Patent Application
20150004659
This disclosure relates to the isolation and sequencing of a nucleic acid molecule that includes a gene cluster comprising 10 genes from a noscapine producing Papaver somniferum [opium poppy] cultivar; transgenic cells transformed with said nucleic acid molecule, sequence variants of the genes; the use of said genes/proteins in the production of opiate alkaloids; and the use of the genes as a marker of P. somniferum plants that synthesize opiate alkaloids, in particular noscapine.
Noscapine belongs to the phthalideisoquinoline subclass of the structurally diverse isoquinoline alkaloids whereas codeine, morphine, thebaine and oripavine belong to the morphinan subclass. While the biosynthesis of morphinans has been elucidated at the molecular level our knowledge of noscapine biosynthesis has not advanced significantly since the demonstration using isotope labeling in the 1960s, that it is derived from scoulerine. Understanding the biochemical genetics underpinning noscapine biosynthesis should enable improved production of noscapine and related molecules both in poppy and other expression systems.
P. somniferum is the plant from which opium is extracted. The opium poppy is the only commercially exploited poppy of the family Papaveraceae and is the principal source of natural opiates. The opium is extracted from latex harvested from the green seed pods. A further source of opiate alkaloids is the poppy straw which is the dried mature plant. P. somniferum is a source of clinically useful opiate alkaloids such as morphine, codeine, thebaine, noscapine [also known as narcotine] and papaverine. The clinical application of these opiate alkaloids and their derivates is broad having use as analgesics, cough suppressants and anti-spasmodics. Although not used as a pharmacological agent in its own right, thebaine is a particularly useful opiate which can be converted into a range of compounds such as hydrocodone, oxycodone, oxymorphone, nalbuphine naltrexone, buprenorphine and etorphine. These intermediates also have broad pharmaceutical applications. For example, oxycodone, oxymorphone and etorphine are widely used as an analgesic for moderate to severe pain and are often combined with other analgesics such as ibuprofen. Buprenorphine is used in the treatment of heroin addiction and chronic pain. Naltrexone is used in the treatment of alcohol and opiate addiction.
This disclosure relates to transcriptomic analysis of P. somniferum noscapine producing cultivars compared to P. somniferum cultivars that are non-noscapine producing. The analysis has revealed the exclusive expression of a group of mostly cytochrome P450 and methyltransferase genes in a poppy variety that produces noscapine. These genes are surprisingly absent from the genomes of two non-noscapine producing varieties. Analysis of an F2 mapping population indicated the genes are tightly linked in the noscapine variety and bacterial artificial chromosome sequencing confirmed they exist as a novel gene cluster for the biosynthesis of opiate alkaloids.
According to an aspect of the invention there is provided an isolated nucleic acid molecule that encodes at least two polypeptides wherein the two polypeptides are selected from the group consisting of a nucleic acid molecule comprising or consisting of a nucleotide sequence selected from:
According to a further aspect of the invention there is provided an isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates, wherein one of said two genes comprises a nucleotide sequence selected from the group consisting of:
According to a further aspect or embodiment of the invention there is provided an isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates, wherein one of said two genes comprises a nucleotide sequence selected from the group consisting of;
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (Allows Sequences that Share at Least 90% Identity to Hybridize)
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 1 wherein said nucleic acid molecule encodes a polypeptide with methyl transferase activity.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 2 wherein said nucleic acid molecule encodes a polypeptide with methyl transferase activity.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 3 wherein said nucleic acid molecule encodes a polypeptide with methyl transferase activity.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 4 wherein said nucleic acid molecule encodes a polypeptide with cytochrome P450 activity.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 5 wherein said nucleic acid molecule encodes a polypeptide with cytochrome P450 activity.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 6 wherein said nucleic acid molecule encodes a polypeptide with cytochrome P450 activity.
In a preferred aspect or embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 7 wherein said nucleic acid molecule encodes a polypeptide with cytochrome P450 activity.
In a preferred aspect or embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 8 wherein said nucleic acid molecule encodes a polypeptide with carboxylesterase activity.
In a preferred aspect or embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 9 wherein said nucleic acid molecule encodes a polypeptide with short-chain dehydrogenase/reductase activity.
