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WO2004099413A1 - Genes de type cad2 de plante et leur utilisation - Google Patents

Genes de type cad2 de plante et leur utilisation Download PDF

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Publication number
WO2004099413A1
WO2004099413A1 PCT/AU2004/000607 AU2004000607W WO2004099413A1 WO 2004099413 A1 WO2004099413 A1 WO 2004099413A1 AU 2004000607 W AU2004000607 W AU 2004000607W WO 2004099413 A1 WO2004099413 A1 WO 2004099413A1
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WIPO (PCT)
Prior art keywords
nucleic acid
plant
cad2l
polypeptide
acid fragment
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PCT/AU2004/000607
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English (en)
Inventor
German Spangenberg
Michael Emmerling
Eng Kok Ong
Timothy Ivor Sawbridge
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Agriculture Victoria Services Pty Ltd
Agresearch Limited
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Priority to NZ542875A priority Critical patent/NZ542875A/en
Priority to AU2004236278A priority patent/AU2004236278B2/en
Publication of WO2004099413A1 publication Critical patent/WO2004099413A1/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
    • 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)
    • C12N15/8241—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
    • C12N15/8255—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 lignin biosynthesis

Definitions

  • the present invention relates to nucleic acid fragments encoding amino acid sequences for lignification-related enzymes in plants, and the use thereof for the modification of plant cell walls and/or defence response in plants.
  • Lignins are complex phenolic polymers that strengthen plant cell walls against mechanical and chemical degradation. The process of lignification typically occurs during secondary thickening of the walls of cells.
  • Biosynthesis of the monolignol precursors is a multistep process beginning with the aromatic amino-acids phenylalanine (and tyrosine in grasses). Lignin biosynthesis is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL).
  • the second enzyme of the pathway is cinnamate-4-hydroxylase (C4H), responsible for the conversion of cinnamate to p-coumarate.
  • C4H cinnamate-4-hydroxylase
  • the second hydroxylation step in the pathway is catalyzed by p-coumarate-3-hydroxylase (C3H) producing caffeic acid.
  • Caffeic acid is then O-methylated by caffeic acid O-methyltransferase (OMT) to form ferulic acid.
  • Ferulic acid is subsequently converted into 5-hydroxyferulate through the last hydroxylation reaction of the general phenylpropanoid pathway catalised by ferulate-5-hydroxylase (F5H).
  • the 5-hydroxyferulate produced by F5H is then O-methylated by OMT, the same enzyme that carries out the O-methylation of caffeic acid.
  • the cinnamic acids are converted by action of the 4-coumarate:CoA ligase (4CL) and caffeoyl-CoA 3-O-mehtyltransferase (CCoAMT) into the corresponding CoA derivatives.
  • CCR cinnamoyl CoA reductase
  • CAD cinnamyl alcohol dehydrogenase
  • the three monolignols, sinapyl, coniferyl and p-coumaryl alcohols, are then polymerised by extracellular peroxidases (PER) and laccases (LAC) to yield lignins.
  • PER extracellular peroxidases
  • LAC laccases
  • the proportions of monolignols incorporated into the lignin polymers vary depending on plant species, tissue, developmental stage and sub-cellular location.
  • Cinnamyl alcohol dehydrogenase governs the last committed step of the lignin biosynthesis pathway, converting the hydroxycinnamaldehydes to their corresponding cinnamyl alcohols (monolignols).
  • CAD1 is monomeric and able to utilise a range of substituted and unsubstituted benzaldehydes.
  • CAD2 is a homo- or heterodimer that has been found in all plants examined and the angiosperm enzyme uses all three cinnamaldehydes whereas the gymnosperm enzyme has a poor affinity for sinapaldehyde.
  • Defense-responsive isoforms CAD3 have also been reported.
  • Dry matter digestibility of forages has been negatively correlated with lignin content.
  • natural mutants of lignin biosynthetic enzymes in maize, sorghum and pearl millet that have higher rumen digestibility have been characterised as having lower lignin content and altered S/G subunit ratio. Lignification of plant cell walls is the major factor identified as responsible for lowering digestibility of forage tissues as they mature.
  • Lignification also affects efficiency of cellulose extraction in the pulping process of wood for paper production.
  • Cell wall digestibility, pulping efficiency and feed (grazed, cut hay, silage) quality can thus be increased by the manipulation of enzymes involved in the biosynthesis of lignins.
