Pathogen-inducible promoter

A pathogen, inducible technology, applied in the field of chimeric DNA sequences, can solve problems such as inability to respond to pathogens

Inactive Publication Date: 2001-05-16
杰尼克莫根有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of some of these promoters is that they are also constitutively active, or they are not reactive with certain types of pathogens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Induction of ms59 messenger in sunflower plants

[0050] 5 sprays of 5 mM salicylic acid (SA), 1 spray of 1 mM salicylic acid, 1 spray of 0.1 mM jasmonic acid (JA), 1 spray of 1 mM ACC (1-aminocyclopropane-1-carboxylic acid, plant hormone ethylene precursor), or wounding, to induce leaves of 7-8 week old sunflower plants (Helianthus annuus cv Zebulon). Leaf samples were collected from induced leaves 24 hours later (1 mM SA, 0.1 mM JA, 1 mM ACC and wounding) and 5 days later (5 mM SA). Control samples were taken in non-induced plants 24 hours after induction.

Embodiment 2

[0052] RNA Extraction and cDNA Synthesis of Sunflower Leaf Tissue

[0053] Total RNA was extracted from 10 g of leaf material by the hot phenol method and purified using the Qiagen RNA buffer series and tip-100 column (Qiagen GmbH, Germany). Contaminating DNA was degraded by DNase I (Gibco BRL) treatment.

[0054] With 1 μg total RNA, 1 μl oligo(dT) as described by the manufacturer 12-18 Primers (500 μg / ml, Gibco BRL) and 200 units Superscript Ⅱ RT RNAse H - cDNA was prepared with reverse transcriptase (GibcoBRL).

Embodiment 3

[0056] Construction of ms59 PCR MIMIC and analysis of samples using competitive RT-PCR

[0057] The transcript level of ms59 was determined by competitive RT-PCR technique. In this technique, competition between the cDNA target and the artificial PCR MIMIC enables the quantification of transcript levels (Paul D. Siebert and James W. Larrick (1992), Nature 359, 557-558).

[0058] For the construction of PCR MIMIC, the following primers were prepared: FR-pUC-208 (SEQ ID NO: 1) 5'GTT CCG GAG GTT GTG ACC GTG GGA TGT GCT GCAAGG CG 3', FR-pUC-209 (SEQ ID NO: 2) 5'CTG GGG AAG CCC GTGTAG TAA AGC CCC CGC GCG TTG GCC GAT TC 3', FR-MS59-47 (SEQ ID NO: 3) 5'CTG GGG AAG CCC GTG TAG TAA AGC 3' and FR-MS59-77 ( SEQ ID NO: 4) 5'GTT CCG GAG GTT GTG ACC GTG3'. Primers FR-pUC-208 and FR-pUC-209 were used by PCR (95°C 1', 55°C 1', 72°C 2', 10 cycles) from plasmid pUC18 (Yanisch-Perron, C., Vieira, J. . and Messing, J. (1985) Gene 33, 103-119) amplified a fragment of 387 bp. 1 µl of this PCR p...

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Abstract

This invention describes pathogen-inducible promoters which normally drive expression of plant hexose oxidases, especially those which can be isolated from Helianthus annuus and Lactuca sativa, more specifically those promoters which naturally are the regulatory regions driving expression of the hexose oxidase MS59 and WL64, respectively. Also claimed are chimeric constructs where these pathogen-inducible promoters drive expression of antipathogenic proteins or of proteins which can elicit a hypersensitive response.

Description

field of invention [0001] The present invention relates to the field of pathogen-inducible promoters and chimeric DNA sequences containing the promoters, especially in the field of plant bioengineering. Background technique [0002] Inducible promoters include any promoter capable of increasing the amount of a gene product produced by a particular gene upon exposure to an inducer. In the absence of an inducer, the DNA sequence is not transcribed. In general, factors that can specifically bind to an inducible promoter to activate transcription exist in an inactive form, and are then directly or indirectly converted into an active form by the inducer. Inducers can be chemical agents such as proteins, metabolites (sugars, alcohols, etc.), growth regulators, herbicides, or phenolic compounds, or physiological stress directly applied by heat, salt, trauma, toxic components, etc., or by pathogens Or physiological stress indirectly imposed by the action of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/82
CPCC12N15/8237
Inventor M·H·斯退弗J·H·H·V·库斯特斯L·H·希蒙斯
Owner 杰尼克莫根有限公司
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