WO2004093530A2 - Generation de vegetaux a contenu d'huile modifie - Google Patents

Generation de vegetaux a contenu d'huile modifie Download PDF

Info

Publication number
WO2004093530A2
WO2004093530A2 PCT/US2004/012274 US2004012274W WO2004093530A2 WO 2004093530 A2 WO2004093530 A2 WO 2004093530A2 US 2004012274 W US2004012274 W US 2004012274W WO 2004093530 A2 WO2004093530 A2 WO 2004093530A2
Authority
WO
WIPO (PCT)
Prior art keywords
plant
sequence
oil
plants
phenotype
Prior art date
Application number
PCT/US2004/012274
Other languages
English (en)
Other versions
WO2004093530A3 (fr
Inventor
Jonathan Lightner
Hein Tsoeng Ng
Original Assignee
Agrinomics Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agrinomics Llc filed Critical Agrinomics Llc
Publication of WO2004093530A2 publication Critical patent/WO2004093530A2/fr
Publication of WO2004093530A3 publication Critical patent/WO2004093530A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8247—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 lipid metabolism, e.g. seed oil composition

Definitions

  • composition of crop seeds has important applications in the agricultural industries, relating both to processed food oils and to oils for animal feeding.
  • Seeds of agricultural crops contain a variety of valuable constituents, including oil, protein and starch.
  • Industrial processing can separate some or all of these constituents for individual sale in specific applications. For instance, nearly 60% of the US soybean crop is crushed by the soy processing industry. Soy processing yields purified oil, which is sold at high value, while the remainder is sold principally for lower value livestock feed (US Soybean Board, 2001 Soy Stats). Canola seed is crushed to produce oil and the co-product canola meal (Canola Council of Canada).
  • compositional alterations include high oleic soybean and corn oil (US Pat Nos 6,229,033 and 6,248,939), and laurate-containing seeds (US Pat No 5,639,790), among others.
  • Work in compositional alteration has predominantly focused on processed oilseeds but has been readily extendable to non-oilseed crops, including corn. While there is considerable interest in increasing oil content, the only currently practiced biotechnology in this area is High-Oil Corn (HOC) technology (DuPont, US PAT NO: 5,704,160).
  • HOC employs high oil pollinators developed by classical selection breeding along with elite (male-sterile) hybrid females in a production system referred to as TopCross.
  • TopCross High Oil system raises harvested grain oil content in maize from -3.5% to -7%, improving the energy content of the grain.
  • T-DNA mutagenesis screens (Feldmann et al, 1989) that detected altered fatty acid composition identified the omega 3 desaturase (FAD3) and delta- 12 desaturase (FAD2) genes (US Pat No 5952544; Yadav et al, 1993; Okuley et al, 1994).
  • a screen which focused on oil content rather than oil quality, analyzed chemically-induced mutants for wrinkled seeds or altered seed density, from which altered seed oil content was inferred (Focks and Benning, 1998).
  • DGAT diacylglycerol acyltransferase
  • seed-specific over-expression of the DGAT cDNA was associated with increased seed oil content (Jako et al, 2001).
  • Activation tagging in plants refers to a method of generating random mutations by insertion of a heterologous nucleic acid construct comprising regulatory sequences (e.g., an enhancer) into a plant genome.
  • the regulatory sequences can act to enhance transcription of one or more native plant genes; accordingly, activation tagging is a fruitful method for generating gain-of -function, generally dominant mutants (see, e.g., Hayashi et al, 1992; Weigel D et al. 2000).
  • the inserted construct provides a molecular tag for rapid identification of the native plant whose mis-expression causes the mutant phenotype.
  • Activation tagging may also cause loss-of-f unction phenotypes.
  • the insertion may result in disruption of a native plant gene, in which case the phenotype is generally recessive.
  • Activation tagging has been used in various species, including tobacco and
  • the invention provides a transgenic plant having a high oil phenotype.
  • the transgenic plant comprises a transformation vector comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a HTO104.3 polypeptide.
  • the transgenic plant is selected from the group consisting of rapeseed, soy, com, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut.
  • the invention further provides a method of producing oil comprising growing the transgenic plant and recovering oil from said plant.
  • the transgenic plant of the invention is produced by a method that comprises introducing into progenitor cells of the plant a plant transformation vector comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a EDO 104.3 polypeptide, and growing the transformed progenitor cells to produce a transgenic plant, wherein the HIO 104.3 polynucleotide sequence is expressed causing the high oil phenotype.
  • vector refers to a nucleic acid construct designed for transfer between different host cells.
  • expression vector refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • a “heterologous” nucleic acid construct or sequence has a portion of the sequence that is not native to the plant cell in which it is expressed.
  • Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating.
  • heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, microinjection, electroporation, or the like.
  • a “heterologous” nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence DNA coding sequence combination found in the native plant.
  • the term "gene” means the segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons) and non-transcribed regulatory sequence.
