# Patent

## Title
Biosynthesis of opiate alkaloids

## Abstract

The disclosure relates to a nucleic acid molecule isolated from a Papaver somniferum cultivar that produces the opiate alkaloid noscapine which comprises 10 genes involved in the biosynthesis of opiate alkaloids.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is the U.S. National Stage of International Application No. PCT/GB2013/050599, filed Mar. 12, 2013, which was published in English under PCT Article 21(2), which in turn claims the benefit of Great Britain Patent Application No. 1204407.09, filed Mar. 13, 2012.
INTRODUCTION
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.
BACKGROUND TO DISCLOSURE
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.
STATEMENTS OF INVENTION
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:

    
    
        i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
        ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
        iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 wherein said nucleic acid molecule encodes polypeptides involved in the biosynthesis of P. somniferum opiate alkaloids or intermediates in the biosynthesis of opiate alkaloids;
        iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
        v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has retained or enhanced opiate alkaloid biosynthetic activity.
    
    


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:

    
    
        i) a nucleotide sequence as set forth in SEQ ID NO: 8;
        ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
        iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence in SEQ ID NO: 8 and which encodes a polypeptide that has carboxylesterase activity; and
        iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 18 or a nucleotide sequence that encodes a polypeptide that has 46% amino acid sequence identity across the full length amino acid sequence set forth in SEQ ID NO: 18 wherein said polypeptide has carboxylesterase activity.
    
    


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;

    
    
        i) a nucleotide sequence as set forth in SEQ ID NO: 9;
        ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
        iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 9 and which encodes a polypeptide that has short-chain dehydrogenase/reductase activity; and
        iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 19 or a nucleotide sequence that encodes a polypeptide that has is 46% amino acid sequence identity across the full length amino acid sequence set forth in SEQ ID NO: 19 wherein said polypeptide has short-chain dehydrogenase/reductase activity.
    
    


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)


    
    
        Hybridization: 5×SSC at 65° C. for 16 hours
        Wash twice: 2×SSC at room temperature (RT) for 15 minutes each
        Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity to Hybridize)

        Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours
        Wash twice: 2×SSC at RT for 5-20 minutes each
        Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity to Hybridize)

        Hybridization: 6×SSC at RT to 55° C. for 16-20 hours
        Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.
    
    


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:

    
    
        i) a polypeptide comprising or consisting of an amino acid sequence as represented in SEQ ID NO: 17; or
        ii) a modified polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO: 17 and which has retained or enhanced cytochrome P450 activity.
    
    


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:

    
    
        i) a polypeptide comprising or consisting of an amino acid sequence as represented in SEQ ID NO:18; or
        ii) a modified polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO: 18 and which has retained or enhanced carboxylesterase activity.
    
    


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:

    
    
        i) a polypeptide comprising or consisting of an amino acid sequence as represented in SEQ ID NO: 19; or
        ii) a modified polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO: 19 and which has retained or enhanced short-chain dehydrogenase/reductase activity.
    
    


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:

    
    
        i) a polypeptide comprising or consisting of an amino acid sequence as represented in SEQ ID NO: 20; or
        ii) a modified polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO: 20 and which has retained or enhanced acetyltransferase activity.
    
    


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:

    
    
        i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 7, 8, 9 or 10;
        ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
        iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 7, 8, 9 or 10 wherein said nucleic acid molecule encodes polypeptides involved in the biosynthesis of P. somniferum opiate alkaloids or intermediates in the biosynthesis of opiate alkaloids;
        iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 17, 18, 19 or 20;
        v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has retained or enhanced opiate alkaloid biosynthetic activity.
    
    


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:

    
    
        i) providing a transgenic plant cell according to the invention;
        ii) cultivating said plant cell to produce a transgenic plant; and optionally
        i) harvesting said transgenic plant, or part thereof.
    
    


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:

    
    
        i) providing a transgenic microbial cell according to the invention that expresses one or more nucleic acid molecules according to the invention in culture with at least one opiate alkaloid or opiate alkaloid intermediate metabolite;
        ii) cultivating the microbial cell under conditions that modify one or more opitate alkaloid or opiate alkaloid intermediate; and optionally
        iii) isolating said opiate alkaloid or opiate alkaloid intermediate from the microbial cell or cell culture.
    
    


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:

    
    
        i) mutagenesis of wild-type seed from a P. somniferum plant that does express said polypeptide;
        ii) cultivation of the seed in i) to produce first and subsequent generations of plants;
        iii) obtaining seed from the first generation plant and subsequent generations of plants;
        iv) determining if the seed from said first and subsequent generations of plants has altered nucleotide sequence and/or altered expression of said polypeptide;
        v) obtaining a sample and analysing the nucleic acid sequence of a nucleic acid molecule selected from the group consisting of:
        
            a) a nucleic acid molecule comprising a nucleotide sequence as represented in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
            b) a nucleic acid molecule that hybridises to the nucleic acid molecule in a) under stringent hybridisation conditions and that encodes a polypeptide with opiate alkaloid biosynthsynthetic activity; and optionally
        
        
        vi) comparing the nucleotide sequence of the nucleic acid molecule in said sample to a nucleotide sequence of a nucleic acid molecule of the original wild-type plant.
    
