WO1999007857A1 - Enzymes degradant la pectine - Google Patents

Enzymes degradant la pectine Download PDF

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Publication number
WO1999007857A1
WO1999007857A1 PCT/GB1998/002350 GB9802350W WO9907857A1 WO 1999007857 A1 WO1999007857 A1 WO 1999007857A1 GB 9802350 W GB9802350 W GB 9802350W WO 9907857 A1 WO9907857 A1 WO 9907857A1
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nucleic acid
plant
cell
sequence
pectate lyase
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PCT/GB1998/002350
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English (en)
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Keith Roberts
Concepcion Domingo Carrasco
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Plant Bioscience Limited
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Priority to AU86390/98A priority Critical patent/AU8639098A/en
Publication of WO1999007857A1 publication Critical patent/WO1999007857A1/fr

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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
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8226—Stem-specific, e.g. including tubers, beets
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • 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
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates broadly to enzymes having pectate lyase activity, genes encoding such enzymes, and methods and materials for isolating and utilising them.
  • Pectate lyases (E.C. 4.2.2.2) have previously been described as microbial extracellular enzymes that assist pathogenesis by cleaving of polygalacturonate blocks in the plant host cell wall (Davis et al . 1984, Collmer and Keen 1986) .
  • a pectate lyase is an enzyme capable of degrading pectin via the hydrolysis of ⁇ -1, 4-galacturonosyl residues by jS-elimination. This results in an unsaturated C-4 - C-5 bond in the galacturonosyl moiety at the non- reducing end of the polysaccharide produced at the cleavage site (Rombouts and Pilnik 1980) .
  • pectin- degrading enzymes pectin lyases
  • pectate lyase is distinguished by its preference for a glycosidic linkage next to a free carboxyl group rather than to an esterified carboxyl group and by its pH optimum (Pilnik 1990) .
  • Microbial pectate lyases show calcium dependence.
  • Microbial pectate lyases usually exist as multiple, independently-regulated isozymes that are 27 to 80 % identical in amino acid sequence, and have subtle differences in substrate specificity (Preston et al . 1992) .
  • cDNAs with homology to microbial pectate lyases have been reported in several plant species, principally in relation to pollen tube growth.
  • Pectate lyase-like genes are expressed in transmitting tissue (McCormick 1991) together with other hydrolases, 3-glucanase, chitinase, proteinase inhibitor, proline-rich proteins and hydroxyproline-rich glycoproteins (Gasser and Robinson- Beers 1993) .
  • LAT 56 and LAT 59 show homology to Erwinia pectate lyases (Wing et al 1989) and are each represented as single-copies in the tomato genome.
  • LAT56 and LAT59 sequences are strikingly similar to the major ragweed pollen allergen (Ambal) (Rafner et al . 1991), to a cDNA clone LMP131 expressed preferentially in anthers of lily (Kim et al . 1994) and to a tomato cDNA that is predominantly expressed in pistils but also detected at low levels in roots (Budelier et al . 1990) .
  • Pectate lyase activity has been demonstrated by Taniguchi et al (1995) . However this was only in pollen, and not in somatic plant cells; nor was it detected in extracts, but only after purification.
  • Authentic plant pectate lyases may be useful, inter alia , for modifying plant cell walls, either in vivo (e.g. to alter storage properties or digestibility) or in vi tro (e.g. as a treatment step during brewing) .
  • the present inventors have used a model system based around mesophyll cells from the leaves of Zinnia elegans c . Envy (see Roberts and Haigler 1994, Stacey et al . 1995) to identify a novel gene with sequence homology to pectate lyase.
  • the gene hereinafter ⁇ ZePel'
  • ⁇ ZePel' has homology to both the pectate lyase-like plant sequences and to the microbial enzymes.
  • pectate lyase activity has been demonstrated in populations of both (a) elongating and (b) differentiating cells following treatment with auxin suggesting a general, widely applicable, utility in the modification of plant cell wall architecture.
  • the mRNA encoding the enzyme is up-regulated in vi tro during both cell elongation and cell differentiation in response to auxin, but in si tu hybridisation suggests that in Zinnia plants it is associated specifically with vascular bundles and shoot primordia rather than with all elongating cells.
  • ZePel protein has pectate lyase activity by using bacterially expressed recombinant enzyme.
  • the recombinant enzyme has been characterised for calcium dependence, Michaelis-Menten constant and pH optimum.
  • the activity of ZePel has been demonstrated at room temperature and within the range of physiological pH. This may be contrasted with the pollen pectate lyase of Taniguchi et al (1995) which apparently had an optimum temperature and pH of 60-70°C and pH 10.
  • nucleic acid molecule encoding a plant pectate lyase wherein the pectate lyase enzyme is obtainable from a plant somatic cell and/or a prokaryotic host transformed with said nucleic acid molecule.
  • Nucleic acid molecules also referred to as nucleic acids
  • vectors may be provided isolated and/or purified from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or origin other than the sequence encoding a polypeptide with the required function.
  • Nucleic acid according to the present invention may include cDNA, RNA, genomic DNA and may be wholly or partially synthetic. Obviously nucleic acid molecules comprising, in addition to those consisting of, the stated sequences are included. The term "isolate" encompasses all these possibilities. Where a DNA sequence is specified, e.g. with reference to a Figure, unless context requires otherwise the RNA equivalent, with U substituted for T where it occurs, is encompassed.
  • the nucleic acid molecule has the nucleotide sequence shown in Seq ID No 1, optionally excluding non-coding regions as appropriate.
  • the nucleotide sequence of the ZePel cDNA (Seq ID No 1) and its predicted amino acid sequence (Hereinafter Seq ID No 2) are presented in Figure 1. Without the polyA tail, the cDNA is 1440 nucleotides long and contains an open reading frame (ORF) for a protein of 401 amino acid residues, beginning with the ATG initiation codon at position 28 and ending with a TGA stop codon at position 1230. The calculated molecular mass for this protein is 44,406 D and the pi is 8.15.
