Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-01-22
2004-03-09
Benzion, Gary (Department: 1634)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S281000, C800S287000, C800S288000, C800S298000, C800S295000
Reexamination Certificate
active
06703539
ABSTRACT:
FIELD OF INVENTION
The present invention provides methods and compositions for the alteration of compounds produced by secondary metabolic pathways in plants. The invention also provides plant cells modified in content of secondary metabolites and plant seed with altered secondary metabolite content. In one embodiment, the content of anti-nutritional secondary metabolic products is altered in plants, plant cells and plant seeds according to the invention. In another embodiment, products found within the phenylpropanoid and sugar alcohol secondary metabolic pathways are altered in plants, plant cells and plant seeds according to the invention. The invention further provides genetic constructs and vectors useful for modifying the secondary metabolite content of plant cells and seeds. The invention further relates to modified seed meal, and to animal feed containing modified seed meal, particularly seed meal in which the secondary metabolite content is reduced or altered.
BACKGROUND OF THE INVENTION
Plants produce a variety of compounds by way of secondary metabolism. While not considered essential to plant metabolism, secondary metabolic pathways often produce unique biochemicals, some of which are considered anti-nutritional or even toxic. The secondary metabolic pathways and the compounds produced by these pathways are be specific to an individual species or genus. Thus manipulation of secondary metabolic pathways can produce novel compositions of biochemicals or produce plant tissue with altered secondary metabolic content. In particular, the manipulation of secondary metabolism for the purpose of alteration of secondary metabolic compounds that are anti-nutritional or toxic in nature can provide unique applications in the food and feed area.
It is desirable to manipulate secondary metabolism without disturbing the biochemical processes considered essential for plant cell growth and survival. The collection of biochemical processes and the compounds involved which are essential for the growth and survival of the plant, are considered primary metabolic pathways and their products. Primary metabolism is generally considered to encompass those biochemical processes that lead to the formation of primary sugars, (such as glucose), amino acids, common fatty acids, nucleotides and the polymers derived from them (polysaccharides such as starch, proteins, lipids, RNA and DNA etc.) Yeoman and Yeoman, Tansley Review No. 90,
Manipulating Secondary Metabolism in Cultured Plant Cells,
New Phytologist, 134:553-569, 1996.
Thus the art recognizes that primary metabolism can be defined as those metabolic processes essential to the survival and growth of all plant cells whereas secondary metabolism can be defined as those biochemical processes that are not essential to all plant cells. For example, secondary metabolic. pathways determine such plant features as colour, taste, morphology, etc. Secondarymetabolism also produces various compounds that are recognized by insects or are involved in pathogen response. Some of these compounds may provide a benefit to some plant species under wild conditions, but under cultivation these compounds may be detrimental to the quality of the harvested product or may restrict the utility of the crop for certain applications. Some of the secondary metabolites are unique compounds that have evolved within a species as a result of specialized biochemical pathways. Secondary metabolism is not characterized by the redundancy in biochemical mechanisms which is typical of primary metabolism, thus, characteristically, the products of secondary metabolism are not produced by multiple pathways in the plant. Secondary metabolites are typically more plant-specific than the ubiquitous biochemicals which are involved in the primary pathways.
Numerous attempts to manipulate primary metabolic pathways have resulted in plant cells with altered starch or oil (lipid) content. However, gross manipulation of primary metabolism can be expected to lead to deleterious effects. For example, the composition of lipids can be changed, but elimination of lipids would obviously be deleterious to cell survival. Manipulation of primary metabolism is not always completely successful because redundant biochemical mechanisms can overcome some attempts at manipulation. Thus primary metabolic pathways in plants are often difficult to manipulate in a fashion that is predictable and provides useful and tangible results under cultivation conditions.
In some instances, primary metabolism has been altered successfully to produce a novel phenotype which represents a compositional change rather than a reduction or elimination of a specific substance. Typically, these manipulations have been accomplished by ectopic expression of a plant gene, such as over-expressing a gene in certain tissues or in a constitutive fashion rather than a regulated fashion, or by inhibition of a specific gene activity by antisense RNA, ribozymes or co-suppression. However, it has been difficult to predict a priori the results of such manipulations.
The expression of a plant enzyme can be modified at many levels. This includes control at the gene expression level, translation, protein processing and allosteric control of protein function. Thus ectopic expression of a plant gene involved in primary metabolism may not overcome the complex biochemical controls on regulation of primary metabolism. Furthermore, redundancy in primary metabolism also poses a difficult hurdle to overcome in these manipulations since primary metabolic pathways are essential to plant growth and survival. Accordingly attempts to alter primary metabolism often fail to provide the intended phenotype. Moreover, the evaluation of these modified plants at the field level, or under a variety of environmental extremes has often led to the discovery that the predictedeffect is not observed or plant performance is compromised. Thus, modification of primary metabolism requires careful consideration ofthe primary metabolic pathway or the discreet step in a pathway in order to achieve a specific phenotype.
The manipulation of secondary metabolic pathways has been complicated by a poor understanding of the biochemistry involved, little information on the genes expressed in secondary metabolic pathways and the complex inter-relationships between biochemical pathways in general.
However, methods to alter secondary metabolism can provide a valuable means to produce novel phenotypes, including those with altered levels of secondary metabolic compounds, for example those considered anti-nutritional in nature. Thus secondary metabolic pathways represent an important target for the genetic manipulation of plants.
Two biochemical pathways in plants that are considered secondary metabolic pathwayshave been the subject of studies aimed at alterationof the levels of the final products. The methods used to manipulate these pathways have not produced the desired results. For example, the phenylpropanoid pathway is involved in the formation of lignin and is considered a secondary metabolic pathway. The biosynthesis of lignin is part of the general phenylpropanoid biosynthetic pathway which produces at least three primary phenolic precursors, coumaric, ferulic and sinapic acids, products of which are polymerized into lignin and other phenolic compounds (see FIG.
2
).
In attempts to alter the secondary metabolic phenylpropanoid pathway, the genes for many of the enzymes involved in the formation of the lignin monomers are currently identified as targets for lignin reduction via antisense or co-suppression technologies (e.g. U.S. Pat. Nos. 5,451,514, 5,633,439, WO 93/05160, WO 94/08036). These target genes include those encoding cinnamyl alcohol dehydrogenase, caffeic acid O-methyl-transferase and phenylalanine ammonia lyase. These techniques are directed to reduction of lignin content as this is assumed to have an overall beneficial effect on processing or digestibility of plants.
However, reduction of lignin by antisense or co-suppression technologies by targeting one of the ge
Datla Raju S. S.
Dong Jin-Zhuo
Georges Fawzy
Hussain Atta A. K.
Keller Wilfred A.
Benzion Gary
Lorusso, Loud & Kelly
National Research Council of Canada
Switzer Juliet C.
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