  • recombinant includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.
  • gene expression refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation; accordingly, “expression” may refer to either a polynucleotide or polypeptide sequence, or both.
  • “Over-expression” refers to increased expression of a polynucleotide and/or polypeptide sequence relative to its expression in a wild-type (or other reference [e.g., non-transgenic]) plant and may relate to a naturally-occurring or non-naturally occurring sequence.
  • “Ectopic expression” refers to expression at a time, place, and/or increased level that does not naturally occur in the non- altered or wild-type plant.
  • "Under-expression” refers to decreased expression of a polynucleotide and/or polypeptide sequence, generally of an endogenous gene, relative to its expression in a wild-type plant.
  • the terms “mis-expression” and “altered expression” encompass over-expression, under-expression, and ectopic expression.
  • a nucleic acid sequence in the context of inserting a nucleic acid sequence into a cell, means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
  • a "plant cell” refers to any cell derived from a plant, including cells from undifferentiated tissue (e.g., callus) as well as plant seeds, pollen, progagules and embryos.
  • mutant and wild-type refers to the form in which that trait or phenotype is found in the same variety of plant in nature.
  • the term "modified" regarding a plant trait refers to a change in the phenotype of a transgenic plant relative to the similar non-transgenic plant.
  • An "interesting phenotype (trait)" with reference to a transgenic plant refers to an observable or measurable phenotype demonstrated by a Tl and/or subsequent generation plant, which is not displayed by the corresponding non-transgenic (i.e., a genotypically similar plant that has been raised or assayed under similar conditions).
  • An interesting phenotype may represent an improvement in the plant or may provide a means to produce improvements in other plants.
  • An “improvement” is a feature that may enhance the utility of a plant species or variety by providing the plant with a unique and/or novel quality.
  • altered oil content phenotype refers to measurable phenotype of a genetically modified plant, where the plant displays a statistically significant increase or decrease in overall oil content (i.e., the percentage of seed mass that is oil), as compared to the similar, but non- modified plant.
  • a high oil phenotype refers to an increase in overall oil content.
  • a "mutant" polynucleotide sequence or gene differs from the corresponding wild type polynucleotide sequence or gene either in terms of sequence or expression, where the difference contributes to a modified plant phenotype or trait.
  • mutant refers to a plant or plant line which has a modified plant phenotype or trait, where the modified phenotype or trait is associated with the modified expression of a wild type polynucleotide sequence or gene.
  • Tl refers to the generation of plants from the seed of TO plants.
  • the Tl generation is the first set of transformed plants that can be selected by application of a selection agent, e.g., an antibiotic or herbicide, for which the transgenic plant contains the corresponding resistance gene.
  • T2 refers to the generation of plants by self-fertilization of the flowers of Tl plants, previously selected as being transgenic.
  • T3 plants are generated from T2 plants, etc.
  • the "direct progeny" of a given plant derives from the seed (or, sometimes, other tissue) of that plant and is in the immediately subsequent generation; for instance, for a given lineage, a T2 plant is the direct progeny of a Tl plant.
  • the "indirect progeny" of a given plant derives from the seed (or other tissue) of the direct progeny of that plant, or from the seed (or other tissue) of subsequent generations in that lineage; for instance, a T3 plant is the indirect progeny of a Tl plant.
  • plant part includes any plant organ or tissue, including, without limitation, seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • Plant cells can be obtained from any plant organ or tissue and cultures prepared therefrom.
  • the class of plants which can be used in the methods of the present invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledenous and dicotyledenous plants.
  • transgenic plant includes a plant that comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide can be either stably integrated into the genome, or can be extra-chromosomal.
  • the polynucleotide of the present invention is stably integrated into the genome such that the polynucleotide is passed on to successive generations.
  • a plant cell, tissue, organ, or plant into which the heterologous polynucleotides have been introduced is considered “transformed”, “transfected", or "transgenic”.
  • Direct and indirect progeny of transformed plants or plant cells that also contain the heterologous polynucleotide are also considered transgenic.
  • the enhancer element can cause up-regulation genes in the vicinity, generally within about 10 kilobase (kb) of the insertion.
  • Tl plants were exposed to the selective agent in order to specifically recover transformed plants that expressed the selectable marker and therefore harbored T-DNA insertions.
  • Samples of approximately 15-20 T2 seeds were collected from transformed Tl plants, and lipids were extracted from whole seeds.
  • Gas chromatography (GC) analysis was performed to determine fatty acid content and composition of seed samples.
  • HIO104.3 genes and/or polypeptides may be employed in the development of genetically modified plants having a modified oil content phenotype ("a HIO104.3 phenotype").