    


In a preferred method of the invention said nucleic acid molecule is analysed by a method comprising the steps of:

    
    
        i) extracting nucleic acid from said mutated plants;
        ii) amplification of a part of said nucleic acid molecule by a polymerase chain reaction;
        iii) forming a preparation comprising the amplified nucleic acid and nucleic acid extracted from wild-type seed to form heteroduplex nucleic acid;
        iv) incubating said preparation with a single stranded nuclease that cuts at a region of heteroduplex nucleic acid to identify the mismatch in said heteroduplex; and
        v) determining the site of the mismatch in said nucleic acid heteroduplex.
    
    


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:

    
    
        i) a nucleic acid molecule comprising a nucleotide sequence as represented in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
        ii) a nucleic acid molecule comprising a nucleotide sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a polypeptide opiate alkaloid biosynthetic activity.
    
    


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.



BRIEF DESCRIPITION OF THE FIGURES
An embodiment of the invention will now be described by example only and with reference to the following figures:
FIGS. 1A-1B: Identification of genes exclusively present in the genome of a noscapine producing poppy variety, HN1 (High Noscapine 1). (A) Relative abundance of the major alkaloids extracted from the capsules of three commercial varieties of poppy, HM1 (High Morphine 1), HT1 (High Thebaine 1) and HN1. M=morphine, C=codeine, T=thebaine, O=oripavine and N=Noscapine. (B) EST libraries from stem and capsule were generated by pyrosequencing and unique contiguous sequences assembled as described in material and methods. Ten genes (PSMT1, PSMT2, PSMT3, CYP82X1, CYP82X2, CYP82Y1, CYP719A21, PSAT1, PSSDR1 and PSCXE1) as defined in the text, were represented only in EST libraries from the HN1 variety. EST abundance of five other functionally characterized P. somniferum genes (BBE, TNMT, SalR, SalAT and T6DM) show them to be expressed in all three varieties and at consistently higher levels in stem compared to capsule as is also the case for the HN1 specific genes as shown in colour code (FIG. 1). PCR on genomic DNA from all three varieties revealed that the ten HN1 specific genes are absent from the genomes of the HM1 and HT1 varieties (FIG. 5A);
FIGS. 2A-2B: Segregation analysis of noscapine content in an F2 mapping population demonstrates requirement for the noscapine gene cluster. (A) Box plot depiction of noscapine levels as percentage dry weight (DW) in glasshouse grown parental lines HN1 and HM1 and the F1 generation. (B) The field grown F2 generation segregated into three classes of zero, low and high noscapine. F2 GC− and F2 GC+ indicate the absence and presence respectively of the noscapine gene cluster. Numbers in brackets indicate number of individuals in each class;
FIG. 3: The HN1 gene cluster. The structure and position of the ten HN1 specific genes expressed in stems and capsule tissues is shown above the central black line which represents 401 Kb of genomic sequence. Exons are represented by filled grey boxes and introns by fine black lines. Arrows indicate the 5′ to 3′ orientation of each gene. Additional open reading frames depicted below the central black line are as defined by the key. None of these ORFs are represented in the stem and capsule EST libraries;
FIGS. 4A-4I: Functional characterisation using virus induced gene silencing of 6 genes from the HN1 gene cluster. (A-F) Results from both leaf latex and capsules are consistent with each of these genes encoding enzymes involved in noscapine biosynthesis. All compounds that accumulate, apart from scoulerine, have been putatively identified on the basis of mass spectra as detailed in FIG. 6A-6F. The mass-to-charge (m/z) value (M) followed by retention time (T) in seconds is shown for each compound on the horizontal axis. (G-I) Proposed pathway for noscapine biosynthesis based on VIGS data. Solid arrows depict steps supported by VIGS data, dotted arrows depict additional proposed steps. For the secoberbine intermediates, R1=H or OH, R2=H or OH and R3=CH2OH or CHO or COOH (FIGS. 6A-F). The noscapine structure is numbered according to the IUPAC convention;
FIGS. 5A-B: The ten genes exclusively expressed in the HN1 variety occur in the genome of HN1 but are absent from that of varieties HT1 and HM1. (A) Amplification of fragments from the ten genes exclusively expressed in HN1 using two different primer pairs. (B) Amplification of fragments of genes from the protoberberine and morphinan branch pathways that are expressed in all three varieties. Primers used are detailed in Table 3; HyperLadder I (Bioline Reagents, London, UK) was used as molecular size standard;
