Patent Application: US-93766500-A

Abstract:
the present invention concerns codeinone reductase from alkaloid poppy plants , the polynucleotides encoding the enzyme , transgenic plants transformed or transfected with polynucleotide encoding codeinone reductase and to the production of alkaloids from transformed or transfected poppy plants .

Description:
cdnas that encode codeinone reductase were isolated . four full - length reading frames and two partial clones ( fig1 to 15 ) were isolated that represent six alleles from a gene family that may have at least 10 members . an analysis of rna and enzyme activity from various stages of developing opium poppy seedlings and roots , stem , leaf and capsule of mature poppy plants indicated that transcript from these alleles is present throughout the plant at all developmental stages , with the highest total enzyme activity being in the capsule after petal fall . this would suggest that morphine biosynthesis occurs in all major plant organs starting within the first seven days after seed germination . biosynthesis of morphine continues throughout the life cycle of this annual with the highest biosynthetic activity taking place in the capsule after petal fall , consistent with the amount of biosynthetic enzyme present . the amount of extractable rna remained high in the capsule until three days after petal fall , after which time the quantity of extractable rna decreased rapidly . a biochemical analysis of four functionally expressed alleles , cor1 . 1 - cor1 . 4 , revealed no significant differences in the temperature or ph optima , k m values or substrate specificity of the isoforms . all isoforms were able to reduce morphinone to morphine . purification and amino acid sequence analysis of opium poppy codeinone reductase codeinone reductase was purified to apparent electrophoretic homogeneity from opium poppy cell suspension cultures and the amino acid sequence of seven endoproteinase lys - c - generated peptides was determined ( fig2 ). a comparison of these amino acid sequences with those available in the genbank / embl sequence database allowed a relative positioning of peptides 7 , 14 and 16 due to sequence homology with an nadph - dependent reductase from members of the fabaceae — alfalfa , glycyrrhiza and soybean ( 6 ′- deoxychalcone synthase ) that synthesizes 4 , 2 ′, 4 ′- trihydroxychalcone in co - action with chalcone synthase ( fig3 ) ( welle et al ., 1991 ). pcr primers were then designed based on the codeinone reductase peptide sequences . the sequences of the primers used in the first round of pcr were : resolution of an aliquot of the first pcr experiment by agarose gel electrophoresis revealed a mixture of dna products , none of which was the expected band of approximately 480 bp . this was presumably due to the relatively low specificity of the degenerate primers coupled to a low abundance of codeinone reductase transcript . another aliquot of the first pcr reaction mixture was , therefore , used as template for nested pcr with the following primers : seq id no : 1 5 ′- gaa ctt ttt ata act tct aa - 3 ′ ( same as pep - g c c c g c tide 14 primer t above ) and seq id no : 3 3 ′- cai cac tta gtt cac ctt tac - 5 ′ ( nested primer g c c derived from peptide 16 ) to yield an approximately 360 bp dna fragment and the following primers to yield an approximately 180 bp dna product : 5 -′ gti gti aac caa gti gaa atg agi cci ac - 3 ′ ( nested primer derived from 3 ′- gtg gtc taa cgt cai cgt tci cct tt - 5 ′ ( same as peptide 7 primer above ) the results from the nested pcr were bands of the expected size . the translation of the nucleotide sequences of these pcr products indicated that they encode codeinone reductase . screening of approximately 200 , 000 clones of a primary cdna library prepared from opium poppy rna isolated from capsule and cell suspension culture did not result in the identification of codeinone reductase clones . likewise , difficulty was also confronted with detecting a band on rna gel blots that corresponds to the size expected for codeinone reductase . in order to overcome the apparent problem of low steady state levels of codeinone reductase transcript , race - pcr was used to generate both the 5 ′- and 3 ′- portions of the cdna ( frohman , 1993 ). a series of non - degenerate primers based on the nucleotide sequence information determined for the pcr product generated as described in the previous section were used for 5 ′- and 3 ′- race . the nucleotide sequence of the resultant 5 ′- and 3 ′- partial clones were thus determined in three major fragments and suggested the presence of isoforms . the full length cdna clones were then generated by rt - pcr using the following primers and rna isolated from opium poppy cell suspension culture as template : 5 ′- atg gag agt aat ggt gta cct - 3 ′ ( located at the 5 ′- terminus ) and 3 ′- tct acc att cac tcc tga cag - 5 ′ ( located in the 3 ′- flanking region ) 5 ′- atg gct agc atg gag agt aat ggt gta cct atg - 3 ′ ( located at the 3 ′- ctt ctc aag acc cta ctc ttc cta cct agg gaa - 5 ′ ( located at the the pcr product was digested with the restriction endonucleases nhe i / bam hi , ligated into nhe i / bam hi digested pcal - c and transformed into escherichia coli bl21 ( de ) plyss . each cdna was hence constructed in frame in front of dna encoding a 25 amino acid long calmodulin - binding peptide to facilitate eventual heterologous protein purification . single colonies were grown in 3 ml medium and were assayed for the ability to reduce codeinone . of forty colonies tested , ten were found to contain functional enzyme . nucleotide sequence determination of these ten cdnas resulted in the identification of four alleles encoding codeinone reductase . the analogous pcr products had also been prepared with the cdnas placed behind the calmodulin - binding peptide gene in pcal - n - ek , but only the c - terminal fusion proteins bound the calmodulin affinity resin , indicating that the amino terminus of the fusion protein lies within the folded polypeptide . by sequence comparison , codeinone reductase clearly belongs to the aldo / keto reductase family , a group of structurally and functionally related nadph - dependent oxidoreductases . members of this family possess three consensus sequences that are also positionally conserved : aldo / keto reductase consensus 1 ( amino terminus )— g ( f , y ) r ( h , a , l )( l , i , v , m , f ) d ( s , t , a , g , c )( a , s ) x x x x x e x x ( l , i , v , m ) g [ cor1 . 1 — g y r h f d t a a a y q t e e c l g ]; aldo / keto reductase consensus 2 ( central )—( l , i , v , m , f , y ) x x x x x x x x x ( k , r , e , q ) x ( l , i , v , m ) g ( l , i , v , m ) ( s , c ) n ( f , y ) [ cor1 . 1 — m e e c q t l g f t r a i g v c n f ]; aldo / keto reductase consensus 3 ( carboxy terminus )—( l , i , v , m ) ( p , a , i , v )( k , r )( s , t ) x x x x r x x ( g , s , t , a , e , q , k ) ( n , s , l ) x x ( l , i , v , m , f , a )[ cor1 . 1 — v v k s f n e a r m k e n l k i ]. this third consensus sequence is centred around a lysine residue , the modification of which has been shown to affect the catalytic efficiency of aldose and aldehyde reductases ( morjana et al ., 1989 ). the four functional full - length cdnas ( cor1 . 1 , cor1 . 2 , cor1 . 3 and cor1 . 4 ) encoding codeinone reductase share approximately 95 – 96 % sequence identity ( fig4 ). these sequences are comprised in microbial deposit nos . dsm 12737 , dsm 12738 , dsm 12739 and dsm 12740 respectively , deposited at deutsche sammlung von mikroorganismen und zellkulturen gmbh ( dsmz ) of mascheroder weg 1b , d - 38124 braunschweig , germany on 16 mar . 1999 . in addition , a similar cdna generated by pcr ( cor2 ) was 70 % identical to the codeinone reductase cdnas , but was not functional . these opium poppy cdnas were 53 % identical to soybean nadph - dependent reductase 6 ′- deoxychalcone synthase ( welle et al ., 1991 ) ( fig4 ), 33 % identical to rat 3 - hydroxysteroid dehydrogenase [ ec 1 . 1 . 1 . 50 ], 38 % identical to bovine prostaglandin f synthase [ ec 1 . 1 . 1 . 188 ], 37 % identical to apple d - sorbitol - 6 - phosphate dehydrogenase [ ec1 . 1 . 1 . 200 ], 38 % identical to bacterial ( pseudomonas putida ) morphine 6 - dehydrogenase [ ec 1 . 1 . 1 . 218 ] and 35 % identical to yeast ( pichia stipitis ) xylose reductase ( amore at al ., 1991 ). genomic dna was used as template for a pcr analysis of cor1 . 1 – cor1 . 4 . each gene was found to contain one intron that was conserved in size ( 443 bp ) and location ( beginning after nucleotide + 561 ) within the open reading frame , but not in nucleotide sequence . in comparison , cor2 contained two introns beginning after nucleotides + 321 and + 514 . genomic dna gel blot analysis using cor1 . 