  • Perennial ryegrass (Lolium perenne L.) is a key pasture grass in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.
  • nucleic acid sequences encoding some of the enzymes involved in lignification have been isolated for certain species of plants, there remains a need for materials useful in the modification of lignification in a wide range of plants, and for methods for their use.
  • the present invention provides substantially isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for novel cinnamyl alcohol dehydrogenase 2 like polypeptides (CAD2L). These CAD2L sequences are identified on the basis of the similarity of their expression pattern to CAD2, not on the basis of sequence similarity. The genes which encode these polypeptides are expressed in a similar manner to CAD2L.
  • CAD2L novel cinnamyl alcohol dehydrogenase 2 like polypeptides
  • CAD2L cinnamonyl alcohol dehydrogenase 2 like polypeptides
  • the present invention also provides substantially isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of polypeptides, which are related to CAD2L.
  • polypeptides are referred to herein as CAD2L-like.
  • This CAD2L-like class of polypeptides includes functionally active fragments or variants of CAD2L polypeptides and non-CAD2L polypeptides having similar functional activity to CAD2L polypeptides.
  • the nucleic acid fragments may be obtained from ryegrass (Lolium) or fescue (Festuca) species. These species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass
  • Nucleic acids according to the invention may be full-length genes or part thereof, and are also referred to as “nucleic acid fragments" and “nucleotide sequences" on this specification.
  • the nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.
  • the RNA is readily obtainable, for example, by transcription of a DNA sequence according to the present invention, to produce a RNA corresponding to the DNA sequence.
  • the RNA may be synthesised in vivo or in vitro or by chemical synthesis to produce a sequence corresponding to a DNA sequence by methods well known in the art. In this specification, where the degree of sequence similarity between an RNA and DNA is such that the strand of the DNA could encode the RNA, then the RNA is said to "correspond" to that DNA.
  • isolated means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid fragment or polypeptide present in a living plant is not isolated, but the same nucleic acid fragment or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such an isolated nucleic acid fragment could be part of a vector and/or such nucleic acid fragments could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
  • the fragment or variant in respect of a nucleotide sequence is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of modifying lignin biosynthesis and/or cellulose degradation in a plant.
  • Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity.
  • Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • the fragment has a size of at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides.
  • the fragment or variant has one or more of the biological properties for the enzymes CAD2L and CAD2L-like. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 60% identity to the functional part of the above mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity.
  • Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.
  • operatively linked is meant that said regulatory element is capable of causing expression of said nucleic acid in a plant cell and said terminator is capable of terminating expression of said nucleic acid in a plant cell.
  • said regulatory element is upstream of said nucleic acid and said terminator is downstream of said nucleic acid.
  • an effective amount is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference. It will also be understood that the term “comprises” (or its grammatical variants) as used in this specification is equivalent to the term “includes” and should not be taken as excluding the presence of other elements or features.
  • the substantially purified or isolated nucleic acid fragment encoding a CAD2L or CAD2L-like protein includes (a) a nucleotide sequence shown in Figures 1 and 5 hereto (SEQ ID NOS 1 and 2); (b) complements of the sequence recited in (a); (c) sequence antisense to the sequences reci ted in (a) or (b); (d) functionally active fragments and variants of the sequences reci ted in (a), (b) and (c); and (e) RNA sequences corresponding to the sequences reci ited in (a), (b), (c) and (d).
  • nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant
  • genes encoding other CAD2L or CAD2L-like enzymes may be isolated directly by using all or a portion of the nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art.
  • Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention can be designed and synthesized by methods known in the art. Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems.
  • primers can be designed and used to amplify a part or all of the sequences of the present invention.
  • the resulting amplification products can be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
  • two short segments of the nucleic acid fragments of the present invention may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al.
  • a substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species selected from the group consisting of CAD2L and CAD2L-like enzymes.
  • the ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue.
  • the species is a ryegrass, more preferably perennial ryegrass (L. perenne).
  • a substantially purified or isolated CAD2L or CAD2L-like polypeptide including an amino acid sequence selected from the group consisting of (a) the amino acid sequence shown in Figure 6 hereto (SEQ ID NO 3); and (b) sequences translated from the nucleotide sequence shown in Figure 1 and 5 hereto (SEQ ID NOS 1 and 2); and functionally active fragments and variants of (a) or (b).
