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Phytate polynucleotides and methods of use

Phytate polynucleotides and methods of use description/claims

This application is a divisional application of U.S. application Ser. No. 10/255,817 filed Sep. 26, 2002, now abandoned, and claims the benefit of U.S. Application Ser. No. 60/325,308 filed Sep. 27, 2001, all of which are herein incorporated by reference.

The present invention relates to the field of animal nutrition. Specifically, the present invention relates to the identification and use of genes encoding enzymes involved in the metabolism of phytate in plants and the use of these genes and mutants thereof to reduce the levels of phytate, and/or increase the levels of non-phytate phosphorus in food or feed.

The role of phosphorous in animal nutrition is well recognized, it is a critical component of the skeleton, nucleic acids, cell membranes and some vitamins. Though phosphorous is essential for the health of animals, not all phosphorous in feed is bioavailable.

Phytates are the major form of phosphorous in seeds, for example phytate represents about 60-80% of total phosphorous in corn and soybean. When seed-based diets are fed to non-ruminants, the consumed phytic acid forms salts with several important mineral nutrients, such as potassium, calcium, and iron, and also binds proteins in the intestinal tract. These phytate complexes cannot be metabolized by monogastric animals and are excreted, effectively acting as anti-nutritional factors by reducing the bioavailability of dietary phosphorous and minerals. Phytate-bound phosphorous in animal excreta also has a negative environmental impact, contributing to surface and ground water pollution.

There have been two major approaches to reducing the negative nutritional and environmental impacts of phytate in seed. The first involves post-harvest interventions, which increase the cost and processing time of feed. Post-harvest processing technologies remove phytic acid by fermentation or by the addition of compounds, such as phytases.

The second is a genetic approach. One genetic approach involves developing crop germplasm with heritable reductions in seed phytic acid. While some variability for phytic acid was observed, there was no change in non-phytate phosphorous. Further, only 2% of the observed variation in phytic acid was heritable, whereas 98% of the variation was attributed to environmental factors.

Another genetic approach involves selecting low phytate lines from a mutagenized population to produce germplasm. Most mutant lines are a loss of function, presumably blocked in the phytic acid biosynthetic pathway, therefore low phytic acid accumulation will likely be a recessive trait. In certain cases, this approach has revealed that homozygosity for substantially reduced phytate proved lethal.

Another genetic approach is transgenic technology, which has been used to increase phytase levels in plants. These transgenic plant tissues or seed have been used as dietary supplements.

The biosynthetic route leading to phytate is complex and not completely understood. Without wishing to be bound by any particular theory of the formation of phytate, it is believed that the synthesis may be mediated by a series of one or more ADP-phosphotransferases, ATP-dependent kinases and isomerases. A number of intermediates have been isolated including, for example, monophosphates such as D-myo-inositol 3-monophosphate, diphosphates (IP2s) such as D-myo-inositol 3,4-bisphosphate, trisphosphates (IP3s) such as D-myo-inositol 3,4,6 trisphosphate, tetraphosphates (IP4s) such as D-myo-inositol 3,4,5,6-tetrakisphosphates, and pentaphosphates (IP5s) such as D-myo-inositol 1,3,4,5,6 pentakisphosphate. The phosphorylation of the IP5 to IP6 is found to be reversible. Several futile cycles of dephosphorylation and rephosphorylation of the IP5 and IP6 forms have been reported as well as a cycle involving glucose-6-phosphate->D-myo-inositol 3-monophosphate->myo-inositol; the last step being completely reversible, indicating that control of metabolic flux through this pathway may be important.

Based on the foregoing, there exists the need to improve the nutritional content of plants, particularly corn and soybean by increasing non-phytate phosphorous and reducing seed phytate. This invention provides tools and reagents that allow the skilled artisan, by the application of, inter alia, transgenic methodologies to influence the metabolic flux in respect to the phytic acid pathway.

Inositol 1,3,4-trisphosphate 5/6-kinases (ITPK) are involved in the phytate biosynthetic pathway. This invention provides nucleic acids and proteins related to inositol 1,3,4-trisphosphate 5/6-kinases as well as recombinant expression cassettes and methods to modulate the level of inositol 1,3,4-trisphosphate 5/6-kinases in host cells, transgenic plants and seeds. The invention also provides the host cells, transgenic plants and transgenic seeds produced by these methods. The invention foresees using these nucleic acids or polypeptides, or variants thereof, to modulate the flux through the phytic acid biosynthetic pathway in order to improve the nutritional quality of feed, corn and soy in particular, and to reduce the environmental impact of animal waste by creating seed with higher available phosphorous or lower phytate levels.

Units, prefixes, and symbols may be denoted in their Si accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.

The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.

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