FIGS. 6A-6F. Evidence for putative identities of intermediates from VIGS experiments. All panels show the mass spectra of the pseudomolecular parent ion at the chromatographic peak apex in black and corresponding MS2 fragmentation spectra in red, scaled to relative abundance. MS2 spectra were generated by targeting the parent ion with an isolation width of 3 m/z and using collisional isolation dissociation energy set to 35%. All mass spectra were obtained at a resolution setting of 7500. Text printed above selected diagnostic ions indicate the exact monoisotopic mass of the ion, the calculated formula within limits C=1:100, O=0:200, N=0:3 and H=1:200, and the number/total number of formulae returned within a 5 ppm error window. Fragments were reconciled against theoretical fragments generated by submitting candidate parent structures to Mass Frontier software (version 5.01.2; HighChem, Bratislava, Slovakia). Candidate parent structures were derived from PubChem searches and the comprehensive review of Papaver spp. alkaloids (Sariyar (2002) Pure Appl. Chem. 74, 557-574). (A) Tetrahydrocolumbamine; this compound was characterized from a peak eluting at 174s from VIGS-silenced CYP719A21. Eight out of ten observed MS2 fragments were calculated as feasible by Mass Frontier; only the two most abundant diagnostic fragments are shown. (B) Secoberbine intermediate 1 (C21H25NO6); this compound was characterized from a peak eluting at 147s from VIGS-silenced CYP82X2. If R1=OH, R2=H, and R3=CH2OH, then this compound is narcotolinol which is consistent with both annotated fragments. Another candidate formula fit would be demethoxylated narcotindiol (R1=H, R2=OH, R3=CH2OH); however this structure would not form the observed fragment at 206.0816. (C) Secoberbine intermediate 2 (C21H23NO6); this compound was characterized from a peak eluting at 103s from VIGS-silenced CYP82X2. If R1=OH, R2=H, and R3=CHO, then this compound would be a desmethylated derivative of macrantaldehyde. (D) Papaveroxine; this compound was characterized from a peak eluting at 214s from VIGS-silenced PSCXE1. The 398.1600 fragment observed is consistent with deacetylation. (E) Narcotinehemiacetal; this compound was characterized from a peak eluting at 121s from VIGS-silenced PSSDR1. (F) Narcotoline (4′-desmethylnoscapine); this compound was characterized from a peak eluting at 208s from VIGS-silenced PSMT2. Other isobaric possibilities were 6- or 7-desmethylnoscapine. However, the 206.0816 fragment observed is consistent with a hydroxylated 4′ position. Alternative structures could be discounted by comparing the candidate fragmentation spectra with that from synthetic 7-desmethylnoscapine, which eluted at a different retention time and lacked the characteristic 206.0816 fragment;
FIGS. 7A-7M are sequences of (A) PSMT1 nucleic acid sequence, SEQ ID NO: 1; (B) PSMT2 nucleic acid sequence, SEQ ID NO: 2; (C) PSMT3 nucleic acid sequence, SEQ ID NO: 3; (D) CYP82X1 nucleic acid sequence, SEQ ID NO: 4; (E) CYP719A21 nucleic acid sequence, SEQ ID NO: 5; (F) CYP82X2 nucleic acid sequence, SEQ ID NO: 6; (G) CYP82Y1 nucleic acid sequence, SEQ ID NO: 7; (H) PSCXE1 nucleic acid sequence, SEQ ID NO: 8; (I) PSSDR1 nucleic acid sequence, SEQ ID NO: 9; (J) PSAT1 nucleic acid sequence, SEQ ID NO: 10; PSMT1 protein sequence, SEQ ID NO: 11; PSMT2 protein sequence, SEQ ID NO: 12; (K) PSMT3 protein sequence, SEQ ID NO: 13; CYP82X1 protein sequence, SEQ ID NO: 14; CYP719A21 protein sequence, SEQ ID NO: 15; CYP82X2 protein sequence, SEQ ID NO: 16; CYP82Y1 protein sequence, SEQ ID NO: 17; (L) PSCXE1 protein sequence, SEQ ID NO: 18; PSSDR1 protein sequence, SEQ ID NO: 19; PSAT1 protein sequence, SEQ ID NO: 20; VIGS PSMT1 protein sequence, SEQ ID NO: 21; VIGS PSMT2 protein sequence, SEQ ID NO: 22; and VIGS CYP82X1 protein sequence, SEQ ID NO: 23; (M) VIGS CYP719A21 protein sequence, SEQ ID NO: 24; VIGS CYP82X2 protein sequence, SEQ ID NO: 25; VIGS CYP82Y1 protein sequence, SEQ ID NO: 26; VIGS PSCXE1 protein sequence, SEQ ID NO: 27; VIGS PSSDR1 protein sequence, SEQ ID NO: 28; VIGS PSAT1 protein sequence, SEQ ID NO: 29; and VIGS PSPDS protein sequence, SEQ ID NO: 30.



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.










TABLE 1






%
Accession



Protein
Identity
number
Annotation







CYP82Y1
54

CYP82X1 from


(SEQ ID


Papaver somniferum


NO 17)
48

CYP82X2 from





Papaver somniferum



39
ABM46919.1
CYP82E3, nicotine demethylase





from Nicotiana tomentosiformis


PSCXE1
45
2O7R_A
AeCXE1, Carboxyl esterase from


(SEQ ID


Actinidia eriantha


NO 18)





PSSDR1
46
AAB41550.1
Vestitone reductase from


(SEQ ID


Medicago sativa


NO 19)
45
ABQ97018.1
Dihydroflavonol 4-reductase from





Saussurea medusa


PSAT1
66
Q94FT4.1
Salutaridinol 7-O-acetyltransferase


(SEQ ID


from Papaver somniferum


NO 20)





