  • HIO104.3 genes may be used in the generation of oilseed crops that provide improved oil yield from oilseed processing and in the generation of feed grain crops that provide increased energy for animal feeding.
  • HIO104.3 genes may further be used to increase the oil content of specialty oil crops, in order to augment yield of desired unusual fatty acids.
  • Transgenic plants that have been genetically modified to express HIO 104.3 can be used in the production of oil, wherein the transgenic plants are grown, and oil is obtained from plant parts (e.g. seed) using standard methods.
  • Arabidopsis HLTO104.3 nucleic acid (genomic DNA) sequence is provided in SEQ ID NO:l and in Genbank entry GI#18390918.
  • the corresponding protein sequence is provided in SEQ ID NO:2 and in GI#15223267.
  • Nucleic acids and/or proteins that are orthologs or paralogs of Arabidopsis HIO104.3, are described in Example 3 below.
  • HJ.O104.3 polypeptide refers to a full-length HIO104.3 protein or a fragment, derivative (variant), or ortholog thereof that is "functionally active,” meaning that the protein fragment, derivative, or ortholog exhibits one or more or the functional activities associated with the polypeptide of SEQ ID NO:2.
  • a functionally active HIO104.3 polypeptide causes an altered oil content phenotype when mis-expressed in a plant.
  • mis- expression of the HIO 104.3 polypeptide causes a high oil phenotype in a plant.
  • a functionally active HIO 104.3 polypeptide is capable of rescuing defective (including deficient) endogenous HIO104.3 activity when expressed in a plant or in plant cells; the rescuing polypeptide may be from the same or from a different species as that with defective activity.
  • a functionally active fragment of a full length HIO104.3 polypeptide i.e., a native polypeptide having the sequence of SEQ TD NO: 2 or a naturally occurring ortholog thereof) retains one of more of the biological properties associated with the full-length HIO104.3 polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity.
  • a HIO104.3 fragment preferably comprises a HIO104.3 domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprises at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a HIO104.3 protein.
  • Functional domains can be identified using the PFAM program (Bateman A et al, 1999 Nucleic Acids Res 27:260-262; website at pfam.wustl.edu).
  • a preferred HIO 104.3 fragment comprises of one or more domains from the group including jmjC domain PF02373; jmjN domain PF02375 SM00545; FY-rich domain C-terminal region SM00542; FY-rich domain N-terminal region SM00541; C5H2 zinc finger
  • variants of full-length HIO 104.3 polypeptides or fragments thereof include polypeptides with amino acid insertions, deletions, or substitutions that retain one of more of the biological properties associated with the full-length HIO 104.3 polypeptide.
  • variants are generated that change the post-translational processing of a HIO 104.3 polypeptide. For instance, variants may have altered protein transport or protein localization characteristics or altered protein half -life compared to the native polypeptide.
  • HIO104.3 nucleic acid encompasses nucleic acids with the sequence provided in or complementary to the sequence provided in SEQ ID NO: 1, as well as functionally active fragments, derivatives, or orthologs thereof.
  • a HLO104.3 nucleic acid of this invention may be DNA, derived from genomic DNA or cDNA, or RNA.
  • a functionally active HIO 104.3 nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active HIO104.3 polypeptide. Included within this definition is genomic DNA that serves as a template for a primary RNA transcript (i.e., an mRNA precursor) that requires processing, such as splicing, before encoding the functionally active HIO104.3 polypeptide.
  • a HIO104.3 nucleic acid can include other non-coding sequences, which may or may not be transcribed; such sequences include 5' and 3' UTRs, polyadenylation signals and regulatory sequences that control gene expression, among others, as are known in the art.
  • HIO 104.3 polynucleotide can also include heterologous coding sequences, for example, sequences that encode a marker included to facilitate the purification of the fused polypeptide, or a transformation marker.
  • a functionally active HIO 104.3 nucleic acid is capable of being used in the generation of loss-of-function HIO104.3 phenotypes, for instance, via antisense suppression, co-suppression, etc.
  • a HIO104.3 nucleic acid used in the methods of this invention comprises a nucleic acid sequence that encodes or is complementary to a sequence that encodes a HIO104.3 polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the polypeptide sequence presented in SEQ ID NO:2.
  • a HJO104.3 polypeptide of the invention comprises a polypeptide sequence with at least 50% or 60% identity to the HIO104.3 polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO104.3 polypeptide sequence of SEQ ID NO:2, such as one or more domains from the group including jmjC domain PF02373; jmjN domain PF02375 SM00545; FY-rich domain C-terminal region SM00542; FY-rich domain N-terminal region SM00541; C5H2 zinc finger PF02928.
  • a HIO104.3 polypeptide comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%, 90% or 95% or more sequence identity to a functionally active fragment of the polypeptide presented in SEQ ID NO:2.
  • a HIO104.3 polypeptide comprises a polypeptide sequence with at least 50%, 60 %, 70%, 80%, or 90% identity to the polypeptide sequence of SEQ ID NO: 2 over its entire length and comprises of one or more domains from the group including jmjC domain PF02373; jmjN domain PF02375 SM00545; FY-rich domain C-terminal region SM00542; FY-rich domain N-terminal region SM00541; C5H2 zinc finger PF02928.
