Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR ENHANCING CELLULOSE AND
MODIFYING LIGNIN BIOSYNTHESIS IN PLANTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application
Serial No. 60/135,280 filed 21 May, 1999.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
Not Applicable
FIELD OF THE INVENTION
This invention relates to polynucleotide molecules encoding cellulose
synthase, promoters of cellulose synthase and cellulose synthase polypeptides,
methods for
genetically altering cellulose and lignin biosynthesis, and methods for
improving strength
properties of juvenile wood and fiber in trees. The invention further relates
to methods for
identifying regulatory elements in a cellulose synthase promoter and
transcription factors
that bind to such regulatory elements, and to methods for augmenting
expression of
polynucleotides operably linked to a cellulose synthase promoter.
BACKGROUND OF THE INVENTION
Lignin and cellulose are the two major building blocks of plant cell walls
that provide mechanical strength and rigidity. In plants, and especially in
trees, these two
organic materials exist in a dynamic equilibrium conferring mechanical
strength, water
transporting ability and protection from biotic and abiotic environmental
stresses.
Normally, oven-dry wood contains 30 to 50% cellulose, 20 to 30% lignin and 20
to 30%
hemicellulose (Higuchi, 1997).
Proportions of lignin and cellulose are known to change with variation in
the natural environment. For example, during the development of compression
wood in
conifers, the percentage of lignin increases from 30 to 40 %, and cellulose
content
proportionally decreases from 40 to 30% (Timmell, 1986). Conversely, in
angiosperm
tension wood the percentage of cellulose increases from 30 to 40%, while
lignin content
decreases from 30 to 20% (Timmell, 1986).
It was recently discovered that the genetic down-regulation of a key tissue-
specific enzyme from the lignin biosynthesis pathway, 4CL, results in
reduction of lignin
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content by up to 45% in transgenic aspen trees (Hu et al., 1999). This down-
regulation is
also associated with a 15% increase in the cellulose content. If the converse
were true, i.e.,
that increasing cellulose content by genetic up-regulation of cellulose
biosynthesis results
in reduction of lignin content, then the pulp yield could be increased. This
would allow
tremendous savings in chemical and energy costs during pulping because, for
example,
lignin must be degraded and removed during the pulping process.
Cellulose is a linear glucan consisting of ~i-D-1,4-linked glucose residues.
It is formed by a cellulose synthase enzyme which catalyzes assembly of UDP-
glucose
units in plasma membrane complexes known as "particle rosettes" (Delmer and
Amor,
1995). Cellulose synthase is thought to be anchored to the membrane by eight
transmembrane binding domains to form the basis of the cellulose biosynthesis
machinery
in the plant cell wall (Pear et al., 1996).
In higher plants, the glucan chains in cellulose microfibrils of primary and
secondary cell walls are different in their degree of polymerization (Brown et
al., 1996).
For example, secondary cell walls are known to contain cellulose having a high
degree of
polymerization, while in primary cell walls the degree of polymerization is
lower. In
another example, woody cell walls suffering from tension stress produce
tension wood on
the upper side of a bent angiosperm tree in response to the stress. In these
cells, there are
elevated quantities of cellulose which have very high crystallinity. The
formation of
highly crystalline cellulose is important to obtain a higher tensile strength
of the wood
fiber. Woody cell walls located at the under side of the same stem experience
a
compression stress, but do not produce highly crystalline cellulose. Such
variation in the
degree of polymerization in cell walls during development is believed to be
due to
different types of cellulose synthases for organizing glucose units into
different
paracrystalline arrays (Haigler and Blanton, 1996). Therefore, it would be
advantageous
to determine the molecular basis for the synthesis of highly crystalline
cellulose so that
higher yields of wood pulp having superior strength properties can be obtained
from
transgenic trees. Production of highly crystalline cellulose in transgenic
trees would also
markedly improve the mechanical strength properties of juvenile wood formed in
normal
trees. This would be a great benefit to the industry because juvenile wood is
generally
undesirable for solid wood applications because it has inferior mechanical
properties.
Since the deposition of cellulose and lignin in trees is regulated in a
compensatory fashion, genetic augmentation of cellulose biosynthesis might
have a
repressive effect on lignin deposition. Since the degree of polymerization and
crystallinity
may depend upon the type of cellulose synthase incorporated in the cellulose
biosynthesis
machinery, the expression of heterologous cellulose synthase or a UDP-glucose
binding
region thereof (e.g., sweetgum protein expression in loblolly pine), could
increase the
quality of cellulose in transgenic plants. Over-expression of a heterologous
cellulose
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synthase may also increase cellulose quantity in transgenic plants. Thus,
genetic
engineering of cellulose biosynthesis can provide a strategy to augment
cellulose quality
and quantity, while reducing lignin content in transgenic plants.
A better understanding of the biochemical processes that lead to wood
formation would enable the pulp and paper industries to more effectively use
genetic
engineering as a tool to meet the increasing demands for wood from a
decreasing
production area. With this objective, many xylem-specific genes, including
most lignin
biosynthesis genes, have been isolated from developing xylem tissues of
various plants
including tree species (Ye and Varner, 1993; Fukuda, 1996; Whetten et al.,
1998). Genes
regulating cellulose biosynthesis in crop plants (Pear et al., 1996 and Arioli
et al., 1998),
versus in trees, have also been isolated. However, isolation of tree genes
which are
directly involved in cellulose biosynthesis has remained a great challenge.
For more than 30 years, no gene encoding higher plant cellulose synthase
(CeIA) was identified. Recently, Pear et al. (1996) isolated the first
putative higher plant
CeIA cDNA, GhCeIA (GenBank No. GHU58283), by searching for UDP-glucose binding
sequences in a cDNA library prepared from cotton fibers having active
secondary wall
cellulose synthesis. GhCeIA was considered to encode a cellulose synthase
catalytic
subunit because it is highly expressed in cotton fibers, actively synthesizes
secondary wall
cellulose, contains eight transmembrane domains, binds UDP-glucose, and
contains two
other domains unique to plants.
Recently, Arioli et al. (1998) cloned a CeIA homolog, RSWI (radial
swelling) (GenBank No. AF027172), from Arabidopsis by chromosome walking to a
defective locus of a temperature sensitive cellulose-deficient mutant.
Complementation of
the rswl mutant with a wild type full-length genomic RSWI clone restored the
normal
phenotype. This complementation provided the first genetic proof that a plant
CeIA gene
encodes a catalytic subunit of cellulose synthase and functions in the
biosynthesis of
cellulose microfibrils. The full-length Arabidopsis RSWl represents the only
known,
currently available cellulose synthase cDNA available for further elucidating
cellulose
biosynthesis in transgenic systems (Wu et al., 1998).
The discovery of the RSWl gene substantiated the belief that the assembly
of a cellulose synthase into the plasma membrane is required for functional
cellulose
biosynthetic machinery and for manufacturing crystalline cellulose
microfibrils in plant
cell walls. Most significantly, a single CeIA gene, e.g. RSWl, is sufficient
for the
biosynthesis of cellulose microfibrils in plants, e.g. Arabidopsis. Thus, RSWI
is a prime
target for engineering augmented cellulose formation in transgenic plants.
Since many of society's fiber, chemical and energy demands are met
through the industrial-scale production of cellulose from wood, genetic
engineering of the
cellulose biosynthesis machinery in trees could produce higher pulp yields.
This would
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allow greater returns on investment by pulp and paper industries. Therefore,
it would be
advantageous to isolate and characterize genes from trees that are involved in
cellulose
biosynthesis in order to improve the properties of wood.
SUMMARY OF THE INVENTION
The present invention relates to polynucleotides comprising a nucleotide
sequence that encodes a cellulose synthase, regulatory sequences, including a
stress-
inducible promoter, of the cellulose s5mthase, a cellulose synthase protein or
a functional
domain thereof and methods for augmenting cellulose biosynthesis in plants.
Thus, in one aspect, the invention provides a polynucleotide comprising a
sequence that encodes a cellulose synthase, or a polynucleotide fragment
thereof, the
fragment encoding a functional domain of cellulose synthase, such as a UDP-
glucose
binding domain. The invention also provides a cellulose synthase or a
functional domain
or fragment thereof, including a UDP-glucose binding domain and at least one
of eight
transmembrane domains. The invention further provides a cellulose synthase
promoter, or
a functional fragment thereof, which fragment contains one or more mechanical
stress
response elements (MSRE).
In another aspect, the present invention is directed to a method of
improving the quality of wood by altering the quantity of cellulose in plant
cells, and
optionally decreasing the content of lignin ~ in the cell. The invention also
relates to a
method of altering the growth or the cellulose content of a plant by
expressing an
exogenous polynucleotide encoding a cellulose synthase or a UDP-glucose
binding
domain thereof in the plant. The invention further provides a method for
causing a stress
induced gene expression in a plant cell by expressing a polynucleotide of
choice using a
stress-inducible cellulose synthase promoter.
In yet another aspect, the invention relates to a method for determining a
mechanical stress responsive element (MSRE) in a cellulose synthase promoters
and a
method for identifying transcription factors that binds to the MSRE.
In a further aspect, the invention provides a method for altering (increasing
or decreasing) i.e., regulating, the expression of a cellulose synthase in a
plant by
expressing an exogenous polynucleotide encoding a transcription factor having
the
property of binding a positive MSRE of a cellulose synthase promoter or by
expressing an
antisense polynucleotide encoding a transcription factor having the property
of binding a
negative MSRE to block the expression of the transcription factor.
Other aspects of the invention will be appreciated by a consideration of the
detailed description of the invention drawings and appended claims.
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DESCRIPTION OF THE DRAWINGS
Fig. 1 represents a nucleic acid sequence encoding a cellulose synthase
from Populus trenauloides [SEQ ID NO: 1] and the protein sequence thereof [SEQ
ID
NO: 2].
Fig. 2 a-c (collectively referred to as Fig. 2) represent a Southern blot
analysis of aspen genomic DNA probed with a fragment of the aspen cDNA
represented in
Fig. 1 under low (panel a) and high stringency conditions (panel b), and a
northern blot
analysis of the total aspen RNA from stem internodes using the same probe
(panel c).
Fig. 3 a-d (collectively referred to as Fig. 3) represent in situ localization
of
the cellulose synthase gene transcripts as shown in the transverse sections
from second
(panel a), fourth (panel b), sixth (panel c) and fifth (panel d) internode.
Fig. 4 represents a nucleic acid sequence of the 5' region of aspen cellulose
synthase gene including the promoter region and the 5' portion of the coding
sequence
[SEQ ID NO: 3].
Fig. 5 a-f (collectively referred to as Fig. 5) represents a histochemical
analysis (panels a-d and f) and fluorescence microscopy (panel e) of
transgenic tobacco for
GUS gene expression driven by a cellulose synthase promoter of the invention.
Fig. 6 a-d (collectively referred to as Fig. 6) represents a histochemical
analysis of GUS gene expression driven by aspen cellulose synthase promoter of
the
invention; tangential and longitudinal sections were harvested before bending
(panel a),
and 4 (panel b), 20 (panel c) and 40 (panel d) hours after bending and stained
for GUS
expression.
Fig. 7 represents a cDNA encoding cellulose synthase isolated from
Arabidopsis [SEQ ID N0:4].
Fig. 8 represents an Arabidopsis cellulose synthase [SEQ ID NO:S]
encoded by the cDNA represented in Fig. 7.
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications and references cited in this specification
are hereby
incorporated herein by reference in their entirety. In case of any
inconsistency, the present
disclosure governs.
Definitions
The terms used in this specification generally have their ordinary meanings
in the art, within the context of the invention, and in the specific context
where each term
is used. Certain terms are discussed below, or elsewhere in the specification,
to provide
additional guidance to the person of skill in the art in describing the
compositions and
methods of the invention and how to make and use them. It will be appreciated
that the
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same thing can be said in more than one way. Consequently, alternative
language and
synonyms may be used for any one or more of the terms discussed herein, nor is
any
special significance to be placed upon whether or not a term is elaborated or
discussed
herein. Synonyms for certain terms are provided. A recital of one or more
synonyms does
not exclude the use of other synonyms. The use of examples anywhere in this
specification, including examples of any terms discussed herein, is
illustrative only, and in
no way limits the scope and meaning of the invention or of any exemplified
term.
Likewise, the invention is not limited to the preferred embodiments.
The term "plant" includes whole plants and portions of plants, including
plant organs (e.g. roots, stems, leaves, etc.).
The term "angiosperm" refers to plants which produce seeds encased in an
ovary. A specific example of an angiosperm is Liquidambar styraciflaca
(L.)[sweetgum].
The term "gymnosperm" refers to plants which produce naked seeds, that
is, seeds which are not encased in an ovary. Specific examples of a gymnosperm
include
Pinus taeda (L.)[loblolly pine].
The term "polynucleotide" or "nucleic acid molecule" is intended to
include double or single stranded genomic and cDNA, RNA, any synthetic and
genetically
manipulated polynucleotide, and both sense and anti-sense strands together or
individually
(although only sense or anti-sense stand may be represented herein). This
includes single-
and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as
well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino
acid
backbone. This also includes nucleic acids containing modified bases, for
example thio-
uracil, thio-guanine and fluoro-uracil.
An "isolated" nucleic acid molecule or polynucleotide refers to a
component that is removed from its original environment (for example, its
natural
environment if it is naturally occurnng). An isolated nucleic acid or
polypeptide may
contains less than about 50%, preferably less than about 75%, and most
preferably less
than about 90%, of the cellular components with which it was originally
associated. A
polynucleotide amplified using PCR so that it is sufficiently and easily
distinguishable (on
a gel, for example) from the rest of the cellular components is considered
"isolated". The
polynucleotides and polypeptides of the invention may be "substantially pure,"
i.e., having
the highest degree of purity that can be achieved using purification
techniques known in
the art.
The term "hybridization" refers to a process in which a strand of nucleic
acid joins with a complementary strand through base pairing. Polynucleotides
are
"hybridizable" to each other when at least one strand of one polynucleotide
can anneal to a
strand of another polynucleotide under defined stringency conditions.
Hybridization
requires that the two polynucleotides contain substantially complementary
sequences;
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depending on the stringency of hybridization, however, mismatches may be
tolerated.
Typically, hybridization of two sequences at high stringency (such as, for
example, in an
aqueous solution of O.SX SSC at 65°C) requires that the sequences
exhibit some high
degree of complementarily over their entire sequence. Conditions of
intermediate
stringency (such as, for example, an aqueous solution of 2X SSC at
65°C) and low
stringency (such as, for example, an aqueous solution of 2X SSC at
55°C), require
correspondingly less overall complementarily between the hybridizing
sequences. (1X
SSC is 0.15 M NaCI, 0.015 M Na citrate.) As used herein, the above solutions
and
temperatures refer to the probe-washing stage of the hybridization procedure.
The term "a
polynucleotide that hybridizes under stringent (low, intermediate) conditions"
is intended
to encompass both single and double-stranded polynucleotides although only one
strand
will hybridize to the complementary strand of another polynucleotide.
A "sequence-conservative variant" is a polynucleotide that contains a
change of one or more nucleotides in a given codon position, as compared with
another
polynucleotide, but the change does not result in any alteration in the amino
acid encoded
at that position.
A "function-conservative variant" is a polypeptide (or a polynucleotide
encoding the polypeptide) having a given amino acid residue that has been
changed
without altering the overall conformation and function of the polypeptide,
including, but
not limited to, replacement of an amino acid with one having similar physico-
chemical
properties (such as, for example, acidic, basic, hydrophobic, and the like).
