Plastidic phosphoglucomutase genes

Abstract

An isolated nucleic acid fragment encoding a plastidic phosphoglucomutase protein is disclosed. Also disclosed is the construction of a chimeric gene encoding all or a substantial portion of the plastidic phosphoglucomutase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the plastidic phosphoglucomutase in a transformed host cell.

1 . An isolated polynucleotide comprising:
 (a) a first nucleotide sequence encoding a first polypeptide having phosphoglucomutase activity, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:8 have at least 95% identity based on the Clustal alignment method,   (b) a second nucleotide sequence encoding a second polypeptide having phosphoglucomutase activity, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 85% identity based on the Clustal alignment method, or   (c) the complement of the first or second nucleotide sequence.   
     
     
         2 . The isolated polynucleotide of  claim 1  wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 90% identity based on the Clustal alignment method. 
     
     
         3 . The isolated polynucleotide of  claim 1 , wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 95% identity based on the Clustal alignment method. 
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:8, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. 
     
     
         5 . The isolated polynucleotide of  claim 1 , wherein the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:7, wherein the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. 
     
     
         6 . A recombinant DNA construct comprising the polynucleotide of  claim 1  operably linked to a regulatory sequence. 
     
     
         7 . A method for transforming a cell comprising transforming a cell with the polynucleotide of  claim 1 . 
     
     
         8 . A cell comprising the recombinant DNA construct of  claim 6 . 
     
     
         9 . A method for producing a transgenic plant comprising transforming a plant cell with the polynucleotide of  claim 1  and regenerating a plant from the transformed plant cell. 
     
     
         10 . A plant comprising the recombinant DNA construct of  claim 6 . 
     
     
         11 . A seed comprising the recombinant DNA construct of  claim 6 . 
     
     
         12 . An isolated polypeptide having phosphoglucomutase activity, wherein the polypeptide comprises:
 (a) a first amino acid sequence, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:8 have at least 95% identity based on the Clustal alignment method, or   (b) a second amino acid sequence, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 85% identity based on the Clustal alignment method.   
     
     
         13 . The polypeptide of  claim 12 , wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 90% identity based on the Clustal alignment method. 
     
     
         14 . The polypeptide of  claim 12 , wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 95% identity based on the Clustal alignment method. 
     
     
         15 . The polypeptide of  claim 12 , wherein the first amino acid sequence comprises the amino acid sequence of SEQ ID NO:8, and wherein the second amino acid sequence comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. 
     
     
         16 . A method of selecting an isolated polynucleotide that decreases the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a plant cell, the method comprising the steps of:
 (a) constructing the isolated polynucleotide comprising a nucleotide sequence of at least 30 contiguous nucleotides derived from the isolated polynucleotide of  claim 1 ;   (b) introducing the isolated polynucleotide into the plant cell;   (c) measuring the level of the polypeptide in the plant cell containing the polynucleotide; and   (d) comparing the level of the polypeptide in the plant cell containing the isolated polynucleotide with the level of the polypeptide in a plant cell that does not contain the isolated polynucleotide; and   
       selecting the isolated polynucleotide that decreases the level of expression of the plastidic polypeptide having phosphoglucomutase activity in the plant cell. 
     
     
         17 . The method of  claim 16  wherein the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3 and 7. 
     
     
         18 . The method of  claim 16  wherein the isolated polynucleotide comprising a nucleotide sequence of at least 30 contiguous nucleotides contains at least 541 nucleotides. 
     
     
         19 . The method of  claim 16  wherein the isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO:15. 
     
     
         20 . A method of selecting an isolated polynucleotide that increases the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a plant cell, the method comprising the steps of:
 (a) constructing the isolated polynucleotide of  claim 1 ;   (b) introducing the isolated polynucleotide into the plant cell;   (c) measuring the level of the polypeptide in the plant cell containing the polynucleotide;   (d) comparing the level of the polypeptide in the plant cell containing the isolated polynucleotide with the level of the polypeptide in a plant cell that does not contain the polynucleotide; and   
       selecting the isolated polynucleotide that increases the level of expression of the plastidic polypeptide having phosphoglucomutase activity in the plant cell. 
     
     
         21 . A method for positive selection of a transformed cell comprising:
 (a) transforming a host cell with the recombinant DNA construct of  claim 6 ; and   (b) growing the transformed host cell under conditions which allow expression of a polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.   
     
     
         22 . The method of  claim 21  wherein the host cell is a plant. 
     
     
         23 . The method of  claim 21  wherein the plant cell is a monocot. 
     
     
         24 . The method of  claim 21  wherein the plant cell is a dicot. 
     