  • a HIO 104.3 polynucleotide sequence is at least 50% to 60% identical over its entire length to the HIO 104.3 nucleic acid sequence presented as SEQ ID NO:l, or nucleic acid sequences that are complementary to such a HIO 104.3 sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO104.3 sequence presented as SEQ TD NQ: 1 or a functionally active fragment thereof, or complementary sequences.
  • percent (%) sequence identity with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et al, J. Mol. Biol. (1997) 215:403-410; website at blast.wustl.edu/blast/README.html) with search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a "% identity value” is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported.
  • Percent (%) amino acid sequence similarity is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • a conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
  • Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that selectively hybridize to the nucleic acid sequence of SEQ TD NO:l.
  • the stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are well known (see, e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al, Molecular Cloning, Cold Spring Harbor (1989)).
  • a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:l under stringent hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 ⁇ g/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 ⁇ g/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.1X SSC and 0.1% SDS (sodium dodecyl sulfate).
  • SSC single strength citrate
  • moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM
  • EDTA 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.
  • low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.
  • a number of polynucleotide sequences encoding a HIO 104.3 polypeptide can be produced.
  • codons may be selected to increase the rate at which expression of the polypeptide occurs in a particular host species, in accordance with the optimum codon usage dictated by the particular host organism (see, e.g., Nakamura et al, 1999).
  • Such sequence variants may be used in the methods of this invention.
  • the methods of the invention may use orthologs of the Arabidopsis HIO104.3.
  • Methods of identifying the orthologs in other plant species are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures.
  • orthologs encompasses paralogs.
  • sequence data is available for a particular plant species, orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.
  • Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al, Genome Research (2000) 10:1204-1210).
  • Programs for multiple sequence alignment such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.
  • Structural threading or other analysis of protein folding e.g., using software by ProCeryon, Biosciences, Salzburg, Austria
  • Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available. Degenerate PCR and screening of cDNA or genomic DNA libraries are common methods for finding related gene sequences and are well known in the art (see, e.g., Sambrook, 1989; Dieffenbach and Dveksler, 1995).
  • HIO104.3 ortholog nucleic acids may hybridize to the nucleic acid of SEQ JD NO: 1 under high, moderate, or low stringency conditions. After amplification or isolation of a segment of a putative ortholog, that segment may be cloned and sequenced by standard techniques and utilized as a probe to isolate a complete cDNA or genomic clone. Alternatively, it is possible to initiate an EST project to generate a database of sequence information for the plant species of interest.
  • antibodies that specifically bind known HIO104.3 polypeptides are used for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999).
  • Western blot analysis can determine that a ELTO104.3 ortholog (i.e., an orthologous protein) is present in a crude extract of a particular plant species.
  • the sequence encoding the candidate ortholog may be isolated by screening expression libraries representing the particular plant species.
  • Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtll, as described in Sambrook, et al, 1989.
  • candidate orthologous sequence are used as bait (the "query") for the reverse BLAST against sequences from Arabidopsis or other species in which HIO104.3 nucleic acid and/or polypeptide sequences have been identified.
  • nucleic acids and polypeptides may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR), as previously described, are well known in the art. Alternatively, nucleic acid sequence may be synthesized. Any known method, such as site directed mutagenesis (Kunkel TA et al, 1991), may be used to introduce desired changes into a cloned nucleic acid.
  • the methods of the invention involve incorporating the desired form of the HIO104.3 nucleic acid into a plant expression vector for transformation of in plant cells, and the HIO104.3 polypeptide is expressed in the host plant.
  • An isolated HIO104.3 nucleic acid molecule is other than in the form or setting in which it is found in nature and is identified and separated from least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the HIO 104.3 nucleic acid.
  • an isolated HIO 104.3 nucleic acid molecule includes HIO104.3 nucleic acid molecules contained in cells that ordinarily express HIO104.3 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • HIO104.3 nucleic acids and polypeptides may be used in the generation of genetically modified plants having a modified oil content phenotype.
  • a "modified oil content phenotype" may refer to modified oil content in any part of the plant; the modified oil content is often observed in seeds.
  • altered expression of the HIO104.3 gene in a plant is used to generate plants with a high oil phenotype.
