WO1993014213A1 - Nouvel element de regulation de genes de plantes - Google Patents

Nouvel element de regulation de genes de plantes Download PDF

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WO1993014213A1
WO1993014213A1 PCT/GB1993/000071 GB9300071W WO9314213A1 WO 1993014213 A1 WO1993014213 A1 WO 1993014213A1 GB 9300071 W GB9300071 W GB 9300071W WO 9314213 A1 WO9314213 A1 WO 9314213A1
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sequence
protein
gene
plant
expression
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PCT/GB1993/000071
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Andrew Peter Goldsbrough
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Unilever Plc
Unilever N.V.
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01039Glucan endo-1,3-beta-D-glucosidase (3.2.1.39)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237Externally regulated expression systems
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01058Glucan 1,3-beta-glucosidase (3.2.1.58)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01073Licheninase (3.2.1.73)

Definitions

  • This invention relates to a novel nucleotide sequence, vectors containing the sequence , substances which recognise said sequence , methods of regulating gene expression and methods of detecting stress in plant cells , tissue cultures or whole plants .
  • promoter which contains specif ic cis-acting regulatory sequence elements
  • enhancer and upstream promoter sequences which also contain specific cis-acting regulatory sequence elements
  • promoters and enhancers are somewhat vague since the specific sequences contained within them may be the same .
  • promoters are always located immediately 5 ' to the start site of transcr iption, whereas enhancers can be located at much greater distances both 5 1 and 3 ' to the transcription start site.
  • Trans-acting transcr iption factors are generally proteins which recognise and bind to the specific cis-acting DNA elements described above . It is the specif ic combination of cis- and trans-acting factors in a given cell at a giver- time or under particular conditions- that determines v?hether or not a gene is expressed.
  • PR proteins pathogenesis related proteins
  • TMV tobacco mosaic virus
  • PAL phenylalanine lyase
  • CHS Chalcone synthase
  • phenylpropanoid metabolism is a common defence response to attempted pathogen attack, light stress and wounding.
  • general phenylpropanoid pathway enzymes and end-product specific branch pathways.
  • " an astonishing array of secondary metabolites are elaborated from phenylalanine - including antimicrobial phytoalexins, UV-protective flavenoids, activators and inhibitors of symbiotic plant- microbe interactions and insect repellants inhibitors of symbiotic plant-microbe interactions and insect repellants and attractan s.
  • the invention provides a nucleotide sequence, comprising substantially the sequence 5'- TCATCTTCTT-3' or functional equivalents thereof.
  • the invention provides a recombi nant vector compr is ing the sequence def ined above .
  • the invention provides the nucleotide sequence def ined above , in isolation from surrounding sequences with which it is naturally associated .
  • the invention provides a recombinant vector comprising the novel sequence def ined above or functional equivalents thereof , in isolation from surrounding sequences with which it is naturally associated .
  • a protein-coding nucleotide sequence may, by means of recombinant DNA technology, be brought into position around and/or proximal and/or adjacent to the novel sequence in such a manner as to cause the expression of the protein-codi ng sequence to be regulated wholly or partly by the novel regulatory sequence.
  • the invention provides a method of regulating the expression of a pr otein-codi ng seque nce comprising the step of positioning a protein-codi ng sequence around and/or proximal and/or adjacent to the novel regulatory sequence.
  • the method described above is reciprocal, in that the expression of a protein-coding sequence may be regulated by positioning the novel regulatory sequence proximal and/or adjacent to and/or within the protein- coding sequence.
  • a barley genomic clone (g19-9) encoding a beta-1,3- glucanase has been isolated and sequenced.
  • a 200bp region shown in Figure 1 and identified as SEQ ID No. 1 in the attached sequence listing
  • approximately 600bp upstream of the putative TATA box of this gene is a 1Obp motif (shown in block capitals in Figure 1 and identified as SEQ ID No. 2 in the attached sequence listing) termed the "TCA" motif, with the consensus sequence 5'- TCATCTTCTT-3 1 , repeated four times.
  • TCA 1Obp motif
  • Table 1 Data obtained from this survey are presented in Table 1.
