Phytate polynucleotides and methods of use

Abstract

This invention relates to newly identified polynucleotides and polypeptides in the phytic acid biosynthetic pathway, variants and derivatives of same; methods for making the polynucleotides, polypeptides, variants, derivatives and antagonists. In particular the invention relates to polynucleotides and polypeptides of the inositol 1,3,4-trisphosphate 5/6-kinase gene family. In particular this invention relates to using the newly identified polynucleotides and polypeptides to modulate the phytic acid biosynthesis in such a way as to decrease phytate and/or increase non-phytate phosphorous, especially in corn or soy animal feedstuffs.

What is claimed is:  
     
         1 . An isolated nucleic acid comprising a member selected from the group consisting of: 
 (a) a polynucleotide having at least 87% sequence identity to SEQ ID NO: 1, or 93% sequence identity to SEQ ID NO: 7, or 80% identity to SEQ ID NOS: 9, 11, or 13; wherein the % sequence identity is determined over the entire length of the coding sequence by the GAP algorithm using default parameters;    (b) a polynucleotide which encodes a polypeptide of SEQ ID NOS: 2, 8, 10, 12, or 14;    (c) a polynucleotide which selectively hybridizes, under stringent conditions and a wash in 0.1×SSC at 60° C., to a polynucleotide of SEQ ID NOS: 1, 7, 9, 11, or 13;    (d) a polynucleotide amplified from a plant nucleic acid library using the primers of SEQ ID NOS: 15, 16, 17, 22, and 27 or primers determined by using Vector NTI Suite, InforMax Version 5, or an equivalent method;    (e) a polynucleotide having the sequence set forth in SEQ ID NOS:1, 7, 9, 11, or 13;    (f) a polynucleotide comprising at least 45 contiguous nucleotides of SEQ ID NOS: 9, 11, or 13; and    (g) a polynucleotide complementary to a polynucleotide of (a) through (f) wherein the isolated nucleic acid modulates the level of ITPK.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the polynucleotide is from a monocot or dicot.  
     
     
         3 . A vector comprising at least one nucleic acid of  claim 1 .  
     
     
         4 . An expression cassette comprising at least one nucleic acid of  claim 1  operably linked to a promoter, wherein the nucleic acid is in sense or antisense orientation.  
     
     
         5 . The expression cassette of  claim 4 , wherein the nucleic acid is operably linked in antisense orientation to the promoter.  
     
     
         6 . A non-human host cell containing at least one nucleic acid of  claim 1 .  
     
     
         7 . The host cell of  claim 6 , wherein the host cell is a plant cell.  
     
     
         8 . A transformed plant comprising at least one nucleic acid of  claim 1 .  
     
     
         9 . The transformed plant of  claim 8 , wherein the plant is corn, barley, soybean, sorghum, wheat, rice, alfalfa, safflower, sunflower, canola, cotton, or millet.  
     
     
         10 . A transformed seed from the transformed plant of  claim 8 .  
     
     
         11 . A method for modulating inositol 1,3,4-trisphosphate 5/6-kinase (ITPK) activity or levels in a host cell, comprising: 
 (a) transforming a host cell with at least one expression cassette of  claim 4;  and    (b) growing the transformed host cell to modulate ITPK activity in the host cell.    
     
     
         12 . The method of  claim 11 , wherein the host cell is a plant cell.  
     
     
         13 . The method of  claim 12 , wherein the plant cell is from a monocot or a dicot.  
     
     
         14 . The method of  claim 12 , further comprising producing a transformed plant from the plant cell.  
     
     
         15 . A transformed plant produced by the method of  claim 14 .  
     
     
         16 . The transformed plant of  claim 15 , wherein the plant is corn, barley, soybean, sorghum, wheat, rice, alfalfa, safflower, sunflower, canola, cotton, or millet.  
     
     
         17 . A transformed seed from the plant of  claim 15 .  
     
     
         18 . The method of  claim 12  wherein the level of phytate is reduced.  
     
     
         19 . The method of  claim 12  wherein the level of non-phytate phosphorous is increased.  
     