  • a polypeptide produced (eg recombinantly) from a nucleic acid according to the present invention.
  • Techniques for recombinantly producing polypeptides are known to those skilled in the art. Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins comprising the amino acid sequences.
  • a genotype is the genetic constitution of an individual or group. Variations in genotype are essential in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers.
  • a genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual.
  • a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait.
  • nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.
  • SNPs single nucleotide polymorphisms
  • a method of isolating a nucleic acid of the present invention including a single nucleotide polymorphism (SNP's), said method including sequencing nucleic acid fragments from a nucleic acid library.
  • the nucleic acid library may be of any suitable type and is preferably a cDNA library.
  • the nucleic acid fragments may be isolated from recombinant plasmids or may be amplified, for example using polymerase chain reaction.
  • the sequencing may be performed by techniques known to those skilled in the art.
  • nucleic acids of the present invention including SNP's, and/or nucleotide sequence information thereof, as molecular genetic markers.
  • nucleic acid according to the present invention and/or nucleotide sequence information thereof, as a molecular genetic marker.
  • nucleic acids according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in ryegrasses and fescues.
  • QTL quantitative trait loci
  • nucleic acids according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in forage and turf grass improvement, e.g.
  • sequence information revealing SNPs in allelic variants of the nucleic acids of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in ryegrasses and fescues.
  • a construct including a nucleic acid according to the present invention may be a vector.
  • the vector may include a regulatory element such as a promoter, a nucleic acid according to the present invention and a terminator; said regulatory element, nucleic acid and terminator being operatively linked.
  • the vector may be of any suitable type and may be viral or non-viral.
  • the vector may be an expression vector.
  • Such vectors include chromosomal, non- chromosomal and synthetic nucleic acid sequences, eg.
  • any other vector may be used as long as it is replicable, or integrative or viable in the plant cell.
  • the regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
  • the vector may include more than one nucleic acid.
  • the nucleic acids within the same vector may have identical or differing sequences.
  • the vector has at least two nucleic acids encoding functionally similar enzymes.
  • a second nucleotide sequence may encode another cinnamyl alcohol dehydrogenase or another lignification-related enzyme.
  • the regulatory element is a promoter.
  • a variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (eg. monocotyledon or dicotyledon).
  • Particularly suitable promoters include but are not limited to the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter and derivatives thereof, the maize Ubiquitin promoter and the rice Actin promoter.
  • terminators which may be employed in the vectors and constructs of the present invention are also well known to those skilled in the art. It may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
  • the vector in addition to the regulatory element, the nucleic acid of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinotricin acetyltransferase (bar or pat) gene and the gentamycin acetyl transferase (aacC1) gene], and reporter genes [such as beta-glucuronidase (GUS) gene (gusA) and green fluorescent protein (gfp)].
  • the vector may also contain a ribosome binding site for translation initiation.
  • the vector may also include appropriate sequences for ampl
  • the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, visual examination including microscopic examination of fluorescence emitted by gfp, northern and Western blot hybridisation analyses.
  • the vectors of the present invention may be incorporated into a variety of plants, including monocotyledons such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, rice, sugarcane, oat, wheat and barley, dicotyledons, such as arabidopsis, tobacco, soybean, canola, cotton, potato, chickpea, medics, white clover, red clover, subterranean clover, alfalfa, eucalyptus poplar, hybrid aspen, and gymnosperms (pine tree).
  • monocotyledons such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses
  • corn rice, sugarcane, oat, wheat and barley
  • dicotyledons such as arabidopsis, tobacco, soybean, canola, cotton, potato, chickpe
  • the vectors are used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including forage- and turf-type cultivars.
  • grass species such as ryegrasses (Lolium species) and fescues (Festuca species)
  • fescues Festuca species
  • a ryegrass most preferably perennial ryegrass, including forage- and turf-type cultivars.
  • Techniques for incorporating the constructs and vectors of the present invention into plant cells are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.
  • Cells incorporating the constructs and vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art.
  • the culture conditions such as temperature, pH and the like, will be apparent to the person skilled in the art.
  • the resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
  • a plant cell, plant, plant seed or other plant part including, e.g.. transformed with, a vector of the present invention.
  • the plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms.
  • the plant cell, plant, plant seed or other plant part is from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably a ryegrass, most preferably perennial ryegrass, including both forage- and turf-type cultivars.