  • the BsPel has three aspartic acid residues in the putative active site cleft: Asp 184 is a ligand to the putative active site calcium ion, the others being Asp 223 and 227.
  • PelE has three Asps also at the active site but PelC has two Asps and a Glu, and the cleft is not so pronounced (Yoder et al .
  • a nucleic acid molecule being a mutant, variant, derivative or allele of the nucleic acid of the first aspect .
  • Preferred mutants, variants, derivatives and alleles are those which encode a product which is homologous to the enzyme encoded by Seq ID No 1, and wherein the product retains all or part of the pectate lyase activity of that enzyme .
  • Particularly desirable mutations may be those which introduce or destroy restriction sites in the sequence.
  • Other mutations may be to remove or introduce Met codons ; signal sequences; putative glycosylation or cleavage sites etc. All of these modifications may facilitate expression of the nucleic in particular hosts or circumstances.
  • Particularly envisaged are the variants discussed hereinafter lacking N-terminal sequence, preferably corresponding to the putative mature protein.
  • Changes to a sequence, to produce a mutant, variant or derivative may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.
  • changes to the nucleic acid which make no difference to the encoded amino acid sequence i.e. 'degeneratively equivalent'
  • Methods for achieving such changes e.g. mutant PCR primers
  • homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine . Similarity may be as defined and determined by the TBLASTN program, of Altschul et al . (1990) J. Mol . Biol .
  • BestFit which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, Wisconsin 53711) . BestFit makes an optimal alignment of the best segment of similarity between two sequences. Optimal alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman
  • altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptides conformation.
  • homologs in having a few non-conservative substitutions.
  • substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide' s three dimensional structure.
  • non-conservative substitutions even in or around the active site may confer slightly advantageous properties on the peptide: in the present context that may mean an altered substrate specificity, increased turnover number, altered pH dependence or temperature optimum etc.
  • a mutant, variant or derivative amino acid sequence in accordance with the present invention may include within the 401 amino acid sequence shown in Figure 1, a single amino acid change with respect to the sequence shown in Figure 1, or 2, 3, 4, 5, 6, 7, 8, or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater than about 50, 60, 70, 80 or 90 changes.
  • a mutant, variant or derivative amino acid sequence may of course include additional amino acids at the C-terminus and/or N-ter inus.
  • a method of identifying and cloning a pectate lyase homolog or allele from a plant species which method employs a nucleotide sequence derived from that shown in Seq ID No 1.
  • nucleotide sequence used in the identification is taken from a portion of Seq ID No 1 not being identical to known pectate lyases, such that molecules can be identified which could not have been identified prior to the work of the present inventors . Even if portions of the sequence are used which are homologous to known pectate lyases, the differences may still mean that novel pectate lyases (particularly those expressed in somatic plant cells, more particularly parenchyma) may be more readily identified.
  • nucleotide sequence information provided herein, or any part thereof may be used in a data-base search to find homologous sequences, expression products of which can be tested for ability to degrade pectins.
  • an allele or homologue in accordance with the present invention is also obtainable by means of a method which includes providing a preparation of nucleic acid, e.g. from arabidopsis, providing a nucleic acid molecule having a nucleotide sequence shown in or complementary to a nucleotide sequence shown in Seq ID No 1, preferably from within the coding sequence (i.e. coding for an amino acid sequence shown in Figure 1) , contacting nucleic acid in said preparation with said nucleic acid molecule under conditions for hybridisation of said nucleic acid molecule to any said gene or homologue in said preparation, and identifying said gene or homologue if present by its hybridisation with said nucleic acid molecule .
  • Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.
  • Test nucleic acid may be provided from a cell as genomic DNA, cDNA or RNA, or a mixture of any of these, preferably as a library in a suitable vector.
  • genomic DNA may allow the identification of non-transcribed regions of DNA e.g. promoter or other control elements. These are discussed in more detail later.
  • Binding of a probe to target nucleic acid may be measured using any of a variety of techniques at the disposal of those skilled in the art.
  • probes may be radioactively, fluorescently or enzymatically labelled.
  • Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR (see below), RN'ase cleavage and allele specific oligonucleotide probing.
  • Probes for use in these methods optionally labelled, form one part of this aspect of the invention.
  • preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further. It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low. Using these conditions nucleic acid libraries, e.g. cDNA libraries representative of expressed sequences, may be searched.
  • the screening is carried out at about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5 X SSC, or a temperature of about 50 °C and a salt concentration of about 2 X SSPE. These conditions will allow the identification of sequences which have a substantial degree of homology
  • Suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na 2 HP0 4 , pH 7.2 , 6.5% SDS , 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65 °C in 0.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0.1X SSC, 0.1% SDS.
  • Hybridisation is generally followed by identification of successful hybridisation and isolation of nucleic acid which has hybridised, which may involve one or more steps of PCR (see below) .
  • hybridisation of nucleic acid molecule to an allele or homologue may be determined or identified indirectly, e.g using a nucleic acid amplification reaction, particularly the polymerase chain reaction (PCR) .
  • PCR requires the use of two primers to specifically amplify target nucleic acid, so preferably two nucleic acid molecules with sequences characteristic of the ZePel sequence are employed.
  • PCR techniques for the amplification of nucleic acid are described in US Patent No. 4,683,195 and Saiki en ai . Science 239: 487-491 (1988). PCR includes steps of denaturation of template nucleic acid (if double- stranded) , annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequences and cDNA transcribed from mRNA. References for the general use of PCR techniques include Mullis et al , Cold Spring Harbor Symp . Quant. Biol .
  • the complexity of a nucleic acid sample may be reduced where appropriate by creating a cDNA library for example using RT-PCR or by using the phenol emulsion reassociation technique (Clarke et al . (1992) NAR 20, 1289-1292) on a genomic library.
