Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1997-11-05
2001-02-06
Bui, Phuong T. (Department: 1645)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S292000, C800S293000, C800S281000, C435S410000, C435S069100, C435S071100, C435S091400, C435S419000, C435S320100, C530S350000, C530S370000, C536S023100, C536S023600, C536S024100
Reexamination Certificate
active
06184437
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the improvement of seed proteins. More particularly, the invention is directed to a lysine-rich protein isolated from winged bean (WBLRP), its associated nucleic acids and their uses, for example, as expressed in transgenic plants to improve their nutritional qualities.
BACKGROUND OF THE INVENTION
Plants are the primary source of all protein consumed by humans and livestock. In 1990, the world protein supply was 150 million tons, of which 65% was obtained directly from plants and 35% through animal protein production. In comparison to meat, plant protein is much more economical to produce, store, and transport. However, when used as a source of dietary protein for human and monogastric animals, most plant proteins are nutritionally incomplete due to their deficiency in several essential amino acids. In general, cereal (e.g., corn, wheat) proteins are low in lysine (Lys) and tryptophan (Trp), while legume (e.g., soybean) and most vegetable proteins are deficient in the sulfur amino acids, methionine (Met) and cysteine (Cys). See, e.g., Sun and Larkins, in
Transgenic Plants
, vol. 1, King and Wu, Ed. at p. 317 (1992).
To overcome amino acid deficiencies, a mixed diet of cereal and legume, or supplementing plant proteins with meat products or synthetic essential amino acids, typically is employed. U.S. growers annually spend over $120 million and $70 million, respectively, on synthetic Met and Lys to supplement the corn and/or soy protein base of swine and poultry diets. In view of such economic factors, a more direct and cost-efficient means of supplying these amino acids in livestock feed would be highly desirable. See, e.g., Beach et al., in
Biosynthesis and Molecular Requlation of Amino Acids in Plants
, Singh et al., Eds., at p. 229 (1992).
Plant geneticists and breeders have made significant efforts in the past to improve the quality of seed proteins and have developed, e.g., mutant high-lysine corn and barley. However, efforts in breeding legumes containing protein with increased levels of sulfur amino acids have not met with significant success.
Various genetic approaches to the selection or engineering of plant improvements include the selection of cultivars with high levels of desired amino acids, modification of amino acid biosynthesis, and modification of existing proteins. Unfortunately, there often are undesirable traits associated with such modified plants, such as lower yields and greater susceptibility to pests and diseases, and these traits have prevented the agronomic utilization of the plants.
With respect to the selection of high seed protein soybean cultivars, Serretti et al.,
Crop Sci
. 34:207-209 (1994) compared the protein and amino acid content of several soybean lines. Similarly, Zarkadas et al.,
J. Agric Food Chem
. 41:6161-623 (1993), compared the total protein amino acids and 4-hydroxyproline-rich glycoproteins of a widely-grown soybean cultivar and a high-protein genotype. In one case, however, a high-lysine protein in barley was found to be an inhibitor of chymotrypsin, arguably not a good candidate for nutritional supplementation because chymotrypsin inhibitors can interfere with digestion.
Similarly, U.S. Pat. No. 5,367,110 (1995) to Galili et al. describes transgenic plants that overexpress threonine and lysine. In such plants, a co-transformation process with chimeric genes introduces aspartate kinase activity as well as dihydrodipicolinate synthase activity. This process is intended to enhance the biosynthesis of aspartate-family amino acids, such as lysine, threonine, methionine and isoleucine, in particular the first two amino acids identified.
Alternatively, U.S. Pat. No. 5,258,300 (1993) to Glassman et al. is directed to methods of inducing lysine overproduction in plants by introducing the gene for dihydrodipicolinic acid synthase (DHDPS) which is substantially resistant to feedback inhibition by endogenously-produced free L-lysine. Attached at the 5′ terminus of this gene is a sequence encoding a chloroplast transit peptide, which serves to localize the DHDPS in the chloroplasts of transformed cells, where it can act to enhance the biosynthesis of free L-lysine.
Other recent advances in agricultural biotechnology offer other molecular approaches to alter the amino acid composition of seed proteins for improved nutritional quality. The transfer and expression of heterologous genes in plant tissues and plant seeds has been considered promising. It has been demonstrated that a seed protein gene can be transferred and expressed in transgenic plants in a developmentally regulated manner, i.e., the protein product generally is synthesized, transported and properly deposited in the seeds. Moreover, the feasibility of transforming a plant with an essential amino acid-rich protein gene also has been demonstrated.
For example, the research group of one of the present inventors (Dr. Samuel Sun) isolated from Brazil nut a gene encoding a sulfur-rich protein (SRP), having 18% Met and 8% Cys. The SRP gene was subsequently (1989) transferred into tobacco plants to test if the expression of this transgene could enhance the Met content in the transgenic seeds. Results showed that it is feasible to increase the Met content of the transgenic tobacco seeds (by up to 30%) through this molecular approach. See, e.g., Altenbach et al.,
Plant Molecular Biology
13:513-522 (1989). The SRP gene was acquired by Pioneer-Hibred International, Inc., patented by Sun et al., and used for crop protein improvement.
However, what has been needed is method to transform plants in order to achieve overexpression of lysine without the problems and disadvantages associated with prior art approaches. With respect to the expression of heterologous genes, the development of such methods will benefit from a better understanding of how gene transcription is regulated and the structure of seed storage proteins, the development of procedures for transforming and regenerating important seed crops, and the identification and isolation of seed storage protein genes that encode proteins containing high levels of a specific essential amino acid or a combination of these amino acids.
SUMMARY OF THE INVENTION
This invention relates to novel lysine rich proteins isolated from winged bean, and to its variants and modifications.
In still other aspects, the invention provides an isolated nucleic acid molecule encoding a WBLRP and to its allelic variants, labeled or unlabeled, and a nucleic acid sequence that is complementary to, or hybridizes under stringent conditions to, a nucleic acid sequence encoding a WBLRP.
Yet additional aspects of the present invention relates to transformed plant cells and intact plants derived from such cells that express elevated levels of lysine in their seeds.
REFERENCES:
patent: 5559223 (1996-09-01), Falco et al.
patent: 94 16078 (1994-07-01), None
patent: 95 15392 (1995-06-01), None
Peyachoknagul et al. Plant Molecular Biology, vol. 12, 1989 p. 51-58.
Yamamoto et al. J. Biochem., vol. 94, 1983, p. 849-863.
Kortt et al. Eur. J. Biochem., vol. 181, 1989, p 403-408.
Liu B et al., “Screening of lysine-rich plant species and identification of the purified proteins”, Chemical Abstracts, vol. 35, No. 1, 1993, pp. 62-68.
Ausubel FM, “Using synthetic oligonucleotides as probes”, Current Protocols in Molecular Biology, vol. 1, 1988, pp. 6.4.01-6.4.10.
Crowell D N et al., “Characterization of a 1,2 stress induced, developmentally regulated gene family from soybean”, Plant Molecular Biology, vol. 18, Feb. 1992, pp. 459-466.
Walter M H et al., “Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitours new class of conserved PR proteins including pollen allergens”, Molecular and General Genetics, vol. 222, Jul. 1990, pp. 353-360.
Liu B et al., Screening of lysine-rich plant species and identification of the purified proteins, ZHIWU XUEBAO, vol. 35, No. 1, 1993, pp. 62-68.
Jing Yuxiang
Liu Bolin
Sun Samuel S. M.
Xiong Liwen
Beijing Vegetable Research Center
Bui Phuong T.
Morrison & Foerster / LLP
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