Amino acids
with have similar physico-chemical properties are well known in the art. For
example,
arginine, histidine and lysine are hydrophilic-basic amino acids and may be
interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be
replaced with
leucine, methionine or valine. Sequence- and function-conservative variants
are discussed
in greater detail below with respect to degeneracy of the genetic code.
A "functional domain" or a "functional fragment" refers to any region or
portion of a protein or polypeptide or polynucleotide which is a region or
portion of a
larger protein or polynucleotide, the region or portion having the specific
activity or
specific function attributable to the larger protein or polynucleotide, e.g.,
a functional
domain of cellulose synthase is the UDP-glucose binding domain.
The term "% identity" refers to the percentage of the nucleotides/amino
acids of one polynucleotide/polypeptide that are identical to the
nucleotides/amino acids of
another sequence of polynucleotide/polypeptide as identified by program GAP
from
Genetics Computer Group Wisconsin (GCG) package (version 9.0) (Madison, WI).
GAP
uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970)
to find the
alignment of two complete sequences that maximizes the number of matches and
minimizes the number of gaps. When parameters required to run the above
algorithm are
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not specified, the default values offered by the program are contemplated. The
following
parameters are used by the GCG program GAP as default values (for
polynucleotides): gap
creation penalty:50; gap extension penalty:3; scoring matrix: nwsgapdna.cpm
(local data
file).
The "% similarity" or "~/o homology" between two polypeptide sequences is
a function of the number of similar positions shared by two sequences on the
basis of the
scoring matrix used divided by the number of positions compared and then
multiplied by
100. This comparison is made when two sequences are aligned (by introducing
gaps if
needed) to determine maximum homology. PowerBlast program, implemented by the
National Center for Biotechnology Information, can be used to compute optimal,
gapped
alignments. GAP program from Genetics Computer Group Wisconsin package
(version
9.0) (Madison, WI) can also be used. GAP uses the algorithm of Needleman and
Wunsch
(J Mol Biol 48: 443-453, 1970) to find the alignment of two complete sequences
that
maximizes the number of matches and minimizes the number of gaps. When
parameters
required to run the above algorithm are not specified, the default values
offered by the
program are contemplated. The following parameters are used by the GCG program
GAP
as default values (for polypeptides): gap creation penalty:l2; gap extension
penalty:4;
scoring matrix:Blosum62.cpm (local data file).
The term "oligonucleotide" refers to a nucleic acid, generally of at least 10,
preferably at least 15, and more preferably at least 20 nucleotides, that is
hybridizable to a
genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene,
mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be
labeled, e.g.,
with 32P-nucleotides or nucleotides to which a label, such as biotin, has been
covalently
conjugated. In one embodiment, a labeled oligonucleotide can be used as a
probe to detect
the presence of a nucleic acid. In another embodiment, oligonucleotides (one
or both of
which may be labeled) can be used as PCR primers, either for cloning full
length or a
fragment of CeIA, or to detect the presence of nucleic acids encoding CeIA. In
a further
embodiment, an oligonucleotide of the invention can form a triple helix with a
CeIA DNA
molecule. In still another embodiment, a library of oligonucleotides arranged
on a solid
support, such as a silicon wafer or chip, can be used to detect various
polymorphisms of
interest. Generally, oligonucleotides are prepared synthetically, preferably
on a nucleic
acid synthesizer. Accordingly, oligonucleotides can be prepared with non-
naturally
occurnng phosphoester analog bonds, such as thioester bonds, etc.
The term "coding sequence" refers to that portion of the gene that contains
the information for encoding a polypeptide. The boundaries of the coding
sequence are
determined by a start codon at the 5' (amino) terminus and a translation stop
codon at the
3' (carboxyl) terminus. A coding sequence can include, but is not limited to,
prokaryotic
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sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic
(e.g., mammalian) DNA, and even synthetic DNA sequences.
A "promoter" is a polynucleotide containing elements (e.g., a TATA box)
which are capable of binding RNA polymerise in a cell and initiating
transcription of a
downstream (3' direction) coding sequence. For purposes of defining the
present
invention, the promoter sequence is bounded at its 3' terminus by the
transcription
initiation site and extends upstream (5' direction) to include the minimum
number of bases
or elements necessary to initiate transcription at levels detectable above
background.
Within the promoter sequence will be found a transcription initiation site
(conveniently
defined for example, by mapping with nuclease S1), as well as protein binding
domains
(consensus sequences) responsible for the binding of RNA polymerise. Examples
of
promoters that can be used in the present invention include PtCeIAP, 4CL-1 and
35S.
The term "constitutive promoter" refers to a promoter which typically, does
not require positive regulatory proteins to activate expression of an
associated coding
sequence, i.e., a constitutive promoter maintains some basal level of
expression. A
constitutive promoter is commonly used in creation of an expression cassette.
An example
of a constitutive promoter are 35S CaMV (Cauliflower Mosaic Virus), available
from
Clonetech, Palo Alto, CA.
The term "inducible promoter" refers to the promoter which requires a
positive regulation to activate expression of an associated coding sequence.
An example
of such a promoter is a stress-inducible cellulose synthase promoter from
aspen described
herein.
A coding sequence is "under the control" of transcriptional and translational
control sequences in a cell when RNA polymerise transcribes the coding
sequence into
mRNA, which is then trans-RNA spliced and translated into the protein encoded
by the
coding sequence.
A "vector" is a recombinant nucleic acid construct, such as plasmid, phage
genome, virus genome, cosmid, or artificial chromosome to which a
polynucleotide of the
invention may be attached. In a specific embodiment, the vector may bring
about the
replication of the attached segment, e.g., in the case of a cloning vector.
The term "expression cassette" refers to a polynucleotide which contains
both a promoter and a protein coding sequence such that expression of a given
protein is
achieved upon insertion of the expression cassette into a cell.
A cell has been "transfected" by exogenous or heterologous polynucleotide
when such polynucleotide has been introduced inside the cell. A cell has been
"transformed" by exogenous or heterologous polynucleotide when the transfected
polynucleotide effects a phenotypic change. Preferably, the transforming
polynucleotide
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should be integrated (covalently linked) into chromosomal DNA making up the
genome of
the cell.
"Exogenous" refers to biological material, such as a polynucleotide or
protein, that has been isolated from a cell and is then introduced into the
same or a
different cell. For example, a polynucleotide encoding a cellulose synthase of
the
invention can be cloned from xylem cells of a particular species of tree,
inserted into a
plasmid and reintroduced into xylem cells of the same or different species.
The species
thus contains an exogenous cellulose synthase polynucleotide.
"Heterologous polynucleotide" refers to an exogenous polynucleotide not
naturally occurnng in the cell into which it is introduced.
"Homologous polynucleotide" refers to an exogenous polynucleotide that
naturally exists in the cells into which it is introduced.
The present invention relates to isolation and characterization of
polynucleotides encoding cellulose synthases from plants, especially trees,
including full
length or naturally occurring forms of cellulose synthases, functional
domains, promoters
and regulatory elements. Therefore, in accordance with the present invention
there may be
employed conventional molecular biology, microbiology, and recombinant DNA
techniques within the skill of the art. Such techniques are explained fully in
the literature.
See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual,
Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New
York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach,
Volumes I
and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984);
Nucleic
Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Tra~zscription And
Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Arcinzal Cell Culture
[R.I.
Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B.
Perbal, A
Practical Guide To Molecular Clorzifzg (1984); F.M. Ausubel et al. (eds.),
Curre~zt
Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
The present invention relates to a novel, full-length cellulose synthase gene
(CeIA), a novel stress inducible promoter of cellulose synthases (CeIAP), and
cellulose
synthase proteins from trees, including UDP-glucose catalytic domains thereof.
The
invention enables the development of transgenic tree varieties having
increased cellulose
content, decreased lignin content and, therefore, improved wood fiber
characteristics.
Production of increased cellulose quantity and quality in multiple varieties
of
commercially relevant, transgenic forest tree species in operational
production scenarios
are further contemplated. The invention further provides a new experimental
system for
study of CeIA gene expression and function in trees.
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Polynucleotides encoding cellulose synthase and fragments thereof
The present invention relates to polynucleotides which comprise the
nucleotide sequence that encodes cellulose synthase of the invention or a
functional
fragment thereof. In a preferred embodiment, the polynucleotide comprises the
sequence
encoding a tree cellulose synthase and most preferrably, the sequence encoding
a cellulose
synthase from aspen. In one embodiment, a polynucleotide of the invention
includes the
entire cellulose synthase coding region, e.g., nucleotides 69 to 3,005 of SEQ
>D NO: 1. In
another aspect of the invention, the polynucleotide encoding an Arabidopsis
cellulose
synthase is provided (see SEQ B7 N0:4 and the translated protein of SEQ ID
NO:S).
Also within the scope of the invention are fragments of the polynucleotides
encoding cellulose synthase of the invention, which fragments encode at least
one
transmembrane domain and/or a LAP-glucose binding domain. For example, a
polynucleotide comprising the nucleotides encoding a UDP-glucose binding
domain of
aspen cellulose synthase (e.g., nucleotides 660 to 2250 of SEQ ID NO:1) or
corresponding
nucleotides of SEQ 117 N0:4 are within the scope of the invention. The
nucleotides
encoding the UDP-glucose binding domain can be determined by, for example,
alignment
of protein sequences as described below.
The invention further relates to sequence conservative variants of the
coding portion of SEQ ID NOS: 1 and 4.
Polynucleotides that hybridize under conditions of low, medium, and high
stringency to SEQ >D NOS: 1 and 4, and their respective coding portions are
also within
the scope of the invention. Preferably, the polynucleotide that hybridizes to
any of SEQ
ID NOS: 1 and 4, or their respective coding portions, is about the same length
as that
sequence, for example, not more than about 10 to about 20 nucleotides longer
or shorter.
In another embodiment of the invention, the hybridizable polynucleotide is at
least 1500
nucleotides long, preferably at least 2500 nucleotides long and most
preferably at least
3000 nucleotides long. In yet another embodiment, the hybridizable
polynucleotide
comprises the UDP-glucose binding domain as found in SEQ ID NO:1 or 4, or at
least the
conserved region QVLRW. Most preferably, the hybridizable polynucleotide has a
UDP
glucose binding activity.
The polynucleotides that occur originally in nature may be isolated from the
organisms that contain them using methods described herein or well known in
the art. The
non-naturally occurnng polynucleotides may be prepared using various
manipulations
known in the field of recombinant DNA. For example, the cloned CeIA
polynucleotide
can be modified according to methods described by Sambrook et al., 1989. The
sequence
can be cleaved at appropriate sites with restriction endonuclease(s), followed
by further
enzymatic modification if desired, isolated, and ligated i~2 vitro. In the
production of the
modified polynucleotides, for example, care should be taken to ensure that the
modified
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polynucleotide remains within the appropriate translational reading frame (if
to be
expressed) or uninterrupted by translational stop signals. As a further
example, a CeIA-
encoding nucleic acid sequence can be mutated in vitro or in vivo, to create
and/or destroy
translation, initiation, and/or termination sequences, or to create variations
in coding
regions and/or form new restriction endonuclease sites or destroy preexisting
ones, to
facilitate further irz vitro modification. Preferably, such mutations enhance
the functional
activity of the mutated CeIA polynucleotide. Any technique for mutagenesis
known in the
art can be used, including but not limited to, in vitro site-directed
mutagenesis
(Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith,
1984, DNA
3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc.
Natl. Acad.
Sci. U.S.A. 83:710), use of TAB linkers (Pharmacia), etc. PCR techniques are
preferred
for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA",
in PCR
Technology: Principles and Applicatiorzs for DNA Amplification, H. Erlich,
ed., Stockton
Press, Chapter 6, pp. 61-70).
The polynucleotides of the present invention may be introduced into
various vectors adapted for plant or non-plant replication. These are well
known in the art,
thus, choice; construction and use of such vectors is well within the skill of
a person
skilled in the art. Possible vectors include, but are not limited to, plasmids
or modified
viruses of plants, but the vector system must be compatible with the host cell
used. An
example of a suitable vector is Ti plasmid. The insertion into a cloning
vector can, for
example, be accomplished by ligating the DNA fragment into a cloning vector
which has
complementary cohesive termini. However, if the complementary restriction
sites used to
fragment the DNA are not present in the cloning vector, the ends of the DNA
molecules
may be enzymatically modified. Alternatively, any site desired may be produced
by
ligating nucleotide sequences (linkers) onto the DNA termini; these ligated
linkers may
comprise specific chemically synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. An expression cassette containing
cellulose synthase
or recombinant molecules thereof can be introduced into host cells via silicon
carbide
whiskers, transformed protoplasts, transformation, e.g., Agrobacteriurn
vectors (discussed
below), electroporation, infection, etc., so that many copies of the gene
sequence are
generated. Preferably, the cloned gene is contained on a shuttle vector
plasmid, which
provides for expansion in a cloning cell, e.g., E. coli, and facile
purification for subsequent
insertion into an appropriate expression cell line, if such is desired. For
example, a shuttle
vector, which is a vector that can replicate in more than one type of
organism, can be
prepared for replication in both E. coli and Saccharomyces cerevisiae by
linking sequences
from an E. coli plasmid with sequences form the yeast 2m plasmid.
Transgenic plants containing the polynucleotides described herein are also
within the scope of the invention. Methods for introducing exogenous
polynucleotides
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into plant cells and regenerating transgenic plants are well known. Some are
provided
below.
In one embodiment, to introduce a plasmid containing a CeIA coding
sequence or promoter of the invention into a plant, a 1:1 mixture of plasmid
DNA
S containing a selectable marker expression cassette and plasmid DNA
containing a
cellulose synthase expression cassette is precipitated with gold to form
microprojectiles.
The microprojectiles are rinsed in absolute ethanol and aliquots are dried
onto a suitable
macrocarrier such as the macrocarrier available from BioRad in Hercules, CA.
Prior to
bombardment, embryogenic tissue is preferably desiccated under a sterile
laminar-flow
hood. The desiccated tissue is transferred to semi-solid proliferation medium.
The
prepared microprojectiles are accelerated from the macrocarner into the
desiccated target
cells using a suitable apparatus such as a BioRad PDS-1000/I~ particle gun. In
a
preferred method, each plate is bombarded once, rotated 180 degrees, and
bombarded a
second time. Preferred bombardment parameters are 1350 psi rupture disc
pressure, 6 mm
distance from the rupture disc to macrocarrier (gap distance), 1 cm
macrocarrier travel
distance, and 10 cm distance from macrocarrier stopping screen to culture
plate
(microcarrier travel distance). Tissue is then transferred to semi-solid
proliferation
medium containing a selection agent, such as hygromycin B, for two days after
bombardment.
Cellulose synthase protein and fragment thereof
A cellulose synthase of the invention is a plant protein that contains a
catalytic subunit which has UDP-glucose binding activity for the synthesis of
glucan from
glucose, and eight transmembrane domains for localizing the cellulose synthase
to the cell
membrane. The cellulose synthase of the invention has eight transmembrane
binding
domains; two at the amino terminal and six at the carboxyl terminal. The UDP-
glucose
binding domain is located between transmembrane domains two and three.