  • a method involving use of PCR in obtaining nucleic acid according to the present invention may include providing a preparation of plant nucleic acid, e.g. from wheat, providing a pair of nucleic acid molecule primers useful in (i.e. suitable for) PCR, at least one said primer having a sequence shown in or complementary to a sequence shown in Seq ID No 1, contacting nucleic acid in said preparation with said primers under conditions for performance of PCR, performing PCR and determining the presence or absence of an amplified PCR product.
  • the presence of an amplified PCR product may indicate identification of a gene of interest or fragment thereof.
  • the methods of the invention may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid.
  • hybridisation will generally be preceded by denaturation to produce single-stranded DNA.
  • the hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR.
  • An example procedure would be a combination of PCR and low stringency hybridisation.
  • a screening procedure chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.
  • An oligonucleotide for use in probing or nucleic acid amplification may have about 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • Generally specific probes/primers are upwards of 14 nucleotides in length.
  • primers of 16-24 nucleotides in length may be preferred. Probes may be much longer e.g. 100 's or even 1000' s of bases long.
  • the sequence of the mutant, variant, derivative, alllele or other homolog related to Seq ID No 1 shares homology with that sequence.
  • Homology may be at the nucleotide sequence and/or amino acid sequence level .
  • the nucleic acid and/or amino acid sequence shares homology with the coding sequence of Seq ID No 1, preferably at least about 50%, or 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology.
  • Homology may be over the full- length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333 or more amino acids or codons, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.
  • the enzyme encoded by the mutant, variant, derivative, allele or other homolog related to Seq ID No 1 has pectate lyase activity.
  • nucleic acid of the present invention which may contain for example DNA encoding the amino acid sequence of Figure 1, as genomic or cDNA, may be in the form of a recombinant and preferably replicable vector.
  • Such replicable vectors form a fourth aspect of the present invention.
  • DNA vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • Vectors may transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication) .
  • Vectors may be introduced into hosts by any appropriate method e.g. conjugation, mobilisation, transformation, transfection, transduction or electoporation.
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in both the actinomycetes and related species and in bacteria and/or eucaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
  • a vector including nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.
  • the ZePel promoter itself which forms a further aspect of the present invention, is described hereinafter.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate .
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate .
  • See, for example Sambrook et al (1989) Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . , John Wiley & Sons, 1992.
  • Selectable genetic markers may be used consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate .
  • the nucleic acid of the present invention may be under the control of an appropriate promoter or other regulatory elements for expression in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • genomic DNA this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell
  • a gene construct preferably a replicable vector, comprising a promoter operatively linked to a nucleotide sequence provided by the present invention, such as the ZePel gene, a homolog from another plant species, or any mutant, variant or allele thereof.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the present invention represents the first demonstration of recombinant expression of a plant pectate lyase in E. coli .
  • the promoter may include one or more sequence motifs or elements conferring developmental and/or tissue- specific regulatory control of expression.
  • Other regulatory sequences may be included, for instance as identified by mutation or digest assay in an appropriate expression system or by sequence comparison with available information, e.g. using a computer to search on-line databases.
  • Suitable promoters for plants include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al , 1990a and 1990b) .
  • CaMV 35S Cauliflower Mosaic Virus 35S
  • the promoter is an inducible oromoter.
  • inducible as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is “switched on” or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
  • the preferable situation is where the level of expression increases upon application of the relevant stimulus by an amount effective to alter a phenotypic characteristic.
  • an inducible (or “switchable” ) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about a desired phenotype (and may in fact be zero) .
  • expression is increased (or switched on) to a level which brings about the desired phenotype.
  • the present invention also provides a host (e.g. plants) transformed with said gene constructs or vectors and methods comprising introduction of such a construct into hosts and/or induction of expression of a construct within a host, by application of a suitable stimulus, an effective exogenous inducer.
  • a host e.g. plants transformed with said gene constructs or vectors and methods comprising introduction of such a construct into hosts and/or induction of expression of a construct within a host, by application of a suitable stimulus, an effective exogenous inducer.
  • GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants.
  • the promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion.
  • the GST-II -27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues .
  • nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned, the target cell type must be such that cells can be regenerated into whole plants (see below) .
  • Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants.
  • DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355 , EP-A-0116718 , NAR 12(22) 8711 - 87215 1984) , particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 32/09696, WO 94/00583, EP 331083, EP 175966, Green et al .
  • a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355 , EP-A-0116718 , NAR 12(22) 8711 - 87215 1984) , particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 32/09696, WO 94/00583, EP 331083, EP 175966
  • Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al . (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plan t Cell Rep . 7, 379-384; Zhang, e t al . (1988) Theor Appl Gene t 76, 835-840; Shimamoto, et al . (1989) Nature 338, 274-276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al .
  • Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective.
  • a combination of different techniques may be employed to enhance the efficiency of the transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233 ) .
  • a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant . Available techniques are reviewd in Vasil et al . , Cell Cul ture and Somatic Cell Genetics of Plants , Vol I, II and III, Laboratory Procedures and Their Applications , Academic Press, 1984, and Weissbach and Weissbach, Methods for Plan t Molecular Biology, Academic Press, 1989.
  • Methods of use of the vectors of the present invention form a further (sixth) ascect of the invention.
  • a method of making plant cell involving introduction of a vector of the present invention into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome.
  • a host cell containing nucleic acid or a vector according to the present invention especially a plant or a microbial cell.
  • the invention further encompasses a host cell transformed with nucleic acid or a vector according to the present invention, especially a plant or a microbial cell .
  • the nucleic acid may be incorporated within the chromosome .
  • a plant cell having incorporated into its genome nucleic acid, particularly heterologous nucleic acid, as provided by the present invention, under operative control of a regulatory sequence for control of expression.
  • the coding sequence may be operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene, such as not naturally associated with the gene for its expression.
