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US 6,639,126 B1Granted
Production of modified polysaccharides
AssigneePioneer Hi-Bred International, Inc.
InventorsVincent J. H. Sewalt, George W. Singletary
Life of this patent
Estimating the term…
Abstract
The invention alters the physical characteristics of storage polyglucans including starch. Methods are provided to modify the polyglucan biosynthesis pathway by simultaneously altering the activity of a pullulanase debranching enzyme and the activity of another polypeptide in the polyglucan biosynthesis pathway. Compositions of the invention include transgenic plants and seeds having a modified polyglucan structure and/or content and elevated phytoglycogen levels. Additional compositions include a grain with increased energy availability for improved feed quality and industrial uses. Further compositions include a polyglucan with improved functional properties useful in a wide range of food and industrial applications.
Claims45
A method for modulating polyglucan biosynthesis in a maize plant comprising stably introducing into the genome of said maize plant at least a first DNA construct and a second DNA construct, wherein
- a) said first DNA construct comprises a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant, and said first nucleic acid molecule comprises a nucleotide sequence comprising at least 20 contiguous nucleotides encoding the maize pullulanase polypeptide;
- and,
- b) said a second DNA construct comprises a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A maize plant having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant and said first nucleic acid molecule comprises a nucleotide sequence comprising at least 20 contiguous nucleotides encoding the maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A maize plant cell having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in said maize plant cell, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant cell and said first nucleic acid molecule comprises a nucleotide sequence comprising at least 20 contiguous nucleotides encoding the maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant cell wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
An expression vector comprising
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in a plant, wherein expression of said first nucleic acid molecule modulates pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in a maize plant and said first nucleic acid molecule comprises a nucleotide sequence comprising at least 20 contiguous nucleotides encoding a maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A method for modulating polyglucan biosynthesis in a maize plant comprising stably introducing into the genome of said maize plant at least a first and a second DNA construct, wherein
- a) said first DNA construct comprises a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant and said first nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:1;
- and,
- b) said second DNA construct comprises a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A method for modulating polyglucan biosynthesis in a maize plant comprising stably introducing into the genome of said maize plant at least a first and a second DNA construct, wherein
- a) said first DNA construct comprises a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant and said first nucleic acid molecule comprises an antisense nucleotide sequence comprising at least 50 contiguous nucleotides complementary to the mRNA encoding a maize pullulanase polypeptide;
- and
- b) said second DNA construct comprises a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A maize plant having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant and said first nucleic acid molecule comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:1;
- and
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to tie second nucleotide sequence in bi).
3 more elements
A maize plant having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in said maize plant, wherein expression of said first nucleic acid molecule decreases pullulanase-type activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant and said first nucleic acid molecule comprises an antisense nucleotide sequence comprising at least 50 contiguous nucleotides complementary to the mRNA encoding the maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A maize plant cell having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to first promoter active in said maize plant cell, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant cell and said first nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:1;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant cell wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
A maize plant cell having stably introduced into its genome
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in said maize plant cell, wherein expression or said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in the maize plant cell and said first nucleic acid molecule comprises an antisense nucleotide sequence comprising at least 50 contiguous nucleotides complementary to the mRNA encoding the maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said maize plant cell wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
An expression vector comprising
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in a plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in a maize plant and said first nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:1;
- and,
- b) i second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
An expression vector comprising
- a) a first DNA construct comprising a first nucleic acid molecule operably linked to a first promoter active in a plant, wherein expression of said first nucleic acid molecule decreases pullulanase activity of a maize pullulanase polypeptide capable of hydrolyzing α(1→6) linkages of pullulan in a maize plant and said first nucleic acid molecule comprises an antisense nucleotide sequence comprising at least 50 contiguous nucleotides complementary to the mRNA encoding the maize pullulanase polypeptide;
- and,
- b) a second DNA construct comprising a second nucleotide sequence operably linked to a second promoter active in said plant wherein expression of said second nucleotide sequence modulates the activity of a polypeptide involved in polyglucan synthesis and said second nucleotide sequence is selected from the group consisting of:
- i) a nucleic acid molecule comprising a nucleotide sequence that encodes the polypeptide involved in polyglucan synthesis;
- and,
- ii) a nucleic acid molecule comprising a nucleotide sequence having an antisense sequence corresponding to the second nucleotide sequence in bi).
3 more elements
Dependencies are inferred from the claim text.