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Pathogen induced promoter PXa13 and application thereof in regulate and control on target gene expression under pathogen induction

A target gene and promoter technology, applied in the field of plant genetic engineering, can solve problems such as reduced transcription efficiency

Inactive Publication Date: 2012-04-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally located around 175bp, once the base of this box is deleted or mutated, the transcription efficiency will be drastically reduced, and the CAAT box may control the frequency of transcription initiation

Method used

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  • Pathogen induced promoter PXa13 and application thereof in regulate and control on target gene expression under pathogen induction
  • Pathogen induced promoter PXa13 and application thereof in regulate and control on target gene expression under pathogen induction
  • Pathogen induced promoter PXa13 and application thereof in regulate and control on target gene expression under pathogen induction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: P Xa13 Structural analysis of promoters

[0021] Using a series of promoter analysis software, such as TSSP software of Softberry website (http: / / www.sofiberry.com), PROSCAN software (http: / / bimas.dcrt.nih.gov / molbio / proscan), PLACE (A Database of Plant Cis-acting Regulatory DNAElements) in the Signal Scan analysis tool ( http: / / www.dna.affrc.go.jp / PLACE / signalscan .html), TESS software ( http: / / searchlauncher.bcm.tmc.edu / seq -search / gene-search.html), Match 1.0 software ( http: / / www.gene-regulation.com / cgi-bin / pub / programs / match / bin / match.cgi), and AliBata2.1 software ( http: / / www.gene-regulation.com / pub / programs / alibata2 / index.html ) etc. analyzed the structure of Xa13 gene promoter. It was found that the -34bp of the Xa13 promoter is a TATA box, and -332bp is a CAAT box related to the transcription frequency (figure 1 A).

Embodiment 2

[0022] Example 2: Analysis of the pathogen-induced functional region of the Xa13 gene promoter

[0023] 1. Isolation of serial truncated fragments of Xa13 gene promoter

[0024] PCR primer Xa13P-F (5'- GGATCC GATGTTGAGCTTTAGGATTAGCGGGTT-3') and X13-promR (5'- GGCAAG CTTGGCCTTGGCCATGGCTCAGT-3') amplifies the complete DNA fragment P of Xa13 gene promoter Xa13 (the sequence length is 1413 bases), its nucleotide sequence is as shown in the sequence table SEQ ID NO: 1, a total of 1413 nucleotides ( figure 1 B). The Xa13 gene promoter specific PCR primer 5'deletion-1147 (5'- GGATCC TCCCTTTTCTTCAAGTTACTCTCTCTC-3'), 5'deletion-608(5'- GGATCC GCATCATTGTCCATGGTTGT-3'), XP-90F(5'- GGATCC GAAATATCAAGCACAAG-3’) and XP-60F(5’- GGATCC CTGTACACCACCAAAAG-3') paired with Xa13 gene promoter specific PCR primer X13-promR to amplify the 5' end truncated Xa13 gene promoter fragment P Xa13-934 (934 nucleotides; the nucleotide sequence shown in 480-1413bp of SEQ ID NO: 1 in the sequence...

Embodiment 3

[0032] Example 3: Functional analysis of Xa13 gene promoter

[0033] In order to verify whether there are other pathogen-specific induction cis-regulatory elements other than the UPT box on the Xa13 gene promoter, the researchers of the present invention mutated the UPT box to study whether the Xa13 gene promoter can also be specifically induced by bacterial blight PXO99.

[0034] 1. Isolation of a point mutant of the Xa13 gene promoter

[0035] P Xa13 The promoter was subjected to site-directed mutation. The basic principle of this kit is to use primers with mutation sites to amplify the methylated template plasmid, electrotransform the amplified product into Escherichia coli, and the McrBC endonuclease in the host will enzymatically digest the methylated template , leaving unmethylated PCR products with mutation sites.

[0036] The following four pairs of primers Xa13PM1F (5'-AAAGCAAAGGTTAGATATT CATCTCCCCCCT-3') / Xa13PM1R(5'-ATATCTAACCTTTGCTTTTTTTTTTC-3'), Xa13PM2F(5'-CAAA...

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Abstract

The invention belongs to the technical field of plant genetic engineering and particularly relates to the analysis of separation cloning, function verification and application prospect of a DNA fragment containing a pathogen induced promoter PXa13. The promoter PXa13A has a specific response reaction to rice infected by xanthomonas oryzae. The expression of genes regulated and controlled by the promoter PXa13 can be rapidly enhanced in the rice infected by the xanthomonas oryzae. The promoter PXa13 can be used for regulating and controlling the expression of resistance genes, the expression of the resistance genes is started only when the pathogen invades, and thus, the resistance capability of the rice against diseases can be enhanced and the rice does not generate redundant proteins.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the functional verification and application prospect analysis of a rice DNA fragment. The DNA fragment contains a rice gene promoter, which can specifically enhance gene expression under the induction of pathogens. Background technique [0002] During the growth of plants, they will be attacked by various pathogens such as viruses, bacteria, molds and nematodes. Pathogens invading plants lead to two results: (1) the pathogens multiply in the host plant and cause related diseases; (2) the host plant produces a disease-resistant response, killing the pathogen or preventing its growth. Using resistance gene resources to improve plant disease resistance is the fundamental way to prevent diseases and protect the environment at the same time. [0003] Using transgenic technology to introduce resistance genes into plants is one of the main ways to improve ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A01H5/00
Inventor 王石平袁婷
Owner HUAZHONG AGRI UNIV
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