  • the nucleic acid according to the invention may be placed under the control of an externally inducible gene promoter to place expression under the control of the user.
  • heterologous may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering, i.e. by human intervention.
  • a transgenic plant cell i.e. transgenic for the nucleic acid in question, may be provided.
  • the transgene may be on an extra-genomic vector or incorporated, preferably stably, into the genome.
  • a heterologous gene may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence .
  • nucleic acid heterologous, or exogenous or foreign, to a plant cell may be non-naturally occuring in cells of that type, variety or species.
  • nucleic acid may include a coding sequence of or derived from a particular type of plant cell or species or variety of plant, placed within the context of a plant cell of a different type or species or variety of plant.
  • nucleic acid sequence to be placed within a cell in which it or a homologue is found naturally, but wherein the nucleic acid sequence is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
  • a sequence within a plant or other host cell may be identifiably heterologous, exogenous or foreign.
  • a plant may be regenerated from one or more transformed plant cells.
  • a plant which includes a plant cell according to the invention. Also embraced is any part or propagule thereof, seed, selfed or hybrid progeny and descendants.
  • a plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders' Rights. It is noted that a plant need not be considered a "plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.
  • the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed.
  • the invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on.
  • the invention further provides a method of influencing or affecting a pectin content of a plant said method comprising the step of causing or allowing expression of a heterologous nucleic acid sequence encoding a pectate lyase in a plant cell of that plant (preferably a somatic cell) .
  • ZePel may be used to assist in the removal and modification of an existing pectin matrix in order to allow the deposition of newly-synthesized wall polymers for a specialised function or to create an architecture that is extensible.
  • This activity can be utilised, for instance, by expressing ZePel protein in response to an applied stimulus in forage crops (e.g. forage maize, grasses, alfalfa etc.) where the induction is timed just prior to harvest, or alternatively where the ZePel is vacuolar targetted and released upon physical disruption of the plant tissue at harvest (e.g. on chopping prior to ensiling) . Breakdown of pectin may allow better access of degrading microorganisms/digestive enzymes to the plant tissue, thereby increasing digestibility of the forage (leading to increased animal productivity.)
  • forage crops e.g. forage maize, grasses, alfalfa etc.
  • Breakdown of pectin may allow better access of degrading microorganisms/digestive enzymes to the plant tissue, thereby increasing digestibility of the forage (leading to increased animal productivity.)
  • the ZePel enzyme may have a number of advantages over pectate lyases in the prior art. For instance (as compared to bacterial enzymes) it may be possible to use it in vivo in plants without triggering a defensive response (by producing pectin degradation products which do not function as elicitors, or are less powerful elicitors than those generated by pathogens) . Conversely it may be used to prime the plants defense mechanisms in certain tissues without causing excessive 'soft rot' type damage to the plant in the process. It is certainly probable (by virtue of its novel sequence) that the enzyme will have different specificity and activity to microbial enzymes, and that this difference may make it more appropriate for use with or in plants.
  • the nucleic acid encodes the amino acid sequence of Figure 1, or a mutant, variant, allele or derivative of the sequence, within cells of the plant (thereby producing the encoded polypeptide) , following an earlier step of introduction of the nucleic acid into a ceil of the plant or an ancestor thereof.
  • over-expression of pectate lyase activity may be achieved by introduction of the nucleotide sequence in a sense orientation.
  • down-regulation of expression of a target gene may be achieved using anti-sense technology or "sense regulation" ("co-suppression") .
  • a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.
  • An alternative is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression.
  • van der Krol et al . (1990) The Plant Cell 2 , 291-299; Napoli et al . , (1990) The Plant Cell 2 , 279- 289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A-5, 231, 020.
  • the complete sequence corresponding to the coding sequence need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence .
  • the sequence employed may be about 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than about 500 nucleotides are preferable where possible, such as longer than about 600 nucleotides, than about 700 nucleotides, than about 800 nucleotides, than about 1000 nucleotides or more.
  • sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild- type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable.
  • the transcribed nucleic acid may represent a fragment of a pectate lyase gene of the present invention, such as including a nucleotide sequence shown in Seq ID No 1, or the complement thereof, or may be a mutant, derivative, variant or allele thereof, in similar terms as discussed above.
  • the homology may be sufficient for the transcribed anti-sense RNA to hybridise with nucleic acid within cells of the plant, though irrespective of whether hybridisation takes place the desired effect is down- regulation of gene expression.
  • Anti-sense or sense regulation may itself be regulated by employing an inducible promoter in an appropriate construct .
  • the present invention also provides a method of influencing or affecting a pectin content of a plant, the method including causing or allowing anti-sense transcription from heterologous nucleic acid according to the invention within cells of the plant . Also provided is a method of influencing or affecting a pectin content of a plant, the method including causing or allowing expression from nucleic acid according to the invention within cells of the plant.
  • Further options for down regulation of gene expression include the use of ribozymes, e.g. hammerhead ribozymes, which can catalyse the site-specific cleavage of RNA, such as mRNA (see e.g.
  • the pectate lyase activity of the plant is preferably suppressed as a result of under-expression within the plant cells. This may be particularly useful in controlling the rate of ripening/softening in fruits from plants in which ZePel homologs have been identified, for instance using the methods of the third aspect.
  • the present invention also encompasses the expression product (generally a pectate lyase, preferably ZePel) of any of the nucleic acid sequences of the invention disclosed above, and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions, which may be in suitable host cells.
  • the product may be isolated from the expression system and may be used as desired, for instance in formulation of a composition including at least one additional component.
  • the method of preparing the expression products of the present invention may particularly involve extraction of inclusion bodies from a microbial host, using detergent (preferably w th n-dodecyl ⁇ -D- maltoside) .