In a preferred aspect or embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented SEQ ID NO: 10 wherein said nucleic acid molecule encodes a polypeptide with acetyltransferase activity.
In a preferred embodiment of the invention said nucleic acid molecule includes SEQ ID NO: 1 and further includes one or more nucleotide sequences selected from the group consisting of: SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In a preferred embodiment of the invention said nucleic acid molecule includes 3, 4, 5, 6, 7, 8 or 9 nucleotide sequences selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In a preferred embodiment of the invention said nucleic acid molecule includes each of the nucleotide sequences as represented in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
According to a further aspect of the invention there is provided an isolated polypeptide selected from the group consisting of:
In a preferred embodiment of the invention said polypeptide comprises or consists of an amino acid sequence that is at least 55% identical to the full length amino acid sequence in SEQ ID NO: 17 and which encodes a polypeptide with cytochrome P450 activity.
According to a further aspect of the invention there is provided an isolated polypeptide selected from the group consisting of:
In a preferred embodiment of the invention said polypeptide comprises or consists of an amino acid sequence that is at least 46% identical to the full length amino acid sequence in SEQ ID NO: 18 and which encodes a polypeptide with carboxylesterase activity.
According to a further aspect of the invention there is provided an isolated polypeptide selected from the group consisting of:
In a preferred embodiment of the invention said polypeptide comprises or consists of an amino acid sequence that is at least 47% identical to the full length amino acid sequence in SEQ ID NO: 19 and which encodes a polypeptide with short-chain dehydrogenase/reductase activity.
According to a further aspect of the invention there is provided an isolated polypeptide selected from the group consisting of:
In a preferred embodiment of the invention said polypeptide comprises or consists of an amino acid sequence that is at least 67% identical to the full length amino acid sequence in SEQ ID NO: 20 and which encodes a polypeptide with acetyltransferase activity.
A modified polypeptide as herein disclosed may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.
In one embodiment, the variant polypeptides have at least 39% to 50% identity, even more preferably at least 55% identity, still more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity, and at least 99% identity with most or the full length amino acid sequence illustrated herein.
According to an aspect of the invention there is provided an isolated nucleic acid molecule comprising or consisting of a nucleotide sequence selected from the group consisting of:
According to a further aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the invention.
Preferably the nucleic acid molecule in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, (e.g. bacterial, yeast), or plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
By “promoter” is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
Constitutive promoters include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christian et al. (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al. (1991) Theor Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. application Ser. No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142, each of which is incorporated by reference.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156, herein incorporated by reference.
Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.
“Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is “under transcriptional initiation regulation” of the promoter. In a preferred aspect, the promoter is a tissue specific promoter, an inducible promoter or a developmentally regulated promoter.
Particular of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).
In a preferred embodiment of the invention said vector is a bacterial artificial chromosome [BACS].
According to a further aspect of the invention there is provided a transgenic cell transformed or transfected with a nucleic acid molecule or vector according to the invention.
In a preferred embodiment of the invention said cell is a plant cell.
In a preferred embodiment of the invention said plant cell is from the genus Papaver.
In a preferred embodiment of the invention said plant cell is a Papaver somniferum cell.
According to a further aspect of the invention there is provided a plant comprising a plant cell according to the invention.
In a preferred embodiment of the invention said plant is from the genus Papaver; preferably Papaver somniferum.
In an alternative preferred embodiment of the invention said cell is a microbial cell; preferably a bacterial or fungal cell [e.g. yeast, Saccharomyces cerevisae].
In a preferred embodiment of the invention said cell is adapted such that the nucleic acid molecule encoding one or more polypeptides according to the invention is over-expressed when compared to a non-transgenic cell of the same species.
According to a further aspect of the invention there is provided a nucleic acid molecule comprising a transcription cassette wherein said cassette includes a nucleotide sequence designed with reference to a nucleotide sequence selected from the group: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and is adapted for expression by provision of at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette.
In a preferred embodiment of the invention said cassette is adapted such that both sense and antisense ribonucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part or all of their length to form a inhibitory RNA or short hairpin RNA.
In a preferred embodiment of the invention said cassette is provided with at least two promoters adapted to transcribe both sense and antisense strands of said ribonucleic acid molecule.
