High thebaine poppy and methods of producing the same

Abstract

This disclosure relates to the production of opium poppy plants having high levels of thebaine. More particularly, the disclosure relates to the production of opium poppies having high levels of thebaine by simultaneously reducing the expression of genes encoding thebaine 6-0-demethylase (T60DM) and codeine 3-0-demethylase (CODM).

1 . A method of increasing accumulation of thebaine in an opium poppy plant, the method comprising genetically modifying the genome of the plant to include one or more stable genetic modifications to simultaneously reduce the activity of thebaine 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) in the poppy plant. 
     
     
         2 . The method of  claim 1 , wherein T6ODM has the amino acid sequence of SEQ ID NO: 1, CODM has the amino acid sequence of SEQ ID NO: 3, or T6ODM has the amino acid sequence of SEQ ID NO: 1 and CODM has the amino acid sequence of SEQ ID NO: 3. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein
 genetically modifying the plant to simultaneously reduce the activity of T6ODM and CODM comprises:   introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding T6ODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding T6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4;   genetically modifying the plant to introduce a loss of function mutation in an endogenous gene encoding T6ODM;   introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding CODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding CODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4;   genetically modifying the plant to introduce a loss of function mutation in an endogenous gene encoding CODM;   introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding T6ODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding T6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4, and introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding CODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding CODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4;   genetically modifying the plant to introduce a loss of function mutation in an endogenous gene encoding T6ODM and introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding CODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding CODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4;   introducing an expression construct to reduce the accumulation of transcripts from an endogenous gene encoding T6ODM, optionally wherein the sequence of the expression construct to reduce the accumulation of transcripts from the endogenous gene encoding T6ODM comprises a portion of SEQ ID NO: 2 or SEQ ID NO: 4, and genetically modifying the plant to introduce a loss of function mutation in an endogenous gene encoding CODM; or   genetically modifying the plant to introduce a loss of function mutation in an endogenous gene encoding T6ODM and a loss of function mutation in an endogenous gene encoding CODM.   
     
     
         5 .- 9 . (canceled) 
     
     
         10 . A genetically modified opium poppy plant having reduced activity of thebaine 6-O-demethylase (T6ODM) and-codeine 3-O-demethylase (CODM) relative to a wild type plant, wherein the genetically modified opium poppy plant comprises one or more stable genetic modifications to reduce expression of T6ODM, CODM, or both. 
     
     
         11 . The plant of  claim 10  comprising a first expression construct for reducing the expression of T6ODM, optionally wherein the first expression construct comprises a first nucleic acid molecule encoding a first hairpin RNA for reducing expression of an endogenous gene encoding T6ODM; and a second expression construct for reducing expression of CODM, optionally wherein the second expression construct comprises a second nucleic acid molecule encoding a second hairpin RNA for reducing expression of an endogenous gene encoding CODM. 
     
     
         12 . (canceled) 
     
     
         13 . The plant ell of  claim 11 , wherein
 the endogenous gene encoding T6ODM encodes an mRNA comprising the sequence of SEQ ID NO: 15 and the endogenous gene encoding CODM encodes an mRNA comprising the sequence of SEQ ID NO: 16; or   the endogenous gene encoding T6ODM encodes an mRNA comprising the sequence of SEQ ID NO: 15 and the endogenous gene encoding CODM encodes an mRNA comprising the sequence of SEQ ID NO: 16.   
     
     
         14 . The plant of  claim 13 , wherein
 the nucleic acid molecule encoding the first hairpin RNA comprises a portion of SEQ ID NO: 2 and the nucleic acid molecule encoding the second hairpin RNA comprises a portion of SEQ ID NO: 4; or   the nucleic acid molecule encoding the first hairpin RNA comprises a portion of SEQ ID NO: 2 and the nucleic acid molecule encoding the second hairpin RNA comprises a portion of SEQ ID NO: 4.   
     
     
         15 .- 17 . (canceled) 
     
     
         18 . The plant of  claim 10 , comprising an expression construct comprising a nucleic acid molecule for reducing the expression of T6ODM and CODM, optionally wherein the nucleic acid molecule encodes: a hairpin RNA for reducing expression of an endogenous gene encoding CODM; a hairpin RNA for reducing expression of an endogenous gene encoding T6ODM; or a single hairpin RNA sufficient to reduce expression of endogenous genes encoding T6ODM and CODM. 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The plant of  claim 18 , wherein the nucleic acid molecule comprises a portion of SEQ ID NO:2, SEQ ID NO:4, or both. 
     
     
         23 . The plant of  claim 18 , wherein the expression construct comprises a first nucleic acid molecule encoding a first hairpin RNA for reducing expression of an endogenous gene encoding T6ODM and a second nucleic acid molecule encoding a second hairpin RNA for reducing expression of an endogenous gene encoding CODM, optionally wherein each of the first nucleic acid molecule and the second nucleic acid molecule comprise a portion of SEQ ID NO: 2, SEQ ID NO: 4, or both. 
     
     
         24 . The plant of  claim 23 , wherein the endogenous gene encoding T6ODM encodes an mRNA comprising the sequence of SEQ ID NO: 15 the endogenous gene encoding T6ODM encodes a polypeptide having the sequence of SEQ ID NO 1, the endogenous gene encoding CODM encodes an mRNA comprising the sequence of SEQ ID NO: 16, the endogenous gene encoding CODM encodes a polypeptide having the sequence of SEQ ID NO 3, or any combination thereof. 
     
     
         25 .- 28 . (canceled) 
     
     
         29 . The plant of  claim 18 , wherein the nucleic acid molecule encoding the hairpin RNA comprises a portion of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         30 . (canceled) 
     
     
         31 . The plant of  claim 11 , wherein the first nucleic acid molecule comprises a portion of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         32 . (canceled) 
     
     
         33 . The plant of  claim 11 , wherein the second nucleic acid molecule comprises a portion of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         34 . The plant of  claim 31 , wherein the second nucleic acid molecule comprises a portion of SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         35 . The plant cell of  claim 10 , wherein the plant is genetically modified to have reduced activity of T6ODM, and wherein reduced activity of CODM is conferred by a mutation in the endogenous gene encoding CODM that was not introduced by genetic modification of the plant, optionally wherein the mutation in the endogenous gene encoding CODM that was not introduced by genetic modification of the plant is the mutation present in seeds of the plant deposited under Patent Deposit Designation PTA-9109, or the plant is genetically modified to have reduced activity of CODM, and wherein reduced activity of T6ODM is conferred by a mutation in the endogenous gene encoding T6ODM that was not introduced by genetic modification of the plant, or the mutation in the endogenous gene encoding T6ODM that was not introduced by genetic modification of the plant is the mutation present in seeds of the plant deposited under Patent Deposit Designation PTA-9110. 
     
     
         36 .- 95 . (canceled) 
     
     
         96 . An isolated nucleic acid molecule, wherein the sequence of the nucleic acid molecule comprises SEQ ID NO:7. 
     
     
         97 . An expression vector for simultaneously reducing the expression of endogenous genes encoding thebaine 6-O-demethylase (T6ODM) and codeine 3-O-demethylase (CODM) in an opium poppy plant, the expression vector comprising an isolated nucleic acid as defined in  claim 96 . 
     
     
         98 . (canceled) 
     
     
         99 . (canceled) 
     
     
         100 . Poppy straw from a plant as defined in  claim 10 . 
     
     
         101 . Latex from a plant as defined in  claim 10 .