  • the expression product is most preferably a pectate lyase which is active at physiologically relevant temperature and pH. It may have modified properties with respect to the authentic ZePel as shown in Figure 1 when expressed in nature, or as expressed in E. coli as described herein. Such modifications are discussed in relation to the second aspect of the invention. They may include alterations for ease of expression, or to change specificity (e.g. for pectins having a pattern of methyl esterification or some other physical characteristic) .
  • Polypeptides containing these motifs or signals form one part the present invention. They can be identified, for instance, by immunolocalisation of ZePel and/or mutants differing in defined regions and/or fusion proteins incorporating defined regions of the ZePel sequence .
  • the expression products of the eleventh aspect may be used to raise antibodies employing techniques which are standard in the art. These antibodies, and polypeptides comprising antigen-binding fragments of antibodies, form a twelth aspect of the invention, and may be used in identifying homologues from other species as discussed further below. They can be used, for instance in labelled form e.g. in immunolocalisation experiments such as those described above.
  • Methods of producing antibodies include immunising a mammal (e.g. human, mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof.
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and might be screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82). Antibodies may be polyclonal or monoclonal .
  • antibodies with appropriate binding specificity may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • Antibodies raised to a polypeptide or peptide can be used in the identification and/or isolation of homologous polypeptides, and then the encoding genes.
  • the present invention embraces a method of identifying or isolating a polypeptide with pectate lyase activity (in accordance with embodiments disclosed herein) , comprising screening candidate polypeptides with a polypeptide comprising the antigen-binding domain of an antibody (for example whole antibody or a fragment thereof) which is able to bind a the ZePel enzyme, or fragment, variant or derivative thereof, and preferably has binding specificity for such a polypeptide.
  • an antibody for example whole antibody or a fragment thereof
  • a method of modifying a substrate by contacting said substrate with a recombinant expression product of the eleventh aspect, optionally in purified or partly purified form.
  • Said substrate in this context may be an extraneous one i.e. not part of the host cell from which the enzyme is derived.
  • Recombinant ZePel (or modified derivatives thereof) , for instance, may be used in microbial fermentation. This may be useful in fruit or vegetable juice extraction or clarification; in brewing; in sugar beet processing, in (paper) pulp processing; in treating silage.
  • the high specificity of pectate lyases can provide advantages over the use of chemical processing. They can thus be used to provide novel precursors for the food, chemical, or pharmaceutical industries.
  • Such products, particularly those from modified ZePel form one part of the present invention.
  • the ZePel could be used in purified or partially purified form, or as a microbial innoculan .
  • a yet further aspect of the present invention concerns the non- coding regions of the ZePel gene.
  • a fourteenth aspect of the invention is a nucleic acid molecule encoding the promoter of the ZePel gene .
  • the present inventors have noted that cells in the Zinnia system cultured in the presence of auxin alone have a higher pectate lyase activity than cells cultured in inductive medium.
  • One possibility is that the cell division in the cultures containing auxin results in a higher cell density in a given culture volume. After eight days of culture in inductive medium a decrease of activity is detected, which may correlate with a decrease in the percentage of viable cells present after tracheary element differentiation.
  • a second possibility is that the auxin- induced expression may be modulated by other growth factors such as cytokinin. It has also been demonstrated that ZePel expression is correlated with sites of vascular differentiation and with cells that are recent products of meristematic divisions.
  • Auxin-inducible promoters may have utility in controlling the expression of particular products e.g. during industrial fermentation products.
  • auxin or late-auxin, inducible promoter, which may be down-regulated by cytokinin (see above) may be used to induce the expression of ZePel or heterologous (to the promoter) nucleic acids in a delayed fashion.
  • the ZePel promoter may be induced preferentially in particular tissues (e.g. roots and stem) and/or stages of development means it may have particular utility in targeting ZePel or heterologous (to the promoter) nucleic acids to particular tissues or expressing them at particular times.
  • the promoter may be advantageous in offering the ability to specifically up- or down-regulate genes in xylogenetic tissue e.g. parenchyma cells surrounding the xylem vessels.
  • This may useful when trying inter alia to modify lignification, affect forage digestibility, improve roughage content, or alter paper-pulping properties. Modification of targets specifically in vascular/lignified tissues without side effects in other tissues may be useful when the expressed factor or enzyme is detrimental to some tissues. For instance the transcription factor myb308 downregulates phenolics and leads to lowered iignin content with desirable results for forages/pulp but its expression in leaves leads to reduced ability to resist oxidative stress - hence its expression would be most beneficial if restricted to parenchyma .
  • Genes which may be usefully operably linked to the promoter include any of those encoding enzymes which are involved in cell wall formation, modification, or degradation (e.g. during growth, differentiation, abscission etc.) . Examples of such enzymes can be found in Brett and Waldron (1996) "Physiology and biochemistry of plant cell walls” Second Edition: Chapman & Hall, London. Particular enzymes may be concerned with the modification of cellulose, pectic polysaccharides, hemicellulose, lignin, plant proteins (including glycoproteins) plus energy storage polysaccharides, lipids, proteins etc.
  • the promoter region may be readily identified using a probe or primer based on Seq ID No 1, as described in relation to the third aspect. This can be used in the identification and isolation of a promoter from a genomic library containing DNA derived from a plant source (i.e. the plant used to generate the ZePel cDNA) . Techniques and conditions for such probing are well known in the art as is discussed above. Following probing, promoter activity is assessed using a test transcription system.
  • Promoter activity is used to refer to ability to initiate transcription.
  • the level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter.
  • the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
  • the reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including ⁇ - galactosidase and luciferase.
  • ?-galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectro-photometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer .
  • Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non- radioactive assays .
  • the presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof.
  • the binding molecule may be labelled directly or indirectly using any standard technique.
  • a promoter which is a mutant, variant, derivative, allele or other homolog of the ZePel promoter. These can be generated or identified as described above; they will share homology with the ZePel promoter and retain promoter activity.