TABLE 2











Expected







segregation






in






F3 if F2 low






noscapine






class is



F3 seed

Observed
heterozygous



family

segregation
and the high


Noscapine class
(obtained
Number
of gene
noscapine


and genotyping
through self-
of F3
cluster in F3
class is


result of F2
pollination of
individuals
progeny
homozygous
Chi-Square















individual
F2 individual)
genotyped
GC+
GC−
GC+
GC−
X-squared
p-value


















low noscapine/GC+
S-111809
28
18
10
21
7
1.714
0.190


low noscapine/GC+
S-111835
26
18
8
19.5
6.5
0.462
0.497


high noscapine/GC−
S-111714
28
28

28


high noscapine/GC−
S-111854
54
54

54






















TABLE 3






Primer sequences
SEQ

SEQ





(5′- to 3′-)
ID

ID




Gene
Forward
NO
Reverse
NO
Notes
Application







PSMT1
GATTCCCGATTTACTCC
31
AACACAAAATACGATTACTT
32
primer pair 1
Primersfor the



TGATG

ACTTTTGTCC


amplification


PSMT1
TGCCTCATGTTATTTCT
33
GCATGAAATGGATGTAGTTA
34
primer pair 2
of fragments



GTTGCC

TCTTGG


from genomic


PSMT2
ATTGATGTCGGTGGTGG
35
ATTCCCGTTCAAGTAAACAT
36
primer pair 1
DNA of HM1,HT1



TCACG

GCGG


and HN1 as shown


PSMT2
GCAACTGTTTCATTAAC
37
CAGTAAATTCACACATTCCG
38
primer pair 2
in FIG. 5



AGGCACATCC

TATCTTCCC





PSMT3
GCTTCAGCATTGGTTAA
39
GAGGGTAAGCCTCAATAACA
40
primer pair 1




CGAGTGC

GACTGG





PSMT3
AGACCGTTTGTACCGAA
41
TCGTTCCATTCGTGAAGAAT
42
primer pair 2




TTCTGC

GC





CYP82X1
GAACCATTAAACACTTG
43
TGCAATTGAATTTAGCTCAT
44
primer pair 1




AGTCATGC

CTCC





CYP82X1
TTGATGAACGACAAGGA
45
ATTCATGATTGTGACCTTTG
46
primer pair 2




ACCG

TAATCC





CYP82X2
ATGTGGAAAACGGTAAG
47
ACGATTCTGTCATCATCATT
48
primer pair 1




CAAGTGG

TTCGC





CYP82X2
CAACCTCAATCTAGCTA
49
CCCAAGATTTTCATATCCTT
50
primer pair 2




GAGTCG

TACAA





CYP82Y1
CAATAATTGAGTAATTT
51
GCTCCGTAAGTGCTCCTGTG
52
primer pair 1




CAGTTCATTCATGG







CYP82Y1
GAATTGTGGTAAAAAAT
53
CCCTTCACATCTACCATCC
54
primer pair 2




TAGATGCAG

CTT





CYP719A21
CAAAGAGTCAATCTGAC
55
CGAGTGCCCATGCAGTGG
56
primer pair 1




TCAAGCTAGC







CYP719A21
TCAAACCCTGCTACTAA
57
CACTCCATCAGACACACAAG
58
primer pair 2




CACTTACTTGC

ACC





PSAT1
TTTTATCGACCTTGAGG
59
AAATGGCAGTTCCACCGC
60
primer pair 1




AACAATTAGG







PSAT1
GACTTCATGATGAAATC
61
CACTGCTGACTTCCATATC
62
primer pair 2




AGATGCAC

AAAGC





PSCXE1
ATGCTGTTGATGCTTTA
63
AGCTGAATTTGTCGATCAA
64
primer pair 1




AACTGGG

TAAGTGG





PSCXE1
AATAAAAATCCAACAAT
65
ACTGGCATGATATGCAACA
66
primer pair 2




GGCAGATCC

TTAGC





PSSDR1
GGAAGATGTGAGCCACC
67
GATACACTGGGAGGAGGAT
68
primer pair 1




TTAAAGC

GGG





PSSDR1
GAGAGTAACCACATCTT
69
CGGCAAAATTCATTCCTTG
70
primer pair 2




TGTTGTCGG

AGC







71

72




BBE
GTTTACTCCCACGTGCA
71
CATTCCTCGTCTAATTCATC
72





TC

TGC





TNMT
GTTTACTCCCACGTGCA
73
GCTTCACTACTTCTTCTTG
74





TC

AAAAG





SalR
AAACAATGCTGGGGTTGC
75
CATTATAATTTCCAATGCCGT
76







AGTTC





SalAT
TAAGAGAGGGAGACCAC
77
CATTCGTTGTTGTTGCTG
78





GAG

GTAAG





T6ODM
CTTATGAAGCTAGGTAA
79
CATCCTCATTGCTTGTGT
80





TGGTATGGA

CC





PSMT1
CTCTAAAATGCCAAACG
81


sequencing primer
Primers used as



CG




sequencing primers


PSMT1
GACCCTTTGGGACTTCC
82


sequencing primer
to obtain genomic



TCG




DNA sequence from


PSMT1
CGTGTTGTTTGGTCCCT
83


sequencing primer
HN1



CG







PSMT1
TGCCTCATGTTATTTCT
84


sequencing primer




GTTGCC







PSMT1
GATTCCCGATTTACTCC
85


sequencing primer




TGATGG







PSMT1
AACACAAAATACGATTA
86


sequencing primer




CTTACTTTTGTCC







PSMT1
TGCCTCATGTTATTTCT
87


sequencing primer




GTTGCC 







PSMT1
GCATGAAATGGATGTAG
88


sequencing primer




TTATCTTGG







PSMT1
AAATCGTTCGCTCTTTA
89


sequencing primer




CCGC







PSMT1
CACACCAAACTTGATCA
90


sequencing primer




TTGTC







PSMT2
ATTGTTGATATTGAATC
91


sequencing primer




AGAAACTTTC







PSMT2
TCAATACCAGTACTGTT
92


sequencing primer




AGTTTCCG







PSMT2
GCAACTGTTTCATTAAC
93


sequencing primer




AGGCACATCC







PSMT2
ATTGATGTCGGTGGTGG
94


sequencing primer




TCACG







PSMT2
GCACACTGTCTTTTTCT
95


sequencing primer




TCCACC







PSMT2
ACCGGAATGAGAATGCA
96


sequencing primer




TAAAGTAAAGG







PSMT2
CCAATACCCAATCAATT
97


sequencing primer




AAACTC







PSMT2
CAGTAAATTCACACATT
98


sequencing primer




CCGTATCTTCCC







PSMT3
ATTGTATAGCCAAAGTT
99


sequencing primer




GCAGGTAGGG







PSMT3
AGACCGTTTGTACCGAA
100


sequencing primer




TTCTGC







PSMT3
GCAGTGAAAGCCATATC
101


sequencing primer




CAAAGC







PSMT3
AACCGTCCCCAAGATGA
102


sequencing primer




TTCC







PSMT3
TCGTTCCATTCGTGAAG
103


sequencing primer




AATGC







PSMT3
GAGGGTAAGCCTCAATA
104


sequencing primer