In an alternative preferred embodiment of the invention said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an ribonucleic acid molecule which forms a double stranded region by complementary base pairing of said first and second parts thereby forming an short hairpin RNA.
A technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as small inhibitory/interfering RNA (siRNA) or short hairpin RNA [shRNA], into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA/shRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically derived from exons of the gene which is to be ablated. The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.
In a preferred embodiment of the invention said nucleic acid molecule is part of a vector adapted for expression in a plant cell.
According to a further aspect of the invention there is provided a plant cell transfected with a nucleic acid molecule or vector according to the invention wherein said cell has reduced expression of a polypeptide according to the invention.
According to an aspect of the invention there is provided a process for the modification of one or more opiate alkaloids comprising:
In a preferred method of the invention said harvested plant material is dried and opiate alkaloid is extracted.
According to an alternative aspect of the invention there is provided a process for the modification of one or more opiate alkaloids or opiate alkaloid intermediate metabolites comprising:
In a preferred method of the invention said microbial cell is a bacterial cell or fungal/yeast cell.
If microbial cells are used as organisms in the process according to the invention they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while gassing in oxygen.
The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The methylated opiate alkaloids produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. To this end, the organisms can advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.
The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.
Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.
Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.
Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.
The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
The culture temperature is normally between 15° C. and 45° C., preferably at from 25° C. to 40° C., and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20° C. to 45° C. and preferably 25° C. to 40° C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.
The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.
However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the opiate alkaloids present therein.
According to a further aspect of the invention there is provided the use of a gene encoded by a nucleic acid molecule as represented by the nucleic acid sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleotide sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and encodes a polypeptide with opiate alkaloid biosynthetic activity as a means to identify a locus wherein said locus is associated with altered expression or activity of said opiate alkaloid biosynthetic activity.
Mutagenesis as a means to induce phenotypic changes in organisms is well known in the art and includes but is not limited to the use of mutagenic agents such as chemical mutagens [e.g. base analogues, deaminating agents, DNA intercalating agents, alkylating agents, transposons, bromine, sodium azide] and physical mutagens [e.g. ionizing radiation, psoralen exposure combined with UV irradiation].
According to a further aspect of the invention there is provided a method to produce a P. somniferum plant that has altered expression of a polypeptide according to the invention comprising the steps of:
In a preferred method of the invention said nucleic acid molecule is analysed by a method comprising the steps of:
In a preferred method of the invention said P. somniferum plant has enhanced opiate alkaloid biosynthetic activity.
In an alternative preferred method of the invention said P. somniferum plant has reduced or abrogated opiate alkaloid biosynthetic activity.
According to a further aspect of the invention there is provided a P. somniferum plant obtained by the method according to the invention.
According to an aspect of the invention there is provided a P. somniferum plant wherein said plant comprises a viral vector that includes all or part of a gene comprising a nucleic acid molecule according to the invention.
In a preferred embodiment of the invention said gene or part is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 21.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 22.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 23.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 24.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 25.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 26.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 27.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 28.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 29.
In a preferred embodiment of the invention said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 30.
According to a further aspect of the invention there is provided a viral vector comprising all or part of a nucleic acid molecule according to the invention.
According to an aspect of the invention there is provided the use of a viral vector according to the invention in viral induced gene silencing in a P. somniferum plant.
Virus induced gene silencing [VIGS] is known in the art and exploits a RNA mediated antiviral defence mechanism. Plants that are infected with an unmodified virus induces a mechanism that specifically targets the viral genome. However, viral vectors which are engineered to include nucleic acid molecules derived from host plant genes also induce specific inhibition of viral vector expression and additionally target host mRNA. This allows gene specific gene silencing without genetic modification of the plant genome and is essentially a non-transgenic modification.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following figures:
Table 1 Illustrates the % identity of CYP82Y1, PSCXE1, PSDFR1 and PSAT1 (SEQ ID 17-20) with their respective closest functionally characterised homologues. Accession numbers given are from GenBank, Swiss-Prot or PDB databases;
Table 2. Genotyping of F3 families derived from two F2 phenotypic classes: low noscapine and high noscapine. The observed versus expected segregation ratios strongly support the hypothesis that individuals in the low noscapine F2 class are heterozygous for the HN1 gene cluster and individuals in the high noscapine class are homozygous;
Table 3. Primer sequences and associated information.