1 as hybridization probe resulted in a complex hybridization pattern that suggests the presence of at least ten genes that could encode codeinone reductase in opium poppy ( fig5 ). from the isolation and nucleotide sequence analysis of cdna clones , it is certain that at least six of these ten genes are expressed in the plant and plant cell suspension culture . ( two additional partial cdnas ( cor1 . 5 and cor1 . 6 ; fig1 and 15 ) were generated by rt - pcr using plant rna as template .) when the peptide sequences presented in fig2 are compared with the translations of the cdna sequences in fig4 , it is clear that a mixture of isoforms was purified for amino acid sequence analysis . from the initial biochemical analysis of codeinone reductase , evidence for only two isoforms in the poppy plant and one isoform in poppy cell suspension culture was observed ( lenz and zenk , 1995b ). rna gel blot analysis indicated the presence of a very weakly hybridizing rna of approximately 1 . 4 kb in poppy leaf , root and stem of a mature plant two days after petal fall ( fig6 ). since cor1 transcript was apparently present at very low levels , further analysis was undertaken by nested rt - pcr . morphinan alkaloids begin to accumulate rapidly in poppy seedlings four to seven days after germination ( rush et al ., 1985 ; wieczorek et al ., 1986 ). an analysis of codeinone reductase enzyme activity and transcript accumulation showed that enzyme activity is at 310 pkat / g dry tissue weight ( dwt ) already at day seven after germination ( table 1 ). this activity remains at that level throughout a three week growth period , then decreases to 148 pkat / g dwt by the eighth week . in comparison , opium poppy cell suspension culture also contains 330 pkat / g dwt enzyme activity . transcript was detected by rt - pcr for cor1 . 1 - cor1 . 4 at all developmental stages . since two pcr amplifications were necessary in order to detect cor1 transcript , a comparative quantitation was not undertaken . the distribution of codeinone reductase enzyme activity and transcript was also investigated in mature opium poppy plants two days after petal fall . on a dry tissue weight basis , most activity was present in the capsule ( 730 pkat / g dwt ), then the lateral root ( 560 pkat / g dwt ) followed by stem and leaf lamina ( table 2 ). again , no differences could be found in the distribution pattern of the four isoforms by rt - pcr . table 2 analysis of codeinone reductase enzyme activity and transcript in developing opium poppy two days after petal fall . plant specific activity total activity transcript part ( pkat / mg ) ( pkat / dwt ) detection a capsule 25 730 + stem b 30 250 + leaf 10 120 + lamina lateral 90 560 + root a presence of transcript in each rna population was determined by performing two nested pcr amplifications as described in the examples . b stem tissue beginning at the receptacle and extending 12 cm downwards was extracted . plants were approximately 120 cm high . the four codeinone reductase isoform - calmodulin - binding peptide fusion proteins were purified from e . coli lysates in one step with a calmodulin affinity column . beginning with 250 mg total protein in the bacterial extract , 10 . 5 mg codeinone reductase with a specific activity of 5 . 2 nkat / mg protein could be obtained in 73 % yield . aliquots from a typical purification analyzed by sds - page are shown in fig7 . codeinone reductase purified by this method is nearly homogeneous and demonstrated properties that compared favourably to those of the native enzyme ( lenz and zenk , 1995b ). the temperature optimum , ph optimum and k m values for codeinone , codeine , nadph and nadp were determined for each of the isoforms ( k m values are indicated in table 3 ). significant differences in these values were not found . for all isoforms , the temperature optimum for reduction ( physiologically forward reaction ) was 28 ° c ., for oxidation ( physiologically reverse reaction ) was 30 ° c ., the ph optimum for reduction was 6 . 8 and for oxidation was 9 . 0 . the isoforms were also tested for their ability to transform morphinan alkaloids structurally related to codeinone and codeine . the reductive reaction with nadph as cofactor functions with morphinone , hydrocodone and hydromorphone as substrate . the oxidative reaction with nadp as cofactor functions with morphine and dihydrocodeine as substrate . the k m values for , and structures of , these additional substrates with cor1 . 