  • restriction enzyme or nucleases may be used to digest a nucleic acid molecule, followed by an appropriate assay (for example using a reporter gene such as luciferase) to determine the sequence required. Nucleic acid comprising these elements or motifs forms one part of the present invention.
  • the promoters of the present invention have the auxin inducible and/or tissue specific and/or developmentally specific promoter activity of the authentic ZePel promoter.
  • nucleic acid construct preferably an expression vector, including the ZePel promoter region or fragment, mutant, allele, derivative or variant thereof able to promote transcription, operably linked to a heterologous gene, e.g. a coding sequence, which is preferably not the coding sequence with which the promoter is operably linked in nature.
  • Figure 1 cDNA and predicted amino acid sequence of ZePel .
  • the amino acid sequence predicted from the cDNA sequence of ZePel is shown in boldface letters .
  • the predicted cleavage site for the mature protein is indicated with an asterisk.
  • the potential N-glycosylation site is underlined.
  • the DNA bases are numbered at the right .
  • the nucleotide and deduced amino acid sequence have been submitted to EMBL, GenBank and DDBJ as accession number Y09541. Residues possibly involved in the active site and the calcium binding are double underlined.
  • Figure 2 Homology of ZePel to conserved regions of other pectate lyases or pectin-degrading enzymes. Shown is a multiple alignment of the predicted amino acid sequence of ZePel, from Serl78 to His277 (numbered according to the 'mature' protein i.e. from the potential Ser-Ser cleavage site) to the predicted amino acid sequences of the most homologous sequences in the EMBL database, including tomato style-expressed gene 9612 (le9612) , tobacco pollen-specific gene G10 (tobGlO) , short ragweed allergen (Amb a I) , pectate lyase from B . Subtilis (BsPel) , E .
  • Pelc carotova pectate lyase
  • Pnl pectin lyase
  • the alignment was created using PILEUP program (Genetics Computer Group) and sequence homology is shown by using the PRETTYBOX program. Dark shading indicates identical residues, and pale shading indicates residues that are conservative substitutions. Gaps introduced to improve the alignment are represented by dots .
  • Figure 3 The pH dependence of recombinant ZePel enzyme activity as discussed in Example 4.
  • the ⁇ and • represent the average enzyme activity in duplicate assays performed on different days.
  • Figure 4 Time courses of pectate lyase activity at pH 10 in cells sampled from cultures in inductive medium, in medium containing 1.0 mgl "1 NAA, or in maintenance medium. The specific activity was expressed in mU of activity per mg of protein (see Example 5) .
  • Zinnia elegans cv. Envy plants were grown from seed in a peat/sand potting compost in a controlled environment room at 20 °C with a 16 h day.
  • Mesophyll cells were isolated from the first true leaves of 14 -day-old seedlings as described previously (Stacey et al . , 1995). Cells were resuspended in culture medium (Fukuda and Komamine, 1980) with 1.0 mg I "1 BAP and 1.0 mg I "1 NAA (inductive medium) or 1 ⁇ g l "1 BAP and 1 ⁇ g l "1 NAA (maintenance medium) or 1.0 mg l "1 BAP or 1.0 mg l "1 NAA. Cultures were maintained at 27 °C in the dark, shaking at 80 rpm, with 3 ml of cell suspension per well in 6-well plates (Sterilin, UK) at a density of 10 s cells ml "1 .
  • a Zinnia cDNA library was constructed in ⁇ ZAP (Stratagene, San Diego, CA) using cells cultured in inductive medium for 72 h. The library was screened in duplicate with radiolabeled single-strand cDNA probes from cells cultured in inductive medium for 24 h or 72 h. One of the phages showing differential hybridization with the cDNA probes was selected and converted to the pBluescript SK form by co-infection with R408 helper phage (Stratagene) . Hybridization and washes of filters were done at 70 °C with the Church and Gilbert (1984) hybridization solution.
  • RNA and DNA Gel Blot Analysis R ⁇ A was purified from different cultured cells and organs as previously described (Shirzadegan et al . , 1991) .
  • R ⁇ A gel blot analysis 15 ⁇ g of total R ⁇ A was electrophoresed on 1% agarose gels containing formaldehyde and blotted onto nylon membranes as described by Sambrook et al . (1989) . Equal loading of RNA was verified by ethidium bromide staining of the gel before transfer to the membrane. DNA was isolated from leaves as described previously (Rogers and Bendich, 1988) .
  • RNA and DNA gel blots were probed with the entire cDNA insert or an EcoRI/HincII released fragment, which was radiolabelled by random priming using T7 polymerase (Pharmacia) . Hybridization and filter- washing conditions were done at high stringency (Church and Gilbert, 1984) .
  • a fragment of 0.9 kb EcoRI-HincII fragment of the ZePel gene was subcloned into Bluescript KS(+) and SK( + ) vectors to provide templates for T7 polymerase to generate sense and antisense R ⁇ A.
  • the probes were labelled with digoxigenin-uridine 5 ' -triphosphate (Boehringer) according to the manufacturer's instructions.
  • the methods for tissue preparation and in situ hybridization were as described by Bradley et al (1993) .
  • Two oligonucleotides binding at the N-terminal end of the reading frame of ZePel were synthesised so as to introduce Ncol sites either over the initiating ATG (5'- AAACCATGGCAACCACAATTCTACC-3' ) or to create a Met-1 and exchange Ser21 to Ala, so as to remove the putative signal sequence (5 ' -GCTTCCATGGCACCAAGTAGAACCCC-3 ' ) .
  • a single terminal oligonucleotide was synthesised to introduce a BamHI site following the stop codon (5'- CTCGGATCCATAATCAACAACGAGACCC-3' ) .
  • Two independent PCR amplifications of the ZePel cDNA were performed using the two alternative N- terminal oligonucleotides and the single C-terminal oligonucleotide (500ng each) with AmpliTaq (Perkin-Elmer, UK) for 25 cycles of 1 min at 94 'C to denature, 1 min at 57 °C to anneal and extension for 2 min at 72 °C.