     
         20 . A method of improving the nutritional value of animal feed, comprising: 
 (a) transforming a plant host cell with at least one expression cassette of  claim 4  to reduce phytate content;    (b) growing the transformed host cell to modulate ITPK activity in the host cell;    (c) generating a plant with the transformed genotype; and    (d) producing animal feed from the plant, wherein the animal feed has improved nutritional value.    
     
     
         21 . The method of  claim 20 , wherein the plant cell is from a monocot or a dicot.  
     
     
         22 . A transformed plant produced by the method of  claim 20 .  
     
     
         23 . A transformed seed from the plant of  claim 22 .  
     
     
         24 . The transformed plant of  claim 22 , wherein the plant is corn, barley, soybean, sorghum, wheat, rice, safflower, sunflower, canola, or millet.  
     
     
         25 . The method of  claim 20 , wherein the level of non-phytate phosphorous is increased.  
     
     
         26 . A method of decreasing the level of phosphorous in non-ruminant animal waste comprising providing said non-ruminant animal said animal feed produced by the method of  claim 20 .  
     
     
         27 . A method of increasing the level of available phosphorous in animal feed, comprising: 
 (a) transforming a plant host cell with at least one expression cassette of  claim 4  to reduce phytate content;    (b) growing the transformed host cell to modulate ITPK activity in the host cell;    (c) generating a plant with the transformed genotype; and    (d) producing animal feed from the plant, wherein the animal feed has an increased level of available phosphorous.    
     
     
         28 . The method of  claim 27 , wherein the plant cell is from a monocot or a dicot.  
     
     
         29 . A transformed plant produced by the method of  claim 27 .  
     
     
         30 . A transformed seed from the plant of  claim 29 .  
     
     
         31 . The transformed plant of  claim 29 , wherein the plant is corn, barley, soybean, sorghum, wheat, rice, safflower, sunflower, or canola.  
     
     
         32 . A method of decreasing the level of phosphorous in non-ruminant animal waste comprising providing said non-ruminant animal said animal feed produced by the method of  claim 27 .  
     
     
         33 . A method of altering plant phenotype comprising: 
 (a) transforming a plant host cell with at least one ITPK polynucleotide of  claim 1  and at least one polynucleotide of interest;    (b) growing the transformed host cell to modulate the activity of ITPK and the polynucleotide of interest in the host cell; and    (c) generating a transformed plant with an altered phenotype.    
     
     
         34 . The method of  claim 33 , wherein the activity of ITPK is downregulated and wherein the activity of the polynucleotide of interest is up-regulated.  
     
     
         35 . The method of  claim 34 , wherein the polynucleotide of interest is myo-inositol monophosphatase (IMP) or phytase.  
     
     
         36 . The method of  claim 33 , wherein the activity of ITPK and the activity of the polynucleotide of interest are each downregulated.  
     
     
         37 . The method of  claim 36 , wherein the polynucleotide of interest is inositol polyphosphate kinase (IPPK) or myo-inositol 1-phosphate synthase (MI 1PS).  
     
     
         38 . A transformed plant produced by the method of  claim 33 .  
     
     
         39 . The transformed plant of  claim 38 , wherein the plant is corn, barley, soybean, sorghum, wheat, rice, alfalfa, safflower, sunflower, canola, cotton, or millet.  
     
     
         40 . A transformed seed from the plant of  claim 38 .  
     
     
         41 . An isolated protein comprising a member selected from the group consisting of: 
 (a) a polypeptide comprising at least 25 contiguous amino acids of SEQ ID NOS: 10, 12, or 14;    (b) a polypeptide comprising at least 94% sequence identity compared to the full-length of SEQ ID NOS: 2 or 8, or 75% sequence identity compared to the full-length of SEQ ID NOS: 10, 12, or 14; wherein the percent sequence identity is based on the entire sequence length and is determined by the GAP algorithm using default parameters;    (c) a polypeptide encoded by the nucleic acid of  claim 1;     (d) a polypeptide encoded by a nucleic acid of SEQ ID NOS:1, 7, 9, 11 or 13; and    (e) a polypeptide having the sequence set forth in SEQ ID NOS: 2, 8, 10, 12, or 14.    
     
     
         42 . An isolated ribonucleic acid sequence encoding the protein of  claim 41.