Genes encoding 4-hydroxyphenylpyruvate dioxygenase (hppd) enzymes for plant metabolic engineering

Abstract

The present invention is in the field of plant genetics and biochemistry. More specifically, the present invention relates to genes and polypeptides associated with the tocopherol biosynthesis pathway, namely those encoding 4-Hydroxyphenylpyruvate Dioxygenase activity, and uses thereof.

1 . A substantially purified polynucleotide molecule comprising:
 (a) a polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 3, a nucleotide sequence having at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, a nucleotide sequence having at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 9, a nucleotide sequence having at least 80% identity to SEQ ID NO: 9, SEQ ID NO: 11, a nucleotide sequence having at least 80% identity to SEQ ID NO: 11, SEQ ID NO: 13, a nucleotide sequence having at least 80% identity to SEQ ID NO: 13, SEQ ID NO: 15, a nucleotide sequence having at least 87% identity to SEQ ID NO: 15, SEQ ID NO: 17, a nucleotide sequence having at least 87% identity to SEQ ID NO: 17, SEQ ID NO: 19, a nucleotide sequence having at least 91% identity to SEQ ID NO: 19, SEQ ID NO: 21, a nucleotide sequence having at least 91% identity to SEQ ID NO: 21, SEQ ID NO: 23, and a nucleotide sequence having at least 90% identity to SEQ ID NO: 23;   (b) a polynucleotide molecule encoding a 4-hydroxyphenylpyruvate dioxygenase (“HPPD”) polypeptide or polypeptide having HPPD activity comprising a sequence selected from the group consisting of: SEQ ID NO: 3, a nucleotide sequence having at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, a nucleotide sequence having at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 9, a nucleotide sequence having at least 80% identity to SEQ ID NO: 9, SEQ ID NO: 11, a nucleotide sequence having at least 80% identity to SEQ ID NO: 11, SEQ ID NO: 13, a nucleotide sequence having at least 80% identity to SEQ ID NO: 13, SEQ ID NO: 15, a nucleotide sequence having at least 87% identity to SEQ ID NO: 15, SEQ ID NO: 17, a nucleotide sequence having at least 87% identity to SEQ ID NO: 17, SEQ ID NO: 19, a nucleotide sequence having at least 91% identity to SEQ ID NO: 19, SEQ ID NO: 21, a nucleotide sequence having at least 91% identity to SEQ ID NO: 21, SEQ ID NO: 23, and a nucleotide sequence having at least 90% identity to SEQ ID NO: 23;   (c) a polynucleotide molecule comprising a polynucleotide sequence encoding a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 4, a polypeptide sequence having at least 58% identity to SEQ ID NO: 4, SEQ ID NO: 6, a polypeptide sequence having at least 58% identity to SEQ ID NO: 6, SEQ ID NO: 10, a polypeptide sequence having at least 58% identity to SEQ ID NO: 10, SEQ ID NO: 12, a polypeptide sequence having at least 58% identity to SEQ ID NO: 12, SEQ ID NO: 16, a polypeptide sequence having at least 80% identity to SEQ ID NO: 16, SEQ ID NO: 18, a polypeptide sequence having at least 80% identity to SEQ ID NO: 18, SEQ ID NO: 20, a polypeptide sequence having at least 93% identity to SEQ ID NO: 20, SEQ ID NO: 22, a polypeptide sequence having at least 79% identity to SEQ ID NO: 22, SEQ ID NO: 24, and a polypeptide sequence having at least 54% identity to SEQ ID NO: 24; or   (d) a polynucleotide molecule comprising a polynucleotide sequence encoding a 4-hydroxyphenylpyruvate dioxygenase (“HPPD”) polypeptide or polypeptide having HPPD activity comprising a sequence selected from the group consisting of: SEQ ID NO: 4, a polypeptide sequence having at least 58% identity to SEQ ID NO: 4, SEQ ID NO: 6, a polypeptide sequence having at least 58% identity to SEQ ID NO: 6, SEQ ID NO: 10, a polypeptide sequence having at least 58% identity to SEQ ID NO: 10, SEQ ID NO: 12, a polypeptide sequence having at least 58% identity to SEQ ID NO: 12, SEQ ID NO: 16, a polypeptide sequence having at least 80% identity to SEQ ID NO: 16, SEQ ID NO: 18, a polypeptide sequence having at least 80% identity to SEQ ID NO: 18, SEQ ID NO: 20, a polypeptide sequence having at least 93% identity to SEQ ID NO: 20, SEQ ID NO: 22, a polypeptide sequence having at least 79% identity to SEQ ID NO: 22, SEQ ID NO: 24, and a polypeptide sequence having at least 54% identity to SEQ ID NO: 24.   
     
     
         2 . A DNA construct comprising the substantially purified polynucleotide molecule of  claim 1 . 
     
     
         3 . The DNA construct of  claim 2 , further comprising a seed-preferred promoter operably linked to the polynucleotide molecule. 
     
     
         4 . The DNA construct of  claim 3 , wherein the seed-preferred promoter is selected from the group consisting of: napin, 7S alpha, 7S alpha′, 7S beta, USP 88, enhanced USP 88, Arcelin 5, and oleosin. 
     
     
         5 . The DNA construct of  claim 2 , further comprising a polynucleotide encoding a chloroplast transit peptide. 
     
     
         6 . A plant cell transformed with the DNA construct of  claim 2 . 
     
     
         7 . The DNA construct of  claim 2  wherein the substantially purified polynucleotide molecule comprises the polynucleotide sequence of SEQ ID NO: 15. 
     