  • the present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.
  • the present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.
  • a method of modifying one or more of lignification, defence response, and cell walls, in a plant including introducing into said plant an effective amount of a nucleic acid and/or a vector according to the present invention.
  • an effective amount is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom.
  • Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
  • plant lignification and defence may be increased or decreased. They may be increased, for example, by incorporating additional copies of a sense nucleic acid of the present invention. They may be decreased, for example, by incorporating an antisense nucleic acid or dsRNA or small interfering RNA (siRNA) derived from the nucleotide sequences of the present invention.
  • siRNA small interfering RNA
  • the number of copies of genes encoding for different enzymes involved in lignification and defence may be manipulated to modify the composition of lignin and plant cell walls.
  • a lignin or modified lignin or a cellulose or modified cellulose substantially or partially purified or isolated from a plant, plant seed or other plant part of the present invention.
  • Such lignins may be modified from naturally occurring lignins in terms of their monomeric composition or ratios of individual monolignols, the presence of novel monolignols, the degree of linkage and/or nature of linkages between lignins and other plant cell wall components.
  • Such cellulose may be modified from naturally occurring cellulose in terms of the degree of polymerisation (number of units), and/or degree of branching and/or nature of linkages between units and/or nature of linkages between cellulose and other plant cell wall components.
  • a preparation for transforming a plant comprising at least one nucleic acid according to the present invention.
  • the preparation may contain vectors or other constructs to facilitate administration to and/or transformation of the plant with the nucleic acid.
  • Figure 1 shows the nucleotide sequence of LpCAD2L EST.
  • Figure 2 shows the microarray based expression profile of LpCAD2 in perennial ryegrass as log ratio of its expression values.
  • Figure 3 shows the expression profiling of LpCAD2L genes using LpCAD2 as template gene in perennial ryegrass as log ratios of genes matching LpCAD2 at an Euclidian distance of 5.665.
  • Figure 4 shows the plasmid map of pGEMLpCAD2La.
  • Figure 5 shows the nucleotide sequence of LpCAD2La.
  • Figure 6 shows the putative amino acid sequence of LpCAD2La.
  • Figure 7 shows the binary transformation vector pPZP221LpCAD2La.
  • FIG. 8 shows Agrobacterium-mediated transformation and selection of Arabidopsis.
  • cDNA libraries representing mRNAs from various organs and tissues of perennial ryegrass were prepared. The characteristics of the libraries are described below (Table 1 ).
  • RNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the Trizol method (Gibco-BRL, USA) or the RNeasy Plant Mini kit (Qiagen, Germany), following the manufacturers' instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech, USA), cDNAs may be amplified by long distance polymerase chain reaction using the Advantage 2 PCR Enzyme system (Clontech, USA), cDNAs may be cleaned using the GeneClean spin column (Bio 101 , USA), tailed and size fractionated, according to the protocol provided by Clontech.
  • Trizol method Gibco-BRL, USA
  • RNeasy Plant Mini kit Qiagen, Germany
  • the cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega, USA) according to the protocol provided by Promega.
  • the cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coll Epicurian coli XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.
  • the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA, USA).
  • the Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene.
  • cDNA inserts will be contained in the plasmid vector pBluescript.
  • the cDNAs may be introduced directly into precut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).
  • plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"). The resulting ESTs are analyzed using an Applied Biosystems ABI 3700 sequence analyser.
  • EXAMPLE 2 Microarray-based expression profiling and identification of CAD2L genes from perennial ryegrass (Lolium perenne) The function of a gene may be inferred by its co-expression with other genes involved in the same cellular processes.
  • the use of cDNA microarrays allows the expression of thousands of genes to be monitored in one experiment. In this technology a microscope slide is spotted with DNA, each spot containing copies of one gene sequence. The spotted DNA is immobilised on the slide and interrogated with labelled cDNA in solution. The cDNA is prepared from RNA extracted from a tissue of interest and fluorescence-labelled. The amount of fluorescence remaining on the spot after hybridisation with the probe and washing is a measure of the mRNA level of that gene in the tissue of interest.
  • the DNA spotted on the perennial ryegrass cDNA slide is derived from EST sequences. Each spot contains an EST that is unique or representative of a unique cluster of ESTs.