  • the PCR product was purified from a 0.8% agarose gel which was then made blunt ended and phosphorylated using klenow fragment and polynucleotide kinase .
  • This product was ligated upon itself using T4 ligase which was then cut with the restriction enzymes Ncol and BamHI before ligation into a modified form of pBluescript similarly digested and transformed into E. coli Sure cells (Stratagene, Cambridge UK) and plated out on L-agar containing ampicillin (50 ⁇ g/ml) , IPTG (1 mM) and X-gal (12.5 mg) .
  • the recombinant plasmids carrying the ZePel cDNA were sequenced and shown to carry the PCR induced mutations and otherwise to retain the native sequence.
  • the ZePel cDNAs were then liberated with Ncol and BamHI and gel-purified before ligation into pET3d (Studier et ai . 1990) previously digested with Ncol and BamHI. These constructs were then transformed into E. coli BL21 cells carrying pLysS and plated out on L-agar with ampicillin (50 ⁇ g/ml) and chloramphenicol (30 ⁇ g/ml) .
  • 500 ml L-broth was inoculated with 1 ml of overnight culture until 0.5 OD 600nm was reached, and then induced with 0.4 mM IPTG until 1 OD 600nm.
  • the periplasmic, cytoplasmic and inclusion body fractions were checked for protein expression using 12% SDS-PAGE.
  • the IPTG-inducible protein bands were blotted to Immobilon-P (Millipore, USA) from which the N-terminal protein sequences were determined using automated Edman degradation.
  • the cells were grown in 500 ml L-broth containing chloramphenicol (30 ⁇ g/ml) and ampicillin (50 ⁇ g/ml) with protein induction as carried out above.
  • the cells were French-pressed using a 50 mM Tris HC1 pH 7.5 buffer and 0.1 M NaCl , 0.1% n-dodecyl ⁇ -D-maltoside .
  • the culture was centrifuged at 12000 rpm at 4°C in a Sorvall SS-34 rotor.
  • the clarified supernatant was initially used to determine pectate lyase activity using 50 mM Tris HCl pH 8.5 buffer, 0.5% polygalacturonic acid and 1 mM CaCl 2 assay conditions (Nasser et al . 1990) .
  • the V max , pH optimum and calcium dependence were initially established using the enriched detergent extract before confirmation following purification by ion-exchange chromatography
  • the enzyme assays were performed with 50 mM Na acetate within the pH ranges 5 to 7; with 50 mM Tris HCl within the pH range 7.2 to 9 and with 50 mM Na 2 C0 3 within the pH range 9 to 11.
  • a cDNA library representing mRNA extracted from cells cultured for 72 h in the TE inductive medium was used to isolate cDNA clones that were induced early in the trans-differentiation process.
  • the library (30,000 recombinant phage) was differentially screened with radiolabeled first strand cDNA using RNA samples from cells cultured for 24 h or 72 h in inductive medium.
  • One of the isolated cDNAs that was abundantly present under inductive conditions at 72 h but not at 24 h, was selected for further study.
  • Example 2 Expression of the Pectate L ase mRNA in Zinnia Mesophyll Cells In Vitro and in Plant Organs
  • Mesophyll cells from the leaves of Zinnia differentiate directly into TEs when cultured in a medium containing a 1:1 ratio of auxin to cytokinin.
  • the mesophyll cells elongate after five days.
  • a time course of ZePel mRNA accumulation in the cells cultured in the TE induction medium showed a large increase in the accumulation of the corresponding transcript after only 48h in culture, at a point when the cells still look indistinguishable from their appearance in the original Zinnia leaf, and coincident with the time at which the cells become committed to TE precursor fate (Stacey et al . 1995) . It was also shown that NAA alone can induce expression of the ZePel gene at the same time or earlier than with the combination of both hormones. The time of appearance of this mRNA is thus much earlier than cell elongation which does not occur until after 120h.
  • ZePel transcripts in si tu were investigated by examining cells in sections of stem and root from three-weeks-old Zinnia plants, using the antisense probe.
  • ZePel mRNA was largely present in the recent products of cambial divisions on both phloem and xylem sides, and conspicuously localized in the xylem parenchyma cells of young vascular bundles.
  • the ZePel mRNA was located in the outer (most recently formed) part of the xylem and in phloem parenchyma cells. When shoot meristems were examined, the ZePel transcript was detected in young leaf primordia.
  • the ZePel cDNA sequence was mutated to produce initiation codons within convenient Ncol sites at the start of the ORF and at the predicted mature N-terminus (Ser 20) .
  • the two constructs were cloned into the pET3d T7 polymerase based vector (Studier et al , 1990) and their expression induced in BL21 (pLysS) cells with IPTG. No protein expression could be obtained from the construct with its native initiation codon. However, with the predicted signal sequence removed, the construct, upon induction, produced a protein of approximately 42 kDa by SDS-PAGE. At this stage no enzyme activity could be detected in protein extracts of this material. E .
  • coli expressing the recombinant protein at 28°C and 37°C were fractionated into cytoplasmic, periplasmic and inclusion body extracts and visualized by SDS-PAGE. Surprisingly at the lower temperature almost all of the recombinant protein was found to be in inclusion bodies. At 37 °C, however, a proportion of the protein was found to be soluble (cytoplasmic protein) . This fraction was shown to contain a low level of pectate lyase activity. However, the cytoplasmic protein was also observed to migrate just in front of the inclusion body material upon SDS-PAGE. These gels were electro-blotted and the corresponding proteins bands excised and N-terminally sequenced.
  • the inclusion body protein proved to have the expected N- terminus (APSRT) accounting for the PCR induced change of the second codon and removal of the initiating methionine by E. coli .
  • the cytoplasmic fraction produced the sequence ASRRN which is consistent with the loss of an 18 amino acid peptide from the anticipated N- terminus, and which correlates with the putative N- glycosylation site. This suggests that the glycosylation may be protecting the peptide from being cleaved in the plant .