     
         25 . A method of increasing the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a host cell comprising:
 (a) transforming a host cell with the recombinant DNA construct of  claim 6 ; and   (b) growing the transformed host cell from step (a) under conditions that are suitable for expression of the recombinant DNA construct; and   (c) selecting a transformed cell wherein expression of the recombinant DNA construct results in production of higher levels of a plastidic polypeptide having phosphoglucomutase activity in the transformed host cell.   
     
     
         26 . A method for suppressing the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity in a transgenic plant, wherein the method comprises:
 (a) transforming a plant cell with a fragment of the isolated polynucleotide of  claim 1 ;   (b) regenerating a transgenic plant from the transformed plant cell of (a); and   (c) selecting a transgenic plant wherein the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity has been suppressed.   
     
     
         27 . A recombinant DNA construct comprising:
 (a) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15;   (b) the complement of (a);   
       wherein (a) or (b) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant. 
     
     
         28 . A method for producing transgenic seed, the method comprising:
 (a) transforming a plant cell with the recombinant DNA construct of  claim 27 ;   (b) regenerating a transgenic plant from the transformed plant cell of (a); and   (c) selecting a transgenic plant that produces a transgenic seed having an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% as compared to seed obtained from a non-transgenic plant.   
     
     
         29 . The method of  claim 28  wherein the increase in the combined oil and protein content is at least 1.8%. 
     
     
         30 . The method of  claim 28  wherein the increase in the combined oil and protein content is at least 2.0%. 
     
     
         31 . A method for producing transgenic seed, the method comprising:
 (a) transforming a plant cell with the recombinant DNA construct of  claim 27 ;   (b) regenerating a transgenic plant from the transformed plant cell of (a); and   (c) selecting a transgenic plant that produces a transgenic seed having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to seed obtained from a non-transgenic plant.   
     
     
         32 . The method of  claim 31  wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 1.6%. 
     
     
         33 . The method of  claim 31  wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 1.8%. 
     
     
         34 . The method of  claim 31  wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 2.0%. 
     
     
         35 . A method for producing defatted meal from transgenic seed, the method comprising:
 (a) transforming a plant cell with the recombinant DNA construct of  claim 27 ;   (b) regenerating a transgenic plant from the transformed plant cell of (a); and   (c) selecting a transgenic plant that produces a transgenic seed wherein said seed is processed into defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% as compared to defatted meal obtained from seed of a non-transgenic plant.   
     
     
         36 . The method of  claim 35  wherein the defatted meal of the transgenic seed has a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to the sucrose to raffinose family oligosaccharide ratio of defatted meal obtained from a non-transgenic seed. 
     
     
         37 . The method of any one of  claims 28 - 36  wherein the transgenic seed is obtained from a transgenic dicot plant comprising in its genome the recombinant construct. 
     
     
         38 . The method of  claim 37  wherein the dicot plant is selected from the group consisting of  Arabidopsis , soybean, oilseed  Brassica , peanut, sunflower, safflower, cotton, tobacco, tomato, potato, and cocoa. 
     
     
         39 . The method of  claim 37  wherein the dicot plant is soybean. 
     
     
         40 . A transgenic seed comprising the recombinant DNA construct of  claim 27  in its genome wherein said transgenic seed has an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% when compared to a non-transgenic seed. 
     
     
         41 . The transgenic seed of  claim 40  wherein the increase in the combined oil and protein content is at least 1.8%. 
     
     
         42 . The transgenic seed of  claim 40  wherein the increase in the combined oil and protein content is at least 2.0%. 
     
     
         43 . A transgenic seed comprising the recombinant DNA construct of  claim 27  in its genome wherein said transgenic seed has a sucrose to raffinose family oligosaccharide ratio of 1.0 or less when compared to a non-transgenic seed. 
     
     
         44 . The transgenic seed of  claim 43  wherein the transgenic seed has an increase in the combined oil and protein content of at least 1.6% when compared to a non-transgenic seed. 
     
     
         45 . The transgenic seed of  claim 43  wherein the transgenic seed has an increase in the combined oil and protein content of at least 1.8% when compared to a non-transgenic seed. 
     
     
         46 . The transgenic seed of  claim 43  wherein the transgenic seed has an increase in the combined oil and protein content of at least 2.0% when compared to a non-transgenic seed. 
     
     
         47 . Transgenic seed comprising the recombinant construct of  claim 27  in its genome wherein said transgenic seed is processed to make defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% when compared to defatted meal obtained from a non-transgenic seed. 
     
     
         48 . The transgenic seed of  claim 47  wherein the defatted meal of the transgenic seed differs from the defatted meal of a non-transgenic seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. 
     
     
         49 . The transgenic seed of any one of  claims 40 - 48  wherein the seed is a soybean seed.