vector for transforming corn, which comprises a corn fungus inducing promoter sequence and a 5' non-translational region of a corn endochitinase A gene, a transgenic corn mature embryo transformed from a plant expression vector, and PCR (Polymerase Chain Reaction) primers which are shown as SEQ ID NO:2 and SEQ ID NO 3 and are suitable for amplifying DNA fragments comprising the SEQ ID NO:1. The corn fungus inducing promoter can be used for promoting high-efficiency expression of a fungus-resistant gene, is applied to plants which are not resistant to fungi to realize fungus-resistant properties of the plants, and plays a positive role in solving the problem of crisis in food in regions where the fungi disease occurs frequently.

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 the stress of fungal diseases. In particular, it relates to a fungus-inducible promoter sequence derived from maize endochitinase A gene ZmECA 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. Plant diseases have always been one of the important restrictive factors for high-quality and high-yield crops. Among plant diseases, 70% to 80% of diseases are caused by pathogenic fungi. Plant fungal diseases not only directly cause the decline of crop yield and quality, but also some pathogenic fungi can secrete and produce a variety of toxins and metabolites harmful to humans and animals during the infection proc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/84A01H5/10C12N15/11C12Q1/68
Inventor 刘金亮陈宣明潘洪玉张世宏余刚赵淑莉贾承国李桂华
Owner JILIN UNIV
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