Tyra genes and uses thereof

Abstract

The present invention is in the field of plant genetics and biochemistry. More specifically, the invention relates to genes associated with the tocopherol biosynthesis pathway. The present invention provides and includes nucleic acid molecules, proteins, and antibodies associated with the genes of the tocopherol biosynthesis pathway. The present invention also provides methods for utilizing such agents, for example in gene isolation, gene analysis and the production of transgenic plants. Moreover, the present invention includes transgenic plants modified to express proteins associated with the tocopherol pathway. In addition, the present invention includes methods for the production of products from the tocopherol biosynthesis pathway.

1 . A substantially purified nucleic acid molecule comprising as operably linked components: (A) a promoter region which functions in a plant cell to cause the production of an mRNA molecule; (B) a heterologous nucleic acid molecule that encodes an enzyme with chorismate mutase and prephenate dehydrogenase activities or a fragment thereof at least 20 contiguous amino acids of said enzyme.  
     
     
         2 . The substantially purified nucleic acid molecule of  claim 1  wherein the heterologous nucleic acid molecule encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and fragments thereof at least 20 contiguous amino acids.  
     
     
         3 . The nucleic acid molecule of  claim 1 , further comprising a 3′ non-translated sequence that functions in said plant cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3′ end of the mRNA molecule.  
     
     
         4 . The nucleic acid molecule of  claim 1 , wherein said heterologous nucleic acid molecule encodes  Erwinia herbicola  tyrA or  Escherichia coli  tyrA.  
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein said heterologous nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3.  
     
     
         6 . The nucleic acid molecule of  claim 1 , wherein said heterologous nucleic acid molecule further comprises an expression cassette which expresses phytyl prenyltransferase.  
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein said heterologous nucleic acid molecule further comprises an expression cassette which expresses hydroxyphenylpyruvate dehydrogenase.  
     
     
         8 . The nucleic acid molecule of  claim 1 , wherein said fragment encodes a polypeptide having prephenate dehydrogenase activity.  
     
     
         9 . The nucleic acid molecule of  claim 1 , wherein said heterologous nucleic acid molecule further comprises two or more expression cassettes, each of which expresses a member selected from the group consisting of slr 1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase.  
     
     
         10 . The nucleic acid molecule of  claim 1 , wherein said heterologous nucleic acid molecule further comprises a nucleic acid sequence encoding HPPD and either slr 1736 or ATPT2.  
     
     
         11 . (canceled)  
     
     
         12 . A nucleic acid molecule comprising as operably linked components: (A) a promoter region which functions in a plant cell to cause the production of an mRNA molecule; (B) a heterologous nucleic acid molecule with a transcribed strand and a non-transcribed strand, wherein said transcribed strand is complementary to a nucleic acid molecule encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4.  
     
     
         13 . A transformed plant comprising the substantially purified nucleic acid molecule of  claim 1 .  
     
     
         14 . The transformed plant of  claim 13 , wherein said plant further comprises an expression cassette which expresses phytyl prenyltransferase.  
     
     
         15 . The transformed plant of  claim 14 , wherein said plant further comprises an expression cassette which expresses hydroxyphenylpyruvate dehydrogenase.  
     
     
         16 . The transformed plant of  claim 13 , wherein said plant further comprises two or more expression cassettes, each of which expresses a member of the group consisting of: sir 1736, ATP2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, sir 1737, and an antisense construct for homogentisic acid dioxygenase.  
     
     
         17 . The transformed plant of  claim 13 , wherein said plant further comprises a nucleic acid sequence encoding HPPD and either sir 1737 or ATPT2.  
     
     
         18 . The transformed plant according to  claim 13 , wherein said plant is selected from the group consisting of canola, maize,  Arabidopsis, Brassica campestris, Brassica napus , soybean,  crambe , mustard, castor bean, peanut, sesame, cottonseed, linseed, safflower, oil palm, flax and sunflower.  
     
     
         19 . The transformed plant according to  claim 13 , wherein said plant is soybean.  
     
     
         20 . The transformed plant according to  claim 13 , wherein said plant is canola.  
     
     
         21 . The transformed plant according to  claim 13 , wherein said plant is  Brassica napus.    
     
     
         22 . The transformed plant according to  claim 13 , wherein said plant exhibits increased tocopherol levels relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         23 - 25 . (canceled)  
     
     
         26 . The transformed plant according to  claim 13 , wherein said plant exhibits increased tocotrienol levels relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         27 - 29 . (canceled)  
     
     
         30 . The transformed plant according to  claim 13 , wherein said plant exhibits increased α-tocopherol levels relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         31 - 33 . (canceled)  
     
     
         34 . The transformed plant according to  claim 13 , wherein said plant exhibits increased α-tocotrienol relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         35 - 37 . (canceled)  
     
     
         38 . The transformed plant according to  claim 13 , wherein said plant exhibits increased γ-tocopherol levels relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         39 - 41 . (canceled)  
     
     
         42 . The transformed plant according to  claim 13 , wherein said plant exhibits increased γ-tocotrienol levels relative to a plant with a similar genetic background but lacking said exogenous nucleic acid molecule.  
     
     
         43 - 45 . (canceled)  
     
     
         46 . The transformed plant of  claim 13 , wherein said nucleic acid molecule further comprises a plastid targeting sequence, wherein said plastid targeting sequence is operably linked to said exogenous nucleic acid molecule to cause a transcript of said exogenous nucleic acid molecule to further encode a plastid peptide target sequence operably linked to said amino acid sequence.  
     