3 are shown in fig8 . in all cases , the physiologically forward reaction yielded lower k m values than the physiologically reverse reaction , with codeinone having the lowest k m value at 48 μm . no differences in temperature or ph optimum were observed whether codeinone or morphinone were used as substrate in the assay . nadh could not substitute for nadph with any of the isoforms . tritium was enzymatically transferred to codeinone from [ 4r - 3 h ] nadph , but not from [ 4s - 3 h ] nadph , indicating that codeinone reductase stereospecifically abstracts the pro - r hydrogen from the cofactor . the reduction of codeinone to codeine is the last of three nadph - dependent reductions that occur along the biosynthetic pathway leading from ( s )- reticuline to morphine in opium poppy . the two other potential substrates for reduction , the 1 , 2 - dehydroreticulinium ion and salutaridine ( fig1 ), or for the physiologically reverse reaction , salutaridinol and ( r )- reticuline , were tested as substrates ; with the codeinone reductase isoforms . none of these alkaloids served as substrate indicating that codeinone reductase can catalyze only one reductive step in morphine biosynthesis . in addition , the following analogs were also inactive : ( s ) and ( r )- norreticuline , ( s )- reticuline and norcodeine . since codeinone reductase showed sequence similarity to several members of the aldo / keto reductase family , a series of substrates were tested to reflect members from carbohydrate and steroid metabolism . d - sorbitol - 6 - phosphate , d - xylose , prostaglandin d1 , 5 - androstene - 3β , 17β - diol , 5α - androstan - 17β - ol - 3 - one , 5α - cholestane - 3β - ol , β - estradiol , cyclohexanone and 2 - cyclohexene - 1 - one were not transformed by codeinone reductase . the highest amino acid sequence identity ( 53 %) was , however , to the reductase subunit of the 6 ′- deoxychalcone synthase complex from soybean ( welle et al ., 1991 ). in order to test for a functional evolutionary relationship between isoflavonoid and alkaloid anabolism , codeinone reductase was analyzed for the ability to substitute for the reductase in the formation of 6 - deoxychalcone in co - action with either native chalcone synthase or native stilbene synthase from pinus sylvestris . in the presence of 4 - coumaryl - coa , malonyl - coa , nadph , chalcone synthase and codeinone reductase or cinnamoyl - coa , malonyl - coa , nadph , stilbene synthase and codeinone reductase , formation of product was not observed . likewise , the reductase of the 6 ′- deoxychalcone synthase complex could neither reduce codeinone in the presence of nadph nor oxidize codeine in the presence of nadp . cell suspension cultures of the opium poppy papaver somniferum were routinely grown in either 1 - liter conical flasks containing 400 ml of linsmaier - skoog medium ( linsmaier and skoog , 1965 ) over 7 days at 23 ° c . on a gyratory shaker ( 100 rpm ) in diffuse light ( 750 lux ). differentiated opium poppy plants were grown outdoors in upper bavaria . seedlings were grown on substrate from 7 to 56 days in a greenhouse at 20 ° c ., 65 % relative humidity and 12 h cycles of light and dark . a mixture of codeinone reductase isoforms was purified from opium poppy cell suspension cultures exactly according to lenz and zenk ( 1995b ). the purified enzyme preparation was subjected to sds / page to remove traces of impurities and the coomassie brilliant blue r - 250 - visualized band representing codeinone reductase was digested in situ with endoproteinase lys - c as reported in ( eckerskorn and lottspeich , 1989 , dittrich and kutchan , 1991 ). the peptide mixture thereby obtained was resolved by reversed phase hplc [ column , merck lichrospher rp18 ; 5 μm ( 4 × 125 mm ); solvent system , ( a ) 0 . 1 % trifluoroacetic acid , ( b ) 0 . 1 % trifluoroacatic : acid / 60 % acetonitrile ; gradient of 1 % per min ; flow rate of 1 ml / min ] with detection at 206 nm . microsequencing of seven of the peptides thus purified was accomplished with an applied biosystems model 470 gas - phase sequencer . partial cdnas encoding codeinone reductases from opium poppy were produced by pcr using cdna produced by reverse transcription of total rna isolated from 3 to 5 - day - old suspension cultured cells . dna amplification using either taq or pfu polymerase was performed under the following conditions : 4 min at 94 ° c ., 35 cycles of 94 ° c ., 30 sec : 45 ° c ., 30 sec ; 72 ° c ., 1 min . at the end of 35 cycles , the reaction mixtures were incubated for an additional 5 min at 72 ° c . prior to cooling to 4 ° c . reamplification of dna using nested primers was performed as above , but the primer annealing temperature was raised from 45 to 55 ° c . the amplified dna was then resolved by agarose gel electrophoresis , the bands of approximately the correct size were isolated and subcloned into pgem - t ( promega ) prior to nucleotide sequence determination . the specific sequences of the oligodeoxynucleotide primers used are indicated above . total rna was isolated and rna gels were run and blotted as previously described ( pauli and kutchan , 1998 ). genomic dna was isolated and dna gels were run and blotted according to bracher and kutchan ( 1992 ). cdna clones were labelled by random - primed labelling with [ α - 32 p ] dctp and oligodeoxynucleotides were end - labelled with [ γ - 32 p ] atp . hybridized rna on northern blots and dna on southern blots were visualised with a raytest bas - 1500 phosphorimager . the entire nucleotide sequence on both dna strands of full - length cdna clones in either pgem - t or pcal - c was determined by dideoxy cycle sequencing using internal dna sequences for the design of deoxyoligonucleotides as sequencing primers . the sequence information requisite to the generation of full - length cdnas was derived from the nucleotide sequences of the partial cdnas generated as described above . the complete nucleotide sequence of one reading frame was determined using codeinone reductase specific oligodeoxynucleotide primers in 5 ′- and 3 ′- race - pcr experiments with a marathon ™ cdna amplification kit ( clontech ). race - pcr was performed using the pcr cycles described above . the amplified dna was then resolved by agarose gel electrophoresis and the band of the approximate expected size was isolated , subcloned into pgem - t and sequenced . nested primer pairs were then used to generate full - length clones for heterologous expression by rt - pcr using opium poppy cell suspension culture rna as template . the final primers used in clone amplification contained the restriction endonuclease recognition sites nhe i and bam hi that were appropriate for subcloning directly into the pcal - c ( stratagene ) expression vector . the specific sequences of these primers are indicated above . rt - pcr was carried out using the pcr cycles given above . the amplified dna was then resolved by agarose gel electrophoresis and the band of the correct size ( 972 bp ) was excised and isolated for further subcloning into the expression vector . full - length cdnas generated by rt - pcr were ligated into p - cal - c and transformed into the e . coli strain bl21 ( de3 ) plyss . for enzyme assays , single colonies were picked and grown in 3 ml luria - bertani medium containing 100 μg / ml ampicillin at 37 ° c . to an od 590 of 0 . 8 . for protein purification , single colonies were picked and grown in 1 l luria - bertani medium containing 100 μg / ml ampicillin at 37 ° c . to an od 590 of 1 . 8 . cells were collected by centrifugation 5 min at 4 , 000 × g and 4 ° c . the bacterial pellet was resuspended in either 0 . 1 m potassium phosphate buffer ph 6 . 8 for the reduction of codeinone or 0 . 1 m glycine buffer ph 9 for the oxidation of codeine . the bacterial pellet from a 3 ml culture was resuspended in 0 . 5 ml buffer and that from a one liter culture in 100 ml buffer . the cells were ruptured by sonication . cellular debris was removed by centrifugation 5 min at 4 , 000 × g and 4 ° c . and the supernatant used directly for either affinity chromatography purification using the affinity ™ protein expression and purification system according to the manufacturer &# 39 ; s instructions ( stratagene ) or for enzyme activity measurements according to lenz and zenk ( 1995b ). the oxidative and reductive reactions catalyzed by codeinone reductase were assayed according to lenz and zenk ( 1995b ). the oxidation of codeine to codeinone by heterologously expressed enzyme in a crude bacterial extract was used for large scale production of enzymic product for structure elucidation by 1 h nmr , 13 c nmr and mass spectrometry . the enzyme assays were extracted twice with two volumes of chcl 3 , the combined organic phase was reduced in vacuo and resolved by semipreparative hplc using the following gradient : [ column , knauer lichrosopher 100 rp18 endcapped ; 5 μm ( 16 × 250 mm ); solvent system , ( a ) 97 . 99 % ( v / v ) h 2 o 2 % ch 3 cn , 0 . 01 % ( v / v ) h 3 po 4 , ( b ) 1 . 