Maize multidrug resistance-associated protein polynucleotides and methods of use

Abstract

Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing nucleic acids comprising multidrug resistance-associated protein (MRP) nucleotide sequences to modulate the expression of MRP(s) in a plant of interest. The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. The invention also finds use in reducing the environmental impact of animal waste.

That which is claimed: 
     
         1 . An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide that modulates the level of phytate in a plant, wherein the nucleotide sequence is selected from the group consisting of:
 a) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 244-4776 of SEQ ID NO: 2;   b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 93% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3 or 5;   c) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 245-4762 of SEQ ID NO: 6;   d) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 7;   e) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 3-1350 of SEQ ID NO: 10;   f) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 11;   g) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 1-465 of SEQ ID NO: 12;   h) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 13;   i) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 71; and   j) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that comprises the sequence set forth in SEQ ID NO: 15.   
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         3 . The nucleic acid molecule of  claim 2 , wherein said nucleotide sequence encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3. 
     
     
         4 . An expression cassette comprising the nucleic acid molecule of  claim 1 , wherein said nucleotide sequence is operably linked to a promoter that drives expression in a microorganism or in a plant cell. 
     
     
         5 . An isolated polypeptide comprising an amino acid sequence which has at least 93% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein said polypeptide modulates the level of phytate in a plant. 
     
     
         6 . An expression cassette comprising a first nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   b) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   c) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and   d) a nucleotide sequence which is the complement of a), b), or c).   
     
     
         7 . A method for producing food or feed with a reduced level of phytate, said method comprising:
 a) transforming a plant with a nucleic acid molecule comprising a first nucleotide sequence selected from the group consisting of:   i) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   ii) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   iii) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and   iv) a nucleotide sequence which is the complement of i), ii), or iii);   b) growing said plant under conditions in which said nucleotide sequence is expressed; and   c) producing food or feed from said plant,   wherein said plant has a reduced level of phytate in comparison to a control plant.   
     
     
         8 . The method of  claim 7 , wherein said first nucleotide sequence has at least 95% sequence identity to the nucleotide sequence set forth in nucleotides 244-4776 of SEQ ID NO: 2. 
     
     
         9 . The method of  claim 7 , wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   b) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   c) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and   d) a nucleotide sequence which is the complement of i), ii), or iii);   wherein said plant has a reduced level of phytate in comparison to a control plant.   
     
     
         10 . The method of  claim 7 , wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of:
 a) an mi1ps nucleotide sequence;   b) an IPPK nucleotide sequence;   c) an ITPK-5 nucleotide sequence;   d) an IP2K nucleotide sequence;   e) an MIK nucleotide sequence;   f) a phytase nucleotide sequence;   g) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68;   h) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68;   i) a nucleotide sequence which is the complement of (a), (b), (c), (d), (e), (g), or (h); and   j) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66.   
     
     
         11 . The method of  claim 7 , wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence conferring a trait of interest on said transformed plant. 
     
     
         12 . The method of  claim 11 , wherein said trait of interest is selected from the group consisting of:
 a) high oil;   b) increased digestibility;   c) high energy;   d) balanced amino acid;   e) high oleic acid;   f) insect resistance;   g) disease resistance;   h) herbicide resistance;   i) drought tolerance; and   j) male sterility.   
     
     
         13 . A transformed plant comprising in its genome at least one stably incorporated nucleic acid molecule having a first nucleotide sequence selected from the group consisting of:
 a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   b) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14;   c) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; and   d) a nucleotide sequence which is the complement of a), b), or c);   wherein said plant has a reduced level of phytate compared to a control plant.   
     
     
         14 . The transformed plant of  claim 13 , wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of:
 a) an mi1ps nucleotide sequence;   b) an IPPK nucleotide sequence;   c) an ITPK-5 nucleotide sequence;   d) an IP2K nucleotide sequence;   e) an MIK nucleotide sequence;   f) a phytase nucleotide sequence;   g) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68;   h) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68;   i) a nucleotide sequence which is the complement of (a), (b), (c), (d), (e), (g) or (h); and   j) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66.   
     
     
         15 . The transformed plant of  claim 13 , wherein said plant is further transformed with a nucleic acid molecule comprising at least one second nucleotide sequence that confers at least one trait of interest on said transformed plant. 
     
     
         16 . The transformed plant of  claim 15 , wherein said trait of interest is selected from the group consisting of:
 a) high oil;   b) increased digestibility;   c) high energy;   d) balanced amino acid composition;   e) high oleic acid;   f) insect resistance;   g) disease resistance;   h) herbicide resistance;   i) drought tolerance; and   j) male sterility.   
     
     
         17 . Transformed seed of the plant of  claim 13 , wherein said seed comprises said first nucleotide sequence. 
     
     
         18 . Food or feed comprising the plant of  claim 13 . 
     
     
         19 . Food or feed comprising the transformed seed of  claim 13 . 
     
     
         20 . The transformed plant of  claim 13 , wherein said plant is a monocot. 
     
     
         21 . A method for producing food or feed with a reduced level of phytate, said method comprising the steps of:
 (a) transforming a plant cell with at least one first polynucleotide comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, or 71;   (b) transforming a plant cell with at least one second polynucleotide having at least 94% sequence identity to the complement of the polynucleotide of step (a);   (c) regenerating a transformed plant from said plant cell; and   (d) producing food or feed from said transformed plant or from seed of said transformed plant;   wherein the level of phytate in said plant is reduced in comparison to a control plant.   
     
     
         22 . A plant containing an Lpa1 insertion mutation comprising a Mu element, wherein said Mu element is inserted in the Lpa1 gene in a location selected from the group consisting of:
 a) in exon 1;   b) at nucleotide 585 in exon 1;   c) at nucleotide 874 in exon 1;   d) in exon 11; and   e) at nucleotide 6069 in exon 11.