ACAGACTGG







CYP82X1
GAACCATTAAACACTTG
105


sequencing primer




AGTCATGC







CYP82X1
TTGATGAACGACAAGGA
106


sequencing primer




ACCG







CYP82X1
TCGACAGCGGTTACGAA
107


sequencing primer




CG







CYP82X1
CAATTATCAAAGAATCA
108


sequencing primer




ATGC







CYP82X1
TGCAATTGAATTTAGCT
109


sequencing primer




CATCT 







CYP82X1
ATTCATGATTGTGACCT
110


sequencing primer




TTGTAATCC







CYP82X1
GACAGAGGGCCCAAGTT
111


sequencing primer




AAGG







CYP82X1
AGCAAACCATTCGTCCA
112


sequencing primer




TCC







CYP82X1
TACGACAGGTTGCTAGC
113


sequencing primer




TTGG







CYP82X2
AATAATGGATCAGTCAC
114


sequencing primer




GGCTTCC







CYP82X2
AATCCATCAGATTTTCA
115


sequencing primer




ACCAGAGAGG







CYP82X2
TGTCAGCCAACCATTCG
116


sequencing primer




TCCATCCTAAC







CYP82X2
GGCTTCCCGGAGATGAC
117


sequencing primer




CCAGATTTTAT







CYP82X2
TTGTTATTTTCATGACT
118


sequencing primer




ATTACCACCAGCTTCCT








CTTA







CYP82X2
AGTGGAGGAGGCACAAA
119


sequencing primer




AGTTAGGATGGAC







CYP82X2
CCATGTCTGATAAATAC
120


sequencing primer




GGGTCGGTGTTC







CYP82X2
TTGTTGATAAGGACGAC
121


sequencing primer




TAAGAATAAGCAGAAGA








TA







CYP82X2
ACGATTCTGTCATCATC
122


sequencing primer




ATTTTCGC







CYP82X2
AGTCGTGTATCGTTCGC
123


sequencing primer




TTAATGC







CYP82X2
CATGCCTATCTATTTCC
124


sequencing primer




TCCCTTGCCCTC







CYP82X2
TGTCAGCCAACCATTCG
125


sequencing primer




TCCATCCTAAC







CYP82X2
TGTTCGATCACGTTGTC
126


sequencing primer




TCTTTTTGCCATAA







CYP82X2
TAACAATAAAAGTACTG
127


sequencing primer




ATAATGGTGGTCGAAGG








AGAA







CYP82Y1
TATTGATGTGGACCAGT
128


sequencing primer




ACC







CYP82Y1
TGTAACTCTTGGTCACA
129


sequencing primer




TGG







CYP82Y1
CGCGTACTTGACATTTA
130


sequencing primer




ACG







CYP82Y1
GGATCATCGCCAAAAGA
131


sequencing primer




AAC







CYP719A21
CAAAGAGTCAATCTGAC
132


sequencing primer




TCAAGCTAGC







CYP719A21
TGAAATGCCTGAGATCA
133


sequencing primer




CTAAAATCG







CYP719A21
TCAAACCCTGCTACTAA
134


sequencing primer




CACTTACTTGC







CYP719A21
TGTAAAGACACTTCATT
135


sequencing primer




GATGGGC







CYP719A21
TTCGATTTGTGTAAACA
136


sequencing primer




TTAATGATATTTGG







CYP719A21
GAGATGATCAAGTGGTT
137


sequencing primer




TAACCATTCC







CYP719A21
CGAGTGCCCATGCAGTGG
138


sequencing primer



PSCXE1
AATAAAAATCCAACAAT
139


sequencing primer




GGCAGATCC







PSCXE1
ATGCTGTTGATGCTTTA
140


sequencing primer




AACTGGG







PSCXE1
GGTTAATCGAGAGATGT
141


sequencing primer




TTTGTGGTAGG







PSCXE1
CGATGACACAGAGCAAG
142


sequencing primer




AACGAC







PSCXE1
CGCGGGTATATGTGTAG
143


sequencing primer




CAATCG







PSCXE1
CGGCAACGCCAGTTCCC
144


sequencing primer



PSSDR1
CTAACAGGCAAACAATA
145


sequencing primer




ACAGGTTGC







PSSDR1
GGAAGATGTGAGCCACC
146


sequencing primer




TTAAAGC







PSSDR1
AAAGGTACTGACAGAAA
147


sequencing primer




GAGCTTGCC







PSSDR1
AGATACACTGGGAGGAG
148


sequencing primer




GATGGG







PSSDR1
CGGCAAAATTCATTCCT
149


sequencing primer




TGAGC







PSSDR1
AACATATAGCCAAAGGA
150


sequencing primer




CTCTTCG







PSAT1
AGGATACACAATGACCC
151


sequencing primer




AAC







PSAT1
TTTTATCGACCTTGAGG
152


sequencing primer




AACAATTAGG







PSAT1
TGTTCACTAGGTGGAAA
153


sequencing primer




GAG







PSAT1
AGTACAATACCGAGAAA
154


sequencing primer




TCCGACAAG







PSAT1
GCTCAATTAATGGAACA
155


sequencing primer




GTAGTTACCC












specific PCR








conditions:



PsMT1
VIC®-CGTGTTGTTTGG
156
GCACACTGTCTTTTTCTTC
157
30 cylces, 20 s 
Primer pairs for



TCCCTCG

CACC

extension at 72°
genotyping of the


PsMT2
VIC®-GCAACTGITTCA
158
GCCAGCGCTAATACAAGGA
159
36 cylces, 50 s
F2 mapping popula-



TTAACAGGCACATCC

TGTGG

extension at 72°
tion


PsMT3
VIC®-GCAGTGAAAGCC
160
TCGTTCCATTCGTGAAGAA
161
30 cylces, 30 s




ATATCCAAAGC

TGC

extension at 72°



CYP82X1
VIC®-GCTACGAAAGAT
162
AGCAAACCATTCGTCCATCC
163
30 cylces, 30 s




AATGGTGCAGC



extension at 72°



CYP82X2
VIC®-ATGTGGAAAACG
164
ACGATTCTGTCATCATCAT
165
30 cylces, 50 s




GTAAGCAAGTGG

TTTCGC

extension at 72°



CYP719A21
VIC®-TGAAATGCCTGA
166
GGAATGGTTAAACCACTTG
167
30 cylces, 30 s




GATCACTAAAATCG

ATCATCTC

extension at 72°



PSCXE1
VIC®-ATGCCAGTTTAA
168
GGGAACTGGCGTTGCCG
169
30 cylces, 30 s




GAGCAATAGAAATGG



extension at 72°



PSSDR1
VIC®-GAAGATGTGAGC
170
GCTCAAGGAATGAATTTTG
171
30 cylces, 30 s




CACCTTAAAGC

CCG

extension at 72°













CYP82X2
GTTGACGCAGGAAGCTT
172
GGAACATAAGATTTAACTC
173
Primer pair for PCR amplification