  • the methods described herein are generally applicable to all plants. Although activation tagging and gene identification is carried out in Arabidopsis, the HIO 104.3 gene (or an ortholog, variant or fragment thereof) may be expressed in any type of plant.
  • the invention is directed to oil-producing plants, which produce and store triacylglycerol in specific organs, primarily in seeds. Such species include soybean (Glycine max), rapeseed and canola (including Brassica napus, B.
  • campest ⁇ s sunflower (Helianthus annus), cotton (Gossypium hirsutum), com (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus conimunis) and peanut (Arachis hypogaea).
  • the invention may also be directed to fruit- and vegetable-bearing plants, grain-producing plants, nut-producing plants, rapid cycling Brassica species, alfalfa (Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae family), other forage crops, and wild species that may be a source of unique fatty acids.
  • the skilled artisan will recognize that a wide variety of transformation techniques exist in the art, and new techniques are continually becoming available. Any technique that is suitable for the target host plant can be employed within the scope of the present invention.
  • the constructs can be introduced in a variety of forms including, but not limited to as a strand of DNA, in a plasmid, or in an artificial chromosome.
  • the introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to Agrobacterium-mediated transformation, electroporation, microinjection, microprojectile bombardment calcium-phosphate-DNA co-precipitation or liposome-mediated transformation of a heterologous nucleic acid.
  • the transformation of the plant is preferably permanent, i.e. by integration of the introduced expression constructs into the host plant genome, so that the introduced constructs are passed onto successive plant generations.
  • a heterologous nucleic acid construct comprising an HIO104.3 polynucleotide may encode the entire protein or a biologically active portion thereof.
  • binary Ti-based vector systems may be used to transfer polynucleotides.
  • Standard Agrob ⁇ cterium binary vectors are known to those of skill in the art, and many are commercially available (e.g., pBI121 Clontech Laboratories, Palo Alto, CA).
  • the optimal procedure for transformation of plants with Agrobacterium vectors will vary with the type of plant being transformed.
  • Exemplary methods for Agrobacterium- ediated transformation include transformation of explants of hypocotyl, shoot tip, stem or leaf tissue, derived from sterile seedlings and/or plantlets. Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • Agrobacterium transformation has been previously described for a large number of different types of plants and methods for such transformation may be found in the scientific literature. Of particular relevance are methods to transform commercially important crops, such as rapeseed (De Block et al, 1989), sunflower (Everett et al, 1987), and soybean (Christou et al, 1989; Kline et al, 1987).
  • HIO 104.3 may be regulated with respect to the level of expression, the tissue type(s) where expression takes place and/or developmental stage of expression.
  • a number of heterologous regulatory sequences e.g., promoters and enhancers are available for controlling the expression of a HIO104.3 nucleic acid. These include constitutive, inducible and regulatable promoters, as well as promoters and enhancers that control expression in a tissue- or temporal-specific manner.
  • Exemplary constitutive promoters include the raspberry E4 promoter (U.S. Patent Nos.
  • tissue-specific promoters include the tomato E4 and E8 promoters (U.S. Patent No. 5,859,330) and the tomato 2AU gene promoter (Van
  • HIO 104.3 expression is under control of regulatory sequences from genes whose expression is associated with early seed and/or embryo development.
  • Legume genes whose promoters are associated with early seed and embryo development include V.faba legumin (Baumlein et al, 1991, Mol Gen Genet 225:121-8; Baumlein et al, 1992, Plant J 2:233-9), V. faba usp (Fiedler et al, 1993, Plant Mol Biol 22:669-79), pea convicilin (Bown et al, 1988, Biochem J 251:717-26), pea lectin (dePater et al, 1993, Plant Cell 5:877-86), P.
  • Cereal genes whose promoters are associated with early seed and embryo development include rice glutelin ("GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol 37:107-11; "GluB-1,” Takaiwa et al, 1996, Plant Mol Biol 30:1207-21; Washida et al, 1999, Plant Mol Biol 40:1-12; "Gt3,” Leisy et al, 1990, Plant Mol Biol 14:41-50), ⁇ ceprolamin (Zhou & Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack et al, 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et al, 1990, Plant Mol Biol 14:323-32), and barley B-hordeins (Entwistle et al, 1991, Plant Mol Biol 17:1217-31).
  • genes whose promoters are associated with early seed and embryo development include oil palm GLO7A (7S globulin, Morcillo et al, 2001, Physiol Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et al, 1987, J Biol Chem 262:12196-201; Stalberg et al, 1993, Plant Mol Biol 1993 23:671-83; Ellerstrom et al, 1996, Plant Mol Biol 32:1019-27), Brassica napus oleosin (Keddie et al, 1994, Plant Mol Biol 24:327-40), Arabidopsis oleosin (Plant et al, 1994, Plant Mol Biol 25:193-205), Arabidopsis FAE1 (Rossak et al, 2001, Plant Mol Biol 46:717-25), Canavalia gladiata conA (Yamamoto et al, 1995, Plant Mol Bio
  • regulatory sequences from genes expressed during oil biosynthesis are used (see, e.g., US Pat No: 5,952, 544).
  • Alternative promoters are from plant storage protein genes (Bevan et al, 1993, Philos Trans R Soc Lond B Biol Sci 342:209-15).
  • exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et ⁇ Z.,1988; van der Krol et al, 1988); co-suppression (Napoli, et al, 1990); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et al, 1998).
  • Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed.
  • Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence.
  • Antisense inhibition may use the entire cDNA sequence (Sheehy et al, 1988), a partial cDNA sequence including fragments of 5' coding sequence, (Cannon et al, 1990), or 3' non-coding sequences (Ch'ng et al, 1989).
  • Cosuppression techniques may use the entire cDNA sequence (Napoli et al, 1990; van der Krol et al, 1990), or a partial cDNA sequence (Smith et al, 1990).
  • stage- and tissue-specific gene expression patterns in mutant versus wild-type lines may be determined, for instance, by in situ hybridization. Analysis of the methylation status of the gene, especially flanking regulatory regions, may be performed. Other suitable techniques include overexpression, ectopic expression, expression in other plant species and gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing [VIGS, see Baulcombe D, 1999]).
  • expression profiling is used to simultaneously measure differences or induced changes in the expression of many different genes.
  • Techniques for microarray analysis are well known in the art (Schena M et al, Science (1995) 270:467-470; Baldwin D et al, 1999; DangondF, Physiol Genomics (2000) 2:53-58; van Hal NL et al, J Biotechnol (2000) 78:271-280; Richmond T and Somerville S, Curr Opin Plant Biol (2000) 3:108-116).
  • Expression profiling of individual tagged lines may be performed. Such analysis can identify other genes that are coordinately regulated as a consequence of the overexpression of the gene of interest, which may help to place an unknown gene in a particular pathway.
  • Analysis of gene products may include recombinant protein expression, antisera production, immunolocalization, biochemical assays for catalytic or other activity, analysis of phosphorylation status, and analysis of interaction with other proteins via yeast two-hybrid assays.
  • Pathway analysis may include placing a gene or gene product within a particular biochemical, metabolic or signaling pathway based on its mis-expression phenotype or by sequence homology with related genes.
  • analysis may comprise genetic crosses with wild-type lines and other mutant lines (creating double mutants) to order the gene in a pathway, or determining the effect of a mutation on expression of downstream "reporter" genes in a pathway.
  • the invention further provides a method of identifying plants that have mutations in endogenous HIO 104.3 that confer altered oil content, and generating altered oil content progeny of these plants that are not genetically modified.
  • TJLLTNG for targeting induced local lesions in genomes
  • mutations are induced in the seed of a plant of interest, for example, using EMS treatment.
  • the resulting plants are grown and self-fertilized, and the progeny are used to prepare DNA samples.
  • HI0103.1- specific PCR is used to identify whether a mutated plant has a HJO104.3 mutation.
  • Plants having HI0104.3 mutations may then be tested for altered oil content, or alternatively, plants may be tested for altered oil content, and then HI0104.3-specific PCR is used to determine whether a plant having altered oil content has a mutated HI0104.3 gene.
  • TILLING can identify mutations that may alter the expression of specific genes or the activity of proteins encoded by these genes (see Colbert et al (2001) Plant Physiol 126:480-484; McCallum et al (2000) Nature Biotechnology 18:455-457).
  • a candidate gene/Quantitative Trait Locus (QTLs) approach can be used in a marker-assisted breeding program to identify alleles of or mutations in the HJ.0104.3 gene or orthologs of HI0104.3 that may confer altered oil content (see Bert et al., Theor Appl Genet. 2003 Jun;107(l): 181-9; and Lionneton et al, Genome. 2002 Dec;45(6):1203-15).
  • a HI0104.3 nucleic acid is used to identify whether a plant having altered oil content has a mutation in endogenous HI0104.3 or has a particular allele that causes altered oil content.
  • T3 seed pools were analyzed by Near Infrared Spectroscopy (NTR) intact at time of harvest.
  • NTR infrared spectra were captured using a Bruker 22 N/F.
  • Bruker Software was used to estimate total seed oil and total seed protein content using data from NTR analysis and reference methods according to the manufacturers instructions. Oil contents predicted by our calibration (PDX Oil 3, Predicts Hexane Extracted Oil) were compared for 40,000 individual ACTTAG lines. To identify high oil lines the NTR oil result was compared to the mean oil result for all ACTTAG lines planted on the same day (Relative oil content). Subsequent to seed compositional analysis ACTTAG flanking sequence was determined. 22,000 lines with recovered flanking sequences were considered in this analysis.
  • Line W000110876 (TN036255) had a NTR determined oil content of 37.6% relative to a planting day average oil content of 34.4% (109% of PDA).
  • Line W000164786 (TN061059) had a NLR determined oil content of 36.1% relative to a planting day average oil content of 33.4%( 108% of PDA).
  • Line W000110876 (TN036255) had a confirmed ACTTAG insertion on Chromosome 1 at bp 2733299.
  • Line W000164786 (TN061059) had a confirmed ACTTAG insertion on Chromosome 1 at bp 2736584.
  • ACTTAG line TN061059 there was sequence identity to nucleotides 34502- 34660 on Arabidopsis BAC clone F22O13 chromosome 1 (GI#9454484), placing the left border junction downstream from 34502 (GI#9454484). The opposite flank (predicted right border junction) of this insert was not determined. Left border TN061059 T-DNA was ⁇ 975 bp 5' of the translation start site.
