Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-05-06
2001-07-03
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S069100, C435S070100, C435S101000, C435S252300, C435S419000, C536S023600, C800S284000
Reexamination Certificate
active
06255114
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in starch biosynthesis in plants and seeds.
BACKGROUND OF THE INVENTION
Starch is an important component of food, feed, and industrial products. Broadly speaking, it consists of two types of glucan polymers: relatively long chained polymers with few branches known as amylose, and shorter chained but highly branched molecules called amylopectin. Its biosynthesis depends on the complex interaction of multiple enzymes (Smith, A. et al., (1995)
Plant Physio
. 107:673-677; Preiss, J., (1988)
Biochemistry of Plants
14:181-253). Chief among these are ADP-glucose pyrophosphorylase, which catalyzes the formation of ADP-glucose; a series of starch synthases which use ADP glucose as a substrate for polymer formation using &agr;-1-4 linkages; and several starch branching enzymes, which modify the polymer by transferring segments of polymer to other parts of the polymer using &agr;-1-6 linkages, creating branched structures. However, based on data from starch forming plants such as potato, and corn, it is becoming clear that other enzymes also play a role in the determination of the final structure of starch. In particular, debranching and disproportionating enzymes not only participate in starch degradation, but also in modification of starch structure during its biosynthesis. Different models for this action have been proposed, but all share the concept that such activities, or lack thereof, change the structure of the starch produced.
This is of applied interest because changes in starch structure, such as the relative amounts of amnylose and amylopectin or the degree and length of branching of amylopectin, alter its function in cooking and industrial processes. For example, starch derived from different naturally occurring mutants of corn can be shown on the one hand to differ in structure and correspondingly to differ in functional assays such as Rapid Visco analysis, which measures changes in viscosity as starch is heated and then cooled (Walker, C. E., (1988)
Cereal Foods World
33:491-494). The interplay of different enzymes to produce different structures, and in turn how different structures correlate with different functionalities, is not yet completely understood. However, it is understood that changing starch structure will result in alteration in starch function which can in turn lead to new applications or reduced processing costs (certain starch functionalities can at present only be attained through expensive chemical modification of the starch).
Glycogen, a non-plant analogue of starch, is synthesized by the concerted actions of glycogen synthase and glycogen branching enzymes in much the same way that starch biosynthesis occurs in plants. Glycogen synthesis requires a primer for the initial action of the glycogen synthase enzyme. This primer function is thought to be provided by self-glucosylating protein called glycogenin in mammals. Inactivation of the two genes that encode this enzyme in yeast has been shown to result in the absence of glycogen. It is evident that a similar primer function may be necessary for starch biosynthesis in plants and the isolation of such a self glucosylating activity has been the subject some study (Singh, D. G. et al., (1995)
FEBS Letters
376:61-64; World Patent Publication No. WO 94/04693). These reports describe the identification and purification a self-glucosylating protein activity from plants that is structurally unrelated to glycogenin. However, these reports provide no direct evidence that this protein is essential for starch biosynthesis. Lastly, the rice gene WSI76 is a gene induced by short term water stress. Its expression is decreased in response to chilling (Plant Mol Biol 1994 October 26(1):339-352). WS176 may be a rice glycogenin because its only homology to a functionally characterized protein is to glycogenin.
Alterations in starch fine structure are known to result in changes to the physiochemical properties of the starch. Because starch fine structure results from the concerted action of several starch synthases, starch branching enzymes and starch debranching enzymes, it is reasonable to suppose that manipulating the amount of substrate for these enzymes may impact on the ultimate structure of the starch granule. Further it is clear that attempts to manipulate starch fine structure through altering expression of starch biosynthetic genes may lower the overall production of starch by reducing the amount of substrate, glucan chains, available to prime synthesis. One useful approach to resolve such difficulties would be the overexpression of a primer protein, glycogenin. Finally, manipulating the expression of the glycogenin primer may be used, for example, to alter the total number of granules initiated in corn endosperm. Increasing or decreasing the number of initial primers for synthesis might reasonably be expected to decrease or increase, respectively, the ultimate size of the synthesized granules. Altering granule size may usefully alter starch functionality and or starch.
The role of glycogenin in starch biosynthesis suggests that over-expression or reduction of expression of genes encoding glycogenin in corn, rice or wheat could be used to alter branch chain distribution of the starch produced by these plants. While glycogenin genes and genes encoding peptides with homology to glycogenin have been described from other organisms (Barbetti, F. et al. (1995)
Diabetologia
38:295; Wilson, R. et al. (1994)
Nature
368:32-38; Takahashi, R. et al. (1994)
Plant Mol. Biol
. 26(1):339-352), a glycogenin gene has yet to be described for corn, rice or wheat.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding corn, rice and wheat glycogenin and water stress proteins. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding corn, rice and wheat glycogenin and water stress proteins.
In another embodiment, the instant invention relates chimeric genes encoding a corn, rice and wheat glycogenin and water stress protein or nucleic acid fragments that are complementary to nucleic acid fragments encoding a corn, rice and wheat glycogenin and water stress protein, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of altered levels of a corn, rice and wheat glycogenin or water stress protein in a transformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding corn, rice and wheat glycogenin or water stress protein, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of altered levels of corn, rice and wheat glycogenin or water stress protein in the transformed host cell. The transformed host cells can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and from seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a corn, rice and wheat glycogenin or water stress protein in a transformed host cell comprising: a) transforming a host cell with the chimeric gene encoding a corn, rice and wheat glycogenin or water stress protein, operably linked to suitable regulatory sequences; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of a corn, rice and wheat glycogenin and water stress protein in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or substantially all
Everard John D.
Lightner Jonathan Edward
E. I. du Pont de Nemours & Company
Fox David T.
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