  • the putative function of the gene spotted has been inferred by homology searching of the EST sequence against public DNA and protein databases. The results of theses searches demonstrated that around 40% of the genes tagged cannot be assigned a function based on a homology search. However, comparisons of the expression profiles of these unknown genes with those of known genes allows an inference of their function to be made.
  • CDNA prepared from 4 day old seedling was used as a common reference probe and used in all the hybridisations.
  • Each of the other treatments was used as a co- hybridisation probe at least once.
  • Fluorescence-labelled cDNA was prepared from each RNA preparation. cDNA prepared from pseudostems was used as a common reference probe and used in all the hybridisations. Each of the other treatments was used as a co- hybridisation probe at least once. The protocols for these processes are described below.
  • RNA isolation and probe preparation 1.1 Total RNA isolation
  • the quality of RNA influences the efficiency of the labelling processes, hybridisation performance and background level.
  • the CTAB protocol has been modified to extract total plant RNA of high purity and quality.
  • the number of extraction steps with chloroform (step 5) is critical to the purity and yield of the isolated RNA.
  • the quality of the isolated RNA is measured using the ratio of absorbance at 230:280, with high quality RNA having a value of around 0.88-1.0.
  • RNA samples that do not meet the quality requirement can be further purified using an RNeasy mini column (Qiagen, Germany) to give microarray quality RNA.
  • these factors may not be universally applicable and methods for extraction of microarray quality RNA should be optimised for each organism or tissue.
  • CTAB protocol A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter (1993) 11 (2): 113-116]:
  • Step 5 Pipette the supernatant (top layer) into a new 50 ml tube. Step 5 is repeated until the interface is clear.
  • RNA is concentrated as follows:
  • Probes are either generated by directly incorporating fluorescent nucleotides during reverse transcription reaction or by indirect methods.
  • Cy3 and Cy5 are generally used as fluorophores as they are compatible with the excitation and emission wavelength of most slide scanners. These fluorochromes are light-sensitive and measures should be taken to minimise their exposure to light.
  • the labelled probes are purified using DyeEx column (Qiagen, Germany) or S-400 column (Amersham BioSciences, UK) according to the manufacturer's instructions.
  • Hybridisations can be carried out under a cover slip placed in a humid hybridisation chamber or a fluidic station.
  • Hybridisation chambers can be obtained from Corning (# 2551 ) or TeleChem International (# AHC-1).
  • Fluidic stations can be purchased from Amersham Pharmacia Biotech, Affymetrix or Genomic
  • Hybridisations carried out under a cover slip use 15-45 ⁇ l of pooled probe depending on the array size.
  • the potential drawback to this method is that the movement of probes is by diffusion.
  • the fluidic station allows a larger volume of pooled probe (100-200 ⁇ l) and agitation during hybridisation.
  • Hybridisations can occur either at 65°C, or 42°C if 50% formamide is included.
  • General and species specific blocking elements such as CoT-1 DNA, yeast tRNA or poly-d(A) should be included in the hybridisation.
  • prehybridisation is used to block DNA void spaces on the slide to prevent non-specific binding of probes.
  • prehybridisation on poly-L- lysine or aminosilane coated slide involves two steps. Firstly, the free amine groups on the slide are blocked using succinic anhydride. A condensation reaction takes place and for every succinic anhydride molecule two peptide bonds are formed with the poly-L-lysine and aminosilane. Secondly, DNA void spaces are blocked using salmon sperm DNA. Non specific hybridisation blocker such as Cot- 1 DNA or poly-d(A) are also included.
  • the volume required for hybridisation is dependent on the size of array used: 15-20 ⁇ l for half a glass slide (25x75 mm) and 30 ⁇ l for a full glass slide.
  • the log ratios from each experiment are then plotted using the time series tool in the GeneSight software. This allows the changes in the expression in the experimental sample relative to the reference sample to be visualised over the time of the experiment.
  • the median signal values are also plotted over time, for both the reference sample and the experimental sample, which allows visual confirmation of the data seen in the log ratio plot.
  • the expression profile of individual genes can be examined by selecting the gene in the software. This produces a line in each graph.
  • the genes showing the most similar pattern of expression ratios to the selected gene can be chosen by specifying a Euclidian distance within which the software will look for matching genes.