Examples of
this protein structure are seen in the aspen cellulose synthase as well as in
those of RSWI
and GhCeIA. The location of the transmembrane domain may be identified as
described
below and as exemplified in the Example. Preferably, the cellulose synthase of
the
invention has an amino acid sequence of a tree cellulose synthase.
In one embodiment, the cellulose synthase protein of the invention is
isolated from aspen. Aspen cellulose synthase contains about 978 amino acids
and has a
molecular weight of about 110 KDa and a pI of about 6.58. In one embodiment,
the aspen
cellulose synthase has the amino acid sequence of SEQ >D N0:2 as represented
in Fig. 1.
In another aspect, the invention relates to cellulose synthase of SEQ ID NO:
5.
The invention further relates to fragments of plant cellulose synthases, such
as fragments containing at least one transmembrane region and/or a UDP-glucose
binding
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domain. The transmembrane regions may be identified as described in the
Example by
using the method of Hoffman and Stoffel (1993).
The cellulose synthase fragment containing the UDP-glucose binding
domain is functional without the presence of the rest of the protein. This
separable activity
is as shown in the Example. This result was surprising and unexpected because
previously
identified UDP-glucose binding domains were not known to be functional when
isolated
from other portions of the protein. Thus, a fragment of any cellulose synthase
(such as
PtCeIA, RSWI, GhCeIA and SEQ )D NO:S) that contains a UDP-glucose binding
domain
and is independently functional is within the scope of the invention. The
function of the
UDP-glucose binding domain may be determined using the assay described in the
Example. The UDP-glucose binding domain of the invention is located between
the
second and third transmembrane region of the cellulose synthase and has
conserved amino
acid sequences for UDP-glucose binding, such as the sequence QVLRW and
conserved D
residues. The UDP-glucose binding domain and the conserved regions therein may
be
located in a cellulose synthase using the guidance of the present
specification and the
general knowledge in the art, for example Brown, 1996. In one embodiment, the
UDP-
glucose binding domain and the conserved regions therein may be identified by
comparing
the amino acid sequence of cellulose synthase of interest with the amino acid
sequence of
aspen cellulose synthase using the algorithms described in the specification
or generally
known in the art. For example, the UDP-glucose binding domain of SEQ ID N0:2
is in
the position amino acids 220 to 749. The conserved QVLRW sequence is located
at
positions 715-719 of SEQ ID N0:2.
Polypeptides having at least 75%, preferably at least 85% and most
preferably at least 95% similarity to the amino acid sequence of SEQ >D NO: 2,
amino
acids 220-749 of SEQ 1D N0:2, SEQ )D NO:S or its UDP-glucose binding domain
using
Power Blast or GAP algorithm described above. In a preferred embodiment, these
polypeptides are of about the same length as the polypeptide of SEQ ID NO: 2
or amino
acids 220-749 of SEQ ID N0:2. For example, the polypeptide may be from about 2-
3 to
about 5-7 and to about 10-15 amino acids longer or shorter. In another
embodiment, the
polypeptides described in this paragraph are not originally found (i.e.,
naturally occurring)
in Arabidopsis or cotton. These polypeptides may be prepared by, for example,
altering
the nucleic acid sequence of a cloned polynucleotide encoding the protein of
SEQ ID
NO:2 or SEQ >D NO:S using the methods well known in the art.
Function conservative variants of cellulose synthase are also within the
scope of the invention and can be prepared by altering the sequence of a
cloned
polynucleotide encoding cellulose synthase or fragments thereof. Conventional
methods
used in the art can be used to make substitutions, additions or deletions in
one or more
amino acids, to provide functionally equivalent molecules. For example, a
function
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conservative variant that has substitutions, deletions and/or additions in the
amino and/or
carboxyl terminus of the protein, outside of the UDP-glucose binding domain is
within the
scope of the invention. Preferably, variants are made that have enhanced or
increased
functional activity relative to native cellulose synthase. Methods of directed
evolution can
be used for this purpose.
The invention also includes function conservative variants which include
altered sequences in which functionally equivalent amino acid residues are
substituted for
residues within the sequence resulting in a conservative amino acid
substitution. For
example, one or more amino acid residues within the sequence can be
substituted by
another amino acid of a similar polarity, which acts as a functional
equivalent, resulting in
a silent alteration. Substitutes for an amino acid within the sequence may be
selected from
other members of the class to which the amino acid belongs. For example, the
nonpolar
(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,
proline,
phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring
structures are phenylalanine, tryptophan, and tyrosine. The polar neutral
amino acids
include glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. The
positively charged (basic) amino acids include arginine, lysine and histidine.
The
negatively charged (acidic) amino acids include aspartic acid and glutamic
acid. Such
alterations will not be expected to affect apparent molecular weight as
determined by
polyacrylamide gel electrophoresis, or isoelectric point. Particularly
preferred
substitutions are: (i) Lys for Arg and vice versa such that a positive charge
may be
maintained; (ii) Glu for Asp and vice versa such that a negative charge may be
maintained;
(iii) Ser for Thr such that a free -OH can be maintained; and (iv) Gln for Asn
such that a
free CONHZ can be maintained. Amino acid substitutions may also be introduced
to
substitute an amino acid with a particularly preferable property. For example,
a Cys may
be introduced a potential site for disulfide bridges with another Cys. A His
may be
introduced as a particularly "catalytic" site (i.e., His can act as an acid or
base and is the
most common amino acid in biochemical catalysis). Pro may be introduced
because of its
particularly planar structure, which induces b-turns in the protein's
structure.
The cellulose synthase of the invention can be isolated by expressing a
cloned polynucleotide encoding the cellulose synthase as well as using direct
protein
purification techniques. These methods will be apparent to those of skill in
the art.
Polmucleotides containing cellulose synthase promoter
The present invention further relates to a cellulose synthase promoter. The
promoter is a stress-inducible promoter and may be used to synthesize greater
quantities of
high crystalline cellulose in plant, and preferably in trees. This permits an
increase in the
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proportion of cellulose in transgenic plants, greater strength of juvenile
wood and fiber,
and acceleration of overall growth rate.
In one embodiment, the promoter of the invention is from aspen and is
represented in Figure 4. The promoter sequence is located within the region of
nucleotides
1-840 of SEQ >D N0:3. A person of skill in the art will appreciate that not
the entire
sequence is required for the promoter function and can easily identify the
critical regions
by looking for conserves boxes and doing routine deletion analysis. Thus,
functional
fragments of SEQ ID NO:1 are within the scope of the invention.
Polynucleotides that hybridize under conditions of low, medium, and high
stringency to SEQ ID N0:3, and its non-coding portion are also within the
scope of the
invention. The hybridizable polynucleotide may be about the same length as the
sequence
to which it hybridizes, for example, not more than about 10 to about 20
nucleotides longer
or shorter. In another embodiment, the hybridizable polynucleotide is at least
about 200
nucleotides long, preferably at least about 400 nucleotides long and most
preferably at
least 500 nucleotides long. In yet another embodiment, the hybridizable
polynucleotide
comprises at least one MSRE element identified according to the method
described below.
A cellulose synthase promoter of the invention typically provides tissue
specific gene regulation in xylem, but also permits up-regulation of gene
expression in
other tissues as well, e.g., phloem under tension stress. Furthermore,
expression of
cellulose synthase is localized to an area of the plant under stress.
This stress-inducible phenomenon is regulated by positive and negative
mechanical stress response elements (MSREs). These MSREs upregulate (positive)
or
downregulate (negative) the expression of a cellulose synthase polynucleotide
under stress
conditions through binding of transcription factors. MSRE-regulated expression
of
cellulose synthase permits synthesis of cellulose with high crystallinity.
The MSREs of cellulose synthase can be modified or employed otherwise
in methods to regulate expression of a polynucleotide, including a cellulose
synthase,
operatively linked to a promoter containing an MSRE in response to mechanical
stress
(e.g., tension or compression) to a transgenic plant.
Negative MSREs of a cellulose synthase promoter can be modified,
removed or blocked to improve expression of a cellulose synthase, and thereby
increase
cellulose production and improve wood quality. Alternatively, positive MSREs
can be
removed or blocked to decrease expression of a cellulose synthase, which
decreases
cellulose production and increases lignin deposition. This is useful for
increasing the fuel
value of wood because lignin has a higher BTU value than cellulose. Moreover,
a
modified cellulose synthase promoter can be operatively linked to a
polynucleotide of
interest to control its expression upon mechanical stress to a plant harboring
it.
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The location of MSRE elements in the SEQ ID N0:3 may be identified, for
example, using promoter deletion analysis, DNAse Foot Print Analysis, and
Southwestern
screening of an expression library for an MSRE. In one embodiment, cellulose
synthase
promoter that has one or more portions deleted, and is operatively linked to a
reporter
sequence, is introduced into a plant or a plant cell. A positive MSRE is
detected by
observing no relative change or increase in the amount of reporter in a
transgenic plant or
tissue, e.g., phloem after inducing a stress to the plant, and a negative MSRE
is detected by
observing increases in the amount of reporter in the plant in the absence of
any stress to
the plant. A positive element is detected when by removing it, GUS expression
goes down
and by adding it kept at the same level or more. The negative element does not
support, or
suppreses, expression of GUS and by removing it, normal or enhanced GUS
expression is
observed as compared to when negative element is present.
Manipulation of a MSRE binding sites and/or providing transcription
factors that bind thereto, provides a mechanism to continuously produce high
crystalline
cellulose in woody plant cell walls of transgenic plants. For example, one
having ordinary
skill in the art can delete or block negative MSRE elements, or provide cDNA
encoding
proteins) that bind the positive MSREs, to enable constitutive expression of a
cellulose
synthase without the requirement of a mechanical stress. The increased
cellulose synthase,
and therefore, increased cellulose content, can improve the strength
properties of juvenile
wood and fiber. It is also contemplated that the positive MSREs can be deleted
or
blocked, or cDNA in an antisense direction, which in the sense direction
encodes a protein
that binds a positive MSRE, can be provided, to reduce cellulose synthase
activity and
decrease cellulose production.
Method of Isolating Polynucleotides Encoding Cellulose Synthase
The invention further relates to identifying and isolating polynucleotides
encoding cellulose synthase in plants, e.g., trees, (in addition to those
polynucleotides
provided in the Example and represented in Fig. 1 and Fig. 7). These
polynucleotides may
be used to manipulate expression of cellulose synthase with an objective to
improve the
cellulose content and properties of wood.
The method comprises identifying a nucleic acid fragment containing a
sequence encoding cellulose synthase or a portion thereof by using a fragment
of SEQ ID
NOS:l or 4 as a probe or a primer. Once identified, the nucleic acid fragment
containing a
sequence encoding cellulose synthase or a portion thereof is isolated.
Polynucleotides encoding cellulose synthases of the invention, whether
genomic DNA, cDNA, or fragments thereof, can be isolated from many sources,
particularly from cDNA or genomic libraries from plants, preferably trees
(e.g. aspen,
sweetgum, loblolly pine, eucalyptus, and other angiosperms and gymnosperms).
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PCT/US00/13637
Molecular biology methods for obtaining polynucleotides encoding a cellulose
synthase
are well known in the art, as described above (see, e.g., Sambrook et al.,
1989, supra).
Accordingly, cells from any species of plant can potentially serve as a
nucleic acid source for the molecular cloning of a polynucleotide encoding a
cellulose
S synthase of the invention. The DNA may be obtained by standard procedures
known in
the art from cloned DNA (e.g., a DNA "library"), and preferably is obtained
from a cDNA
library prepared from tissues with high level expression of a cellulose
synthase (e.g.,
xylem tissue, since cells in this tissue evidence very high levels of
expression of CeIA), by
chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or
fragments
thereof, purifred from a desired cell (see, for example, Sambrook et al.,
1989, supra;
Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,
Oxford,
U.K. Vol. I, II). Clones derived from genomic DNA may contain regulatory and
intron
DNA regions in addition to coding regions; clones derived from cDNA will not
contain
intron sequences. Whatever the source, a polynucleotide should be molecularly
cloned
into a suitable vector for its propagation.
In another embodiment for the molecular cloning of a polynucleotide
encoding a cellulose synthase of the invention from genomic DNA, DNA fragments
are
generated from a genome of interest, such as from a plant, or more
particularly a tree
genome, part of which will correspond to a desired polynucleotide. The DNA may
be
cleaved at specific sites using various restriction enzymes. Alternatively,
one may use
DNAse in the presence of manganese to fragment the DNA, or the DNA can be
physically
sheared, as for example, by sonication. The linear DNA fragments can then be
separated
according to size by standard techniques, including but not limited to,
agarose and
polyacrylamide gel electrophoresis and column chromatography.
Once the DNA fragments are generated, identification of the specific DNA
fragment containing a desired CeIA sequence may be accomplished in a number of
ways.
For example, if an amount of a portion of a CeIA sequence or its specific RNA,
or a
fragment thereof, is available and can be purified and labeled, the generated
DNA
fragments may be screened by nucleic acid hybridization to a labeled probe
(Benton and
Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad.
Sci.
U.S.A. 72:3961). For example, a set of oligonucleotides corresponding to the
partial
amino acid sequence information obtained for a CeIA protein from trees can be
prepared
and used as probes for DNA encoding cellulose synthase, or as primers for cDNA
or
mRNA (e.g., in combination with a poly-T primer for RT-PCR). Preferably, a
fragment is
selected that is highly unique to a cellulose synthase of the invention, such
as the UDP-
glucose binding regions. Those DNA fragments with substantial homology to the
probe
will hybridize. As noted above, the greater the degree of homology, the more
stringent
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hybridization conditions can be used. In a specific embodiment, stringency
hybridization
conditions can be used to identify homologous CeIA sequences from trees or
other plants.
Thus, in one embodiment, a labeled cellulose synthase cDNA from, e.g.,
Populus tremuloides (PtCeIA), can be used to probe a library of genes or DNA
fragments
from various species of plants, especially angiosperm and gymnosperm, to
determine
whether any bind to a CeIA of the invention. Once genes or fragments are
identified, they
can be amplified using standard PCR techniques, cloned into a vector, e.g.,
pBluescript
vector (StrataGene of LaJolla, CA), and transformed into a bacteria, e.g.,
DHSa E. coli
strain (Gibco BRL of Gaithersburg, MD). Bacterial colonies are typically
tested to
determine whether any contains a cellulose synthase-encoding nucleic acid.
Once a
positive clone is identified through binding, it is sequenced from an end,
preferably the 3'
end.
cDNA libraries can be constructed in various hosts, such as lambda ZAPII,
available from Stratagene, LaJolla, CA, using poly(A) +RNA isolated from aspen
xylem,
according to the methods described by Bugos et al. (Biotechniques 19:734-737,
1995 ).
The above mentioned probes are used to assay the aspen cDNA library to locate
cDNA
which codes for enzymes involved in production of cellulose synthases. Once a
cellulose
synthase sequence is located, it is then cloned and sequenced according to
known methods
in the art.
Further selection can be carned out on the basis of the properties of the
gene, e.g., if the gene encodes a protein product having the isoelectric,
electrophoretic,
hydropathy plot, amino acid composition, or partial amino acid sequence of a
cellulose
synthase protein of the invention, as described herein. Thus, the presence of
the gene may
be detected by assays based on the physical, chemical, or immunological
properties of its
expressed product. For example, cDNA clones or DNA clones which hybrid-select
the
proper mRNAs can be used to produce a protein that has similar properties
known for
cellulose synthases of the invention. Such properties may include, for
example, similar or
identical electrophoretic migration patterns, isoelectric focusing or non-
equilibrium pH gel
electrophoresis behavior, proteolytic digestion maps, hydropathy plots, or
functional
properties (such as isolated, functional UDP-glucose binding domains).
