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
1996-05-06
2001-04-10
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
C435S069100, C435S252300, C435S254200, C435S320100, C435S419000, C435S468000, C435S477000, C435S483000, C536S023200, C536S023600, C800S278000, C800S286000
Reexamination Certificate
active
06215044
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleotide sequences encoding a plant enzyme, vectors containing said nucleotide sequences, hosts containing said nucleotide sequences, amino acid sequences encoded by said nucleotide sequences, recombinant DNA methods of producing the enzyme, and a method of altering the properties of a plant.
BACKGROUND OF THE INVENTION
Fruit and vegetable cell walls are largely polysaccharide, the major components being pectin, cellulose and xylogucan (Selvendran & Robertson, IFR Report 1989). Numerous cell wall models have been proposed which attempt to incorporate the essential properties of strength and flexibility (e.g. Albersheim, 1975 Sci. Am. 232, 81-95; Albersheim, 1976 “The primary cell wall”, Plant Biochemistry, 3rd Edition, Academic Press; and Hayashi, 1989, Ann. Rev. Plant Physiol. & Plant Mol. Biol. 40, 139-168).
Xyloglucans are 1,4-&bgr;-glucans that are extensively substituted with &agr;-1,6-xylosyl side chains, some of which are 1,2 &bgr;-galactosylated. They are found in large amounts in the primary cell walls of dicots but also in certain seeds, where they serve different roles.
Primary cell wall xyloglucan is tightly hydrogen bonded to cellulose microfibrils and requires concentrated alkali or strong swelling agents to release it. Xyloglucan is thought to form cross-bridges between cellulose microfibrils, the cellulose/xyloglucan network forming the major load-bearing/elastic network of the wall. DCB mutated suspension culture cells (cell walls lacking cellulose) release xyloglucan into their media, suggesting that xyloglucan is normally tightly bound to cellulose.
Hydrolysis of primary cell wall xyloglucan has been demonstrated in segments of dark grown squash hypocotyls, during IAA induced growth (Wakabayashi et al., 1991 Plant Physiol. 95. 1070-1076). Endohydrolysis of wall xyloglucan is thought to contribute to the wall loosening which accompanies cell expansion (Hayashi, cited previously). The average molecular weight of xyloglucan has also been shown to decrease during tomato fruit ripening and this may contribute to the tissue softening which accompanies the ripening process (Huber, 1983 J. Amer. Soc. Hort. Sci. 108, 405-409).
Certain seeds, e.g. nasturtium, contain up to 30% by weight of xyloglucan, stored in thickened cotyledonary cell walls, which serves as a reserve polysaccharide and is rapidly depolymerised during germination.
An endo 1,4 &bgr;-D glucanase which specifically acts on xyloglucan (i.e. a xyloglucanase) has been isolated and purified to apparent homogeneity from germinating nasturtium (
Tropaeolum majus
L.) seeds (Edwards et al., 1986 J. Biol. Chem. 261, 9494).
The purified xyloglucanase gives a single polypeptide band on SDS polyacrylamide gel electrophoresis, (apparent molecular weight, 29-31 kDa) and isoelectric focusing (isoelectric point, 5.0). The enzyme displays an absolute specificity for xyloglucan and an endo mode of action, as determined by end product analysis following hydrolysis of tamarind seed xyloglucan (Wakabayashi et al., cited above). Although the natural substrate of the enzyme is nasturtium cotyledonary reserve xyloglucan, it has also been shown to hydrolyse primary cell wall xyloglucans in vitro (Edwards et al., cited previously). At high substrate concentrations, xyloglucan endo-transglycosylase (XET) activity has been demonstrated (Fanutti et al., 1993 The Plant Journal 3, 691-700).
The nucleotide sequence of this nasturtium xyloglucanase/XET enzyme has been determined and is disclosed in International Patent Application No. WO 93/17101 and by de Silva et al., (1993 The Plant Journal 3, 701-711).