  • XnsNF _RAT 19024 arab-Arabidopsis thaliana UV hypersensitive protein (UVH3) mRNA, complete cds
  • Table 3 Identification of representative ESTs and size of EST cluster
  • Candidate ESTs identified by microarray expression profiling were further analysed by visual inspection of their sequencing trace files and CAD2L genes were selected based on sequence quality assessment. Table 4 summarises the selected cDNA clone (indicating EST code on microarray) coding for CAD2L enzyme identified by microarray expression profiling and provides the sequence name for the perennial ryegrass CAD2L cDNA sequence.
  • the original plasmids identified by microarray as described in Example 2 were used to transform chemically competent XL-1 cells (prepared in-house, CaCI protocol). After colony PCR (using HotStarTaq, Qiagen) a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result.
  • oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases the sequencing can be performed from both 5' and 3' end. In this instance, however, an extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5' end.
  • the sequences of the oligonucleotide primers are shown in Table 5.
  • Contigs were then assembled in Sequencher.
  • the contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRI cut site both at the 5' and 3' end.
  • pKYLX71 :35S 2 was cut with Clal. The 5' overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After cutting with EcoRI, the 2kb fragment with an EcoRI-compatible and a 3'-A tail was gel-purified.
  • pPZP221 was cut with Hindi 11 and the resulting 5' overhang filled in and T-tailed with Taq polymerase. The remainder of the original pPZP221 multi-cloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRI site and the 3' T overhang restoring the Hindlll site.
  • This binary vector contains between the left and right border the plant selectable marker gene aacC1 under the control of the 35S promoter and 35S terminator and the pKYLX71 :35S 2 - derived expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator.
  • a GATEWAY ® cloning cassette (Invitrogen) was introduced into the multicloning site of the pPZP221 :35S 2 vector obtained as described following the manufacturer's protocol.
  • the LpCAD2La cDNA fragment was generated by high fidelity PCR with a proofreading DNA polymerase using the original pGEM-T Easy plasmid cDNA as a template.
  • the primers used (Table 6) contained attB sequences for use with recombinases utilising the GATEWAY ® system (Invitrogen).
  • the resulting PCR fragment was used in a recombination reaction with pDONR ® vector (Invitrogen) to generate an entry vector.
  • the cDNA encoding the open reading frame sequence was transferred from the entry vector to the GATEWAY ® -enabled pPZP221 :35S 2 vector.
  • the orientation of the construct was checked by restriction enzyme digest and sequencing which also confirmed the correctness of the sequence.
  • a set of transgenic Arabidopsis plants carrying the chimeric perennial ryegrass gene CAD2La were produced.
  • a pPZP221 -based transformation vector with LpCAD2La cDNA comprising the full open reading frame sequence in sense orientation under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 ) was generated as detailed in Example 6.
  • transgenic Arabidopsis plants carrying the perennial ryegrass CAD2La cDNA under the control of the CaMV 35S promoter with duplicated enhancer region (35S 2 ) is described here in detail.
  • the overnight cultures were centrifuged for 15 min at 5500 xg and the supernatant discarded.
  • the cells were resuspended in 1 I of infiltration medium [5% (w/v) sucrose, 0.03% (v/v) Silwet-L77 (Vac-ln-Stuff, Lehle Seeds #VIS-01 )] and immediately used for infiltration.
  • the Agrobacterium suspension was poured into a container (Decor Tellfresh storer, #024) and the container placed inside the vacuum desiccator (Bel Art, #42020-0000).
  • a punnet with Arabidopsis plants was inverted and dipped into the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 2 min. After infiltration, the plants were returned to the growth room where they were kept away from direct light overnight. The next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed. The plants were then allowed to dry out, the seed collected from the siliques and either stored at room temperature in a dry container or used for selection of transformants.
  • 150 mm petri dish (approximately 40 mg or 2000 seeds) were placed in a 1.5 ml microfuge tube.
  • 500 ⁇ l 70% ethanol were added for 2 min and replaced by 500 ⁇ l sterilisation solution (H 2 O:4% chlorine:5% SDS, 15:8:1).
  • 500 ⁇ l sterilisation solution H 2 O:4% chlorine:5% SDS, 15:8:1.
  • the tube was left for 10 min after which time the sterilisation solution was replaced with 500 ⁇ l sterile water.
  • the tube was shaken and spun for 5 sec to sediment the seeds. The washing step was repeated 3 times and the seeds were left covered with approximately 200 ⁇ l sterile water.