A cellulose synthase polynucleotide of the invention can also be identified
by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro
translation. In
this procedure, nucleotide fragments are used to isolate complementary mRNAs
by
hybridization. Such DNA fragments may represent available, purified CeIA DNA,
or may
be synthetic oligonucleotides designed from the partial amino acid sequence
information.
Functional assays (e.g., UDP-glucose activity) of the in vitro translation
products of the
products of the isolated mRNAs identifies the mRNA and, therefore, the
complementary
DNA fragments, that contain the desired sequences.
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A radiolabeled CeIA cDNA can be synthesized using a selected mRNA as a
template. The radiolabeled mRNA or cDNA may then be used as a probe to
identify
homologous CeIA DNA fragments from amongst other genomic DNA fragments.
It will be appreciated that other polynucleotides, in addition to a CeIA of
the
invention can be operatively linked to a CeIA promoter to control expression
of the
polynucleotide upon application of a mechanical stress.
Expression of CeIA Polypeptides
The nucleotide sequence coding for CeIA, or a functional fragment,
derivative or analog thereof, including chimeric proteins, can be inserted
into an
appropriate expression vector, i.e., a vector which contains the necessary
elements for the
transcription and translation of the inserted protein-coding sequence.
Preferably, an
expression vector includes an origin of replication. The elements are
collectively termed
herein a "promoter." Thus, a nucleic acid encoding CeIA of the invention can
be
operatively associated with a promoter in an expression vector of the
invention. Both
cDNA and genomic sequences can be cloned and expressed under control of such
regulatory sequences. The necessary transcriptional and translational signals
can be
provided on a recombinant expression vector, or they may be supplied by the
native gene
encoding CeIA and/or its flanking regions.
In addition to a CeIAP, expression of cellulose synthase can be controlled
by any promoter/enhancer element known in the art, but these regulatory
elements must be
functional in the host selected for expression. Promoters which may be used to
control
CeIA polynucleotide expression include, constitutive, development-specific and
tissue-
specific. Examples of these promoters include 35S Cauliflower Mosaic Virus,
terminal
flower and 4CL-1. Thus, there are various ways to alter the growth of a plant
using
different promoters, depending on the needs of the practitioner.
The nucleotide sequence may be inserted in a sense or antisense direction
depending on the needs of the practitioner. For example, if augmentation of
cellulose
biosynthesis is desired then polynucleotides encoding, e.g., cellulose
synthase, can be
inserted into the expression vector in the sense direction to increase
cellulose synthase
production and thus cellulose biosynthesis. Alternatively, if it is desired
that cellulose
biosynthesis is reduced or lignin content is increased, then polynucleotides
encoding, e.g.,
cellulose synthase ,can be inserted in the antisense direction so that upon
transcription the
antisense mRNA hybridizes to other complementary transcripts in the sense
orientation to
prevent translation. In other embodiments, the polynucleotide encodes a UDP-
glucose
binding domain and is used in a similar manner as described.
A recombinant CeIA protein of the invention, or functional fragment,
derivative, chimeric construct, or analog thereof, may be expressed
chromosomally, after
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integration of the coding sequence by recombination. In this regard, any of a
number of
amplification systems for plants may be used to achieve high levels of stable
gene
expression, as discussed above. Any of the methods previously described for
the insertion
of DNA fragments into a cloning vector may be used to construct expression
vectors
containing a gene consisting of appropriate transcriptional/translational
control signals and
the protein coding sequences. These methods may include in vitro recombinant
DNA and
synthetic techniques and in vivo recombination (genetic recombination).
Expression vectors containing a nucleic acid encoding a CeIA of the
invention can be identified by four general approaches: (a) PCR amplification
of the
desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c)
presence or
absence of selection marker gene functions, (d) analyses with appropriate
restriction
endonucleases, and (e) expression of inserted sequences. In the first
approach, the nucleic
acids can be amplified by PCR to provide for detection of the amplified
product. In the
second approach, the presence of a foreign gene inserted in an expression
vector can be
detected by nucleic acid hybridization using probes comprising sequences that
are
homologous to an inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the presence or
absence of
certain "selection marker" gene functions (e.g., ~i-glucuronidase activity,
resistance to
antibiotics, transformation phenotype, etc.) caused by the insertion of
foreign genes in the
vector. In another example, if the nucleic acid encoding CeIA is inserted
within the
"selection marker" gene sequence of the vector, recombinants containing the
CeIA insert
can be identified by the absence of the CeIA gene function. In the fourth
approach,
recombinant expression vectors are identified by digestion with appropriate
restriction
enzymes. In the fifth approach, recombinant expression vectors can be
identified by
assaying for the activity, biochemical, or immunological characteristics of
the gene
product expressed by the recombinant, provided that the expressed protein
assumes a
functionally active conformation.
After a particular recombinant DNA molecule is identified and isolated,
several methods known in the art may be used to propagate it. Once a suitable
host system
and growth conditions are established, recombinant expression vectors can be
propagated
and prepared in quantity. As previously explained, the expression vectors
which can be
used include, but are not limited to those vectors or their derivatives
described above.
Vectors are introduced into the desired host cells by methods known in the
art, e.g., Agrobacteriunz-mediated transformation (described in greater detail
below),
transfection, electroporation, microinjection, transduction, cell fusion, DEAE
dextran,
calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene
gun, or a
DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-
967; Wu and
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Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent
Application
No. 2,012,311, filed March 15, 1990).
The cell into which the recombinant vector comprising the nucleic acid
encoding CeIA is cultured in an appropriate cell culture medium under
conditions that
provide for expression of CeIA by the cell. In addition, a host cell strain
may be chosen
which modulates the expression of the inserted sequences, or modifies and
processes the
gene product in a specific fashion desired. Different host cells have
characteristic and
specific mechanisms for the translational and post-translational processing
and
modification (such as glycosylation, cleavage, e.g., of a signal sequence) of
proteins.
Appropriate cell lines or host systems can be chosen to ensure the desired
modification
and processing of the foreign protein expressed.
AQrobacterium-mediated transformation and inducing somatic embryos
The culture media used in the invention, and for transforming
Agrobacterium, contain an effective amount of each of the medium components
(e.g. basal
medium, growth regulator, carbon source) described above. As used in
describing the
present invention, an "effective amount" of a given medium component is the
amount
necessary to cause a recited effect. For example, an effective amount of a
growth hormone
in the primary callus growth medium is the amount of the growth hormone that
induces
callus formation when combined with other medium components. Other compounds
known to be useful for tissue culture media, such as vitamins and gelling
agents, may also
be used as optional components of the culture media of the invention.
Transformation of cells from plants, e.g., trees, and the subsequent
production of transgenic plants using Agrobacteriunz-mediated transformation
procedures
known in the art, and further described herein, is one example of a method for
introducing
a foreign gene into trees. Transgenic plants may be produced by various
methods, such as
by the following steps: (i) culturing Agrobacterium in low-pH induction medium
at low
temperature and preconditioning, i.e., coculturing bacteria with wounded
tobacco leaf
extract in order to induce a high level of expression of the Agrobacterium vir
genes whose
products are involved in the T-DNA transfer; (ii) coculturing a desired plant
tissue
explants, including zygotic and/or somatic embryo tissues derived from
cultured explants,
with the incited Agrobacterium; (iii) selecting transformed callus tissue on a
medium
containing antibiotics; and (v) and converting the embryos into plantlets.
Any non-tumorigenic A. tumefaciens strain harboring a disarmed Ti
plasmid may be used in the method of the invention. Any Agrobacterium system
may be
used. For example, Ti plasmid/binary vector system or a cointegrative vector
system with
one Ti plasmid may be used. Also, any marker gene or polynucleotide conferring
the
ability to select transformed cells, callus, embryos or plants and any other
gene, such as,
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for example ,a gene conferring resistance to a disease, or one improving
cellulose content,
may also be used. Any promoter desired can be used, such as, for example, a
PtCeIAP of
the invention, and those promoters as described above. A person of ordinary
skill in the
art can determine which markers and genes are used depending on particular
needs.
For purposes of the present invention, "transformed" or "transgenic" means
that at least one marker gene or polynucleotide confernng selectable marker
properties is
introduced into the DNA of a plant cell, callus, embryo or plant.
Additionally, any gene
may also be introduced.
To increase the infectivity of the bacteria, Agrobacterium is cultured in
low-pH induction medium, i.e., any bacterium culture media with a pH value
adjusted to
from 4.5 to 6.0, most preferably about 5.2, and at low temperature such as for
example
about 19-30°C, preferably about 21-26°C. The conditions of low-
pH and low temperature
are among the well-defined critical factors for inducing virulence activity in
Agrobacterium (e.g., Altmorbe et al., Mol. Plant-Microbe. Interac. 2: 301,
1989; Fullner et
al., Science 273: 1107, 1996; Fullner and Nester, J. Bacteriol. 178: 1498,
1996).
The bacteria is preconditioned by coculturing with wounded tobacco leaf
extract (prepared according to methods known generally known in the art) to
induce a high
level of expression of the Agrobacteriacm vir genes. Prior to inoculation of
plant somatic
embryos, Agrobacterium cells can be treated with a tobacco extract prepared
from
wounded leaf tissues of tobacco plants grown in vitro. To achieve optimal
stimulation of
the expression of Agrobacterium vir genes by wound-induced metabolites and
other
cellular factors, tobacco leaves can be wounded and pre-cultured overnight.
Culturing of
bacteria in low pH medium and at low temperature can be used to further
enhance the
bacteria vir gene expression and infectivity. Preconditioning with tobacco
extract and the
vir genes involved in the T-DNA transfer process are generally known in the
art.
Agrobacterium treated as described above is then cocultured with a plant
tissue explant, such as for example zygotic and/or somatic embryo tissue. Non-
zygotic
(i.e., somatic) or zygotic tissues can be used. Any plant tissue may be used
as a source of
explants. For example, cotyledons from seeds, young leaf tissue, root tissues,
parts of
stems including nodal explants, and tissues from primary somatic embryos such
as the root
axis may be used. Generally, young tissues are a preferred source of explants.
The invention also relates to methods of altering the growth of a plant by
expressing the polynucleotide of the invention, which as a result alters the
growth of the
plant. The polynucleotide used in the method may be a homologous
polynucleotide or a
heterologous polynucleotide and are described in detail above. For example,
both full-
length and UDP-glucose binding region containing fragments may be expressed.
Additionally, depending on the aim of the method, the polynucleotide may be
introduced
into the plant in the sense or in the antisense orientation. Any suitable
promoter may be
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used to provide expression. The promoter or a functional fragment thereof is
operatively
linked to the polynucleotide. The promoter may be a constitutive promoter, a
tissue-
specific promoter or a development-specific plant promoter. Examples of
suitable
promoters are Cauliflower Mosaic Virus 35S, 4CL, cellulose synthase promoter,
PtCeIAP
and terminal flower promoter.
The invention further relates to a method of altering the cellulose content in
a plant by expressing the polynucleotide of the invention as described above.
The method
may be used to increased the ratio of cellulose to lignin in the plant that
have an exogenous
polynucleotide of the invention introduced therein.
The invention further relates to a method for altering expression of a
cellulose synthase in a plant cell by introducing into the cell a vector
comprising a
polynucleotide of the invention and expressing the polynucleotide. The
polynucleotides
and promoters described above may be used.
A method for causing stress-induced gene expression in a plant cell is also
within the scope of the invention. The method comprises (i) introducing into
the plant or a
plant cell an expression cassette comprising a cellulose synthase promoter or
a functional
fragment thereof or providing a plant or a plant cell that comprises the
expression cassette
(The promoter of the cassette is operatively linked to a coding sequence of
choice.); and
(ii) applying mechanical stress to the plant to induce expression of the
desired coding
sequence.
A method for determining a positive mechanical stress responsive element
(MSRE) in a cellulose synthase promoter is also within the scope of the
invention and
comprises (i) making serial deletions in the cellulose synthase promoter, such
as for
example, SEQ >D N0:3; (ii) introducing the deletion linked to a polynucleotide
encoding a
reporter sequence into a plant cell, and (iii) detecting a decrease in the
amount of reporter
in the plant after inducing a stress to the plant. Similarly, a method for
determining a
negative MSRE in a cellulose synthase promoter is provided. It comprises (i)
making
serial deletions in the cellulose synthase promoter, such as for example, SEQ
>D N0:3; (ii)
introducing the deletion linked to a polynucleotide encoding a reporter
sequence into a
plant cell, and (iii) detecting an increase in the amount of reporter in the
plant after
inducing a stress to the plant.
The following methods are also within the scope of the invention: a
method for expressing cellulose synthase in a tissue-specific manner
comprising
transforming a plant with a tissue specific promoter operatively linked to a
polynucleotide
encoding a cellulose synthase; a method for inducing expression of a cellulose
synthase in
a plant comprising introducing into a plant a cDNA encoding a protein that
binds to a
positive MSRE of a cellulose synthase promoter, thereby resulting in increased
expression
of cellulose in the plant, wherein the binding to the positive MSRE results in
expression of
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a cellulose synthase; a method for reducing expression of a cellulose synthase
comprising
introducing into a plant a cDNA in an antisense orientation, wherein the cDNA
in a sense
orientation encodes a protein that binds to a positive MSRE and results in
expression of a
cellulose synthase; a method for increasing cellulose biosynthesis in a plant
comprising
introducing into a plant a cDNA encoding a protein that binds to a positive
MSRE of a
cellulose synthase promoter, whereby binding of the protein to the positive
MSRE results
in expression of a cellulose synthase, and A method for reducing cellulose
biosynthesis in
a plant comprising introducing into a plant a cDNA in an antisense
orientation, wherein
the cDNA in a sense orientation encodes a protein that binds to a positive
MSRE of a
cellulose synthase promoter.
EXAMPLE
Molecular cloning of cellulose s nt
This Example describes the first tree cellulose synthase cDNA (PtCeIA,
GenBank No. AF072131) cloned from developing secondary xylem of aspen trees
using
RSWI cDNA.
Prior to the present invention, only partial clones of cellulose synthases
from crop species and cotton GhCeIA have been discovered, which have
significant
homology to each other. The present inventors have discovered and cloned a new
full-
length cellulose synthase cDNA, AraxCelA (GenBank No. AF062485) (Fig. 7, [SEQ
ID
NO: 4]), from an Arabidopsis primary library. AraxCelA is a new member of
cellulose
synthase and shows 63-85% identity and 72-90% similarity in amino acid
sequence with
other Arabidopsis CeIA members.
Another cellulose synthase was cloned in aspen using a 32P-labeled 1651-by
long EcoRI fragment of Arabidopsis CeIA cDNA, which encodes a centrally
located UDP-
glucose binding domain, was used as a probe to screen about 500,000 pfu of a
developing
xylem cDNA library from aspen (Populus tremuloides) (Ge and Chiang, 1996).
Four
positive clones were obtained after three rounds of plaque purification.
Sequencing the 3'
ends of these four cDNAs showed that they were identical clones. The longest
cDNA
clone was fully sequenced and determined to be a full-length cDNA having a
3232 by
nucleotide sequence (Fig. 1) [SEQ ID NO: 1], which encodes a protein of 978
amino acids
[SEQ ID NO: 2].
