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Corn drought inducible gene promoters and activity analysis thereof

A drought-inducible, promoter-sequencing technology, applied in the field of cloning drought-inducible gene promoters in maize, can solve the problem of few inducible promoters

Inactive Publication Date: 2011-08-17
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 drought inducible gene promoters and activity analysis thereof
  • Corn drought inducible gene promoters and activity analysis thereof
  • Corn drought inducible gene promoters and activity analysis thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Cloning of the drought-inducible promoter of the maize CBL-interacting protein kinase gene (ZmCIPK16)

[0048] The promoter of the protein kinase gene (ZmCIPK16) of corn CBL interaction (promoter sequence comprises the DNA nucleotide sequence of the -1bp to -1773bp region relative to the transcription initiation site of SEQ ID NO: 1), in the corn ZmCIPK16 gene identified in the sequence of the 5' region.

[0049] The corn CBL-interacting protein kinase gene (ZmCIPK16) is registered in NCBI GenBank (accession number: EU907939.1), and the sequence listing shows the DNA sequence of the plant drought-inducible promoter and 5' untranslated region of the above-mentioned gene of the present invention. exist figure 1 In , the start codon ATG for protein synthesis is underlined, and the base A of the transcription start site is shown with +1. And use the promoter analysis website to analyze the core elements of the promoter. Promoter analysis website http: / / www.dna...

Embodiment 2

[0054] Example 2: Construction of Plant Drought Inducible Vector

[0055] The promoter of the corn CBL-interacting protein kinase gene (ZmCIPK16) cloned in Example 1 and the 5' untranslated region ZmCIPK16Pro (see sequence listing) of 216 bp were inserted into the vector to construct a plant drought-inducible vector.

[0056] More specifically, the plant expression vector pCAMBIA 1301 and the recombinant plasmid pMD 18-T::ZmCIPK16Pro were digested with EcoRI and NcoRI respectively ( Figure 6 ), and then they were inserted into the EcoRI and NcoRI restriction sites of the vector pCAMBIA1301. This vector is called pCAMBIA 1301::ZmCIPK16Pro, and is used to drive the expression of GUS gene, which was identified by enzyme digestion ( Figure 7 ) obtained the promoter fragment, which was the same as the expected result.

[0057] exist Figure 8 Among them, the gene GUS encoding β-glucuronidase was used as the reporter gene, and the selection marker was hygromycin resistance gene...

Embodiment 3

[0058] Example 3: Identification of the activity of the maize drought-inducible promoter of the present invention

[0059] By the electric shock transformation method, the carrier pCAMBIA 1301::ZmCIPK16Pro constructed in embodiment 2 is transferred in Agrobacterium tumefaciens EHA105, extracts plasmid ( Figure 9 ) and identified by enzyme digestion ( Figure 10 ).

[0060] In order to identify the drought-inducible activity of the promoter, the method of Jefferson et al. (EMBO J, 1987) was used to dry the embryos of maize and then detect the activity of GUS.

[0061] More specifically, soak the corn seeds to accelerate germination, then cut the seeds longitudinally, cut them in half, induce culture with 20% PEG for 24 hours, put the corn seeds 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), 10mM EDTA, 0.5mM potassium ferricyanide, 0.5mM Potass...

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Abstract

The invention discloses corn drought inducible gene promoters and activity analysis thereof, which belong to the technical field of bioengineering. The invention provides the sequences of the obtained corn drought inducible promoters and provides a drought inducible plant expression vectors for corn transfer, and a transgenic plant transferred by the plant expression vector and polymerase chain reaction (PCR) primers suitable for amplifying a DNA fragment having a sequence SEQ ID No.1. The prompters can be used for promote the high-efficiency expression of an anti-drought gene, are used in drought-resistance transgenic plants and have an active significance for solving food crisis, ecological deterioration and the like in drought areas.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to the cloning of a corn drought-inducible gene (ZmCIPK16) promoter. Background technique [0002] The drought ecological crisis has seriously affected the sustainable development of human beings. 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 finely adjust the effect of breeding by substituting the gene level for the original genome level. [0003] The regulation of gene expression in higher plants has become a hot spot in the field of molecular biology research, and the promoter is an important element of gene expression regulation. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11A01H5/00
Inventor 潘洪玉胡瑞学张世宏刘金亮贾承国李桂华
Owner JILIN UNIV
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