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Corn plants and seed enhanced for asparagine and protein

Corn plants and seed enhanced for asparagine and protein description/claims

This application claims the priority of U.S. Provisional application Ser. No. 60/912,909, filed Apr. 19, 2007, the entire disclosure of which is incorporated herein by reference.

1. Field of the Invention

The present invention relates generally to the field of plant biotechnology and more specifically to enhancing asparagine and protein in plants and seeds.

2. Description of Related Art

Farmers and consumers desire crop plants with improved agronomic traits such as increased yield, increased seed protein production, and improved nutritional composition. Desirable nutritional components of crop plants include, among others, fiber, antioxidants such as Vitamin E, selenium, iron, magnesium, zinc, B vitamins, lignans, phenolic acids, essential amino acids, and phytoestrogens. Although considerable efforts in plant breeding have provided some gains in these desired traits, the ability to introduce specific non-host DNA into a plant genome provides further opportunities for generation of plants with these traits. In particular, while the yield of conventional corn has steadily increased over the years, there has not been a similar increase in the capacity of corn plants to assimilate nitrogen more efficiently or to increase seed protein content.

Availability of nitrogen has a significant positive impact on plant productivity, biomass, and crop yield including the production of seed protein. In plants, inorganic nitrogen is assimilated from the soil, reduced to ammonia, and incorporated into organic nitrogen in the form of the nitrogen-transporting amino acids asparagine, glutamine, aspartic acid and glutamic acid. Asparagine (Asn) is the preferred amide transport molecule because of its high nitrogen to carbon ratio (2N:4C versus 2N:5C) and because it is relatively inert. Asn and other amino acids are also used as building blocks for protein synthesis.

In plants, Asn is synthesized from glutamine, aspartate and ATP, in a reaction catalyzed by the enzyme asparagine synthetase (AsnS). Glutamate, AMP and pyrophosphate are formed as by-products. Two forms of AsnS have been described: a glutamine-dependent form and an ammonia-dependent form. The glutamine-dependent AsnS can catalyze both the glutamine-dependent and ammonia-dependent reactions although glutamine is the preferred nitrogen source.

High concentration of protein is considered an important quality trait for most major crops, including soybean, corn, and wheat. Varieties of high protein corn, wheat, and soybeans, for example, have been identified through traditional breeding. However, most of the high protein lines developed this way have yield drag or other agronomic disadvantages. It would be desirable if the protein content of crops, especially corn, could be increased above the presently available levels, both for human consumption and for use of the product in animal feeds. This would offer the benefit of greatly enhanced nutrient value when the crop is used as food and feed for humans and animals.

The invention provides methods and compositions for development of crops and plant products to increase the protein and amino acid content. The methods and compositions increase the level of free amino acids and protein in plant tissues, particularly in seeds. More specifically, transgenic plants and seeds are provided that contain heterologous DNA compositions that expresses a gene product involved in increased asparagine and increased protein biosynthesis. The expression of the product enhances the nutritional value of food corn and feed corn sources and processed products derived from the transgenic corn seed or parts thereof.

In one aspect, the invention provides methods for increasing protein content in a corn plant. DNA constructs comprising a polynucleotide sequence encoding polypeptides with asparagine synthetase activity are also provided.

In another embodiment, the present invention comprises a corn plant cell transformed with the heterologous DNA composition encoding an asparagine synthetase identified herein. In specific embodiments, the heterologous expression of a corn AsnS4 (asparagine synthetase isozyme 4) polynucleotide molecule is provided, for example, to result in elevated asparagine and/or protein in a transgenic plant, including in the seeds, relative to a plant of the same variety not expressing the heterologous corn AsnS2 polynucleotide molecule.

In particular embodiments, a nucleic acid sequence is provided, as well as methods of use thereof, wherein the sequence is selected from the group consisting of: (a) a nucleic acid sequence comprising the sequence of SEQ ID NO:50; (b) a nucleic acid sequence with at least about 90% identity to the sequence of SEQ ID NO:50, wherein the nucleic acid sequence encodes a polypeptide comprising asparagine synthetase activity; (c) a nucleic acid sequence that encodes the polypeptide sequence of SEQ ID NO:51; (d) a nucleic acid sequence that encodes a polypeptide sequence with at least about 90% identity to the sequence of SEQ ID NO:51, wherein the polypeptide comprises asparagine synthetase activity; and (e) a nucleic acid sequence that hybridizes to a sequence of (a)-(d) under high stringency conditions of about 0.2×SSC and 65° C., wherein the nucleic acid sequence encodes a polypeptide comprising asparagine synthetase activity. In particular emnbodiments, nucleic acids are provided having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO:50. In further embodiments, nucleic acids are provided encoding a polypeptide sequence having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO:51. The invention also provides isolated polypeptide sequences comprising SEQ ID NO:51, as well as sequences having at least 90%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

The present invention also relates to food or animal feed prepared from a seed provided by the invention with increased protein and/or amino acid content, or a processed product of such seed, for example, a meal. Accordingly, the present invention also encompasses a corn seed containing an asparagine synthetase enzyme produced by expression of a heterologous DNA construct comprising a DNA molecule encoding a corn asparagine synthetase enzyme. One embodiment of such a seed is harvested grain, the present invention also encompasses meal, gluten and other corn products made from such grain.

The present invention includes isolated nucleic acid primer sequences comprising one or more of SEQ ID NOs:52-59, or the complement thereof. The present invention includes a method to detect or identify, in the genome of a transformed plant or progeny thereof, a heterologous polynucleotide molecule encoding a plant AsnS polypeptide, or a plant polypeptide having AsnS activity of the present invention, comprising a polynucleotide molecule selected from the group consisting of SEQ ID NOs:52-59, wherein said polynucleotide molecule is used as a DNA primer in a DNA amplification method.

FIG. 1 illustrates the plasmid map of pMON79706.

• Provisional Patent
• Utility Patent

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