WO1993024639A1 - Expression of genes in transgenic plants - Google Patents

Expression of genes in transgenic plants Download PDF

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Publication number
WO1993024639A1
WO1993024639A1 PCT/GB1993/001099 GB9301099W WO9324639A1 WO 1993024639 A1 WO1993024639 A1 WO 1993024639A1 GB 9301099 W GB9301099 W GB 9301099W WO 9324639 A1 WO9324639 A1 WO 9324639A1
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promoter
gene
exogenous
malic enzyme
plant
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PCT/GB1993/001099
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French (fr)
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Jörg SCHAAF
Michael Herbert Walter
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Zeneca Limited
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Publication of WO1993024639A1 publication Critical patent/WO1993024639A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible

Definitions

  • the present invention relates to the regulation of gene expression in transgenic plants.
  • it is concerned with the isolation and use of DNA sequences which control the expression of foreign genes in response to wounding and elicitor.
  • An object of the present invention is to provide a promoter for use in the regulation of exogenous genes in plants.
  • a DNA construct for use in transforming plant cells which comprises an exogenous coding sequence under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology to a promoter of a gene involved in respiration and/or intracellular pH regulation.
  • Malic enzyme is found in several compartments of the plant cell such as the cytosol, the chloroplast and the mitochondria.
  • the gPvMEl gene product is cytosolic because approximately 50 N-terminal amino acids which are found in other malic enzyme forms corresponding to signal peptide ⁇ in chloroplast ME are missing.
  • the cytosolic form is believed to be involved in regulation of intracellular pH and in other metabolic processes. It may, in analogy to animal systems (Ayala et.al. FEBS Lett. 202, 102-106 (1986)) also be involved in detoxification and defence mechanisms and the expression of the plant gene may be regulated accordingly.
  • Evidence for the identity of the gPvMEl gene as malic enzyme is the significant degree of homology between the malic enzyme encoded by this gene and those from maize, mouse, rat and man. It also shows between 72 and 79% homology to malic - A -
  • malic enzyme mRNA is expressed preferentially in vascular tissue. Expression of malic enzyme mRNA could also be induced by wounding, elicitor and other defence-related stimuli.
  • the invention also comprises the use the promoters of malic enzyme and similar genes to control the expression of novel and exogenous proteins and genes in response to such stimuli.
  • promoters having similar action to that of the malic enzyme promoter, for use in the invention may be derived from genes such as chitinase, phenylalanine ammonia lya ⁇ e and chalcone synthase. Such promoters may be isolated from genomic libraries by the use of cDNA probes, as has been done in the case of pMEl. We particularly prefer to use the promoter of the malic enzyme gene.
  • the present invention provides a gene promoter comprising the promoter of a gene encoding malic enzyme.
  • the promoter may be isolated from any source thereof, including plants, animals and prokaryote ⁇ .
  • the invention provides a gene promoter having the nucleotide sequence of bases 1 to approximately 2640 shown in Figure 2. The precise location of the transcription start site is still to be determined.
  • the invention includes modifications and the use of only particular parts of the the said sequence which, while retaining sufficient homology to the sequence shown in order to maintain functionality, enhance the activity of the promoter or alter its tissue-specificity or response to external stimuli.
  • the invention further provides a recombinant gene construct comprising, in sequence, a promoter according to the invention, a coding region and a terminator sequence.
  • the invention also provides a recombinant genome comprising the said construct.
  • the downstream (3') terminator sequences may also be derived from the malic enzyme gene or they may be derived from other genes. Many possibilities are available from the literature: the selection of the terminator being of rather lesser importance.
  • 'exogenous coding sequence' we indicate a sequence of DNA, other than that which follows the promoter region in the natural ME gene, that is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase.
  • RNA which affects the biochemistry of the cell: it may for example be mRNA which is translated into protein by ribosomes; or antisen ⁇ e RNA which inhibits the translation of mRNA complementary (or otherwise related) to it into protein.
  • ribosomes ribosomes
  • antisen ⁇ e RNA which inhibits the translation of mRNA complementary (or otherwise related) to it into protein.
  • any exogenous coding sequence may be used in the present invention.
  • this protein may be of bacterial origin (such a ⁇ enzyme ⁇ involved in poly ⁇ accharide etaboli ⁇ m and cell wall metaboli ⁇ m) , of eukaryotic origin ( ⁇ uch a ⁇ pharmaceutically active polypeptides) or of plant origin (such as other enzymes involved in re ⁇ piration or genes or parts thereof in sen ⁇ e and anti ⁇ ense orientation.
  • bacterial origin such as a ⁇ enzyme ⁇ involved in poly ⁇ accharide etaboli ⁇ m and cell wall metaboli ⁇ m
