Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-08-16
2004-03-16
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C536S023200, C435S419000
Reexamination Certificate
active
06706951
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the genetic manipulation of plants, particularly to the expression and regulation of GDP-mannose pyrophosphorylase in transformed plants.
BACKGROUND OF THE INVENTION
Gums are derived from the seeds of plants which accumulate certain polysaccharides as storage polymers. They are capable of forming gels or highly viscous solutions at low concentrations in solvents and have many industrial applications. Because gums can absorb large quantities of water, they are used as food additives to provide texture, prevent ice crystal formation, maintain crispness and retain moisture.
Gums also have uses outside of the food industry. For example, gums are used in the textile industry as dyeing and printing aids; in the petroleum industry as drilling agents for petroleum and gas wells; in the paper industry as binders and hardeners; in the mining industry for separation of minerals from crude ores; in the explosive industry to thicken explosive slurries and as desiccants; and in the cosmetic industry to thicken shampoos and conditioners. A new and rapidly emerging area for gum use is the pharmaceutical industry where gums have been reported to have applications as soluble fibers that lower cholesterol and blood pressure; as weight loss facilitators; as aids for the controlled release of drugs; in improving the microflora of the digestive system; in lowering blood glucose; and for prolonging the release of sugar during strenuous physical exercise.
Industrial gums are currently prepared from bacteria or from plant seeds. The main sources of seed-derived industrial gums are the subtropical plants Guar (
Cyamopsis tetragonoloba
), Locust Bean or carob (
Ceratonia siliqua
), Tara (
Caesalpina spinosa
), and Fenugreek (
Trigonella foenumgraecum
). More than 70% of natural plant gum is produced from Guar and Locust Bean.
Seed-derived gums are classified into two main categories: galactomannans and xyloglucans. Galactomannan, a linear polymer of mannosyl residues, substituted to varying degrees by galactosyl residues, is a major constituent of the seeds of the leguminous plants Guar, Fenugreek and Locust Bean. The differences in the properties of the galactomannan gums are determined by the mannose:galactose ratio in the polymer, which ranges from a low of 2 in Guar to a high of 4 in Locust Bean. Locust bean gum is considered to be of the highest quality for industrial applications and is the most expensive of all the plant seed gums.
Another class of plant gums, xyloglucans, has not received much attention, perhaps due to the very low yield of the source plants. Xyloglucans are the predominant storage polymers in the seeds of Nasturtium (
Tropaeoleum majus
), Tamarind (
Tamarindus indica
), and Balsam (
Impatiens balsamina
). Xyloglucan consists of a linear glucan backbone substituted with xylosyl residues. The xylosyl residues may be substituted with galactosyl residues.
Due to the high cost of gum derived from the seeds of subtropical plants, it is desirable to engineer transgenic plants which over-produce the gums galactomannan and/or xyloglucan. The present invention provides compositions and methods for the overexpression of enzymes and substrates required for the synthesis of the gum galactomannan.
SUMMARY OF THE INVENTION
The synthesis of the gum galactomannan is catalyzed by the enzymes mannan synthase and galactosyl transferase, from the substrates GDP-mannose and UDP-galactose (FIG.
1
). The formation of the substrate GDP-mannose, from mannose-1-phosphate and GTP, is catalyzed by the enzyme GDP-mannose pyrophosphorylase. The present invention provides compositions and methods for manipulating the levels of enzymes in the galactomannan biosynthetic pathway in order to regulate gum production in plants, plant cells and plant tissues.
Protein glycosylation is required for cell growth. GDP-mannose is a substrate for glycosylation of proteins. Therefore, down-regulation of GDP-mannose pyrophosphorylase will result in decreased levels of GDP-mannose and concomitant decreases in protein glycosylation. Thus, by down-regulating GDP-mannose pyrophosphorylase, cell growth may be inhibited. The present invention includes antisense nucleic acids for GDP-mannose pyrophosphorylase which have use in decreasing GDP-mannose pyrophosphorylase and GDP-mannose levels. Also provided are expression cassettes encoding truncated GDP-mannose pyrophosphorylase for reduction of GDP-mannose pyrophosphorylase levels by cosuppression. Such methods and compositions inhibit cell growth, and are therefore useful in the production of dwarf plants, including ornamental Bonsai type plants.
Thus, it is an object of the invention to provide nucleotide sequences encoding GDP-mannose pyrophosphorylase gene and related genes. The sequences are useful in transforming plants for expression of GDP-mannose pyrophosphorylase or the antisense RNAs thereof Such sequences find use in regulating the levels of GDP-mannose, a substrate for the formation of the gum galactomannan and for protein glycosylation. Expression cassettes comprising GDP-mannose pyrophosphorylases and antisense RNAs thereto, are provided. Additionally provided are plants, plant cells, and plant tissues.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses compositions and methods for the synthesis of the gum galactomannan in non-legume plants, plant cells and specific tissues, as well as for the increased expression in leguminous plants, plant cells and specific tissues. The methods involve modulation of the levels of enzymes in the galactomannan biosynthetic pathway. The synthesis of the gum galactomannan is catalyzed by the enzymes mannan synthase and galactosyl transferase, from the substrates GDP-mannose and UDP-galactose. The formation of the substrate GDP-mannose, from mannose-1-phosphate and GTP, is catalyzed by the enzyme GDP-mannose pyrophosphorylase.
The methods of the invention involve manipulating the pathway for gum production by over-expressing or up-regulating at least one enzyme in the biosynthetic pathway. By overexpression or up-regulation is meant causing an increase of 0.2-200 fold in the level of an RNA, enzyme or substrate in a transformed plant, as compared with the non-transformed plant. Preferably, the increase is 0.5-150 fold, and more preferably 1-100 fold. A key step in production of the gum galactomannan is providing an adequate supply of substrates, which include GDP-mannose and UDP-galactose. Thus, in particular the nucleotide sequence for maize GDP-mannose pyrophosphorylase is provided for use in priming gum production. It is recognized that if other enzymes in the galactomannan biosynthetic pathway are identified, they can be used to manipulate gum production or precursor accumulation in plants of interest.
Compositions also comprise antisense constructs for enzymes of the galactomannan biosynthetic pathway. Such compositions have use in down-regulating the levels of enzymes and/or substrates of the galactomannan biosynthetic pathway. When a plant carries a transgenic copy of an endogenous or foreign gene, both genes may be silenced. This phenomenon is termed cosuppression. Accordingly, constructs encoding are truncated enzymes of the GDP-mannose biosynthetic pathway, preferably encoding a truncated GDP-mannose pyrophosphorylase, are provided for use in cosuppression of the enzymes in the pathway. By down-regulation is meant causing a decrease of about 0.1-1000 fold, preferably about 1-500 fold, and more preferably about 5-100 fold, in a RNA, enzyme or substrate. In a preferred embodiment, compositions encoding antisense RNA to mRNA for GDP-mannose pyrophosphorylase are provided. Such compositions have use in decreasing levels of GDP-mannose, a substrate for protein glycosylation. The decrease in a substrate for protein glycosylation will result in the inhibition of cell growth or in cell death. By inhibiting cell growth, dwarf, miniature and bonsai plants may be produced.
The present invention is further drawn to compositions and methods for manipulating the levels of
Bowen Benjamin A.
Dhugga Kanwarpal S.
Wang Xun
Kubelik Anne
Lappegard Kathryn K.
Nelson Amy J.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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