TTGC

CGCCTC

of the BAC library screening probe













PSMT1
aaactcgagaagctTGG
174
aaaggtaccCATGTACTAC
175

Primer pairs for



TCATAATCATCAATCAG

TACATCATCTCC


the amplification


PSMT2
aaactcgagaagcttGT
176
aaaggtaccACTTGAATAT
177

and cloning of



GTAACTAAGCCAGCGC

ATCACCGC


fragments selected


CYP82X1
aaaggatccTTTGAGTA
178
aaaggtaccAACATCTACT
179

for VIGS



ATGGTGAAAAGA

CTCGAGGATTG





CYP82X2
aaactcgagaagcttTA
180
aaaggtaccTTAACTCCGC
181





GGAGGGTATGTCCGGC

CTCGGCTCC





CYP82Y1
aaaggatccTTCAGTT
182
aaaggtaccGTTCATAGTAA
183





CATTCATGGCG

ATAATAACAGGCG





CYP719A21
aaactcgagaagcttAT
184
aaaggtaccCCAACAGGCCA
185





GATCATGAGTAACTTAT

TTCCGTTG






GGA







PSCXE1
aaaggatccTGGCAGAT
186
aaaggtaccTTATGATAGGA
187





CCTTATGAATTCC

AGCAGCTTATTC





PSSDR1
aaaggatccGAAATTGAC
188
aaaggtaccCATTCAAAAAC
189





GAGACAATATGG

GAATATGTGTGC





PSAT1
aaaggatccCCTAAGAGA
190
aaaggtaccAATACAAGTAT
191





GATCCTCCAACTG

GAAAACAAGAGAATAA





PSPDS
GAGGTGTTCATTGCCATG
192
GTTTCGCAAGCTCCTGCATA
193





TCAA

GT









Materials and Methods

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) RootrainersTM (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. SEQ ID NO 194). 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:

    
    
        i. upper stem, 1-3 days after petal fall
        ii. upper stem, 4-6 days after petal fall
        iii. capsule, 1-3 days after petal fall
        iv. capsule, 4-6 days after petal fall
    
    


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 FIG. 1B was counted for each EST library. EST numbers were normalised on the basis of total number of ESTs obtained per library. For each variety, EST counts were combined for the two developmental stages from both stems and capsules. Differences in candidate gene expression levels between organs and varieties were visualised as a heat map using Microsoft Excel.
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.

Genotyping of the HN1×HM1 F2 Mapping Population for the Presence or Absence of the HN1-Specific Candidate Genes 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 GeneMarkerTM 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:













TIME (minutes)
% Eluent A
% Eluent B
Flow (ml/min)


















0.0
98.
2.0
0.50


0.2
98.0
2.0
0.50


0.5
60.0
40
0.50


4.0
20.0
80.0
0.50


4.5
20.0
80.0
0.50









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.
Bacterial Artificial Chromosome (BAC) Library Construction 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 Construction and Screening 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 FIG. 3 since they are not represented in any of the EST libraries. The predicted function of all ORFs was evaluated by BLAST analysis (Altschul et al (1997) Nucleic Acids Res. 25, 3389-3402) and those ORFs with significant hits (e-value less than 1e−8) were included in FIG. 3.
Generation of Plasmid Constructs for Virus Induced Gene Silencing (VIGS) 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.
EXAMPLE 1
Transcriptomic Analysis Reveals the Exclusive Expression of 10 Genes Encoding Five Distinct Enzyme Classes in a High Noscapine Producing Poppy Variety, HN1. These Genes are Absent from the Genome of Two Noscapine Non-Producing Varieties
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 (FIG. 1A) underwent metabolite profiling. Noscapine was found to be unique to HN1 relative to HM1 and HT1. Roche 454 pyrosequencing was performed on cDNA libraries derived from stem and capsule tissue from all three varieties. Analysis of Expressed Sequence Tag (EST) abundance led to the discovery of a number of previously uncharacterized genes that are expressed in the HN1 variety but are completely absent from the HM1 and HT1 EST libraries (FIG. 1B). The corresponding genes were putatively identified as three O-methyltransferases (PSMT1, PSMT2, PSMT3), four cytochrome P450s (CYP82X1, CYP82X2, CYP82X3 and CYP719A21), an acetyltransferase (PSAT1), a carboxylesterase (PSCXE1) and a short-chain dehydrogenase/reductase (PSSDR1). In contrast a number of other functionally characterized genes associated with benzylisoquinoline alkaloid synthesis, including Berberine Bridge Enzyme (BBE), Tetrahydroprotoberberine cis-N-MethylTransferase (TNMT), Salutaridine Reductase (SalR), Salutaridinol 7-O-AcetylTransferase (SalAT) and Thebaine 6-O-demethylase (T6ODM) were expressed in all three varieties (FIG. 1B). PCR analysis on genomic DNA from all three varieties revealed that the genes exclusively expressed in the HN1 variety are present as expected in the genome of HN1 but absent from the genomes of the HM1 and HT1 varieties (FIG. 1B and FIGS. 5A-5B).