Papaver somniferum
Papaver somniferum
Actinidia eriantha
Medicago sativa
Saussurea medusa
Plant Material
Three GSK Australia poppy varieties that predominantly accumulate either noscapine (High Noscapine, HN1), morphine (High Morphine, HM1) or thebaine (High Thebaine HT1), were grown in Maxi (Fleet) Rootrainers™ (Haxnicks, Mere, UK) under glass in 16 hour days at the University of York horticulture facilities. The growth substrate consisted of 4 parts John Innes No. 2, 1 part Perlite and 2 parts Vermiculite. The HM1×HN1 F2 mapping population was grown at the GlaxoSmithKline Australia field-trial site, Latrobe, Tasmania from September 2009 to February 2010.
Crossing and Selfing
Crosses were carried out between HN1 and HM1 individuals to generate F1 hybrid seed. At the hook stage of inflorescence development, immature stamens were removed from selected HN1 flower buds. HN1 stigmas were fertilized with pollen from synchronously developing HM1 flowers shortly after onset of anthesis. To prevent contaminating pollen from reaching the receptive stigmas, emasculated flowers were covered with a muslin bag for four days after pollination. Both the F1 and F2 generations were self-pollinated to produce F2 and F3 seed, respectively. Self-pollination was ensured by covering the flowers shortly before onset of anthesis with a muslin bag.
RNA Isolation and cDNA Synthesis
Upper stems (defined as the 2 cm section immediately underneath the capsule) and whole capsules were harvested at two developmental stages represented by 1-3 days and 4-6 days, after petal fall. Five plants were used per developmental stage and cultivar. The material was ground to a fine powder in liquid nitrogen using a mortar and pestle. RNA was isolated from the powder using a CTAB-based extraction method (Chang et al (1993) Plant Mol. Biol. Rep. 11, 113-116) with small modifications: (i) three sequential extractions with chloroform:isoamylalcohol (24:1) were performed and (ii) the RNA was precipitated overnight with lithium chloride at 4° C. After spectrophotometric quantification, equal amounts of RNA were pooled from five plants per cultivar, development stage and organ. The pooled samples underwent a final purification step using an RNeasy Plus MicroKit (Qiagen, Crawley, UK). RNA was typically eluted in 30-100 μl water. cDNA was prepared with the SMART cDNA Library Construction Kit (Clontech, Saint-Germainen-Laye, France) according to the manufacturer's instructions but using SuperScript II Reverse Transcriptase (Invitrogen, Paisley, UK) for first strand synthesis. The CDSIII/3′PCR primer was modified to: 5′ ATT CTA GAT CCR ACA TGT TTT TTT TTT TTT TTT TTT TVN 3′ where R=A or G, V=A, C or G; N=A/T or C/G. Following digestion with MmeI (New England Biolabs, Hitchin, UK) the cDNA was finally purified using a QIAquick PCR Purification kit (Qiagen, Crawley, UK).
cDNA Pyrosequencing: Pyrosequencing was performed on the Roche 454 GS-FLX sequencing platform (Branford, Conn.) using cDNA prepared from the following four samples of each of the three varieties:
Raw Sequence Analysis, Contiguous Sequence Assembly and Annotation
The raw sequence datasets were derived from parallel tagged sequencing on the 454 sequencing platform (Meyer et al (2008) Nature Prot. 3, 267-78). Primer and tag sequences were first removed from all individual sequence reads. Contiguous sequence assembly was only performed on sequences longer than 40 nucleotides and containing less than 3% unknown (N) residues. Those high quality Expressed Sequence Tag (EST) sequences were assembled into unique contiguous sequences with the CAPS Sequence Assembly Program (Huang and Madan (1999) Genome Res. 9, 868-877), and the resulting contigs were annotated locally using the BLAST2 program (Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402) against the non-redundant peptide database downloaded from the NCBI.
Expression profiling: The number of ESTs associated with a specific consensus sequence representing each of the candidate genes detailed in
Preparation of Genomic DNA from Glasshouse Grown Plants
In order to amplify and obtain genomic sequences of the candidate genes 30-50 mgs of leaf material was collected from 4-6 week old glasshouse-grown seedlings from each of the three varieties. Genomic DNA was extracted using the BioSprint 96 Plant kit on the BioSprint 96 Workstation (Qiagen, Crawley, UK) according to the manufacturer's protocol. Extracted DNA was quantified using Hoescht 33258 and normalized to 10 ng/ul.