  • the seeds were then evenly spread on 150 mm petri dishes containing germination medium (4.61 g Murashige & Skoog salts, 10 g sucrose, 1 ml 1 M KOH, 2 g Phytagel, 0.5 g MES and 1 ml 1000x Gamborg's B-5 vitamins per litre) supplemented with 250 ⁇ g/ml timetin and 75 ⁇ g/ml gentamycin.
  • germination medium 4.61 g Murashige & Skoog salts, 10 g sucrose, 1 ml 1 M KOH, 2 g Phytagel, 0.5 g MES and 1 ml 1000x Gamborg's B-5 vitamins per litre
  • Genomic DNA was analysed for the presence of the transgene by real-time
  • PCR primer pairs (Table 7) were designed using Primer Express 1.5. The forward primer was located within the 35S 2 promoter region and the reverse primer within the transgene to amplify products of approximately 150 bp as recommended. The positioning of the forward primer within the 35S 2 promoter region guaranteed that homologous genes in Arabidopsis were not detected.
  • each genomic DNA sample was run in a 50 ⁇ l PCR reaction including SYBR Green on an ABI7700 (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control).
  • Plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin. The transformation and selection process is shown in Figure 8.

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Abstract

La présente invention se rapporte à des polynucléotides codant des polypeptides de type cinnamyl alcool déshydrogénase 2 (CAD2L). Les polypeptides CAD2L sont produits dans une plante dans les mêmes organes et aux mêmes étapes de mise au point et procédés que CAD2 et risquent d'être impliqués dans les mêmes procédés de mise au point que les enzymes CAD. Ils peuvent être utilisés pour la modification, entre autre la lignification, la dégradation de la cellulose, la modification des parois cellulaires de la plante ou de la réponse défensive de la plante.
PCT/AU2004/000607 2003-05-07 2004-05-07 Genes de type cad2 de plante et leur utilisation WO2004099413A1 (fr)

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NZ542875A NZ542875A (en) 2003-05-07 2004-05-07 Nucleic acid fragments encoding amino acid sequences (CADL2) for lignifications-related enzymes in plants, and the use thereof for the modification of plant cell walls and/or defence response in plants
AU2004236278A AU2004236278B2 (en) 2003-05-07 2004-05-07 Plant CAD2-like genes and their use

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8129588B2 (en) 2004-04-20 2012-03-06 Syngenta Participations Ag Regulatory sequences for expressing gene products in plant reproductive tissue
US9238818B2 (en) 2004-04-20 2016-01-19 Syngenta Participations Ag Methods and genetic constructs for modification of lignin composition of corn cobs

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2001095702A1 (fr) * 2000-06-14 2001-12-20 Molecular Plant Breeding Nominees Ltd Modification de la biosynthese de la lignine
WO2002026994A1 (fr) * 2000-09-29 2002-04-04 Agriculture Victoria Services Pty Ltd Manipulation de parois de cellules vegetales
WO2003040306A2 (fr) * 2001-11-07 2003-05-15 Genesis Research And Development Corporation Limited Compositions a partir des graminees lolium perenne et festuca arundinacea

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2001095702A1 (fr) * 2000-06-14 2001-12-20 Molecular Plant Breeding Nominees Ltd Modification de la biosynthese de la lignine
WO2002026994A1 (fr) * 2000-09-29 2002-04-04 Agriculture Victoria Services Pty Ltd Manipulation de parois de cellules vegetales
WO2003040306A2 (fr) * 2001-11-07 2003-05-15 Genesis Research And Development Corporation Limited Compositions a partir des graminees lolium perenne et festuca arundinacea

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8129588B2 (en) 2004-04-20 2012-03-06 Syngenta Participations Ag Regulatory sequences for expressing gene products in plant reproductive tissue
US8597913B2 (en) 2004-04-20 2013-12-03 Syngenta Participations Ag Method of constructing an expression cassette comprising regulatory sequences of a target gene of a plant for expressing gene products
US8679844B2 (en) 2004-04-20 2014-03-25 Syngenta Participations Ag MADS gene regulatory sequences for expressing gene products in plant reproductive tissue
US9238818B2 (en) 2004-04-20 2016-01-19 Syngenta Participations Ag Methods and genetic constructs for modification of lignin composition of corn cobs

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