Characterization of a cellulose synthase from as en
The first AUG codon of PtCeIA was in the optimum context for initiation of
transcription on the basis of optimal context sequence described by Joshi
(1987a) and
Joshi et al. (1997). A putative polyadenylation signal (AATACA) was found 16
by
upstream of a polyadenylated tail of 28 bp, which is similar to the proposed
plant structure
(Joshi, 1987b). The 5' untranslated leader was determined to have 68 by and
the 3'
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untranslated traitor was 227 bp. Both of these regions have a typical length
observed in
many plant genes (Joshi, 1987a and Joshi, 1987b). This cDNA clone exhibited
90%
amino acid sequence similarity with cellulose synthase from cotton (GhCeIA,)
and 71°70
with cellulose synthase from Arabidopsis (RSWl ), suggesting that this
particular tree
homolog also encodes a cellulose synthase.
The full length cDNA was designated PtCeIA, and encodes a 110,278 Da
polypeptide having an isoelectric point (pI) of 6.58 and 8 charged molecules.
The
hydropathy curve indicated that this particular cellulose synthase has eight
transmembrane
binding domains; two at the amino terminal and six at the carboxyl terminal,
using the
method of Hoffman and Stoffel (1993). This protein structure is analogous to
those of
RSW1 and GhCeIA. All of the conserved domains for UDP-glucose binding, such as
QVLRW and conserved D residues, are also present in a cellulose synthase of
the
invention, e.g., PtCeIA (Brown et al., 1996). Thus, based on sequence and
molecular
analyses, it was concluded that PtCeIA encodes a catalytic subunit which, like
RSWI in
Arabidopsis, is essential for the cellulose biosynthesis machinery in aspen.
ha situ localization of PtCeIA mRNA transcripts along the developmental
gradient defined by stem primary and secondary growth demonstrated that
cellulose
synthase expression is confined exclusively to developing xylem cells
undergoing
secondary wall thickening. This cell-type-specific nature of PtCeIA gene
expression was
also consistent with xylem-specific activity of cellulose synthase promoter
(PtCeIAP)
based on heterologous promoter-13-glucuronidase (GUS) fusion analysis.
Overall, the
results provide several lines of evidence that cellulose synthase is the gene
primarily
responsible for cellulose biosynthesis during secondary wall formation in
woody xylem of
trees, such as aspen. Previous results by the inventors (Hu et al., 1999)
showed that
cellulose and lignin are deposited in a compensatory fashion in wood. The
discovery of a
cellulose synthase in trees, such as aspen, permits the up-regulation of the
protein to
elevate cellulose production. Surprisingly, expression of CeIA in trees
suppressed lignin
biosynthesis to further improve wood properties of trees.
Preparation of transgenic plants
The UDP-glucose binding sequence was subcloned into pBI121, which was
used to prepare transgenic tobacco plants (Hu et al., 1998). The expression of
a
heterologous UDP-glucose binding sequence resulted in a remarkable growth-
accelerating
effect. This was surprising because current knowledge of the function of plant
cellulose
synthases teaches that a UDP-glucose sequence must remain intact with other
functional
domains in CeIA, e.g., the transmembrane domains, in order for cellulose
synthase to
initiate cellulose biosynthesis. The remarkable growth and tremendous increase
in plant
biomass observed in transgenic tobacco was due likely to an augmented
deposition of
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cellulose, indicating that the UDP-glucose domain alone is sufficient for
genetic
augmentation of cellulose biosynthesis in plants.
Genome organization and expression of a novel cellulose synthase
To confirm that the cDNA clone of Fig. 1 [SEQ >D NO: 1] was a cellulose
synthase, genomic Southern blot analysis was performed under both high and low
stringency conditions using the cDNA. Genomic DNA from aspen was digested with
PstI
(lane P), HindIII (lane H) and EcoRI (lane E), and probed using a lkb 32P-
labeled
fragment from the 5' end of a cellulose synthase of Fig. 1. The Southern blot
suggested
the presence of a small family of cellulose synthase genes in aspen genome
(Fig. 2, panels
a and b). Repeated screening of the aspen xylem cDNA library with various
plant CeIA
gene-related probes always resulted in the isolation of the same cellulose
synthase cDNA
clone. This suggested that the cellulose synthase cDNA cloned (Fig. 1) [SEQ >D
NO: 1],
represents the primary and most abundant cellulose synthase-encoding gene in
developing
xylem of trees, such as aspen, where active cellulose deposition takes place.
It also
indicates that manipulation of cellulose synthase gene expression can have a
profound
influence on cellulose biosynthesis in trees.
In situ hybridization
Northern blot analysis of total RNA from the internodes of aspen seedling
stems (Fig. 2, panel c) using the labeled probe (as described above) revealed
the near
absence of cellulose synthase transcripts in tissues undergoing primary growth
(internodes
1 to 4), and that the presence of cellulose synthase transcripts occurs during
the secondary
growth of stem tissues (internodes 5 to 11). However, weak northern signals in
primary
growth may only suggest that cellulose synthase gene expression is specific to
xylem, of
which there is little in primary growth tissue.
Xylogenesis in higher plants offers a unique model that involves sequential
execution of cambium cell division, commitment to xylem cell differentiation,
and
culmination in xylem cell death (Fukuda, 1996). Although primary and secondary
xylem
cells originate from different types of cambia, namely procambium and
inter/intrafasicular
cambium, both exhibit conspicuous secondary wall development with massive
cellulose
and lignin deposition (Esau, 1965). To further investigate spatial and
temporal cellulose
synthase gene expression patterns at the cellular level, in situ hybridization
was used to
localize cellulose synthase mRNA along the developmental gradient defined by
stem
primary and secondary growth.
Localization of cellulose synthase gene transcripts (RNA) in stem at
various growth stages was also observed. Fig. 3 shows transverse sections from
2°d, 4th
and 6'h internodes hybridized with digoxygenin (DIG)-labeled cellulose
synthase antisense
or sense (control) RNA probes, as described.
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PtCeIA transcripts were detected in young aspen stem sections by in sitar
hybridization with transcripts of highly variable 5' region of PtCeIA cDNA (a
771 by long
fragment generated from PstI and SacI). This region was first subcloned in the
plasmid
vector, pGEM,-3Zf (+) (Promega) for the production of digoxygenin (DIG)-
labeled
transcripts using T7 (for antisense transcripts) and SP6 (for sense
transcripts) RNA
polymerase (DIG system: Boehringer Mannheim). Probes were subjected to mild
alkaline
hydrolysis by incubation in 100 m'VI NaHC03, pH 10.2 at 60 °C, which
produced
approximately 200 by fragments.
Aspen young stems were prepared for sectioning by fixation in 4% (w/v)
paraformaldehyde in 100 mM phosphate buffer (pH 7.0) at 4 °C overnight,
dehydrated
through an ethanol series on ice, and embedded in Paraplast medium (Sigma).
Ten ~.m
sections were mounted on Superfrost/plus (Fisher) slides at 42 °C
overnight, dewaxed and
then rehydrated through a descending ethanol series. The sections were
incubated with
proteinase K (10 ~,g/ml in 100 mM Tris-HCI, 50 mM EDTA, pH 7.5) for 30 min and
were
post-fixed with FAA. The sections were acetylated with 0.33% (v/v) acetic
anhydride in
0.1 M triethanolamine-HCl (pH 8.0) prior to hybridization. The sections were
then
incubated in a hybridization mixture (approximately 2 ~,g/ml DIG-labeled
probes, 50%
(v/v) formamide, 2 X SSPE, 10% (w/v) dextran sulfate, 125 ~,g/ml tRNA, pH 7.5)
at 45 °C
for 12-16 hrs. Nonhybridized single-stranded RNA probe was removed by
treatment with
20 ~.g/ml RNase A in TE buffer with 500 mM NaCI. The sections were washed at
50 °C.
Hybridized DIG-labelled probe was detected on sections using anti-digoxygenin
antiserum
at a 1:1500 dilution, as described in the manufacturer's instruction (DIG
system:
Boehringer Mannheim). Sections were examined by Eclipse 400 light microscope
(Nikon)
and photographed.
During the primary growth stage (Fig. 3, panels a and b), strong expression
of cellulose synthase was found localized exclusively to primary xylem (PX)
cells. At this
stage, young internodes are elongating, resulting in thickening of primary
xylem cells
through formation of secondary walls (Esau, 1968). The concurrence of shoot
elongation
with high expression of cellulose synthase strongly suggests the association
of cellulose
synthase protein with secondary cell wall cellulose synthesis. Later stages of
primary
growth (Fig. 3, panel b) are characterized by the appearance of an orderly
alignment of
primary xylem cells. Active cellulose biosynthesis accompanies cell elongation-
induced
wall thickening, as indicated by the strong expression of cellulose synthase
in these
primary xylem cells.
At the beginning of secondary growth in older internodes, it was observed
that expression of cellulose synthase is also exclusively localized to xylem
cells (Fig. 3,
panel c). Instead of elongation in internodes distal to the meristematic
activity, growth at
this stage is mainly radial due to thickening in secondary cell walls of
secondary xylem.
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At the same time, expression of PtCeIA gene becomes localized to the secondary
developing xylem cells (SX in Fig. 3, panel c), which is again consistent with
the idea that
PtCeIA encodes a secondary cell wall cellulose synthase. At this stage,
secondary xylem
cells cover the elongated and differentiated primary xylem cells in which
PtCeIA gene
expression is no longer detectable (Fig. 3, panel c). These results
demonstrate that
expression of PtCeIA gene is xylem-specific and the cellulose synthase of Fig.
1 [SEQ ID
NO: 1] encodes a cellulose synthase associated with cellulose biosynthesis in
secondary
walls of xylem cells. To further confirm xylem-specific expression of
cellulose synthase,
a cellulose synthase gene promoter sequence was cloned and characterized for
regulatory
activities.
Characterization of expression regulated by cellulose synthase promoter
A 5' 1,200 by cDNA fragment of a cellulose synthase of Fig. 1 [SEQ >D
NO: 1] was used as a probe to screen an aspen genomic library for 5'
regulatory sequences
of a novel cellulose synthase gene, PtCeIA. The library was constructed by
cloning aspen
genomic DNA fragments, generated from an Sau3AI partial-digest and sucrose
gradient-
selected, into the BamHI site of a Lambda DASH II vector (Stratagene, La
Jolla, CA).
Five positive clones were obtained from about 150,000 pfu and Lambda DNA was
purified. One clone having about a 20 kb DNA insert size was selected for
restriction
mapping and partial sequencing. This resulted in the identification of a 5'
flanking region
of PtCeIA gene of approximately 1 kb. This genomic fragment, designated
PtCeIAP (Fig.
4) [SEQ ID NO: 3], contained about 800 by of promoter sequence, 68 by of 5'
end
untranslated region and 160 by of coding sequence. To investigate regulation
of tissue-
specific cellulose synthase expression at the cellular level, promoter
activity was analyzed
in transgenic tobacco plants by histochemical staining of a GUS protein. A
PtCeIAP-GUS
fusion binary vector was constructed in pBIl21 with the 35S promoter replaced
with
PtCeIAP [SEQ ID NO: 3] and introduced into tobacco (Nicotiaoa tabacum) as per
Hu et
al. (1998).
Eleven independent transgenic lines harboring a CeIAP-GUS fusion were
generated. Fig. 5 shows a histochemical analysis of GUS expression driven by a
cellulose
synthase promoter of the invention in transgenic tobacco plants. Transverse
sections from
the 3rd (panel a), 5th (panel b), 7th (panel c), and 8th (panels d and f)
internodes were
stained from GUS activity, and fluorescence microscopy was used to visualize
expression
under UV radiation.
GUS staining was detected exclusively in xylem tissue of stems, roots and
petioles. In stems, strong GUS activity was found localized to xylem cells
undergoing
primary (Fig. 5, panel a) and secondary growth (Fig. 5 panels b-d and f). GUS
expression
was confined to xylem cells in the primary growth stage and became more
localized in
developing secondary xylem cells during secondary growth. An entire section
from the
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8th internode stained for GUS activity (Fig. 5, panel f). These results are
consistent with
the in vivo expression patterns of cellulose synthase in aspen stems. Lignin
autofluorescence was visualized after UV radiation. Phloem fibers, which are
also active
in cellulose and lignin biosynthesis (Fig. 5, panels d and e), did not show
GUS activity,
suggesting that cellulose synthase gene expression is not associated with
cellulose
biosynthesis in cell types other than xylem. Examination of GUS activity in
roots, stems,
leaves, anthers and fruit also showed GUS expression in xylem tissue of all
these organs
suggesting that cellulose synthases of the invention are xylem-specific
cellulose and
expressed in all plant organs.
Characterization of promoter activity and cellular expression of a cellulose
synthase of the invention from one particular source (aspen) indicated hat
expression
produces a protein that encodes a secondary cell wall-specific cellulose
synthase and is
specifically compartmentalized in developing xylem cells. Characterization of
the
cellulose synthase gene promoter sequence not only confirms cell type-specific
expression
of cellulose synthase, but also provides a method for over-expressing
cellulose synthase in
a tissue-specific manner to augment cellulose production in xylem.
Expression of cellulose synthase under tension stress
As described earlier, a cellulose synthase promoter of the invention is
involved in a novel gene regulatory phenomenon of cellulose synthase. To
further
characterize a cellulose synthase of the invention, GUS expression driven by
an aspen
cellulose synthase promoter (PtCeIAP) was observed in transgenic tobacco
plants without
or under tension stress. The stress was induced by bending and affixing the
plants to
maintain the bent position (e.g., tying) over a 40 hour period. Tangential and
longitudinal
sections were taken before bending, and 4 hrs, 20 hrs and 40 hrs after bending
(panels a-d,
respectively).
The cellulose synthase promoter-GUS fusion binary constructs showed
exclusive xylem-specific expression of GUS without any tension stress (Fig. 6,
panel a).
However, under tension stress conditions endured by angiosperms in nature, the
transgenic
tobacco plants induced xylem and phloem-specific expression on the upper side
of the
stem within the first four hours of stress (Fig. 6, panel b).
This observation was surprising because during tension wood development
fibers produce highly crystalline cellulose in order to provide essential
mechanical strength
to a bending stem. The present observation was the first showing of
transcriptional up-
regulation of a cellulose synthase, mediated through a cellulose synthase
promoter that is
directly responsible for development of highly crystalline cellulose in trees.
Furthermore,
after 20 hrs of tension stress, both xylem and phloem exhibited GUS
expression, but only
on the upper side of the stem that was under tensile stress, i.e., GUS
expression on the
lower side was inhibited (Fig. 6, panel c). With extended stress (up to 40
hrs), GUS
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expression was restricted to only one small region on the upper side of the
stem where
maximum tension stress was present (Fig. 6, panel d). Based on the observation
of GUS
signal in woody cells upon tension stress and the absence of GUS under
compression or no
stress, it was concluded that a cellulose synthase promoter of the invention
has mechanical
stress responsive elements (MSREs) that turn cellulose synthase genes on and
off
depending on the presence and type of stress to the stem.
The results indicate that positive MSREs exist in a cellulose synthase
promoter of the invention to bind transcription factors in response to tension
stress for
regulating the expression of cellulose synthase and increasing biosynthesis of
higher
crystalline cellulose. This is evident based on the expression of GUS in xylem
and phloem
tissue at the upper side of the stem subjected to tension stress, but not when
tissue on the
lower side was subjected to compression or no stress. Furthermore, the tissue
at the lower
side of the stem, which was subjected to compression stress, showed no GUS
expression,
i.e., expression was turned off. This indicated the presence of negative
MSREs, which
bind transcription factors to turn off expression of cellulose synthase at the
lower side of
the stem. Negative MSREs likely suppress development of highly crystalline
cellulose in
normal wood.