Amplification and Sequencing of Candidate Genes from Genomic DNA
Primers for amplification and Sanger-sequencing of the candidate genes from genomic DNA were based on the respective contiguous sequences assembled from the ESTs or on BAC sequences. The primer sequences are shown in Table 3. PCR amplifications were performed on pools of genomic DNA comprising DNA from four individuals. Amplification was typically carried out on 10 ng genomic DNA in 1× Phusion High Fidelity Buffer supplemented with 200 nM forward and reverse primers, 0.2 mM dNTPs, 0.02 units/μl Phusion Hot Start DNA Polymerase (Finnzymes, Vantaa, Finnland). Standard PCR conditions were used throughout with annealing temperatures and times dependent on primers and PCR equipment.
DNA Extraction from the Field-Grown F2 Mapping Population
40-50 mg of leaf tissue was harvested from F2 plants at the ‘small rosette’ growth stage (˜10 leaves present on each plant) into 1.2 ml sample tubes. A 3 mm tungsten carbide bead was added to each tube and samples were kept at −80° C. for a minimum of two hours prior to freeze-drying for 18 hours. Following freeze drying, samples were powdered by bead-milling (Model TissueLyser, Qiagen, Hilden, Germany) at 30 Hz for two 60 s cycles separated by plate inversion. DNA extraction was performed with the Nucleospin Plant II kit (Macherey-Nagel, Duren, Germany) using the supplied Buffer Set PL2/3 following the manufacturer's protocol for centrifugal extraction. DNA was quantified by UV-spectroscopy.
Plants of the F2 mapping population were genotyped for the presence or absence of eight candidate genes. The gene primer pairs (Table 3) were designed with fluorescent tags (5′-VIC®-labeled) for use on the ABI 3730xl capillary apparatus (Applied Biosystems, Foster City, Calif.). PCR amplifications were typically carried out on 10 ng genomic DNA in 1×GoTaq buffer supplemented with 1 mM MgCl2, 500 nM forward and reverse primer, 0.125 mM dNTPs, 0.1 U GoTaq (Promega, Southampton, UK). The amplification conditions were: 1 min 94° C., 30-36 cycles of 30 s denaturation at 94° C., 30 s annealing at 62° C. and 20-50 s extension at 72° C., followed by a final extension for 5 min at 72° C. Cycle number and extension times depended on the candidate gene (Table 3). Amplification products were diluted 1:20 in H2O and fractionated on an ABI 3730xl capillary sequencer (Applied Biosystems, Foster City, Calif.). Data were scored using GeneMarker™ software (Softgenetics, State College, Pa.).
Poppy Straw Analysis from Field Grown F2 Plants
Poppy capsules were harvested by hand from the mapping population once capsules had dried to approximately 10% moisture on the plant. After manually separating the seed from the capsule, the capsule straw samples (Poppy Straw) were then ground in a ball mill (Model MM04, Retsch, Haan, Germany) into a fine powder. Samples of ground poppy straw were then weighed accurately to 2±0.003 g and extracted in 50 ml of a 10% acetic acid solution. The extraction suspension was shaken on an orbital shaker at 200 rpm for a minimum of 10 min, then filtered to provide a clear filtrate. The final filtrate was passed through a 0.22 μm filter prior to analysis. The loss on drying (LOD) of the straw was determined by drying in an oven at 105° C. for 3 hours.
All solutions were analysed using a Waters Acquity UPLC system (Waters Ltd., Elstree, UK). fitted with a Waters Acquity BEH C18 column, 2.1 mm×100 mm with 1.7 micron packing. The mobile phase used a gradient profile with eluent A consisting of 10 mM ammonium bicarbonate of pH 10.2 and eluent B methanol. The mobile phase gradient conditions used are as listed in the table below with a linear gradient. The flow rate was 0.5 ml per minute and the column maintained at 60° C. The injection volume was 2 μl and eluted peaks were ionised in positive APCI mode and detected within 5 ppm mass accuracy using a Thermo LTQ-Orbitrap. The runs were controlled by Thermo Xcalibur software (Thermo Fisher Scientific Inc., Hemel Hempstead, UK).