99 % ( v / v ) h 2 o , 98 % ch 3 cn , 0 . 01 % h 3 po 4 ; gradient : 0 – 9 min 0 – 8 % b , 9 – 24 min 8 % b , 24 – 45 min 8 – 25 % b , 45 – 75 min 25 % b , 75 – 75 . 3 min 25 – 0 % b , 75 . 3 – 90 min 0 % b ; flow 4 . 5 ml / min ] with detection at 204 nm using authentic codeine ( retention time , 38 min ) and codeinone ( retention time , 49 min ) as reference materials . in this manner , 10 mg codeinone was enzymically produced and purified . codeinone — 1 h ( 360 mhz , cdcl 3 ) 1 . 87 ( 1h , dd j 15a / 15e 12 . 2 , j 15c / 15a 3 . 1 , h - 1 se ), 2 . 08 ( 1h , ddd , j 15a / 16a 4 . 5 , j 15a / 15e 12 . 2 , h - 15 a ) 2 . 29 ( 1h , ddd , j 15a / 16a 12 . 3 , j 15e / 16a 3 . 1 , j 16a / 16c 3 . 1 , j 16a / 16e 11 . 8 , h - 16 a ), 2 . 35 ( 1h , dd , j 10a / 10e 18 . 5 , j 9 / 10a 5 . 9 , h - 10 a ), 2 . 47 ( 3h , s , ch 3 n —), 2 . 63 ( 1h , dd , j 16a / 16c 11 . 8 j 15a / 16c 4 . 5 , h - 16 e ), 3 . 12 ( 1h , d , j 10a / 10c 18 . 5 , h - 10 c ), 3 . 21 ( 1h , m , h - 14 ), 3 . 43 ( 1h , m , h - 9 ), 3 . 85 ( 3h , s , ch 3 o —), 4 . 71 ( 1h , s , h - 5 ), 6 . 09 ( 1h , dd , j 7 / 8 10 . 1 , j 7 / 14 2 . 8 , h - 7 ), 6 . 62 ( 1h , d , j 1 / 2 8 . 3 , h - 1 ), 6 . 66 ( 1h , dd , j 7 / 8 10 . 1 , j 8 / 14 1 . 5 , h - 8 ), 6 . 68 ( 1h , d , j 1 / 2 8 . 3 , h - 2 ); 13 c ( 90 . 6 mhz , cdcl 3 ) 20 . 5 ( c - 10 ), 33 . 8 ( c - 15 ), 41 . 3 ( c - 14 ), 42 . 8 ( nme ), 43 . 0 ( c - 13 ), 46 . 8 ( c - 16 ), 56 . 8 ( ome ), 59 . 1 ( c - 9 ), 88 . 0 ( c - 5 ), 114 . 8 ( c - 2 ), 119 . 9 ( c - 1 ), 125 . 7 ( c - 11 ), 128 . 9 ( c - 12 ), 132 . 6 ( c - 7 ), 142 . 6 ( c - 3 ), 144 . 9 ( c - 4 ) 148 . 7 ( c - 8 ), 194 . 4 ( c - 6 ); ei - ms ( 70 ev ), m / z 297 ( m + , 100 %, 282 ( 8 ), 268 ( 9 ), 254 ( 8 ), 238 ( 9 ), 229 ( 23 ), 214 ( 17 ), 188 ( 15 ) 165 ( 11 ), 152 ( 13 ), 139 ( 16 ), 128 ( 22 ), 115 ( 41 ). transformation of plants with nucleotide sequences from genes encoding codeinone reductase proteins two plant lines were used in transformation experiments . these were nicotiana tabacum line wisconsin38 , and papaver somniferum line c048 . preparation of plant materials and tissue culture and transformation conditions were as described in an et . al ( 1986 ), hooykaas and schilperoort ( 1992 ) and pct application pct / au99 / 00004 , all of which are incorporated herein by reference . the disarmed agrobacterium tumefaciens strain lba4404 was used in transformation experiments . dna constructs capable of expressing the codeinone reductase genes were prepared in a binary vector containing a 35 s - nptii selectable marker , and transformed into the n . tabacum and p . somniferum lines . successful transformation of these plant lines was achieved as judged by ( a ) regeneration of n . tabacum plants on medium containing 100 mg / l kanamycin indicating expression of the nptii selectable marker , which was verified by nptii enzyme assays . coexpression of the codeinone reductase gene was determined by rt - pcr ( reverse transcriptase polymerase chain reaction ) assay . ( b ) successful selection of transformed cell cultures of p . somniferum using the same nptii selectable marker indicative of expression from the vector , followed by the generation of typei and typeii embroyogenic callus prior to the production of transformed plants . thus , the identification and cloning of genes for codeinone reductase from p . somniferum now provides a means by which alteration of the enzymatic step ( s ) involving this can be achieved . the overexpression of these sequences can be achieved using vectors which express one or more of the codeinone reductase alleles , while downregulation of general codeinone reductase activity or the activity of specific alleles can be achieved using vectors expressing antisense , ribozymes , plus - sense cosuppression or rnai sequences from regions conserved between the codeinone reductase alleles or other sequences which are unique to each allele . these genes encoding the sense , antisense , ribozyme , rnai or other such sequences can be delivered as transgenes stably integrated into the poppy genome or transiently in the form of a viral vector . although the invention has been described with reference to specific embodiments , modifications that are within the knowledge of those skilled in the art are also contemplated as being within the scope of the present invention . amore , r ., koetter , p ., kuester , c ., cirlacy , m ., and hollenberg , c . p . 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