Similar enzyme activity has been detected in other plant tissue and shown to be positively correlated with growth rate in different zones of the pea stem (Fry et al., 1992 Biochem. J. 282, 821-829). It has been proposed that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that this causes the wall-loosening required for plant cell expansion. XET activity has also been demonstrated in tomato fruit (xyloglucanase apparently activatable by xyloglucan oligosaccharides) where it is reportedly highest at the 2 days post-“breaker” stage of ripening (Machlachlan & Brady, 1992 Aust. J. Plant Physiol. 19, 137-146) and may be involved in the softening process.
This application describes the isolation from tomato plants of a nucleotide sequence which encodes an XET activity.
SUMMARY OF THE INVENTION
In a first aspect the invention provides a polypeptide having xyloglucan endo-transglycosylase (XET) activity, comprising substantially residues 21-289 of the amino acid sequence of
FIG. 1
(Seq ID No. 2), or functional equivalents thereof.
The term “functional equivalent” as used herein and applied to an amino acid sequence is intended to refer to those amino acid sequences having the same functional characteristics as the sequence of the invention but which have slightly different sequences. Generally, for example, it is well known that one may make a “conservative” substitution, exchanging one amino acid residue in a sequence for another residue with very similar properties, without having any significant effect on the function of the polypeptide.
An example of a functionally equivalent polypeptide sequence is shown in
FIG. 5
(Seq ID No. 8). The amino acid sequences shown in
FIGS. 1 and 5
are directly compared in FIG.
6
.
Further, one or more amino acid residues may be deleted or added without significant deleterious effect. It will be apparent to those skilled in the art that such changes may particularly be made at points in the sequence which are not evolutionarily conserved. Enzyme precursors are also included within the meaning of “functional equivalents” and also mature, processed enzymes lacking signal sequences. In the present case amino acid residues 1-20 of
FIG. 1
are believed to constitute such a signal sequence and are therefore not essential for enzyme activity. Likewise, amino acid residues 1-18 of the sequence shown in
FIG. 5
are thought to comprise a non-essential signal sequence.
In specific embodiments therefore the invention may provide a polypepide having XET activity and comprising residues 1-289 of the sequence shown in
FIG. 1
; or residues 19-287 of the amino acid sequences shown in
FIG. 5
; or residues 1-287 of the amino acid sequence shown in FIG.
5
.
Preferably such functional equivalents will exhibit at least 80% homology, and more preferably at least 85% homology, and most preferably at least 90% homology, with the amino acid sequence of the invention.
The present inventors have, by analysis of the amino acid sequence data, identified a number of peptide sequences which are thought to be relatively conserved within functional equivalents of the sequence of the invention.
Preferably, functional equivalents will comprise substantially one or more of the following peptide sequences (which will generally be perfectly conserved but may contain up to one or, at most, two amino acid differences):
DEIDFEFLGN; SLWNADDWAT; FYSKNEYLFG; and GTVTTFYLSS; (Seq ID Nos. 3-6 respectively).
Desirably the polypeptide is substantially in isolation from other plant derived substances.
In a second aspect the invention provides a nucleotide sequence encoding the amino acid sequence of
FIG. 1
(Seq ID No. 1), or functional equivalents thereof.
Preferably the nucleotide sequence comprises the nucleotide sequence of
FIG. 1
or other functionally equivalent sequences obtainable from tomato plants. The term “functional equivalent” as used herein and applied to nucleotide sequences is intended to refer to nucleotide sequences encoding a polypeptide having the same functional characteristics as the amino acid sequences of the first aspect and includes nucleotide sequences which will hybridize under standard conditions (described by Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual) to the complement of the nucleotide sequence of FIG.
1
. Preferably the functionally equivalent nucleotide sequence will exhibit at least 70% homology,
Arrowsmith David A.
de Silva Jacqueline
Hellyer Susan A.
Whiteman Sally A.
Nelson Amy J.
Pillsbury & Winthrop LLP
Unilever Patent Holdings B.V.
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