Gradient Flow Program:
Mass spectra were collected over the 150-900 m/z range at a resolution setting of 7500. All data analysis was carried out in the R programming language in a 64-bit Linux environment (R 2.11). Peak-picking was performed using the Bioconductor package, XCMS (Smith et al (2006) Anal. Chem. 78, 779-787), employing the centWave algorithm (Tautenhahn et al (2008) BMC Bioinformatics 9, 504). Redundancy in peak lists was reduced using the CAMERA package (Kuhl et al (2012) Anal. Chem. 84, 283-289). Alkaloids were identified by comparing exact mass and retention time values to those of standards and quantified by their pseudomolecular ion areas using custom R scripts.
The HN1 BAC library was constructed from high molecular weight (HMW) genomic DNA processed at Amplicon Express, Inc. (Pullman, Wash.) from four week old seedlings using the method described (Tao et al (2002) Theor. Appl. Genet. 105, 1058-1066). The HMW DNA was partially digested with the restriction enzyme HindIII and size selected prior to ligation of fragments into the pCC1BAC vector (Epicentre Biotechnologies, Madison, Wis.) and transformation of DH10B E. coli cells, which were then plated on Luria-Bertani (LB) agar with chloramphenicol, X-gal and IPTG at appropriate concentrations. Clones were robotically picked with a Genetix QPIX (Molecular Devices, Sunnyvale, Calif.) into 240 384-well plates containing LB freezing media. Plates were incubated for 16 hours, replicated and then frozen at −80° C. The replicated copy was used as a source plate for nylon filters that were made and used for screening using the PCR DIG Probe Synthesis Kit (Roche Applied Science, Indianapolis, Ind.). To estimate insert sizes, DNA aliquots of 10 BAC minipreps were digested with 5 U of NotI enzyme for 3 hours at 37° C. The digestion products were separated by pulsed-field gel electrophoresis (CHEF-DRIII system, Bio-Rad, Hercules, Calif.) in a 1% agarose gel in TBE. Insert sizes were compared to those of the Lambda Ladder MidRange I PFG Marker (New England Biolabs, Ipswich, Mass.). Electrophoresis was carried out for 18 hours at 14° C. with an initial switch time of 5 s, a final switch time of 15 s, in a voltage gradient of 6 V/cm. The average BAC clone size for the library was found to be 150 Kb.
Filter design and screening was carried out at Amplicon Express, Inc. (Pullman, Wash.). Bioassay dishes containing LB agar plate media and 12.5 μg/mL chloramphenicol were prepared. Positively charged nylon Amersham Hybond-N+ membrane (GE Healthcare Bio-Sciences, Piscataway, N.J.) was applied to the media surface and the GeneMachines G3 (Genomics Solutions, Bath, UK) was used to robotically grid 18,432 clones in duplicate on filters. The filters were incubated at 37° C. for 12 to 14 hours. The filters were processed using the nylon filter lysis method (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, ed. 3, vol. 1, chap. 1) with slight modifications. Following processing, the DNA was linked to the hybridization membrane filters according to the Hybond N+ manual by baking at 80° C. for 2 hours. To screen the library a 643 bp digoxigenin (DIG)-labeled probe representing position 2161-2803 in the genomic sequence of CYP82X2 (SEQ ID NO 6) was generated from 1.5 ng gDNA by PCR reaction using the primers shown in Table 3 and the PCR DIG synthesis kit (Roche Applied Science, Indianapolis, Ind.) according to the manufacturer's instructions. A non-labeled probe was amplified, diluted and spotted to each filter in the following dilutions of 2 ng, 1 ng, 0.1 ng and 0.0 ng as a positive control. The controls were baked at 80° C. for 30 min. Following a 30 min prehybridizing wash in DIG EasyHyb solution at 45° C. approximately 0.5 μl of denatured DIG labeled PCR product was added per ml of hybridization solution with the nylon filters and incubated with gentle shaking overnight at 45° C. The nylon filters were washed twice in a 2× standard sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) buffer at room temperature for 5 min each, and twice with a 0.5×SSC, 0.1% SDS buffer at 65° C. for 15 minutes each. The hybridized probe was detected using NBT/BCIP stock solution according to the manufacturer's instructions (Roche Applied Science, Indianapolis, Ind.) and was found to hybridize to six BAC clones.