  • eukaryotic origin ⁇ uch a ⁇ pharmaceutically active polypeptides
  • plant origin such as other enzymes involved in re ⁇ piration or genes or parts thereof in sen ⁇ e and anti ⁇ ense orientation.
  • malic enzyme gene promoter to respond to exogenously supplied ethylene, glutathione, elicitor and wounding.
  • exogenous coding sequences A wide variety of exogenous coding sequences is known from the literature, and the present invention is applicable to these.
  • the promoter may be used to express genes such as antiviral protein, antifungal protein, or antibacterial proteins as well as in ⁇ ecticidal proteins.
  • the exogenous gene may code for RNA that interferes with the function of any kind of mRNA produced by the plant cell: for example, antisen ⁇ e RNA complementary to mRNA for gene involved in pathogen ⁇ u ⁇ ceptibility, or genes involved in plant development.
  • vector ⁇ and con ⁇ truct ⁇ of the present invention will be described in more detail in the Examples below. For convenience it will be generally found suitable to use promoter sequences (upstream - i.e. 5' - of the coding sequence of the gene) of between 100 and 2000 bases in length.
  • Plant cells according to the invention may be transformed with construct ⁇ of the invention according to a variety of known methods (Agrobacterium Ti plasmid ⁇ , electroporation, microinjection, microprojectile bombardment, etc).
  • the tran ⁇ formed cell ⁇ may then be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in thi ⁇ way.
  • tran ⁇ formation and regeneration methods employed are not particularly germane to this invention and may simply be selected from those available from the literature
  • genetically modified plants according to the pre ⁇ ent invention include tomatoes, fruit ⁇ ⁇ uch a ⁇ mangoes, peaches, apple ⁇ , pear ⁇ , strawberries, bananas and melons; and field crops such as maize (corn), sunflowers, sugarbeet, canola, and small grained cereals such as wheat, barley and rice, ornamental plants such carnations, petunias, roses, chrysanthemums etc.
  • Plants produced by the proces ⁇ of the invention may contain more than one recombinant con ⁇ truct.
  • a ⁇ well a ⁇ one or more con ⁇ tructs containing the malic enzyme promoter they may contain a wide variety of other recombinant con ⁇ truct ⁇ , for example con ⁇ tructs having different effects on respiration and intracellular and intra- organellar pH.
  • a further aspect of the present invention is a proce ⁇ of activating exogenou ⁇ coding ⁇ equence ⁇ in plant ⁇ under the control of the malic enzyme promoter which compri ⁇ e ⁇ the application of exogenou ⁇ ethylene.
  • Genomic clones representing one individual gene has been isolated and characterised by DNA sequence analy ⁇ i ⁇ .
  • the clone gPvMEl repre ⁇ ent ⁇ a gene with exon ⁇ equence identical to the clone pMEl except for very few mismatches which can be ascribed to cultivar differences.
  • the genomic clones described in the Examples cover 5' end of the coding region and the complete transcriptional initiation region of the malic enzyme gene.
  • Figure 1 show ⁇ a re ⁇ triction map of the clone gPvMEl and the ⁇ tructure of the ME gene.
  • Figure 2 ⁇ how ⁇ the nucleotide ⁇ equence of approximately 2.6kb of promoter fragment, functionally investigated in transgenic tobacco, and some 3' adjacent sequence.
  • Figure 3 illu ⁇ trate ⁇ a scheme for construction of a plant transformation vector EXAMPLE
  • a library was constructed from bean (Phaseolus vulgari ⁇ var. Processor) genomic DNA which was partially dige ⁇ ted with Mbol and cloned into lambda EMBL3 .
  • the library wa ⁇ ⁇ creened with a pMEl cDNA fragment of 1.4 kb and po ⁇ itive phage ⁇ were purified by four ⁇ ucces ⁇ ive cycles of plaque purification.
  • One positive clone was isolated. This clone was characterised by DNA ⁇ equence analysi ⁇ . The overall ⁇ tructure of the gene is shown in Figure 1.
  • the promoter sequence of the malic enzyme gene was determined and is shown in Figure 2 together with leader sequence and transcription and translation initiation regions down to the first intron of the coding region.
  • the vector pPvMEl Prom-GUSl (from Example 1.3) was transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologist ⁇ ) and i ⁇ u ⁇ ed to transform tobacco plants. Transformation of tobacco leaf disk ⁇ follow ⁇ ⁇ tandard protocol ⁇ . Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.
  • a number of independently transformed tobacco plant ⁇ tran ⁇ genic in the pPvMEl Prom-GUSl construct were analy ⁇ ed by fluorimetric and hi ⁇ tochemical a ⁇ says for the expression of the GUS reporter gene driven by the PvMEl promoter.
  • Base level activity in particular tissues is high in stems, lower in roots and very low in leaves. Histochemical staining using the X-gluc ⁇ ub ⁇ trate of GAS ⁇ howed ⁇ trong expre ⁇ sion in the vascular bundles of the stem, particularly at the inner parts corresponding to the xyle regions. ME gene activation by external stimuli was measured in leaves. Wounding of leaves activates
  • ME gene expres ⁇ ion Thi ⁇ induction can be ⁇ trongly enhanced by the addition of glutathione, elicitor from the fungal bean pathogen Colletotrichum lindemuthianum, mercuric chloride, cellula ⁇ e and Etephone (a compound which relea ⁇ es ethylene).
  • glutathione elicitor from the fungal bean pathogen Colletotrichum lindemuthianum, mercuric chloride, cellula ⁇ e and Etephone (a compound which relea ⁇ es ethylene).
  • the preferential localisation of expre ⁇ sion is in leaf veins and spread acros ⁇ the leaf rather than being adjacent to the wound or inducer application ⁇ ite.