These results provide a mechanism for genetically engineering synthesis of
highly crystalline cellulose in juvenile wood for enhancing strength
properties, and for
synthesizing a higher percentage of cellulose in reaction wood. The positive
MSREs and
their cognate transcription factors are important in the synthesis of highly
crystalline
cellulose of high tensile strength, as are the negative MSREs and inhibition
of cognate
transcription factors thereto. The present invention thus provides a starting
point for
cloning cDNAs for the transcription factors that bind to positive and negative
MSREs
according to methods known in the art. Constitutive expression of cDNAs for
positive
MSRE transcription factors allows the continuous production of highly
crystalline
cellulose in transgenic trees, while expression of antisense cDNAs for
negative MSRE
transcription factors inhibits those transcription factors so that cellulose
synthase cannot
turn off. This combination will assure continuous production of highly
crystalline
cellulose in trees.
Genetic en ing~ Bering of cellulose synthase in trans eg nic plants
As discussed above, the nucleotide sequence of a cellulose synthase of the
invention, e.g., PtCeIA cDNA from aspen, shows significant homology with other
polynucleotides encoding cellulose synthase proteins that have been suggested
as authentic
cellulose synthase clones. To further characterize the activity of a cellulose
synthase, four
constructs were prepared in a PBI121 plasmid.
1) A constitutive plant promoter Cauliflower mosaic Virus 35S was
operatively linked to PtCeIA (35SP-PtCeIA-s) and overexpressed in transgenic
plants.
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This causes excess production of cellulose, resulting in a reduction in lignin
content.
Tobacco and aspen have been transformed with this construct.
2) Cauliflower mosaic Virus 35S was operatively linked to antisense
RNA from PtCeIA (35S-PtCeIA-a) and constitutively expressed to reduce
production of
cellulose and increase lignin content in transgenic plants. This negative
control construct
may not result in healthy plants since cellulose is essential for plant growth
and
development. Aspen plants have been transformed with this construct.
3) Aspen 4CL-1 promoter (Hu et al., 1998) was operatively linked to
PtCeIA (Pt4CLP-PtCeIA) (the 35S promoter of PBI121 was removed in this
construct) and
expressed in a tissue-specific manner in developing secondary xylem of
transgenic aspen.
This expression augments the native cellulose production and reduces lignin
content of
angiosperm tissues. Tobacco and aspen have transformed with this construct.
4) The cytoplasmic domain of PtCeIA which contains three conserved
regions thought to be involved in UDP-glucose binding during cellulose
biosynthesis, was
linked to a 35S promoter to produce binary constructs (35S-PtCeIA UDP-
glucose).
Expression by this promoter permits constitutive expression of a UDP glucose
binding
domain of PtCeIA in transgenic plants. Tobacco and aspen have been transformed
with
this construct.
35S-GUS constructs (pBI121, ClonTech, CA) were used as controls for
each experiment with the constructs. Transgenic tobacco plants were
transformed with the
constructs. The following table shows the general growth measurements of the
TO tobacco
plants. Plants carrying a PtCeIA construct grew much faster than control
plants carrying a
pBI121 (control) construct. In comparing developmental 4CL and constitutive
35S
promoter control of PtCeIA expression, the 35S was more effective, permitting
faster
growth of transgenic tobacco plants. The fastest growth was seen in transgenic
plants
carrying a 35S promoter driven UDP-G domain from PtCeIA.
It is noted that TO generation plants can have carry over effects from their
tissue culture treatments. Therefore, seeds were collected for testing this
growth
phenomenon in T1 generations. The transgenic tobacco plants were analyzed for
presence
of the transferred genes and all tested positive for the respective gene
constructs.
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
-33-
TABLE
Transgenic tobacco plant measurements
after transfer in soil for about 1.5 months (N = 2)
Construct Height Diameter Internode lengthNo. of Longest
leaves leaf
35S-GUS 17 0.5 1 11 17
35S-PtCeIA 77 1.0 6 13 37
35S-UDPG 83 1.0 6 13 37
4CLP-PtCeIA 41 0.8 5 10 29
Note: All values were measured in centimeters, excluding cumber of leaves.
It will be appreciated by persons of ordinary skill in the art that the
examples and preferred embodiments herein are illustrative, and that the
invention may be
practiced in a variety of embodiments which share the same inventive concept.
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
-34-
BIBLIOGRAPHY
Hu et al., 1999, Nature Biotechnology, In Press
Whetten et al., 1998, Ann Rev Pl Physiol Pl Mol Biol, 49: 585-609
Arioli et al., 1998, Science, 279: 717-720
Wu et al., 1998, PI Physiol, 117: 1125
Hu et al., 1998, PNAS, 95: 5407-5412
Joshi et al., 1997, PMB, 35: 993-1001
Fukuda, 1996, Ann Rev Pl Physiol Pl Mol Biol, 47: 299-325
Pear et al., 1996, PNAS, 93: 12637-12642
Haigler and Blanton, 1996, PNAS, 93: 12082-12085
Ge and Chiang, 1996, Pl Physiol, 112: 861
Brown et al., 1996, Trends Pl Sci., l: 149-156
Delmer and Amor, 1995, PI Cell, 7: 987-1000
Hoffman and Stoffel, 1993, Biol Chem, Hoppe-Seyler 374: 166
Joshi, 1987, NAR, 15: 6643-6653
Joshi, 1987, NAR, 15: 9627-9640
Timmell, 1986, Compression Wood in Gymnopserms, Springer Verlag
Esau, 1967, Plant Anatomy, Wiley and sons, NY
Higuchi, 1997, Biochemistry and Molecular Biology of Wood, Springer Verlag
CA 02371659 2001-11-19
INTERNATIONAL SEARCH REPORT International application
No.
PCT/US00/13637
Bo: I Observations where certain claims were
found unsearchable (Continuation of item
1 of first sheet)
This international report has not been established
in respect of certain claims under Article
17(2xa) for the following reasons:
1. ~ Claims Nos.:
because they relate to subject matter not
requmed to be searched by this Authority,
namely:
2. ~ Claims Nos.:
because they relate to parts of the international
application that do not comply with the
prescribed requirements to such
an extent that no meaningful international
search can be cartied out, specifically:
3. ~ Claims Nos.:
because they are dependent claims and are
not drafted in accordance with the second
and third sentences of Rule 6.4(a).
Boz II Observations where unity of invention
is lacking (Continuation of item 't of first
sheet)
This International Searching Authority found
multiple inventions in this international
application, as follows:
Please See Extra Sheet.
1. ~ As all required additional search fees
were timely paid by the applicant, this
international search report covers all searchable
claims.
2. Q As all searehable claims could be searched
without effort justifying an additional
fee, this Authority did not invite payment
of any additional fee.
3. ~ As only some of the required additional
searcl. fees were timely paid by the applicant,
this international search report covers
only those claims for which fees were paid,
specifically claims Nos.:
4. ~ No required additional search fees were
timely paid by the applicant. Consequently,
this international search report is
restricted to the invention first mentioned
in the claims; it is covered by claims Nos.:
1-4, 9-16, 25-31, 35, 40, 42-43, 45-46
Remark on Protest ~ The additional search
fees were accompanied by the applicant's
protest.
No protest accompanied the payment of additional
search fees.
Form PCT/ISA/210 (continuation of first sheet(1)) (July 1998)*
CA 02371659 2001-11-19
INTERNATIONAL SEARCH REPORT ~ International application No.
PCT/US00/13637
A. CLASSIFICATION OF SUBJECT MATTER:
US CL
800/278, 286, 287, 295, 298; 435/G9.1, 320.1, 419; 536/23.2, 23.6, 24.1, 24.5
BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING
This ISA found multiple inventions as follows:
This application contains the following inventions or groups of inventions
which are not so linked as to form a singe
inventive concept under PCT Rule 13.1. In order for all inventions to be
searched, the appropriate additional search fees
must be paid.
Group I, claims) 1-4, 9-16, 25-31, 35, 40, 42-43, 45-46, drawn to
polynucleotide having specific nucleic acid sequence
or a fragment thereof encoding a functional domain of a cellulose synthase,
methods of altering cellulose content of a
transgenic plant.
Group II , claim(s)5-7, 32, 36-39, 41 44, drawn to cellulose synthase
promoters and methods of their use.
Group III, claims) 8, drawn to cellulose synthase polypeptides
Group IV , claim(s) 17, 19-24 , drawn to a polynucleotide encoding UDP-glucose
binding domain, and transgenic plants
expressing it.
Group V , claim(s)18, drawn to UDP-glucose polypeptide .
Group VI, claims) 33-34 , drawn to a method of identifying regulatory elements
in a cellulose synthase promoter.
The inventions listed as Groups 1-VI do not relate to a single inventive
concept under PCT Rule 13.1 because, under
PCT Rule 13.2, they lack the same or corresponding special technical features
for the following reasons:
The claimed polynucleotide sequences or a fragment thereof encoding a
functional domain of a cellulose synthase is
anticipated by Stalker et al (WO 98/18949) who teach plant cDNAs encoding
functional units of cellulose synthase, and
so do not constitute a single special technical feature which would be an
advance over the prior art.
The invention of Group I, drawn to a polynucleotide encoding cellulose
synthase, reguires a polynucleotide with speci5c
sequence and transgenic plants expressing it, which are not reguired by any of
the other groups.
The invention of Group 11, drawn to cellulose synthase promoter and a
mechanical stress to a plant which are not
reguired by any of the other groups.
The invention of Group III reguires isolated cellulose synthase polypeptides
which are not reguired by any of the other
groups.
The invention of Group IV reguires polynucleotides encoding UDP-glucose
binding domain which are not reguired by
any of the other groups.
The invention of Group V reguires UDP-glucose polypeptide which is not
reguired by any of the other groups.
The invention of Group VI reguires methods for identifying regulatory elements
which are not reguired by any of the
other groups.
Form PCT/ISA/210 (extra sheet) (July 1998)*
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
SEQUENCE LISTING
<110> Board of Control of Michigan Technological Univers
<120> METHOD FOR ENHANCING CELLULOSE AND MODIFYING LIGNIN
BIOSYNTHESIS IN PLANTS
<130> 66040/9675
<140>
<141>
<150> 60/135,280
<151> 1999-05-21
<160> 6
<170> PatentIn Ver. 2.1
<210> 1
<211> 3232
<212> DNA
<213> Populus tremuloides
<220>
<221> CDS
<222> (69)..(3002)
<400> 1
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Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glu
1 5 10
cag gtg ggg cat gat gca aat ggg gag cta ttt gtg get tgc cat gag 158
Gln Val Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His Glu
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tgt agc tat ccc atg tgc aag tct tgt ttc gag ttt gaa atc aat gag 206
Cys Ser Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Asn Glu
35 40 45
ggc cgg aaa gtt tgc ttg cgg tgt ggc tcg cca tat gat gag aac ttg 254
Gly Arg Lys Val Cys Leu Arg Cys Gly Ser Pro Tyr Asp Glu Asn Leu
50 55 60
ctg gat gat gta gaa aag aag ggg tct ggc aat caa tcc aca atg gca 302
Leu Asp Asp Val Glu Lys Lys Gly Ser Gly Asn Gln Ser Thr Met Ala
65 70 75
tct cac ctc aac gat tct cag gat gtc gga atc cat get aga cat atc 350
Ser His Leu Asn Asp Ser Gln Asp Val Gly Ile His Ala Arg His Ile
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agt agt gtg tcc act gtg gat agt gaa atg aat gat gaa tat ggg aat 398
Ser Ser Val Ser Thr Val Asp Ser Glu Met Asn Asp Glu Tyr Gly Asn
1
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
95 100 105 110
ccaatttggaagaat cgggtg aagagctgt aaggataaa gagaacaag 446
ProIleTrpLysAsn ArgVal LysSerCys LysAspLys GluAsnLys
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aagaaaaagagaagt cctaag getgaaact gaaccaget caagttcct 494
LysLysLysArgSer ProLys AlaGluThr GluProAla GlnValPro
130 135 140
acagaacagcagatg gaagag aaaccgtct gcagagget tcggagccg 542