     
         8 . A method of producing a transformed plant cell, plant tissue, plant organ, or plant comprising at least one of an increased tocopherol level and increased tocotrienol level relative to a wild type plant cell, plant tissue, plant organ, or plant comprising: (1) transforming a plant cell, plant tissue, plant organ, or plant with an introduced polynucleotide molecule encoding a 4-hydroxyphenylpyruvate dioxygenase (“HPPD”) polypeptide or polypeptide having HPPD activity comprising:
 (a) a polynucleotide molecule comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 3, a nucleotide sequence having at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, a nucleotide sequence having at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 7, a nucleotide sequence having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 9, a nucleotide sequence having at least 80% identity to SEQ ID NO: 9, SEQ ID NO: 11, a nucleotide sequence having at least 80% identity to SEQ ID NO: 11, SEQ ID NO: 13, a nucleotide sequence having at least 80% identity to SEQ ID NO: 13, SEQ ID NO: 15, a nucleotide sequence having at least 87% identity to SEQ ID NO: 15, SEQ ID NO: 17, a nucleotide sequence having at least 87% identity to SEQ ID NO: 17, SEQ ID NO: 19, a nucleotide sequence having at least 91% identity to SEQ ID NO: 19, SEQ ID NO: 21, a nucleotide sequence having at least 91% identity to SEQ ID NO: 21, SEQ ID NO: 23, and a nucleotide sequence having at least 90% identity to SEQ ID NO: 23;   (b) a polynucleotide molecule comprising a polynucleotide sequence encoding a HPPD polypeptide or polypeptide having HPPD activity comprising a sequence selected from the group consisting of: SEQ ID NO: 3, a nucleotide sequence having at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 5, a nucleotide sequence having at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 7, a nucleotide sequence having at least 80% identity to SEQ ID NO: 7, SEQ ID NO: 9, a nucleotide sequence having at least 80% identity to SEQ ID NO: 9, SEQ ID NO: 1, a nucleotide sequence having at least 80% identity to SEQ ID NO: 11, SEQ ID NO: 13, a nucleotide sequence having at least 80% identity to SEQ ID NO: 13, SEQ ID NO: 15, a nucleotide sequence having at least 87% identity to SEQ ID NO: 15, SEQ ID NO: 17, a nucleotide sequence having at least 87% identity to SEQ ID NO: 17, SEQ ID NO: 19, a nucleotide sequence having at least 91% identity to SEQ ID NO: 19, SEQ ID NO: 21, a nucleotide sequence having at least 91% identity to SEQ ID NO: 21, SEQ ID NO: 23, and a nucleotide sequence having at least 90% identity to SEQ ID NO: 23;   (c) a polynucleotide molecule comprising a polynucleotide sequence encoding a polypeptide sequence selected from the group consisting of: SEQ ID NO: 4, a polypeptide sequence having at least 58% identity to SEQ ID NO: 4, SEQ ID NO: 6, a polypeptide sequence having at least 58% identity to SEQ ID NO: 6, SEQ ID NO: 8, a polypeptide sequence having at least 58% identity to SEQ ID NO: 8, SEQ ID NO: 10, a polypeptide sequence having at least 58% identity to SEQ ID NO: 10, SEQ ID NO: 12, a polypeptide sequence having at least 58% identity to SEQ ID NO: 12, SEQ ID NO: 16, a polypeptide sequence having at least 80% identity to SEQ ID NO: 16, SEQ ID NO: 18, a polypeptide sequence having at least 80% identity to SEQ ID NO: 18, SEQ ID NO: 20, a polypeptide sequence having at least 93% identity to SEQ ID NO: 20, SEQ ID NO: 22, a polypeptide sequence having at least 79% identity to SEQ ID NO: 22, SEQ ID NO: 24, and a polypeptide sequence having at least 54% identity to SEQ ID NO: 24;   (2) regenerating a plant from the cell, tissue, organ or plant; and   (3) growing the plant.   
     
     
         9 . The method of  claim 8 , wherein the substantially purified polynucleotide molecule is operably linked to a polynucleotide encoding a chloroplast transit peptide. 
     
     
         10 . The method of  claim 8  further comprising introducing at least one additional polynucleotide molecule encoding an enzyme selected from the group consisting of MT1, tMT2, GMT, tyrA, HPT, tocopherol cyclase, chlorophyllase, dxs, dxr, GGPPS, AANT1, LITT1, IDI, and GGH. 
     
     
         11 . The method of  claim 10 , wherein the at least one additional nucleotide molecule is selected from the group consisting of: SEQ ID NOs: 25, 26, and 27. 
     
     
         12 . The method of  claim 8 , wherein said plant is selected from the group consisting of  Brassica campestris , canola, oilseed rape, and soybean. 
     
     
         13 . The method of  claim 8 , wherein said plant tissue is seed tissue. 
     
     
         14 . The method of  claim 8 , wherein the polynucleotide sequence comprises SEQ ID NO: 15. 
     
     
         15 . The method of sequence 10 wherein the additional polynucleotide molecule comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 25, 26, and 27. 
     
     
         16 . A method for increasing the ability of a plant to withstand a stress comprising transforming the plant with a DNA construct comprising a polynucleotide encoding an HPPD, wherein expression of the polynucleotide increases the ability of the plant to withstand the stress. 
     
     
         17 . The method of  claim 16  wherein said polynucleotide encoding an HPPD is selected from the group consisting of: SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. 
     
     
         18 . The method of  claim 16  wherein the stress is selected from the group consisting of salt, drought, or cold. 
     
     
         19 . A transformed plant produced from the method of  claim 16 , wherein the plant comprises the DNA construct comprising a polynucleotide encoding an HPPD. 
     
     
         20 . A seed from the transformed plant of  claim 19 , wherein the seed comprises the DNA construct comprising a polynucleotide encoding an HPPD.