  • the list comprises genes for the pathogenesis related (PR) family of proteins from tobacco and parsle ; enzymes of the phenylpropanoid and flavenoid biosynthetic pathways; several wound-inducible genes from potato and tomato (Win!, Wun1 and proteinase inhibitors I and II); ubiquitin, which is known to be a stress related protein throughout the biological kingdom; a heat shock protein from soybean; nitrite and nitrate reductase genes which are induced by water stress; an osmotic stress-inducible Abscissic acid-responsive gene, and an Arabidopsis sp. alcohol dehydrogenase gene which is induced under anaerobic stress.
  • PR pathogenesis related
  • the motif is present in genes from both dicotyledonous and monocotyledonous plants. In some genes it is present as a single copy (eg tobacco PR-1 [Payne et al. , 1989]) and in others it is repeated several times - up to seven in the case of 4-CL (Douglas et al. , 1987). With the exception of one of the parsley PR2 sequences, all those listed in Table 1 share at least 8 nucleotides in common with the consensus sequence. In some of the examples given (those marked with an asterisk in Table 1), the motif occurs on the complementary DNA strand. In one case it occurs 3' to the coding sequence and in two cases it is found within an intron. It is also frequently observed within the transcribed region upstream of the ATG initiator codon. The motif itself does not show dyad symmetry and has an unusual base composition, being 5'-TC-3' rich in its forward orientation.
  • Tobacco (Nicotiana tabacum cv Samsun NN) was selected as a model system for these experiments because methods for nuclear protein extraction and induction of PR proteins using salicylic acid (5m ) from this species are well documented (Jensen et al., 1988). Salicylic acid has recently been confirmed as a systemic signal and inducer of PR proteins in virus-infected tobacco (Yalpani et al., 1991).
  • a 357 bp Aval-BamHI restriction fragment (termed "AB"), incorporating several copies of the TCA motif, was excised from the promoter region of a barley B-1 ,3-glucanase gene (g19-9) ( Figure 1). This was made blunt using Klenow polymerase and dNTPs, and subcloned into the Smal site of pBI221.8 (a pUC19 derivative containing the polylinker region and CaMV-GUS-Nos cassette from the vector pBI121.8 [Goldsbrough ⁇ - Bevan, 1991]) to produce the plasmid pAG281.8. The fragment was subsequently excised from pAG281.8 as an Asp718-BamHI fragment and purified from a gel.
  • the cloned AB fragment contains 4 copies of the TCA motif: two are identical to the consensus sequence and two have 8 bases in common with the consensus sequence.
  • the AB fragment could be 3' end labelled (specific activity 10'cpm/ug) using alpha J P dCTP (purchased from Amersham International) and Klenow polymerase.
  • Radiolabelled AB fragment was incubated with 10ug of nuclear proteins extracted from leaves of salicylic acid- treated tobacco plants and water-treated control plants prepared using the method of Jensen et al., (1988). Protein/DNA complexes were analysed by polyacrylamide gel electrophoresis ( Figure 2).
  • the AB probe reacted to form complexes with nuclear proteins from both control and salicylic acid-treated plants ( Figure 2, lanes 2 & 3), but the complex was more predominant when extracts from salicylic acid-treated plants were used. No complexes were observed when the nuclear extracts were boiled or treated with proteinase K prior to reacting with the labelled probe, confirming that the complex is the result of a DNA/protein interaction.
  • the invention provides a substance which recognises the TCA motif or functional equivalents thereof.
  • the substance which recognises the TCA motif is proteinaceous. Typically it is a nuclear protein.
  • Such ubstances might have the ability of modulating the resp .siveness to stress of certain gene expression systems.
  • TCA motif is the specific binding site for the nuclear protein which binds to the beta-1 ,3-glucanase promoter fragment.
  • further gel retardation assays were carried out using a cloned synthetic 82 bp DNA fragment incorporating six copies of the TCA motif.
  • two 26bp complementary oligonucleotides incorporating two copies of the TCA sequence were synthesised, using a P armacia Gene Assembler Plus. The oligonucleotides were annealed and ligated into the BamHI site of p ⁇ C19.
  • pAG280.1 contained 3 copies of the annealed fragment and thus contained 6 copies of the TCA motif.
  • TCAx ⁇ Hindlll - Asp718 fragment
  • TCAx ⁇ fragment was 3' end labelled in the same manner as AB fragment.
  • the sequence of this DNA is presented in Figure 3 (and is identified as SEQ ID No. 3 in the attached sequence listing).
  • the invention provides a plant cell nuclear protein having a molecular weight of substantially 40 kDa as judged by SDS-PAGE and having the ability to recognise specifically the TCA motif or functional equivalents thereof.