ThrGluGlnGlnMet GluGlu LysProSer AlaGluAla SerGluPro
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ctttcaattgtttat ccaatt ccacgcaac aagctcaca ccatacaga 590
LeuSerIleValTyr ProIle ProArgAsn LysLeuThr ProTyrArg
160 165 170
gcagtgatcattatg cgactg gtcattctg ggcctcttc ttccacttc 638
AlaValIleIleMet ArgLeu ValIleLeu GlyLeuPhe PheHisPhe
175 180 185 190
agaataacaaatcct gtcgat agtgccttt ggcctgtgg cttacttct 686
ArgIleThrAsnPro ValAsp SerAlaPhe GlyLeuTrp LeuThrSer
195 200 205
gtcatatgtgagatc tggttt gcattttct tgggtgttg gatcagttc 734
ValIleCysGluIle TrpPhe AlaPheSer TrpValLeu AspGlnPhe
210 215 220
cccaagtggaatcct gtcaat agagaaacg tatatcgaa aggctgtcg 782
ProLysTrpAsnPro ValAsn ArgGluThr TyrIleGlu ArgLeuSer
225 230 235
gcaaggtatgaaaga gagggt gagccttct cagcttget ggtgtggat 830
AlaArgTyrGluArg GluGly GluProSer GlnLeuAla GlyValAsp
240 245 250
tttttcgtgagtact gttgat ccgctgaag gaaccgcca ttgatcact 878
PhePheValSerThr ValAsp ProLeuLys GluProPro LeuIleThr
255 260 265 270
gccaatacagtcctt tccatc cttgetgtg gactatccc gtcgataaa 926
AlaAsnThrValLeu SerIle LeuAlaVal AspTyrPro ValAspLys
275 280 285
gtctcctgctacgtg tctgat gatggtgca getatgctt tcatttgaa 974
ValSerCysTyrVal SerAsp AspGlyAla AlaMetLeu SerPheGlu
290 295 300
tctcttgtagaaaca getgag tttgcaagg aagtgggtt ccgttctgc 1022
SerLeuValGluThr AlaGlu PheAlaArg LysTrpVal ProPheCys
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aaaaaattctcaatt gaacca agagcaccg gagttttac ttctcacag 1070
LysLysPheSerIle GluPro ArgAlaPro GluPheTyr PheSerGln
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2
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/ITS00/13637
aaa att gat tac ttg aaa gac aag gtt caa cct tct ttc gtg aaa gaa 1118
Lys Ile Asp Tyr Leu Lys Asp Lys Val Gln Pro Ser Phe Val Lys Glu
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Arg Arg Ala Met Lys Arg Asp Tyr Glu Glu Tyr Lys Val Arg Val Asn
355 360 365
gcc ctg gta gca aag get cag aaa aca cct gaa gaa gga tgg act atg 1219
Ala Leu Val Ala Lys Ala Gln Lys Thr Pro Glu Glu Gly Trp Thr Met
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caa gat gga aca cct tgg cct ggg aat aac aca cgt gat cac cct ggg 1262
Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly
385 390 395
cat gat tca ggt ctt cct tgg gaa ata ctg gga get cgt gac att gaa 1310
His Asp Ser Gly Leu Pro Trp Glu Ile Leu Gly Ala Arg Asp Ile Glu
900 905 910
gga aat gaa cta cct cgt cta gta tat gtc tcc agg gag aag aga cct 1358
Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro
915 920 925 430
ggc tac cag cac cac aaa aag get ggt gca gaa aat get ctg gtg aga 1406
Gly Tyr Gln His His Lys Lys Ala Gly Ala Glu Asn Ala Leu Val Arg
935 990 445
gtg tct gca gta ctc aca aat get ccc tac atc ctc aat gtt gat tgt 1454
Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Ile Leu Asn Val Asp Cys
950 955 460
gat cac tat gta aac aat agc aag get gtt cga gag gca atg tgc atc 1502
Asp His Tyr Val Asn Asn Ser Lys Ala Val Arg Glu Ala Met Cys Ile
465 470 475
ctg atg gac cca caa gta ggt cga gat gta tgc tat gtg cag ttc cct 1550
Leu Met Asp Pro Gln Val Gly Arg Asp Val Cys Tyr Val Gln Phe Pro
980 485 490
cag agg ttt gat ggc ata gat aag agt gat cgc tac gcc aat cgt aac 1598
Gln Arg Phe Asp Gly Ile Asp Lys Ser Asp Arg Tyr Ala Asn Arg Asn
495 500 505 510
gta gtt ttc ttt gat gtt aac atg aaa ggg ttg gat ggc att caa gga 1696
Val Val Phe Phe Asp Val Asn Met Lys Gly Leu Asp Gly Ile Gln Gly
515 520 525
cca gta tac gta gga act ggt tgt gtt ttc aac agg caa gca ctt tac 1699
Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Gln Ala Leu Tyr
530 535 540
ggc tac ggg cct cct tct atg ccc agc tta cgc aag aga aag gat tct 1792
Gly Tyr Gly Pro Pro Ser Met Pro Ser Leu Arg Lys Arg Lys Asp Ser
545 550 555
3
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/i1S00/13637
tcatcctgcttc tcatgttgc tgcccctca aagaagaag cctgetcaa 1790
SerSerCysPhe SerCysCys CysProSer LysLysLys ProAlaGln
560 565 570
gatccagetgag gtatacaga gatgcaaaa agagaggat ctcaatget 1838
AspProAlaGlu ValTyrArg AspAlaLys ArgGluAsp LeuAsnAla
575 580 585 590
gccatatttaat cttacagag attgataat tatgacgag catgaaagg 1886
AlaIlePheAsn LeuThrGlu IleAspAsn TyrAspGlu HisGluArg
595 600 605
tcaatgctgatc tcccagttg agctttgag aaaactttt ggcttatct 1934
SerMetLeuIle SerGlnLeu SerPheGlu LysThrPhe GlyLeuSer
610 615 620
tctgtcttcatt gagtctaca ctaatggag aatggagga gtacccgag 1982
SerValPheIle GluSerThr LeuMetGlu AsnGlyGly ValProGlu
625 630 635
tctgccaactca ccaccattc atcaaggaa gcgattcaa gtcatcggc 2030
SerAlaAsnSer ProProPhe IleLysGlu AlaIleGln ValIleGly
640 645 650
tgtggctatgaa gagaagact gaatgggga aaacagatt ggttggata 2078
CysGlyTyrGlu GluLysThr GluTrpGly LysGlnIle GlyTrpIle
655 660 665 670
tatgggtcagtc actgaggat atcttaagt ggcttcaag atgcactgc 2126
TyrGlySerVal ThrGluAsp IleLeuSer GlyPheLys MetHisCys
675 680 685
cgaggatggaga tcaatttac tgcatgccc gtaaggcct gcattcaaa 2174
ArgGlyTrpArg SerIleTyr CysMetPro ValArgPro AlaPheLys
690 695 700
ggatctgcaccc atcaacctg tctgataga ttgcaccag gtcctccga 2222
GlySerAlaPro IleAsnLeu SerAspArg LeuHisGln ValLeuArg
705 710 715
tgggetcttggt tctgtggaa attttcttt agcagacac tgtcccctc 2270
TrpAlaLeuGly SerValGlu IlePhePhe SerArgHis CysProLeu
720 725 730
tggtacgggttt ggaggaggc cgtcttaaa tggctccaa aggcttgcg 2318
TrpTyrGlyPhe GlyGlyGly ArgLeuLys TrpLeuGln ArgLeuAla
735 740 745 750
tatataaacacc attgtgtac ccatttaca tccctccct ctcattgcc 2366
TyrIleAsnThr IleValTyr ProPheThr SerLeuPro LeuIleAla
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tattgcacaatt cctgcagtt tgtctgctc accggaaaa ttcatcata 2414
TyrCysThrIle ProAlaVal CysLeuLeu ThrGlyLys PheIleIle
770 775 780
cca acg ctc tca aac ctg gca agc atg ctg ttt ctt ggc ctc ttt atc 2462
4
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
ProThrLeuSer AsnLeuAlaSer MetLeu PheLeuGly LeuPheIle
785 790 795
tccatcattgta actgcggtgctt gagcta agatggagc ggtgtcagc 2510
SerIleIleVal ThrAlaValLeu GluLeu ArgTrpSer GlyValSer
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attgaagattta tggcgtaatgaa caattc tgggtgatc ggaggtgtt 2558
IleGluAspLeu TrpArgAsnGlu GlnPhe TrpValIle GlyGlyVal
815 820 825 830
tcagcccatctc tttgcggtcttc caggga ttcttaaaa atgttgget 2606
SerAlaHisLeu PheAlaValPhe GlnGly PheLeuLys MetLeuAla
835 840 845
ggcatcgatacg aacttcactgtc acagca aaagcagcc gaagatgca 2654
GlyIleAspThr AsnPheThrVal ThrAla LysAlaAla GluAspAla
850 855 860
gaatttggggag ctatatatggtc aagtgg acaacactt ttgattcct 2702
GluPheGlyGlu LeuTyrMetVal LysTrp ThrThrLeu LeuIlePro
865 870 875
ccaaccacactt ctcattatcaat atgtcg ggttgtget ggattctct 2750
ProThrThrLeu LeuIleIleAsn MetSer GlyCysAla GlyPheSer
880 885 890
gatgcactcaac aaaggatatgaa gcatgg gggcctctc tttggcaag 2798
AspAlaLeuAsn LysGlyTyrGlu AlaTrp GlyProLeu PheGlyLys
895 900 905 910
gtgttctttget ttctgggtgatt cttcat ctctatcca ttccttaaa 2846
ValPhePheAla PheTrpValIle LeuHis LeuTyrPro PheLeuLys
915 920 925
ggtctaatgggt cgccaaaaccta acacca accattgtt gttctctgg 2894
GlyLeuMetGly ArgGlnAsnLeu ThrPro ThrIleVal ValLeuTrp
930 935 940
tcagtgctgttg gcctctgtcttc tctctc gtttgggtc aagatcaat 2942
SerValLeuLeu AlaSerValPhe SerLeu ValTrpVal LysIleAsn
945 950 955
ccattcgttaac aaagttgataac accttg gttgcggag acctgcatt 2990
ProPheValAsn LysValAspAsn ThrLeu ValAlaGlu ThrCysIle
960 965 970
tccattgattgc tgagctacct ccaataagtc 3042
tctcccagta
ttttggggtt
SerIleAspCys
975
acaaaacctt tgggaattgg aatatgatcc tcgttgtagt ttccctcaag aaagcacata 3102
tcgctgtcag tatttaaatg aactgcaaga tgattgttct ctatgaagtt ttgaacagtt 3162
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SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
aaaaaaaaaa
<210> 2
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<213> Populus tremuloides
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Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glv _
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Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His G1~
20 25 3'
Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Asn Glv
35 40 45
Lys Val Cys Leu Arg Cys Gly Ser Pro Tyr Asp Glu Asn Lei. _
50 55 60
Asp Val Glu Lys Lys Gly Ser Gly Asn Gln Ser Thr Met Al~
65 70 75
Leu Asn Asp Ser Gln Asp Val Gly Ile His Ala Arg His Ile _
85 90
Val Ser Thr Val Asp Ser Glu Met Asn Asp Glu Tyr Gly As:. _
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Trp Lys Asn Arg Val Lys Ser Cys Lys Asp Lys Glu Asn Lye _
115 120 125
Lys Arg Ser Pro Lys Ala Glu Thr Glu Pro Ala Gln Val Prc _
130 135 140
Gln Gln Met Glu Glu Lys Pro Ser Ala Glu Ala Ser Glu Prc -
145 150 155
Ile Val Tyr Pro Ile Pro Arg Asn Lys Leu Thr Pro Tyr Arc_ __
165 170
Ile Ile Met Arg Leu Val Ile Leu Gly Leu Phe Phe His Phe -
180 185 19~_
Thr Asn Pro Val Asp Ser Ala Phe Gly Leu Trp Leu Thr Ser -
195 200 205
Cys Glu Ile Trp Phe Ala Phe Ser Trp Val Leu Asp Gln Ph=
210 215 220
Trp Asn Pro Val Asn Arg Glu Thr Tyr Ile Glu Arg Leu Ser _
225 230 ~ 235
Tyr Glu Arg Glu Gly Glu Pro Ser Gln Leu Ala Gly Val Asp -
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6
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn
260 265 270
Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser
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Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Ser Phe Glu Ser Leu
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Val Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys
305 310 315 320
Phe Ser Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Ile
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Asp Tyr Leu Lys Asp Lys Val Gln Pro Ser Phe Val Lys Glu Arg Arg
340 345 350
Ala Met Lys Arg Asp Tyr Glu Glu Tyr Lys Val Arg Val Asn Ala Leu
355 360 365
Val Ala Lys Ala Gln Lys Thr Pro Glu Glu Gly Trp Thr Met Gln Asp
370 375 380
Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly His Asp
385 390 395 400
Ser Gly Leu Pro Trp Glu Ile Leu Gly Ala Arg Asp Ile Glu Gly Asn
405 410 415
Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr
420 425 430
Gln His His Lys Lys Ala Gly Ala Glu Asn Ala Leu Val Arg Val Ser
435 440 445
Ala Val Leu Thr Asn Ala Pro Tyr Ile Leu Asn Val Asp Cys Asp His
450 455 460
Tyr Val Asn Asn Ser Lys Ala Val Arg Glu Ala Met Cys Ile Leu Met
465 470 475 480
Asp Pro Gln Val Gly Arg Asp Val Cys Tyr Val Gln Phe Pro Gln Arg
485 490 495
Phe Asp Gly Ile Asp Lys Ser Asp Arg Tyr Ala Asn Arg Asn Val Val
500 505 510
Phe Phe Asp Val Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val
515 520 525
Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Gln Ala Leu Tyr Gly Tyr
530 535 540
Gly Pro Pro Ser Met Pro Ser Leu Arg Lys Arg Lys Asp Ser Ser Ser
545 550 555 560
7
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Cys Phe Ser Cys Cys Cys Pro Ser Lys Lys Lys Pro Ala Gln Asp Pro
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Ala Glu Val Tyr Arg Asp Ala Lys Arg Glu Asp Leu Asn Ala Ala Ile
580 585 590
Phe Asn Leu Thr Glu Ile Asp Asn Tyr Asp Glu His Glu Arg Ser Met
595 600 605
Leu Ile Ser Gln Leu Ser Phe Glu Lys Thr Phe Gly Leu Ser Ser Val
610 615 620
Phe Ile Glu Ser Thr Leu Met Glu Asn Gly Gly Val Pro Glu Ser Ala
625 630 635 640
Asn Ser Pro Pro Phe Ile Lys Glu Ala Ile Gln Val Ile Gly Cys Gly
645 650 655
Tyr Glu Glu Lys Thr Glu Trp Gly Lys Gln Ile Gly Trp Ile Tyr Gly
660 665 670
Ser Val Thr Glu Asp Ile Leu Ser Gly Phe Lys Met His Cys Arg Gly
675 680 685
Trp Arg Ser Ile Tyr Cys Met Pro Val Arg Pro Ala Phe Lys Gly Ser
690 695 700
Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala
705 710 715 720
Leu Gly Ser Val Glu Ile Phe Phe Ser Arg His Cys Pro Leu Trp Tyr
725 730 735
Gly Phe Gly Gly Gly Arg Leu Lys Trp Leu Gln Arg Leu Ala Tyr Ile
740 745 750
Asn Thr Ile Val Tyr Pro Phe Thr Ser Leu Pro Leu Ile Ala Tyr Cys
755 760 765
Thr Ile Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr
770 775 780
Leu Ser Asn Leu Ala Ser Met Leu Phe Leu Gly Leu Phe Ile Ser Ile
785 790 795 800
Ile Val Thr Ala Val Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Glu
805 810 815
Asp Leu Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala
820 825 830
His Leu Phe Ala Val Phe Gln Gly Phe Leu Lys Met Leu Ala Gly Ile
835 840 845
Asp Thr Asn Phe Thr Val Thr Ala Lys Ala Ala Glu Asp Ala Glu Phe
850 855 860
8
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Gly Glu Leu Tyr Met Val Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr
865 870 875 880
Thr Leu Leu Ile Ile Asn Met Ser Gly Cys Ala Gly Phe Ser Asp Ala
885 890 895
Leu Asn Lys Gly Tyr Glu Ala Trp Gly Pro Leu Phe Gly Lys Val Phe
900 905 910