EXAMPLE 2
Analysis of an F2 Mapping Population Shows the Genes are Tightly Linked in HN1 and their Presence is Associated with the Production of Noscapine
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 (FIG. 2B). Analysis of noscapine levels in field grown F2 capsules revealed that individuals containing this putative gene cluster fall into two classes. The first class containing 150 individuals, have relatively low levels of noscapine and the second class containing 63 individuals exhibit the high noscapine trait of the parental HN1 variety (FIG. 2B). The 58 F2 individuals that lack the putative gene cluster contain undetectable levels of noscapine (FIG. 2B). F3 family analysis confirmed that F2 individuals exhibiting the high noscapine trait were homozygous for the gene cluster while those exhibiting the low noscapine trait were heterozygous (Table 2). Noscapine levels in both the F1 population (FIG. 2A) and the heterozygous F2 class are much lower than the intermediate levels expected for a semi-dominant trait, suggesting involvement of some form of repression. The step change to high noscapine in homozygous F2 class suggests this trait is linked to the gene cluster locus rather than spread quantitatively among other loci.
EXAMPLE 3
Bacterial Artificial Chromosome Sequencing Confirms that the 10 Genes Exist as a Complex Gene Cluster
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 (FIG. 3). Only one other homologous gene, a carboxylesterase (PSCXE2), was found in the genomic sequence flanking the gene cluster (FIG. 3) but PSCXE2 was not represented in any of our EST libraries. Interspersed among the ten genes are both retrotransposon and DNA transposable element (TE) sequences (FIG. 3), which may have some function in gene rearrangement for cluster formation as thought to be the case for the thalianol and marneral clusters from A. thaliana (Field et al (2011) PNAS 108, 16116-16121).
EXAMPLE 4
Virus Induced Gene Silencing Results in Accumulation of Pathway Intermediates Allowing Gene Function to be Linked to Noscapine Synthesis and a Novel Bifurcated Biosynthetic Pathway to be Proposed
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 (FIGS. 4A-4F). Silencing PSMT1 resulted in accumulation of scoulerine in capsules and also low levels of reticuline in latex, indicating that this gene product is responsible for the first committed step in the pathway to noscapine synthesis (FIG. 4A). The predicted product of PSMT1 is tetrahydrocolumbamine (FIG. 6A), which accumulated in seedlings and capsules that were silenced for CYP719A21 (FIG. 4B). CYP719A21 shows high homology to cytochrome P450 oxidases that act as methylenedioxy bridge-forming enzymes (Diaz Chavez et al (2011) Arch. Biochem. Biophys. 507, 186193; Ikezawa et al (2009) Plant Cell Rep. 28, 123-133). Therefore CYP719A21 may encode a canadine synthase. FIG. 6). Silencing of a second cytochrome P450 gene, CYP82X2, resulted in accumulation of several secoberbine intermediates some of which may represent side products to the main synthetic pathway (FIG. 4C, FIGS. 6B-6C). Silencing of the carboxylesterase gene PSCXE1 resulted in accumulation of up to 20% total alkaloid content of putative papaveroxine (FIG. 6D) implying acetylation of a secoberbine intermediate as depicted in FIGS. 4G-4I. The PSAT1 gene from the HN1 cluster is an obvious candidate for this reaction. Silencing of PSSDR1 resulted in accumulation of what was putatively identified as narcotinehemiacetal (FIG. 6E), an immediate precursor of noscapine (FIGS. 4G-4I). These data support a biosynthetic route to noscapine that involves early O-methylation of a secoberbine intermediate at the position equivalent to the C4′ hydroxyl group of noscapine (FIGS. 4G-4I). However, silencing PSMT2, resulted in accumulation of up to 20% narcotoline, indicating that O-methylation at the C4′ hydroxyl group can also occur as a final step in noscapine production (FIG. 4F). These results imply bifurcation of the main pathway at the secoberbine intermediate stage with PSMT2 being responsible for both the O-methylation of a secoberbine intermediate and narcotoline. Silencing PSMT2 results in accumulation of high levels of narcotoline as flux is directed down the desmethyl branch of the pathway (FIG. 4F and FIG. 6F).