  • the invention provides a method for detecting and/or assaying the stress to which a plant or plant cell is exposed comprising the steps of placing a reporter gene under the regulatory control of the novel regulatory sequence or functional equivalent thereof, inserting the reporter gene and regulatory sequence (in functional relationship) into a plant or plant cell and detecting or measuring the level of expression of the reporter gene in the plant or plant cell.
  • Insertion of the 10 bp novel regulatory element into a promoter which already confers some form of specificity (eg tissue specificity) and/or regulation may render that promoter stress inducible under the specific circumstances as defined by that promoter.
  • the CaMV 35S promoter A domain confers root tip specificity of expression in potatoes. Insertion of the TCA motif upstream of the A domain may result in a large induction of root tip specific expression under conditions of stress.
  • TCA motif upstream of the patatin promoter, the rubisco small sub ' unit promoter, the rape acyl carrier protein promoter or the wheat high molecular weight glutenin promoter may result in those promoters being highly inducible in tubers, leaves, seeds and endosperm respectively under conditions of stress.
  • increasing the level of TCA-1 protein in plants under conditions of stress may lead to enhanced induction of defence-related proteins or an increased response to stress-
  • One possible method of demonstrating the utility of this technique would be to clone the gene encoding the TCA-1 protein and use it to transform potato plants where it is expressed under the control of a fully active, constitutive CaMV 35S promoter.
  • the potato plant would then be co-transformed with a reporter gene cassette comprising a reporter gene (eg GUS, a reporter gene well known to those skilled in the art) under the joint control of any of the promoters detailed above and the TCA motif.
  • a reporter gene eg GUS, a reporter gene well known to those skilled in the art
  • GUS reporter gene
  • the TCA-1 protein is expressed continuously under the control of the CaMV 35S promoter.
  • the CaMV 35S promoter in the system described above is replaced with the WIN2 (potato wound inducible promoter 2)
  • GUS expression would be- induced to very high levels in a tissue specific manner, but only under conditions of stress. This is because the TCA-1 protein would only be synthesised when the WIN2 promoter is activated, the WIN2 promoter being itself stress- responsive.
  • Figure 1 shows the nucleotide sequence of the AB ragment
  • Figure 2 is an autoradiograph of a gel retardation assay.
  • FIG. 3 shows the nucleotide sequence of TCAx ⁇
  • FIGS 4 and 5 show autoradiographs of gel retardation assays
  • Figure 6 is a photograph of a South- estern blot.
  • This figure shows the results of an experiement to investigate the binding of tobacco (Samsun NN) leaf nuclear proteins to the barley glucanase (g19-9) AB fragment.
  • Lane 1 contains 1 ng of the AB probe alone.
  • Lane 2 contains 1ng of AB probe and 1Oug of nuclear extracts from salicylic acid-treated tobacco leaves.
  • Lane 3 contains Ing of AB probe and 10ug of nuclear extracts from water-treated (control) tobacco leaves.
  • the arrow indicates the position of the retarded complex.
  • Figure 3 This shows the DNA sequence of the cloned TCAx ⁇ fragment.
  • Lanes 1-8 contain 1ng of AB probe. Lane 1 is AB probe alone. Lanes 2-8 contain 10ug of nuclear extracts from salicylic acid- treated tobacco leaves. Lanes 3 and 4 contain 50x and 100x molar excess of unlabelled AB fragment. Lanes 5 and 6 contain 50x and 100x molar excess of an unlabelled 100bp fragment from the coding region of a barley beta-1 ,3-1 ,4- glucanase gene. Lanes 7 and 8 contain 50x and 100x molar excess of unlabelled TCAx ⁇ fragment. The arrow indicates the position of retarded complexes.
  • Lanes 1-8 contain 1ng of TCAx ⁇ probe. Lane 1 is TCAx ⁇ probe alone. Lanes 2-8 contain 1Oug of nuclear extracts from salicylic acid- treated tobacco leaves. Lanes 3 and 4 contain 50x and 100x molar excess of unlabelled TCAx ⁇ fragment. Lanes 5 and 6 contain 50x and 100x molar excess of an unlabelled 100bp fragment from the coding region of a barley beta- 1 ,3-1 ,4-glucanase gene. Lanes 7 and 8 contain 50x and 100x molar excess of unlabelled AB fragment. The arrow indicates the position of retarded complexes.
  • Lam E. et al., (1989). Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc. Natl. Acad. Sci. 86, 7890-7894.
  • a tobacco bZip transcription activator (TAF-1) binds to a G-box-like motif conserved in plant genes. EMBO J. J_0_, 1793-1802.
  • a virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene. Molecular Plant-Microbe Interactions J_, 107-112.
  • Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. Plant Cell 3, 809-818.