BAC sequencing and automated sequence assembly: The six positive BAC clones from the BAC library were sequenced at Amplicon Express, Inc. (Pullman, Wash.) by Focused Genome Sequencing (FGS) with an average depth of 100× coverage. FGS is a Next Generation Sequencing (NGS) method developed at Amplicon Express that allows very high quality assembly of BAC clone sequence data using the Illumina HiSeq platform (Illumina, Inc, San Diego, Calif.). The proprietary FGS process makes NGS tagged libraries of BAC clones and generates a consensus sequence of the BAC clones with all reads assembled at 80 bp overlap and 98% identity. The gapped contiguous sequences were ordered and orientated manually based on mate pair sequences from four libraries of insert size 5000, 2000, 500 and 170 bp. Overlapping BAC clones, PS_BAC193L09, PS_BAC179L19, PS_BAC150A23 and PS_BAC164F07, which together encoded all 10 genes from the HN1 cluster, were selected for further sequence assembly. Where possible, gaps and ambiguous regions on both BAC clones were covered by primer walking with traditional Sanger sequencing to validate the assembly. Combination of the four overlapping BAC sequences gave a single continuous consensus sequence assembly of 401 Kb. The sequences of the 10 genes from the HN1 cluster were determined independently by Sanger sequencing and the 100% agreement of the Sanger determined gene sequences with the assembly from FGS provided quality assurance for the whole assembly.
Annotation of the assembled sequence: The sequences of the four BAC clones were annotated with an automated gene prediction program FGENESH (Salamov and Solovyev (2002) Genome Res. 10, 516-522). The gene structure including exon-intron arrangement for the 10 genes in the HN1 cluster was validated by comparison with cDNA sequence for each gene. cDNA sequence was not available for any of the remaining ORFs detailed in
The tobacco rattle virus (TRV) based gene silencing system (Liu et al (2002) Plant J. 30, 415-422) was used to investigate the gene function of PSMT1, PSMT2, CYP719A21, CYP82X2, PSSDR1 and PSCXE1. DNA fragments selected for silencing were amplified by PCR and cloned into the silencing vector pTRV2 (GenBank accession no: AF406991). They were linked to a 129 bp-long fragment (SEQ ID NO: 30) of the P. somniferum PHYTOENE DESATURASE gene (PSPDS) in order to simultaneously silence the respective candidate genes and PSPDS. Plants displaying the photo-bleaching phenotype resulting from PSPDS silencing (Hileman et al (2005) Plant J. 44, 334-341) were identified as plants successfully infected with the respective silencing constructs and selected for further analysis.
Generation of the pTRV2:PDS construct: A 622 bp fragment of PSPDS was amplified from cDNA prepared from HN1 using primers shown in Table 3. Sau3Al digestion of the 622 bp PCR product yielded among others a fragment of 129 bp (SEQ ID NO: 30) which was cloned into the BamHI site of the pTRV2 vector. The orientation and fidelity was confirmed by sequencing and the resulting pTRV2:PDS vector was used in the generation of the VIGS construct for each candidate gene. The pTRV2:PDS construct also served as the control in the VIGS experiments.
DNA fragments selected for silencing the respective candidate genes were amplified from either HN1 genomic or cDNA. Primers used for amplification as well as the positions of the selected sequences within the respective open reading frames are shown in Table 3. The PSMT1, CYP719A21 and CYP82X2 fragments were first cloned into pTV00 (Ratcliff et al (2001) Plant J., 237-245) using HindIII and KpnI and then subcloned into pTRV2:PDS using BamHI and KpnI. PSMT2, PSCXE1 and PSSDR1 fragments were cloned directly into pTRV2:PDS using BamHI and KpnI. The orientation and fidelity of all constructs was confirmed by sequencing.
Transformation of Agrobacterium tumefaciens with VIGS constructs: VIGS constructs were propagated in E. coli strain DH5α and transformed into electrocompetent Agrobacterium tumefaciens (strain GV3101) by electroporation.