Abstract

A promoter sequence isolated from a malic enzyme gene, having the nucleotide sequence shown in Figure 2, is used to drive expression of an exogenous gene in a recombinant plant genome. Expression of an exogenous gene placed under the control of the promoter is inducible by the application of external chemical stimuli.

Description

EXPRESSION OF GENES IN TRANSGENIC PLANTS
The present invention relates to the regulation of gene expression in transgenic plants. In particular it is concerned with the isolation and use of DNA sequences which control the expression of foreign genes in response to wounding and elicitor.
The ability to isolate and manipulate plant genes has opened the way to gain understanding about the mechanisms involved in the regulation of plant gene expression. This knowledge is important for the exploitation of genetic engineering techniques to practical problems such as the expression of genes in genetically manipulated crop plants exhibiting improved quality and production characteristics.
Many examples have been reported in the literature of plant DNA sequences which have been used to drive the expression of foreign genes in plants. In most instances the regions immediately 5' to the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. They may be derived from plant DNA; or from other sources, eg, viruses. It has been demonstrated that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. The types of regulation exemplified include tissue- specificity, regulation by external factors such as light, heat treatment, chemicals, hormones, and developmental regulation. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants.
These experiments have largely been carried out using gene fusions between the promoter sequences and foreign structural genes such as bacterial genes, etc. This has led to the identification of useful promoter sequences. An object of the present invention is to provide a promoter for use in the regulation of exogenous genes in plants.
According to the present invention we provide a DNA construct for use in transforming plant cells which comprises an exogenous coding sequence under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology to a promoter of a gene involved in respiration and/or intracellular pH regulation.
We further provide novel plant cells, and plants transformed with constructs according to the present invention. The invention is illustrated hereinafter by the use of tobacco as a model plant species.
We further provide a process for stimulating the expression of exogenous coding sequences in plants by applying exogenous stimuli such as ethylene, fungal elicitor, glutathione or other chemical stimuli to plants transformed with constructs according to the invention.
In work leading to the present invention we have identified a gene which expresses an enzyme involved in a wide range of metabolic processes from photosynthetic carbon fixation in C4 plants to intracellular pH regulation. The protein coding region of the gene in question is encoded completely in the clone pMEl disclosed by Walter et al (Proc. Natl. Acad. Sc. USA.; jJ^, 5546-5550 (1988) Plant Mol Biol 15:525-526, (1990)). Hereinafter this gene is referred to as the malic enzyme or ME gene. It is known that the protein encoded by the malic enzyme gene, which is the subject of the present invention is involved in the oxidative decarboxylation of malate to pyruvate. We now disclose the structure of this gene and its transcriptional control sequences, in particular its promoter.
Malic enzyme is found in several compartments of the plant cell such as the cytosol, the chloroplast and the mitochondria.
It is believed that the gPvMEl gene product is cytosolic because approximately 50 N-terminal amino acids which are found in other malic enzyme forms corresponding to signal peptideε in chloroplast ME are missing. The cytosolic form is believed to be involved in regulation of intracellular pH and in other metabolic processes. It may, in analogy to animal systems (Ayala et.al. FEBS Lett. 202, 102-106 (1986)) also be involved in detoxification and defence mechanisms and the expression of the plant gene may be regulated accordingly. Evidence for the identity of the gPvMEl gene as malic enzyme is the significant degree of homology between the malic enzyme encoded by this gene and those from maize, mouse, rat and man. It also shows between 72 and 79% homology to malic - A -
enzyme from other plants such as poplar and Flaveria trinervia.