Abstract

L'invention se rapporte à un nouvel élément de régulation de gènes de plantes possédant pratiquement la séquence 5'-TCATCTTCTT-3' qui confère une réactivité au stress lors de l'expression de séquences codant la protéine, liées fonctionnellement. On décrit également des substances qui reconnaissent le nouvel élément de régulation. L'invention se rapporte à un procédé de régulation de l'expression des séquences codant la protéine et du contrôle du stress auquel une plante est exposée.
PCT/GB1993/000071 1992-01-14 1993-01-14 Nouvel element de regulation de genes de plantes WO1993014213A1 (fr)

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GB929200710A GB9200710D0 (en) 1992-01-14 1992-01-14 Novel plant gene regulatory element
GB9200710.3 1992-01-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6329952B1 (en) 1999-02-17 2001-12-11 Anritsu Company Automobile radar antenna alignment system using transponder and lasers

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330479A2 (fr) * 1988-02-26 1989-08-30 Lubrizol Genetics Inc. Elément régulateur universel de plantes fonctionnant sous influence du stress
EP0332104A2 (fr) * 1988-03-08 1989-09-13 Ciba-Geigy Ag Sèquences d'ADN et gènes chimiquement regulables, et leur emploi
WO1989012230A1 (fr) * 1988-05-20 1989-12-14 The Salk Institute Biotechnology/Industrial Associ Analyse selective de produits agrochimiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330479A2 (fr) * 1988-02-26 1989-08-30 Lubrizol Genetics Inc. Elément régulateur universel de plantes fonctionnant sous influence du stress
EP0332104A2 (fr) * 1988-03-08 1989-09-13 Ciba-Geigy Ag Sèquences d'ADN et gènes chimiquement regulables, et leur emploi
WO1989012230A1 (fr) * 1988-05-20 1989-12-14 The Salk Institute Biotechnology/Industrial Associ Analyse selective de produits agrochimiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS vol. 92 , 1991, Philadelphia, PA, US; abstract no. 45503, JUTIDAMRONGPHAN,W., ET AL. 'Induction of a beta-1,3-glucanase in barley in response to infection by fungal pathogens' *
PLANT MOLECULAR BIOLOGY. vol. 15, 1990, DORDRECHT, THE NETHERLANDS. pages 941 - 946 OHME-TAKAGI, M., ET AL. 'Structure and expression of a tobacco beta-1,3-glucanase gene' *
THE PLANT CELL. vol. 2, no. 12, December 1990, ROCKVILLE, MD, USA. pages 1131 - 1143 CASTRESENA, C., ET AL. 'Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia beta-1,3-glucanase gene' *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6329952B1 (en) 1999-02-17 2001-12-11 Anritsu Company Automobile radar antenna alignment system using transponder and lasers
US6335705B1 (en) 1999-02-17 2002-01-01 Anritsu Company Automotive radar antenna alignment system

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