Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1998-12-23
2002-12-17
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C536S023100, C536S023200, C536S023600, C536S024310, C536S023740, C536S023740, C435S006120, C435S069100, C435S468000, C435S410000, C435S419000, C435S320100, C800S278000, C800S290000, C800S295000, C800S298000, C800S306000, C800S314000, C800S320200, C800S320300, C800S320000, C800S319000
Reexamination Certificate
active
06495740
ABSTRACT:
The present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis and transgenic organisms expressing same in sense or antisense orientation, or as ribozymes, co-suppression or gene-targeting molecules. More particularly, the present invention is directed to a nucleic acid molecule isolated from
Arabidopsis thaliana, Oryza sativa,
wheat, barley, maize, Brassica ssp.,
Gossypium hirsutum
and Eucalyptus ssp. which encode an enzyme which is important in cellulose biosynthesis, in particular the cellulose synthase enzyme and homologues, analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identity numbers (SEQ ID Nos.) for the nucleotide and amino acid sequences referred to in the specification are defined after the bibliography.
Throughout the specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Cellulose, the world's most abundant biopolymer, is the most characteristic component of plant cell walls in so far as it forms much of the structural framework of the cell wall. Cellulose is comprised of crystalline &bgr;-1,4-glucan microfibrils. The crystalline microfibrils are extremely strong and resist enzymic and mechanical degradation, an important factor in determining the nutritional quantity, digestibility and palatability of animal and human foodstuffs. As cellulose is also the dominant structural component of industrially-important plant fibres, such as cotton, flax, hemp, jute and the timber crops such as Eucalyptus ssp. and Pinus ssp., amongst others, there is considerable economic benefit to be derived from the manipulation of cellulose content and/or quantity in plants. In particular. the production of food and fibre crops with altered cellulose content are highly desirable objectives.
The synthesis of cellulose involves the &bgr;-1,4-linkage of glucose monomers, in the form of a nucleoside diphospoglucose such as UDP-glucose, to a pre-existing cellulose chain, catalysed by the enzyme cellulose synthase.
Several attempts to identify the components of the functional cellulose synthase in plants have failed, because levels of &bgr;-1,4-glucan or crystalline cellulose produced in such assays have hitherto been too low to permit enzyme purification for protein sequence determination. Insufficient homology between bacterial &bgr;-1,4-glucan synthase genes and plant cellulose synthase genes has also prevented the use of hybridisation as an approach to isolating the plant homologues of bacterial &bgr;-1,4-glucan (cellulose) synthases.
Furthermore, it has not been possible to demonstrate that the cellulose synthase enzyme from plants is the same as, or functionally related to, other purified and characterised enzymes involved in polysaccharide biosynthesis. As a consequence, the cellulose synthase enzyme has not been isolated from plants and, until the present invention, no nucleic acid molecule has been characterised which functionally-encodes a plant cellulose synthase enzyme.
In work leading up to the present invention, the inventors have generated several novel mutant
Arabidopsis thaliana
plants which are defective in cellulose biosynthesis. The inventors have further isolated a cellulose synthase gene designated RSW1, which is involved in cellulose biosynthesis in
Arabidopsis thaliana,
and homologous sequences in
Oryza sativa,
wheat, barley, maize, Brassica ssp.,
Gossypium hirsutum
and Eucalyptus ssp. The isolated nucleic acid molecules of the present invention provide the means by which cellulose content and structure may be modified in plants to produce a range of useful fibres suitable for specific industrial purposes, for example increased decay resistance of timber and altered digestibility of foodstuffs, amongst others.
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes, or is complementary to a sequence which encodes a polypeptide of the cellulose biosynthetic pathway or a functional homologue, analogue or derivative thereof.
The nucleic acid molecule of the invention may be derived from a prokaryotic source or a eukaryotic source.
Those skilled in the art will be aware that cellulose production requires not only the presence of a catalytic subunit, but also its activation and organisation into arrays which favour the crystallization of glucan chains. This organisation is radically different between bacteria, which possess linear arrays, and higher plants, which possess hexameric clusters or “rosettes”, of glucan chains. The correct organisation and activation of the bacterial enzyme may require many factors which are either not known, or alternatively, not known to be present in plant cells, for example specific membrane lipids to impart an active conformation on the enzyme complex or protein, or the bacterial c-di-GMP activation system. Accordingly, the use of a plant-derived sequence in eukaryotic cells such as plants provides significant advantages compared to the use of bacterially-derived sequences.
Accordingly, the present invention does not extend to known genes encoding the catalytic subunit of
Agrobacterium tumefaciens
or
Acetobacter xylinum
or
Acetobacter pasteurianus
cellulose synthase, or the use of such known bacterial genes and polypeptides to manipulate cellulose.
Preferably, the subject nucleic acid molecule is derived from an eukaryotic organism.
In a more preferred embodiment of the invention, the isolated nucleic acid molecule of the invention encodes a plant cellulose synthase or a catalytic subunit thereof, or a homologue, analogue or derivative thereof.
More preferably, the isolated nucleic acid molecule encodes a plant cellulose synthase polypeptide which is associated with the primary cell wall of a plant cell. In an alternative preferred embodiment, the nucleic acid molecule of the invention encodes a plant cellulose synthase or catalytic subunit thereof which is normally associated with the secondary cell wall of a plant cell.
In a more preferred embodiment, the nucleic acid molecule of the invention is a cDNA molecule, genomic clone, mRNA molecule or a synthetic oligonucleotide molecule.
In a particularly preferred embodiment, the present invention provides an isolated nucleic acid molecule which encodes or is complementary to a nucleic acid molecule which encodes the
Arabidopsis thaliana, Gossypium hirsutum
(cotton),
Oryza sativa
(rice), Eucalyptus ssp., Brassica ssp. wheat, barley or maize cellulose synthase enzyme or a catalytic subunit thereof or a polypeptide component, homologue, analogue or derivative thereof.
As exemplified herein, the present inventors have identified cellulose biosynthesis genes in maize, wheat, barley, rice, cotton, Brassica ssp. and Eucalyptus ssp., in addition to the specific
Arabidopsis thaliana
RSW1 gene sequence which has been shown to be particularly useful for altering cellulose and/or &bgr;-1,4-glucan and/or starch levels in cells.
Hereinafter the term “polypeptide of the cellulose biosynthetic pathway” or similar term shall be taken to refer to a polypeptide or a protein or a part, homologue, analogue or derivative thereof which is involved in one or more of the biosynthetic steps leading to the production of cellulose or any related &bgr;-1,4-glucan polymer in plants. In the present context, a polypeptide of the cellulose biosynthetic pathway shall also be taken to include both an active enzyme which contributes to the biosynthesis of cellulose or any related &bgr;-1,4-glucan polymer in plants and to a polypeptide component of such an enzyme. As
Arioli Antonio
Betzner Andreas S.
Peng Liangcai
Williamson Richard E.
Bui Phuong T.
Greenlee Winner and Sullivan P.C.
The Austrailian National University
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