We have shown in work leading to this invention that malic enzyme mRNA is expressed preferentially in vascular tissue. Expression of malic enzyme mRNA could also be induced by wounding, elicitor and other defence-related stimuli.
Thus, the invention also comprises the use the promoters of malic enzyme and similar genes to control the expression of novel and exogenous proteins and genes in response to such stimuli.
Other promoters, having similar action to that of the malic enzyme promoter, for use in the invention may be derived from genes such as chitinase, phenylalanine ammonia lyaεe and chalcone synthase. Such promoters may be isolated from genomic libraries by the use of cDNA probes, as has been done in the case of pMEl. We particularly prefer to use the promoter of the malic enzyme gene.
Thus the present invention provides a gene promoter comprising the promoter of a gene encoding malic enzyme. The promoter may be isolated from any source thereof, including plants, animals and prokaryoteε.
Further, the invention provides a gene promoter having the nucleotide sequence of bases 1 to approximately 2640 shown in Figure 2. The precise location of the transcription start site is still to be determined. The invention includes modifications and the use of only particular parts of the the said sequence which, while retaining sufficient homology to the sequence shown in order to maintain functionality, enhance the activity of the promoter or alter its tissue-specificity or response to external stimuli.
The invention further provides a recombinant gene construct comprising, in sequence, a promoter according to the invention, a coding region and a terminator sequence.
The invention also provides a recombinant genome comprising the said construct. The downstream (3') terminator sequences may also be derived from the malic enzyme gene or they may be derived from other genes. Many possibilities are available from the literature: the selection of the terminator being of rather lesser importance. By the term 'exogenous coding sequence' we indicate a sequence of DNA, other than that which follows the promoter region in the natural ME gene, that is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase. Functional RNA is RNA which affects the biochemistry of the cell: it may for example be mRNA which is translated into protein by ribosomes; or antisenεe RNA which inhibits the translation of mRNA complementary (or otherwise related) to it into protein. In principle any exogenous coding sequence may be used in the present invention.
Where the exogenous coding sequence codes for mRNA for a protein, this protein may be of bacterial origin (such aε enzymeε involved in polyεaccharide etaboliεm and cell wall metaboliεm) , of eukaryotic origin (εuch aε pharmaceutically active polypeptides) or of plant origin (such as other enzymes involved in reεpiration or genes or parts thereof in senεe and antiεense orientation. Of particular interest is the ability of the malic enzyme gene promoter to respond to exogenously supplied ethylene, glutathione, elicitor and wounding.
A wide variety of exogenous coding sequences is known from the literature, and the present invention is applicable to these. The promoter may be used to express genes such as antiviral protein, antifungal protein, or antibacterial proteins as well as inεecticidal proteins. As well as functional mRNA, the exogenous gene may code for RNA that interferes with the function of any kind of mRNA produced by the plant cell: for example, antisenεe RNA complementary to mRNA for gene involved in pathogen εuεceptibility, or genes involved in plant development.
The construction of vectorε and conεtructε of the present invention will be described in more detail in the Examples below. For convenience it will be generally found suitable to use promoter sequences (upstream - i.e. 5' - of the coding sequence of the gene) of between 100 and 2000 bases in length. Plant cells according to the invention may be transformed with constructε of the invention according to a variety of known methods (Agrobacterium Ti plasmidε, electroporation, microinjection, microprojectile bombardment, etc). The tranεformed cellε may then be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in thiε way. The tranεformation and regeneration methods employed are not particularly germane to this invention and may simply be selected from those available from the literature Examples of genetically modified plants according to the preεent invention include tomatoes, fruitε εuch aε mangoes, peaches, appleε, pearε, strawberries, bananas and melons; and field crops such as maize (corn), sunflowers, sugarbeet, canola, and small grained cereals such as wheat, barley and rice, ornamental plants such carnations, petunias, roses, chrysanthemums etc.