  • the contigged sequence is presented as SEQ TD NO: 4.
  • Atlg08620 has homology to a large number of plants, animal and fungal proteins, only the closest homologue for human, mouse and fission yeast are listed. The top 10 BLAST results for Atlg08620 are listed below and are included in the "Orthologue Table.
  • sequences are other redundant entries of Atlg08620. However, they differ from the sequences listed above by a few nucleotides. This is likely to be the result of sequencing errors or of single nucleotide polymorphisms with little or no effect on activity.
  • the following sequence is another redundant entry of At4g20400. However, it differs from the sequences listed above by a few nucleotides. This is likely to be the result of sequencing errors or of single nucleotide polymorphisms with little or no effect on activity.
  • At2g38950/T7F6.12 [Arabidopsis thaliana] >gi
  • sequences are other redundant entries of At2g38950. However, they differ from the sequences listed above by a few nucleotides. This is likely to be the result of sequencing errors or of single nucleotide polymorphisms with little or no effect on activity.
  • sequences are other redundant entries of Atlg30810. However, they differ from the sequences listed above by a few nucleotides. This is likely to be the result of sequencing errors or of single nucleotide polymorphisms with little or no effect on activity.
  • retinoblastoma- binding protein 2 [Homo sapiens] >gi
  • the following sequence is another redundant entry of At4g20400. However, it differs from the sequences listed above by a few nucleotides. This is likely to be the result of sequencing errors or of single nucleotide polymorphisms with little or no effect on activity.
  • Retinoblastoma binding-like protein from fission yeast >gi
  • At2g34880 Arabidopsis >gi
  • Length 806 BLASTP thaliana >gi
  • At4g20400 Arabidopsis >gi
  • 152370711 Length 872 BLASTP thaliana >gi
  • Atlg08620 is a non-secretory protein that lacks transmembrane domain (predicted by TMHMM) and signal peptide (predicted by SignalP). Atlg08620 is likely to be nuclear (72% nuclear, 16% cytoplasmic by Psort2). Pfam analysis showed that Atlg08620 has several conserved domains (jmjC domain PF02373; jmjN domain PF02375 SM00545; FY-rich domain C-terminal region SM00542; FY-rich domain N-terminal region SM00541; C5H2 zinc finger PF02928).
  • the C5H2 zinc finger, jmjC and jmjN domains were initially identified in the mouse jumonji transcription factor, which is required for neural tube formation (Balciunas and Ronne 2000; Takeuchi et al 1995).
  • the C5H2 zinc finger domain is likely to have DNA-binding activity.
  • the jmjN domain whose functions remain undetermined, is found in the jumonji family of eukaryotic factors, which also has the jmjC domain.
  • the jmjC domain is also found in proteins lacking the jmjN domain.
  • jmjC domain may have enzymatic function (e.g. NAD+ generation) and is often present in DNA- or chromatin- binding proteins Clissold and Ponting 2001).
  • FY-rich domain N- and C- terminal regions (SM00541, SM00542) have unknown function, but occurs frequently in chromatin-associated proteins like trithorax and its homologues.
  • the invention further provides a method of identifying plants that have mutations in, or an allele of, endogenous HIO104.3 that confer a HIO104.3 phenotype, and generating progeny of these plants that also have the HIO 104.3 phenotype and are not genetically modified.
  • TTLLTNG for targeting induced local lesions in genomes
  • mutations are induced in the seed of a plant of interest, for example, using EMS treatment.
  • the resulting plants are grown and self -fertilized, and the progeny are used to prepare DNA samples.
  • HIO104.3-specific PCR is used to identify whether a mutated plant has a HIO104.3 mutation.
  • Plants having HIO104.3 mutations may then be tested for the HT.O104.3 phenotype, or alternatively, plants may be tested for the KLTO104.3 phenotype, and then ELT.O104.3-specific PCR is used to determine whether a plant having the HIO104.3 phenotype has a mutated HIO104.3 gene.
  • TTLLTNG can identify mutations that may alter the expression of specific genes or the activity of proteins encoded by these genes (see Colbert et al (2001) Plant Physiol 126:480-484; McCallum et al. (2000) Nature Biotechnology 18:455-457).
  • a candidate gene/Quantitative Trait Locus (QTLs) approach can be used in a marker-assisted breeding program to identify alleles of or mutations in the HIO104.3 gene or orthologs of HIO104.3 that may confer the ELTO104.3 phenotype (see Foolad et al, Theor Appl Genet. (2002) 104(6-7):945-958; Rothan et al, Theor Appl Genet (2002) 105(1): 145-159); Dekkers and Hospital, Nat Rev Genet. (2002) Jan;3(l):22- 32).
  • QTLs Quality of Traitative Trait Locus
  • a HTO104.3 nucleic acid is used to identify whether a plant having a HJ.O104.3 phenotype has a mutation in endogenous HIO104.3 or has a particular allele that causes the HIO104.3 phenotype compared to plants lacking the mutation or allele, and generating progeny of the identified plant that have inherited the HIO104.3 mutation or allele and have the HIO104.3 phenotype.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Nutrition Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne des végétaux qui présentent un phénotype de contenu d'huile modifié provoqué par l'expression modifiée d'un acide nucléique HIO104.3. Ladite invention a aussi trait à des méthodes d'élaboration de végétaux dotés d'un phénotype de contenu d'huile modifié.
PCT/US2004/012274 2003-04-22 2004-04-19 Generation de vegetaux a contenu d'huile modifie WO2004093530A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46442803P 2003-04-22 2003-04-22
US60/464,428 2003-04-22