Infiltration of plants: Separate overnight liquid cultures of A. tumefaciens containing individual VIGS constructs (each consisting of a selected DNA fragment from the target gene linked to the 129 bp-long fragment from the P. somniferum PHYTOENE DESATURASE gene) were used to inoculate LB medium containing 10 mM MES, 20 μM acetosyringone and 50 μg/ml kanamycin. Cultures were maintained at 28° C. for 24 hours, harvested by centrifugation at 3000×g for 20 min, and resuspended in infiltration solution (10 mM MES, 200 μM acetosyringone, 10 mM MgCl2) to an OD600 of 2.5. A. tumefaciens harbouring the individual VIGS constructs including the control, pTRV2:PDS, were each mixed 1:1 (v/v) with A. tumefaciens containing pTRV1 (GenBank accession no: AF406990), and incubated for two hours at 22° C. prior to infiltration. Two week old seedlings of HN1 grown under standard greenhouse conditions (22° C., 16 h photoperiod), with emerging first leaves, were infiltrated as described (Hagel and Facchini (2010) Nat. Chem. Biol. 6, 273-275).
Latex and capsule analysis of silenced plants: Leaf latex of infiltrated plants displaying photo-bleaching as a visual marker for successful infection and silencing was analyzed when the first flower buds emerged (˜7 week old plants). Latex was collected from cut petioles, with a single drop dispersed into 500 μl of 10% acetic acid. This was diluted 10× in 1% acetic acid to give an alkaloid solution in 2% acetic acid for further analysis. Capsules were harvested from the same plants used for latex analysis and single capsules were ground to a fine powder in a ball mill (Model MM04, Retsch, Haan, Germany). Samples of ground poppy straw were then weighed accurately to 10±0.1 mg and extracted in 0.5 ml of a 10% acetic acid solution with gentle shaking for 1 h at room temperature. Samples were then clarified by centrifugation and a 50 μl subsample diluted 10× in 1% acetic acid to give an alkaloid solution in 2% acetic acid for further analysis. All solutions were analyzed as described for the poppy straw analysis from field grown F2 plants. Likewise, all data analysis was carried out using the R programming language. Putative alkaloid peaks were quantified by their pseudomolecular ion areas using custom scripts. Peak lists were compiled and any peak-wise significant differences between samples were identified using 1-way ANOVA with p-values adjusted using the Bonferroni correction for the number of unique peaks in the data set. For any peak-wise comparisons with adjusted p-values <0.05, Tukey's HSD test was used to identify peaks that were significantly different between any given sample and the control. Alkaloids were identified by comparing exact mass and retention time values to those of standards. Where standards were not available, the Bioconductor rcdk package (Smith et al (2006) Anal. Chem. 78, 779-787) was used to generate pseudomolecular formulae from exact masses within elemental constraints C=1 100, H=1 200, O=0 200, N=0 3 and mass accuracy <5 ppm. The hit with the lowest ppm error within these constraints was used to assign a putative formula.
Capsule extract from three opium poppy varieties developed in Tasmania for alkaloid production designated as High Morphine 1 (HM1), High Thebaine 1 (HT1) and High Noscapine 1 (HN1) on the basis of the most abundant alkaloid in each case (
An F2 mapping population of 271 individuals was generated using HN1 and HM1 as parents. Genotyping of the field grown F2 population revealed that the HN1 specific genes are tightly linked and associated with the presence of noscapine suggesting they occur as a gene cluster involved in noscapine biosynthesis (
To Further Characterize the Putative Noscapine Gene Cluster, a Bacterial Artificial Chromosome (BAC) Library was Prepared from Genomic DNA Isolated from HN1 and Six Overlapping BACs Containing Genes from the Cluster were Identified.
Next generation and Sanger sequencing was used to generate a high quality assembly of 401 Kb confirming the arrangement of the 10 genes in a cluster spanning 221 Kb (
In order to functionally characterize the genes in the HN1 cluster Virus Induced Gene Silencing (VIGS) was performed on poppy seedlings. VIGS in poppy seedlings persists through to mature plant stages (Hileman et al (2005) Plant J. 44, 334-341), and therefore both leaf latex and capsule extracts were routinely assayed (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1204407.9 | Mar 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/050599 | 3/12/2013 | WO | 00 |