Plants produced by the procesε of the invention may contain more than one recombinant conεtruct. Aε well aε one or more conεtructs containing the malic enzyme promoter, they may contain a wide variety of other recombinant conεtructε, for example conεtructs having different effects on respiration and intracellular and intra- organellar pH. A further aspect of the present invention is a proceεε of activating exogenouε coding εequenceε in plantε under the control of the malic enzyme promoter which compriεeε the application of exogenouε ethylene. We now deεcribe the iεolation of genomic cloneε from a bean library encoding the malic enzyme gene and related εequences. Genomic clones representing one individual gene has been isolated and characterised by DNA sequence analyεiε. The clone gPvMEl repreεentε a gene with exon εequence identical to the clone pMEl except for very few mismatches which can be ascribed to cultivar differences. The genomic clones described in the Examples cover 5' end of the coding region and the complete transcriptional initiation region of the malic enzyme gene. The clone gPvMEl 1-4.1HH haε been deposited at the National Collections of Industrial and Marine Bacteria (NCIB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, on 2nd April 1992 under the reference NCIB Number 40498. The invention will be further described with reference to the following drawings, in which:
Figure 1 showε a reεtriction map of the clone gPvMEl and the εtructure of the ME gene.
Figure 2 εhowε the nucleotide εequence of approximately 2.6kb of promoter fragment, functionally investigated in transgenic tobacco, and some 3' adjacent sequence. Figure 3 illuεtrateε a scheme for construction of a plant transformation vector EXAMPLE
1.1 isolation of gPvMEl
A library was constructed from bean (Phaseolus vulgariε var. Processor) genomic DNA which was partially digeεted with Mbol and cloned into lambda EMBL3 . The library waε εcreened with a pMEl cDNA fragment of 1.4 kb and poεitive phageε were purified by four εuccesεive cycles of plaque purification. One positive clone was isolated. This clone was characterised by DNA εequence analysiε. The overall εtructure of the gene is shown in Figure 1.
1.2 Characterisation of the malic enzyme gene promoter The promoter sequence of the malic enzyme gene was determined and is shown in Figure 2 together with leader sequence and transcription and translation initiation regions down to the first intron of the coding region. 1.3 Construction of plant transformation vector - pPvMEl Prom-GUSl gPvMEl 1-4.1HH, a plasmid subclone of gPVMEl compriεing the promoter and part of the coding region was uεed to iεolate a 2821 bp Hindlll-BcllI fragment containing approximately 2.65 kb of the PvMEl promoter and leader εequences down to the Bell site immediately upstream of the ATG translation εtart codon. Thiε fragment waε inserted into the binary plant transformation vector pBl 101.4 between the Hindlll and the BamHI site of the polylinker, which has an overhang compatibility with Bell cut DNA. This conεtruct was named pPvMEl Prom-GUSl. Fusion borders have been confirmed by DNA sequencing.
1.4 Generation of transformed plants
The vector pPvMEl Prom-GUSl (from Example 1.3) was transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologistε) and iε uεed to transform tobacco plants. Transformation of tobacco leaf diskε followε εtandard protocolε. Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.
1.5 Analysis of transformed plants
A number of independently transformed tobacco plantε tranεgenic in the pPvMEl Prom-GUSl construct were analyεed by fluorimetric and hiεtochemical aεsays for the expression of the GUS reporter gene driven by the PvMEl promoter.
Base level activity in particular tissues is high in stems, lower in roots and very low in leaves. Histochemical staining using the X-gluc εubεtrate of GAS εhowed εtrong expreεsion in the vascular bundles of the stem, particularly at the inner parts corresponding to the xyle regions. ME gene activation by external stimuli was measured in leaves. Wounding of leaves activates
ME gene expresεion. Thiε induction can be εtrongly enhanced by the addition of glutathione, elicitor from the fungal bean pathogen Colletotrichum lindemuthianum, mercuric chloride, cellulaεe and Etephone (a compound which releaεes ethylene). The preferential localisation of expreεsion is in leaf veins and spread acrosε the leaf rather than being adjacent to the wound or inducer application εite.