## Claims

1. A method to produce a P. somniferum plant that has altered expression of a polypeptide, comprising:
i) mutagenizing a wild-type seed from a wild-type P. somniferum plant that expresses said polypeptide, thereby generating mutagenized seed;
ii) cultivating the mutagenized seed to produce first and subsequent generations of mutagenized plants;
iii) obtaining seed from the first and subsequent generations of mutagenized plants;
iv) determining if the plants from the seed from said first and subsequent generations of mutagenized plants have an altered nucleotide sequence, altered expression of said polypeptide or both, an altered nucleotide sequence and altered expression of said polypeptide by obtaining a nucleic acid sample from the plant of said first or subsequent generation of mutagenized plants and analysing the nucleic acid sample for presence of a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 7, 8, 9 or 10 and that encodes a polypeptide with opiate alkaloid biosynthetic activity; and optionally
v) comparing the nucleotide sequence of the nucleic acid molecule in said sample to a nucleotide sequence of a nucleic acid molecule of the wild-type plant.

2. The method according to claim 1 wherein said nucleic acid sample obtained from the mutagenized seed or plant is analysed by a method comprising:
i) extracting nucleic acid molecules from said first and subsequent generations of mutagenized plants;
ii) amplifying a part of said extracted nucleic acid molecules by a polymerase chain reaction, thereby generating amplified nucleic acid comprising a part of a nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 7, 8, 9 or 10;
iii) forming a preparation comprising the amplified nucleic acid and nucleic acid extracted from wild-type plants to form heteroduplex nucleic acid;
iv) incubating said preparation with a single stranded nuclease that cuts at a region of heteroduplex nucleic acid to identify a mismatch in said heteroduplex; and
v) determining the site of the mismatch in said nucleic acid heteroduplex.

3. The method according to claim 1, wherein said P. somniferum plant has enhanced opiate alkaloid biosynthetic activity.

4. The method according to claim 1, wherein said P. somniferum plant has reduced or abrogated opiate alkaloid biosynthetic activity.

5. A P. somniferum plant obtained by the method of claim 1.

6. The method of claim 1, wherein the nucleic acid sample is analysed for the presence of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 7, 8, 9 or 10.