  • the recombinant ZePel protein was found to be considerably more soluble in low concentrations of detergent than other proteins present in the inclusion body material. Extraction of the inclusion bodies with n- dodecyl ⁇ -D-maltoside produced a solution enriched for the ZePel protein that also proved to be enzymatically active. The presence of this detergent was shown, unexpectedly, to be necessary for the recovery of the activity under the conditions used by the present inventors . The enzyme activity could be eliminated by the addition of lOO ⁇ M EDTA indicating its expected calcium dependence. The enzyme was purified using conventional ion-exchange chromatography in the presence of detergent. The pH optimum of the enzyme was found to be 10, as shown in Figure 3.
  • the V max was 0.12 ⁇ 0.015 ⁇ mol min "1 and a Km was 0.9 ⁇ 0.23 g/100 ml as shown in Figure 5.
  • the specific activity of recombinant ZePel was found to be 250 ⁇ mol min "1 mg "1 .

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Abstract

La présente invention concerne des acides nucléiques codant des pectate lyases provenant de cellules somatiques végétales (par exemple Seq. ID N° 1 de Zinnia elegans c. Envy) et leurs promoteurs. L'invention se rapporte également à des acides nucléiques variants (par exemple, des allèles, des homologues, des dérivés) et à des procédés et des matériaux permettant d'obtenir ces derniers, au moyen de sondes ou par ACP. L'invention concerne aussi des vecteurs, des cellules hôtes, des plantes et des parties de plantes transgéniques et leur descendance présentant une activité de pectate lyase modifiée, ainsi que des procédés et des matériaux permettant de produire ces derniers. L'invention se rapporte enfin aux pectate lyases elles-mêmes et aux anticorps spécifiques à celles-ci, ainsi qu'à des pectines modifiées et à d'autres substrats contenant du polygalacturonate.
PCT/GB1998/002350 1997-08-07 1998-08-05 Enzymes degradant la pectine WO1999007857A1 (fr)

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AU86390/98A AU8639098A (en) 1997-08-07 1998-08-05 Pectin degrading enzymes

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029230A2 (fr) * 1999-10-15 2001-04-26 Carnegie Institution Of Washington Ingenierie de resistance aux maladies a l'aide de genes de type pectate lyase
WO2001059137A1 (fr) * 2000-02-10 2001-08-16 Biologic A/S Procede de remodelage des structures de polysaccharide de paroi cellulaire dans les plantes
WO2002050291A1 (fr) * 2000-12-18 2002-06-27 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Region regulatrice de la peroxyde lyase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995002043A1 (fr) * 1993-07-06 1995-01-19 Novo Nordisk A/S ADN CODANT UNE ENZYME A ACTIVITE D'ENDOGLUCANASE ET ETANT ISOLEE A PARTIR DE $i(TRICHODERMA HARZIANUM)
WO1998011228A2 (fr) * 1996-09-10 1998-03-19 Zeneca Limited Modulation genetique du murissement de fruits

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995002043A1 (fr) * 1993-07-06 1995-01-19 Novo Nordisk A/S ADN CODANT UNE ENZYME A ACTIVITE D'ENDOGLUCANASE ET ETANT ISOLEE A PARTIR DE $i(TRICHODERMA HARZIANUM)
WO1998011228A2 (fr) * 1996-09-10 1998-03-19 Zeneca Limited Modulation genetique du murissement de fruits

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BUDELIER K. ET AL.: "Regulation of a stylar transmitting tissue-specific gene in wild-type and transgenic tomato and tobacco", MOLECULAR AND GENERAL GENETICS, vol. 224, no. 2, November 1990 (1990-11-01), pages 183 - 192, XP002087369 *
DOMINGO C ET AL: "A pectate lyase from Zinnia elegans is auxin inducible.", PLANT JOURNAL, (1998 JAN) 13 (1) 17-28. JOURNAL CODE: BRU. ISSN: 0960-7412., ENGLAND: United Kingdom, XP002087373 *
DOMINGUEZ-PUIGJANER E ET AL: "A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases.", PLANT PHYSIOLOGY, (1997 JUL) 114 (3) 1071-6. JOURNAL CODE: P98. ISSN: 0032-0889., United States, XP002087368 *
KULIKAUSKAS R. AND MCCORMICK S. ET AL.: "Identification of the tobacco and Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato", PLANT MOLECULAR BIOLOGY, vol. 34, no. 5, July 1997 (1997-07-01), pages 809 - 814, XP002087370 *
MEDINA-ESCOBAR N ET AL: "Cloning, molecular characterization and expression pattern of a strawberry ripening-specific cDNA with sequence homology to pectate lyase from higher plants.", PLANT MOLECULAR BIOLOGY, (1997 AUG) 34 (6) 867-77. JOURNAL CODE: A6O. ISSN: 0167-4412., Netherlands, XP002087371 *
STACEY N. ET AL.: "Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements", PLANT JOURNAL, vol. 8, no. 6, 1995, pages 891 - 906, XP002087372 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029230A2 (fr) * 1999-10-15 2001-04-26 Carnegie Institution Of Washington Ingenierie de resistance aux maladies a l'aide de genes de type pectate lyase
WO2001029230A3 (fr) * 1999-10-15 2002-01-17 Carnegie Inst Of Washington Ingenierie de resistance aux maladies a l'aide de genes de type pectate lyase
WO2001059137A1 (fr) * 2000-02-10 2001-08-16 Biologic A/S Procede de remodelage des structures de polysaccharide de paroi cellulaire dans les plantes
WO2002050291A1 (fr) * 2000-12-18 2002-06-27 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Region regulatrice de la peroxyde lyase
US7223855B2 (en) 2000-12-18 2007-05-29 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Hydroperoxide Lyase regulator region

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AU8639098A (en) 1999-03-01

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