Claims

A DNA conεtruct for uεe in transforming plant cells which compriseε an exogenouε coding εequence under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology to a promoter of a gene involved in respiration and/or intracellular pH regulation.
A DNA construct as claimed in claim 1 in which the promoter iε the promoter of a gene encoding malic enzyme.
A malic enzyme gene promoter, having the nucleotide εequence of bases 1 to approximately 2640 shown in Figure 2 and modificationε of the εaid εequence which retain sufficient homology to the sequence shown in order to maintain or enhance the functionality of the promoter.
A plant cell and plantε transformed with the construct claimed in claim 1.
A procesε for εtimulating the expression of exogenous coding sequenceε in plantε compriεing providing a plant the genome of which has been stably transformed with the construct claimed in claim 1 and applying to the plant an exogenous stimuluε to induce expression of the exogenous gene under control of the promoter.
6. A process as claimed in claim 5, in which the exogenous stimulus is ethylene, fungal elicitor, glutathione.
7. A recombinant plant genome comprising a malic enzyme promoter located to drive expression of an exogenous gene.
8. The promoter εequence of a malic enzyme gene contained within the insert in clone gPvMEl 1-4.IHH which haε been depoεited at the National Collections of Industrial and Marine Bacteria (NCIMB), 23 St. Machar Drive,
Aberdeen AB2 IRY, Scotland, on 2nd April 1992 under the Accession Number NCIMB 40498.
PCT/GB1993/001099 1992-05-29 1993-05-27 Expression of genes in transgenic plants WO1993024639A1 (en)

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EP0690919A4 (en) * 1992-10-15 1995-11-16 Gen Hospital Corp Crucifer acc synthase and uses thereof
US6410241B1 (en) 1999-03-24 2002-06-25 Board Of Regents, The University Of Texas System Methods of screening open reading frames to determine whether they encode polypeptides with an ability to generate an immune response

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JOURNAL OF BIOLOGICAL CHEMISTRY vol. 265, no. 14, 1990, BALTIMORE, MD US pages 7927 - 7932 STRUVE, I., ET AL. 'Structure and function of the promoter of the carrot V-type H+ -ATPase catalytic subunit gene' *
PLANT MOLECULAR BIOLOGY. vol. 15, 1990, DORDRECHT, THE NETHERLANDS. pages 525 - 526 WALTER, M.H., ET AL. 'Extensive sequence similarity of the bean CAD4 (cinnamyl-alcohol dehydrogenase) to a malic enzyme' cited in the application *
PLANT PHYSIOLOGY. vol. 96, 1991, ROCKVILLE, MD, USA. pages 1385 - 1386 VAN DOORSSELAERE, J., ET AL. 'Nucleotide sequnece of a cDNA encoding malic enzyme from poplar' *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 85, no. 8, 1988, WASHINGTON US pages 5546 - 5550 WALTER, M.H., ET AL. 'Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor' cited in the application *

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EP0690919A4 (en) * 1992-10-15 1995-11-16 Gen Hospital Corp Crucifer acc synthase and uses thereof
EP0690919A1 (en) * 1992-10-15 1996-01-10 The General Hospital Corporation Crucifer acc synthase and uses thereof
US5908971A (en) * 1992-10-15 1999-06-01 The General Hospital Corporation Crucifer ACC synthase and uses thereof
US6410241B1 (en) 1999-03-24 2002-06-25 Board Of Regents, The University Of Texas System Methods of screening open reading frames to determine whether they encode polypeptides with an ability to generate an immune response
US6900018B2 (en) 1999-03-24 2005-05-31 Board Of Regents, The University Of Texas System Method of screening for a biological response using linear and circular expression elements
US7018833B2 (en) 1999-03-24 2006-03-28 Board Of Regents, The University Of Texas System Linear and circular expression elements
US7049098B2 (en) 1999-03-24 2006-05-23 Board Of Regents, The University Of Texas System Method of vaccination comprising linear and circular expression elements

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