Phe Ala Phe Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu
915 920 925
Met Gly Arg Gln Asn Leu Thr Pro Thr Ile Val Val Leu Trp Ser Val
930 935 940
Leu Leu Ala Ser Val Phe Ser Leu Val Trp Val Lys Ile Asn Pro Phe
945 950 955 960
Val Asn Lys Val Asp Asn Thr Leu Val Ala Glu Thr Cys Ile Ser Ile
965 970 975
Asp Cys
<210> 3
<211> 1010
<212> DNA
<213> Populus tremuloides
<220>
<221> CDS
<222> (841)..(1008)
<220>
<223> 5' flanking region of PtCelA coding sequence
<400> 3
gaattcgccc ttttgaattc aggagacgat agtttccggt tcgttgaatg gctttgttca 60
cttctggtct agcaatttgc aaaagaagtt acaaaacaaa tgcatattat gtaaatttaa 120
caagagatgg gttctatggt cacttattta tgcccatcat ttgttctggg gttactcttt 180
atagtctgat tcgaagttgc aaactgccgt ttctggtatt gcaattatgt agccataaac 240
tgttaatcct gtagctatta gcggaccaac aaccagatat acgggatcag cgtcgtaaaa 300
gagatctcca ttctacgttt ctttctaatt tttccgtttc agtgagagaa ttaccctgat 360
acattgacat gatgattgat gattatggga accattccga tgttagacac gagaccatct 420
ggatcctgcc agttttcagt tcacatggca tctcagccca agatcatgtg tttatacgcc 480
taatgacttg tattgaaagt ttggtaagtt gaagatgtgc tctgcccaac agaaaccttc 540
9
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
cttaaatttc cagcaaatct ttcaaacttg gccttacacc ccgaaaatag acgtgcttct 600
acttgggttc ttggaaacca tgcaccaacc gccatacccc accaacccac caccctcaac 660
cttctcttcg ccattacaaa aatgtcagta ccaccctctg aaagacacca acacacccta 720
gctttggtta gggtatttga tataaaaaca aggccaaaac aaaagattgg aaggaagcag 780
aggaagaccc tcttgaaaga attgaagttg taaagagctg gtaaagtggt aataagcaag 840
atg atg gaa tct ggg get cct ata tgc cat acc tgt ggt gaa cag gtg 888
Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glu Gln Val
1 5 10 15
ggg cat gat gca aat ggg gag cta ttt gtg get tgc cat gag tgt agc 936
Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His Glu Cys Ser
20 25 30
tat ccc atg tgc aag tct tgt ttc gag ttt gaa atc aaa gag ggc cgg 984
Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Lys Glu Gly Arg
35 40 45
aaa gtt tgc ttg cgg tgt ggc tcg ag 1010
Lys Val Cys Leu Arg Cys Gly Ser
50 55
<210> 4
<211> 56
<212> PRT
<213> Populus tremuloides
<223> 5' flanking region of PtCelA coding sequence
<400> 4
Met Met Glu Ser Gly Ala Pro Ile Cys His Thr Cys Gly Glu Gln Val
1 5 10 15
Gly His Asp Ala Asn Gly Glu Leu Phe Val Ala Cys His Glu Cys Ser
20 25 30
Tyr Pro Met Cys Lys Ser Cys Phe Glu Phe Glu Ile Lys Glu Gly Arg
35 40 45
Lys Val Cys Leu Arg Cys Gly Ser
50 55
<210> 5
<211> 3444
<212> DNA
<213> Arabidopsis thaliana
<220>
<223> cellulose synthase mRNA
<400> 5
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
gcggccgcgg ttaatcgccg gttctcacaa caggaatgag tttgtcctca ttaatgccga 60
tgagaatgcc cgaataagat cagtccaaga gctgagtgga cagacatgtc aaatctgcag 120
agatgagatc gaattgactg ttgatggaga accgtttgtg gcatgtaacg aatgtgcatt 180
ccctgtgtgt agaccttgct atgagtacga aagacgagaa ggcaatcaag cttgtccaca 240
gtgcaaaacc cgtttcaaac gtcttaaagg aagtccaaga gttgaaggtg atgaagagga 300
agatgacatt gatgatttag acaatgagtt tgagtatgga aataatggga ttggatttga 360
tcaggtttct gaaggtatgt caatctctcg tcgcaactcc ggtttcccac aatctgattt 420
ggattcagct ccacctggct ctcagattcc attgctgact tacggcgacg aggacgttga 480
gatttcttct gatagacatg ctcttattgt tcctccttca cttggtggtc atggcaatag 540
agttcatcct gtttctcttt ctgacccgac cgtggctgca catcgaaggc tgatggtacc 600
tcagaaagat cttgcggttt atggttatgg aagtgtcgct tggaaagatc ggatggagga 660
atggaagaga aagcagaatg agaaacttca ggttgttagg catgaaggag atcctgattt 720
tgaagatggt gatgatgctg attttccaat gatggatgag ggaaggcagc cattgtctat 780
gaagatacca atcaaatcga gcaagataaa tccttaccgg atgttaattg tgctacgtct 840
tgtgattctt ggtctcttct ttcactaccg tattcttcac cccgtcaaag atgcatatgc 900
tttgtggctt atttctgtta tatgtgagat atggtttgct gtttcatggg ttcttgatca 960
gttccctaaa tggtacccta tcgagcgaga aacgtacttg~gaccgactct cattaagata 1020
tgagaaagaa gggaaaccgt cgggactatc ccctgtggat gtatttgtta gtacagtgga 1080
tccattgaaa gagcctccgc ttattactgc aaatactgtc ttgtctattc ttgctgttga 1140
ttatcctgtc gataaggttg cttgttacgt atctgatgat ggtgctgcta tgcttacttt 1200
cgaagctctt tctgagaccg ctgaattcgc aaggaaatgg gttcctttct gcaagaaata 1260
ttgtattgag cctcgtgctc ccgaatggta tttctgccat aaaatggact acttgaagaa 1320
taaagttcat cccgcatttg ttagggagcg gcgagccatg aagagagatt atgaagaatt 1380
caaagtaaag atcaatgctt tagtagcaac agcacagaaa gtgcctgagg atggttggac 1440
tatgcaagac ggtacacctt ggcccggtaa tagtgtgcga gatcatcctg gcatgattca 1500
ggtcttcctt ggaagtgacg gtgttcgtga tgtcgaaaac aacgagttgc ctcgattagt 1560
ttacgtttct cgtgagaaga gacccggatt tgatcaccat aagaaggctg gagctatgaa 1620
ttccctgata cgagtctctg gggttctatc aaatgctcct taccttctga atgtcgattg 1680
tgatcactac atcaacaata gcaaagctct tagagaagca atgtgtttca tgatggatcc 1740
tcagtcagga aagaaaatct gttatgttca gttccctcaa aggttcgatg ggattgatag 1800
gcacgatcga tactcaaatc gcaatgttgt gttctttgat atcaatatga aaggtttgga 1860
tgggctacaa gggcctatat acgtcggtac aggttgtgtt ttcaggaggc aagcgcttta 1920
cggatttgat gcaccgaaga agaagaaggg cccacgtaag acatgcaatt gctggccaaa 1980
atggtgtctc ctatgttttg gttcaagaaa gaatcgtaaa gcaaagacag tggctgcgga 2040
taagaagaag aagaataggg aagcgtcaaa gcagatccac gcattagaaa atatcgaaga 2100
gggccgcggt cataaagttc ttaacgtaga acagtcaacc gaggcaatgc aaatgaagtt 2160
gcagaagaaa tatgggcagt ctcctgtatt tgttgcatct gcgcgtctgg agaatggtgg 2220
gatggctaga aacgcaagcc cggcttgtct gcttaaagaa gccatccaag tcattagtcg 2280
cggatatgaa gataaaactg aatggggaaa agagattggg tggatctatg gttctgttac 2340
cgaagatatt cttacgggtt ctaagatgca ttctcatggt tggagacatg tttattgtac 2400
accaaagtta gcggctttca aaggatcagc tccaatcaat ctttcggatc gtctccatca 2460
agttcttcga tgggcgcttg ggtcggttga gattttcttg agtaggcatt gtcctatttg 2520
gtatggttat ggaggtgggt tgaaatggct tgagcggttg tcctacatta actctgtggt 2580
ttacccgtgg acctctctac cgctcatcgt ttactgttct ctccctgcca tctgtcttct 2640
cactggaaaa ttcatcgttc ccgagattag caactatgcg agtatcctct tcatggcgct 2700
cttctcgtcg attgcaataa cgggtattct cgagatgcaa tggggcaaag ttgggatcga 2760
tgattggtgg agaaacgaac agttttgggt cattggaggt gtttctgcgc atctgtttgc 2820
tctcttccaa ggtctcctca aggttcttgc tggtgtcgac actaacttca cagtcacatc 2880
aaaagcagct gatgatggag agttctctga cctttacctc ttcaaatgga cttcacttct 2940
catccctcca atgactctac tcatcataaa cgtcattgga gtcatagtcg gagtctctga 3000
tgccatcagc aatggatacg actcgtgggg accgcttttc ggaagactgt tctttgcact 3060
ttgggtcatc attcatcttt acccgttcct taaaggtttg cttgggaaac aagatagaat 3120
gccaaccatt attgtcgtct ggtccatcct cctggcctcg attcttacac ttctttgggt 3180
ccgggttaat ccgtttgtgg cgaaaggcgg tcctattctc gagatctgtg gtttagactg 3240
cttgtgattc gattgaccgg tggatgggtt ggtgaaaaag gtttaattcc cacggatcaa 3300
agagaggtaa gagagatatt gttttacctc taaaagactc cttcattgtg ttcattagat 3360
gatgaaaaat gaaaagaaaa agaagattta attttgttac gagaattgtt atttttgcaa 3420
11
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
gaatgtgttg tagatagcgg ccgc 3444
<210> 6
<211> 1080
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> cellulose synthase
<400> 6
Arg Pro Arg Leu Ile Ala Gly Ser His Asn Arg Asn Glu Phe Val Leu
1 5 10 15
Ile Asn Ala Asp Glu Asn Ala Arg Ile Arg Ser Val Gln Glu Leu Ser
20 25 30
Gly Gln Thr Cys Gln Ile Cys Arg Asp Glu Ile Glu Leu Thr Val Asp
35 40 45
Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro Val Cys Arg
50 55 60
Pro Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Asn Gln Ala Cys Pro Gln
65 70 75 80
Cys Lys Thr Arg Phe Lys Arg Leu Lys Gly Ser Pro Arg Val Glu Gly
85 90 95
Asp Glu Glu Glu Asp Asp Ile Asp Asp Leu Asp Asn Glu Phe Glu Tyr
100 105 110
Gly Asn Asn Gly Ile Gly Phe Asp Gln Val Ser Glu Gly Met Ser Ile
115 120 125
Ser Arg Arg Asn Ser Gly Phe Pro Gln Ser Asp Leu Asp Ser Ala Pro
130 135 140
Pro Gly Ser Gln Ile Pro Leu Leu Thr Tyr Gly Asp Glu Asp Val Glu
145 150 155 160
Ile Ser Ser Asp Arg His Ala Leu Ile Val Pro Pro Ser Leu Gly Gly
165 170 175
His Gly Asn Arg Val His Pro Val Ser Leu Ser Asp Pro Thr Val Ala
180 185 190
Ala His Arg Arg Leu Met Val Pro Gln Lys Asp Leu Ala Val Tyr Gly
195 200 205
Tyr Gly Ser Val Ala Trp Lys Asp Arg Met Glu Glu Trp Lys Arg Lys
210 215 220
Gln Asn Glu Lys Leu Gln Val Val Arg His Glu Gly Asp Pro Asp Phe
225 230 235 240
12
SUBSTITUTE S~IEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Glu Asp Gly Asp Asp Ala Asp Phe Pro Met Met Asp Glu Gly Arg Gln
245 250 255
Pro Leu Ser Met Lys Ile Pro Ile Lys Ser Ser Lys Ile Asn Pro Tyr
260 265 270
Arg Met Leu Ile Val Leu Arg Leu Val Ile Leu Gly Leu Phe Phe His
275 280 285
Tyr Arg Ile Leu His Pro Val Lys Asp Ala Tyr Ala Leu Trp Leu Ile
290 295 300
Ser Val Ile Cys Glu Ile Trp Phe Ala Val Ser Trp Val Leu Asp Gln
305 310 315 320
Phe Pro Lys Trp Tyr Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg Leu
325 330 335
Ser Leu Arg Tyr Glu Lys Glu Gly Lys Pro Ser Gly Leu Ser Pro Val
340 345 350
Asp Val Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile
355 360 365
Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp
370 375 380
Lys Val Ala Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe
385 390 395 400
Glu Ala Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe
405 410 415
Cys Lys Lys Tyr Cys Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Cys
420 425 430
His Lys Met Asp Tyr Leu Lys Asn Lys Val His Pro Ala Phe Val Arg
435 440 445
Glu Arg Arg Ala Met Lys Arg Asp Tyr Glu Glu Phe Lys Val Lys Ile
450 455 460
Asn Ala Leu Val Ala Thr Ala Gln Lys Val Pro Glu Asp Gly Trp Thr
465 470 475 480
Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Ser Val Arg Asp His Pro
485 490 495
Gly Met Ile Gln Val Phe Leu Gly Ser Asp Gly Val Arg Asp Val Glu
500 505 510
Asn Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro
515 520 525
Gly Phe Asp His His Lys Lys Ala Gly Ala Met Asn Ser Leu Ile Arg
530 535 540
13
SUBSTITUTE SHEET (RULE 26)
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Val Ser Gly Val Leu Ser Asn Ala Pro Tyr Leu Leu Asn Val Asp Cys
545 550 555 560
Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys Phe
565 570 575
Met Met Asp Pro Gln Ser Gly Lys Lys Ile Cys Tyr Val Gln Phe Pro
580 585 590
Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ser Asn Arg Asn
595 600 605
Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Leu Gln Gly
610 615 620
Pro Ile Tyr Val Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly
625 630 635 640
Phe Asp Ala Pro Lys Lys Lys Lys Gly Pro Arg Lys Thr Cys Asn Cys
645 650 655
Trp Pro Lys Trp Cys Leu Leu Cys Phe Gly Ser Arg Lys Asn Arg Lys
660 665 670
Ala Lys Thr Val Ala Ala Asp Lys Lys Lys Lys Asn Arg Glu Ala Ser
675 680 685
Lys Gln Ile His Ala Leu Glu Asn Ile Glu Glu Gly Arg Gly His Lys
690 695 700
Val Leu Asn Val Glu Gln Ser Thr Glu Ala Met Gln Met Lys Leu Gln
705 710 715 720
Lys Lys Tyr Gly Gln Ser Pro Val Phe Val Ala Ser Ala Arg Leu Glu
725 730 735
Asn Gly Gly Met Ala Arg Asn Ala Ser Pro Ala Cys Leu Leu Lys Glu
740 745 750
Ala Ile Gln Val Ile Ser Arg Gly Tyr Glu Asp Lys Thr Glu Trp Gly
755 760 765
Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr
770 775 780
Gly Ser Lys Met His Ser His Gly Trp Arg His Val Tyr Cys Thr Pro
785 790 795 800
Lys Leu Ala Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg
805 810 815
Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Leu
820 825 830
Ser Arg His Cys Pro Ile Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Trp
835 840 845
14
SUBSTITUTE SFtEET (RULE 26,j
CA 02371659 2001-11-19
WO 00/71670 PCT/US00/13637
Leu Glu Arg Leu Ser Tyr Ile Asn Ser Val Val Tyr Pro Trp Thr Ser
850 855 860
Leu Pro Leu Ile Val Tyr Cys Ser Leu Pro Ala Ile Cys Leu Leu Thr
865 870 875 880
Gly Lys Phe Ile Val Pro Glu Ile Ser Asn Tyr Ala Ser Ile Leu Phe
885 890 895
Met Ala Leu Phe Ser Ser Ile Ala Ile Thr Gly Ile Leu Glu Met Gln
900 905 910
Trp Gly Lys Val Gly Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp
915 920 925
Val Ile Gly Gly Val Ser Ala His Leu Phe Ala Leu Phe Gln Gly Leu
930 935 940
Leu Lys Val Leu Ala Gly Val Asp Thr Asn Phe Thr Val Thr Ser Lys
945 950 955 960
Ala Ala Asp Asp Gly Glu Phe Ser Asp Leu Tyr Leu Phe Lys Trp Thr
965 970 975
Ser Leu Leu Ile Pro Pro Met Thr Leu Leu Ile Ile Asn Val Ile Gly
980 985 990
Val Ile Val Gly Val Ser Asp Ala Ile Ser Asn Gly Tyr Asp Ser Trp
995 1000 1005
Gly Pro Leu Phe Gly Arg Leu Phe Phe Ala Leu Trp Val Ile Ile His
1010 1015 1020
Leu Tyr Pro Phe Leu Lys Gly Leu Leu Gly Lys Gln Asp Arg Met Pro
1025 1030 1035 1040
Thr Ile Ile Val Val Trp Ser Ile Leu Leu Ala Ser Ile Leu Thr Leu
1045 1050 1055
Leu Trp Val Arg Val Asn Pro Phe Val Ala Lys Gly Gly Pro Ile Leu
1060 1065 1070
Glu Ile Cys Gly Leu Asp Cys Leu
1075 1080
SUBSTITUTE SHEET (RULE 26)