     
         47 . The transformed plant of  claim 13 , further comprising an expression cassette which expresses phytyl prenyltransferase.  
     
     
         48 . The transformed plant of  claim 47 , wherein said nucleic acid molecule further comprises said expression cassette.  
     
     
         49 . The transformed plant of  claim 13 , wherein said nucleic acid molecule encodes a fragment of SEQ ID NOs: 2 or 4, wherein said fragment has prephenate dehydrogenase activity.  
     
     
         50 . A transformed plant comprising a nucleic acid molecule comprising as operably linked components: (A) an exogenous promoter region which functions in a plant cell to cause the production of an mRNA molecule; and (B) a heterologous nucleic acid molecule with a transcribed strand and a non-transcribed strand, wherein said transcribed strand is complementary to the substantially purified nucleic acid molecule of  claim 1 .  
     
     
         51 . A method of producing a plant having increased tocopherol levels comprising: 
 (A) transforming said plant with the substantially purified nucleic acid molecule of  claim 1;  and    (B) growing said plant.    
     
     
         52 . The method of  claim 51 , wherein said plant further comprises an expression cassette which expresses phytyl prenyltransferase.  
     
     
         53 . The method of  claim 52 , wherein said plant further comprises an expression cassette which expresses hydroxyphenylpyruvate dehydrogenase.  
     
     
         54 . The method of  claim 51 , wherein said plant further comprises two or more expression cassettes, each of which expresses a member selected from the group consisting of slr1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr 1737, and an antisense construct for homogentisic acid dioxygenase.  
     
     
         55 . The method of  claim 51 , wherein said plant further comprises a nucleic acid sequence encoding HPPD and either slr1736 or ATPT2.  
     
     
         56 . The method of  claim 51 , wherein said substantially purified nucleic acid molecule is linked to a 3′ non-translated sequence that functions in the plant to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3′ end of an mRNA molecule, and wherein said expression of said substantially purified nucleic acid molecule results in overexpression of said protein.  
     
     
         57 . The method of producing a plant according to  claim 51 , wherein said plant is selected from the group of canola, maize,  Arabdopsis, Brassica campestris, Brassica napus , soybean,  crambe , mustard, castor bean, peanut, sesame, cottonseed, linseed, safflower, oil palm, flax and sunflower.  
     
     
         58 . The method of producing a plant according to  claim 51 , wherein said plant is canola.  
     
     
         59 . The method of producing a plant according to  claim 51 , wherein said plant is soybean.  
     
     
         60 . The method of producing a plant according to  claim 51 , wherein said plant is  Brassica napus.    
     
     
         61 . The method of producing a plant according to  claim 51 , wherein said plant exhibits increased α-tocopherol levels relative to a plant with a similar genetic background but lacking said substantially purified nucleic acid molecule.  
     
     
         62 . The method of producing a plant according to  claim 51 , wherein said plant exhibits increased γ-tocopherol levels relative to a plant with a similar genetic background but lacking said substantially purified nucleic acid molecule.  
     
     
         63 . The method of producing a plant according to  claim 51 , wherein said plant exhibits increased tocopherol levels relative to a plant with a similar genetic background but lacking said substantially purified nucleic acid molecule.  
     
     
         64 . The method of producing a plant according to  claim 51 , wherein said plant exhibits increased tocotrienol levels relative to a plant with a similar genetic background but lacking said substantially purified nucleic acid molecule.  
     
     
         65 . A method for reducing tocopherol levels in a plant comprising: 
 (A) transforming said plant with a nucleic acid molecule, wherein said nucleic acid molecule comprises as operably linked components an exogenous promoter region which functions in a plant cell to cause the production of an mRNA molecule, a heterologous nucleic acid molecule having a transcribed strand and a non-transcribed strand, wherein said transcribed strand is complementary to the substantially purified nucleic acid molecule of  claim 1;  and wherein said nucleic acid molecule is linked to a 3′ non-translated sequence that functions in the plant cells to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3′ end of the mRNA sequence; and    (B) growing said transformed plant.    
     
     
         66 . A method for screening for increased tocopherol levels in a plant comprising interrogating genomic DNA for the presence or absence of a marker molecule that specifically hybridizes to the substantially purified nucleic acid molecule of  claim 1  and complements thereof; and detecting said presence or absence of said marker.  
     
     
         67 . A cell comprising the substantially purified nucleic acid molecule of  claim 1 .  
     
     
         68 . The cell of  claim 67  wherein said cell further comprises an expression cassette which expresses phytyl prenyltransferase.  
     
     
         69 . The cell of  claim 68 , wherein cell further comprises an expression cassette which expresses hydroxyphenylpyruvate dehydrogenase.  
     
     
         70 . The cell of  claim 67 , wherein said cell further comprises two or more expression cassettes, each of which expresses a member from the group consisting of: slr1736, ATPT2, dxs, dxr, GGH, GGPPS, HPPD, MT1, TMT2, GMT, AANT1, slr1737, and an antisense construct for homogentisic acid dioxygenase.  
     
     
         71 . The cell according to  claim 67 , wherein said cell further comprises a nucleic acid sequence encoding HPPD and either slr1736 or ATPT2.  
     
     
         72 . The cell according to  claim 67 , wherein said cell is a bacterial cell.  
     
     
         73 . The cell according to  claim 67 , wherein said cell is a blue green algae cell.  
     
     
         74 . Oil derived from seed of the transformed plant of  claim 13.