Publications (2)

Publication Number Publication Date
WO2004093530A2 true WO2004093530A2 (fr) 2004-11-04
WO2004093530A3 WO2004093530A3 (fr) 2005-04-28

Family

ID=33310886

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/012274 WO2004093530A2 (fr) 2003-04-22 2004-04-19 Generation de vegetaux a contenu d'huile modifie

Country Status (1)

Country Link
WO (1) WO2004093530A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2316956A3 (fr) * 2007-05-03 2011-08-24 BASF Plant Science GmbH Installations dotées de caractéristiques de rendement améliorées et procédé de fabrication de celles-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN [Online] 27 February 2003 ZHU ET AL, XP002983992 Database accession no. ABP81176 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2316956A3 (fr) * 2007-05-03 2011-08-24 BASF Plant Science GmbH Installations dotées de caractéristiques de rendement améliorées et procédé de fabrication de celles-ci

Also Published As

Publication number Publication date
WO2004093530A3 (fr) 2005-04-28

Similar Documents

Publication Publication Date Title
ZA200508444B (en) Generation of plants with altered oil content
WO2004093528A2 (fr) Génération de plantes à teneur modifiée en huile
WO2004093532A2 (fr) Generation de plantes avec un contenu en huile modifie
ZA200505147B (en) Generation of plants with altered oil content
US8168866B2 (en) Generation of plants with altered oil content
US7528295B2 (en) Generation of plants with altered oil content
US20060174375A1 (en) Generation of plants with altered oil content
ZA200604965B (en) Generation of plants with altered oil content
WO2004093530A2 (fr) Generation de vegetaux a contenu d'huile modifie
US7750205B2 (en) Generation of plants with altered oil content
WO2005006845A2 (fr) Generation de vegetaux a teneur en huile modifiee
WO2004054350A2 (fr) Production de vegetaux ayant une teneur en huile modifiee
WO2004093529A2 (fr) Generation de plantes a teneur modifiee en huile
US20060174374A1 (en) Generation of plants with altered oil content
WO2005006846A2 (fr) Production de vegetaux a teneur en huile modifiee
WO2004056173A2 (fr) Production de plantes a teneur en huile modifiee
WO2005058020A2 (fr) Generation de plantes presentant une teneur en huile modifiee

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase