Coniferin beta-glucosidase cDNA for modifying lignin content in plants

A cDNA molecule encoding coniferin beta-glucosidase is disclosed. This enzyme catalyzes one of the last steps in the synthesis of lignin in plants. Plants having modified lignin content may be produced by transformation with this cDNA (or parts of the cDNA), for example, in either sense or antisense orientation. The invention includes methods of altering-lignin content in plants using this cDNA, as well as transformed plants, such as conifers, having modified lignin content.

TECHNICAL FIELD 
This invention relates to DNA molecules cloned from plants and methods of 
using such DNA molecules to produce transgenic plants with altered lignin 
content. 
BACKGROUND 
Lignin is the second most abundant organic material in the biosphere, and 
is a major component of cell walls of woody plants (such as poplar and 
pine species) and fodder crops (such as maize, wheat and barley). The 
quantity of lignin in plant material affects characteristics that are 
agronomically important. For example, in fodder crops the amount of lignin 
present determines how easily the crop may be digested by animals; 
relatively small increases in lignin content may produce large decreases 
in the digestibility of the crop. Therefore, reducing lignin content would 
enhance digestibility, facilitating a more efficient use of such crops. In 
the timber industry, producing wood pulp for papermaking requires the 
removal of lignin to release the cellulosic content of the timber. The 
process of removing the lignin consumes large amounts of energy and 
produces environmentally harmful lignin waste liquors which must be 
treated prior to disposal. It has also been suggested that residual lignin 
in paper pulp may produce toxic polychlorinated biphenols when the lignin 
interacts with chlorine used in the bleaching process. Thus, decreasing 
lignin content in wood products would be advantageous for papermaking. On 
the other hand, increasing the lignin content of timber offers the 
possibility of increased wood strength. 
Accordingly, modification of quality and quantity of lignin in plants has 
been a long-standing interest among breeders and, more recently, among 
molecular biologists. Recent molecular approaches towards methods for 
reducing lignin content in plants are typified by: U.S. Pat. No. 
5,451,514, "Modification of Lignin Synthesis in Plants"; Canadian Patent 
No. 2,005,597, "Plants Having Reduced Lignin or Lignin of Altered 
Quality"; and International Patent Application Publication No. WO 
94/23044. 
Lignin is a complex polymer of three cinnamyl alcohols, p-coumaryl, 
coniferyl and sinapyl, all products of phenylpropanoid metabolism. 
Depending on the plant species or tissue, the relative proportion of the 
different monomers in lignin can vary significantly. In gymnosperms for 
example, lignin is predominantly composed of coniferyl alcohol monomer 
units, whereas angiosperms have significant proportions of sinapyl 
moieties. The metabolism of lignin production involves many intermediates, 
enzymatic pathways and, correspondingly, genes. Accordingly, there are 
several gene/enzyme targets that might be selected to manipulate lignin 
production through genetic engineering. 
Alteration of lignin levels by antisense and sense suppression of gene 
expression has already been attempted for several enzymes in the 
phenylpropanoid pathway including (Elkind et al. 1990), CAD (Schuch 
1993; Canadian patent 2,005,597; U.S. Pat. No. 5,541,514), 4CL (Lee and 
Douglas 1994) and COMT (WO 94/23044). However, all of these attempts to 
modify lignin synthesis are directed at early stages in the synthetic 
pathway and are therefore likely to interfere with other metabolic 
processes which share these intermediate steps. It is clear, for example, 
that interference with early steps in the phenylpropanoid pathway can have 
undesirable pleiotropic effects (Elkind et al., 1990). In addition, 
modulating biosynthetic enzymes that act early in the pathway may not be 
effective because alternative synthetic routes may be available. A better 
approach to modulating lignin synthesis would be to regulate later stages 
in the lignin biosynthesis pathway: this would minimize or avoid 
pleiotropic effects and would likely provide a greater degree of effective 
control. 
The present invention is directed towards the identification of a gene that 
regulates a later step in the lignin biosynthesis pathway, and the use of 
this gene to modify lignin content in plants. 
SUMMARY OF THE INVENTION 
The inventors have determined that the gene encoding coniferin 
beta-glucosidase would be an excellent target gene for modifying lignin 
content in plants, particularly in trees such as conifers. The coniferin 
beta-glucosidase enzyme catalyzes the hydrolysis of the 4-O-glucoside of 
coniferyl alcohol, coniferin, which is one of the last steps in the 
biosynthesis of lignin. Thus, the level of coniferin beta-glucosidase 
activity directly affects lignin synthesis and, therefore, the quantity of 
lignin in the plant tissue. Coniferin accumulates in conifer xylem during 
cambium reactivation, consistent with a role as the dominant lignin 
precursor in these species (Freudenberg and Harkin 1963, Savidge 1989). 
Beta-glucosidases capable of hydrolyzing coniferin have been detected in 
suspension culture systems (Hosel et al. 1982, Hosel and Todenhagen 1980) 
and seedlings (Marcinowski and Grisebach 1978), and a coniferin 
beta-glucosidase has been purified from differentiating xylem in trees 
(Dharmawardhana et al., 1995). However, to date, the genetic manipulation 
of coniferin beta-glucosidase has not been possible because the gene 
encoding the enzyme has not been cloned. 
To that end, the inventors have cloned and sequenced a complementary DNA 
(cDNA) sequence from the conifer tree species Pinus contorta. The 
provision of this cDNA sequence enables, for the first time, the 
regulation of coniferin beta-glucosidase activity in plants through 
genetic engineering. Specifically, the invention provides genetic 
constructs, such as plant transformation vectors, that include various 
forms of the coniferin beta-glucosidase cDNA or sequences that are 
homologous to this cDNA. Depending on the specific nature of these 
constructs, they may be introduced into plants in order to increase or 
reduce the production of the coniferin beta-glucosidase enzyme, and 
therefore to regulate lignin synthesis. 
Transformation vectors according to this invention preferably include a 
recombinant DNA sequence that comprises all or part of the coniferin 
beta-glucosidase cDNA. Depending on the nature of the promoter sequence 
selected, such constructs may be used to modify lignin content throughout 
the plant or in a tissue-specific manner and either constitutively or at 
certain stages of plant development. The availability of inducible plant 
promoters also offers the possibility of changing lignin biosynthesis in a 
plant at desired times by application of the chemical or physical agent 
that induces transcription from the promoter. 
In one embodiment, transformation vectors may be constructed to 
over-express the coniferin beta- glucosidase enzyme ("sense" orientation). 
Enhanced lignin synthesis may be achieved by introducing such vectors into 
plants. Examples of the application of this approach to modify plant 
phenotypes include U.S. Pat. No. 5,268,526, "Overexpression of Phytochrome 
in Transgenic Plants", U.S. Pat. No. 4,795,855, "Transformation and 
Foreign Gene Expression in Woody Species", and U.S. Pat. No. 5,443,974 
(over-expression of stearoyl-ACP desaturase gene). 
Alternatively, such over-expression vectors may be used to suppress 
coniferin beta-glucosidase enzyme activity through sense-suppression, as 
described in U.S. Pat. Nos. 5,034,323 and 5,283,184, both entitled 
"Genetic Engineering of Novel Plant Phenotypes". 
In another embodiment, constructs may be designed to express plus-sense 
untranslatable coniferin beta-glucosidase RNA, using methodologies 
described in U.S. Pat. No: 5,583,021, "Production of Virus Resistant 
Plants". Constructs of this type may be used to reduce the expression of 
the native coniferin beta-glucosidase gene, thereby reducing coniferin 
beta-glucosidase enzyme activity and, as a result, lignin content. 
In other embodiments, the present invention provides genetic constructs 
designed to express antisense versions of the coniferin beta-glucosidase 
RNA. "Antisense" RNA is an RNA sequence that is the reverse complement of 
the mRNA encoded by a target gene. Examples of the use of antisense RNA to 
inhibit expression of target plant genes include U.S. Pat. No. 5,451,514, 
"Modification of Lignin Synthesis in Plants" (use of antisense RNA to 
regulate CAD), U.S. Pat. No. 5,356,799, "Antisense Gene Systems of 
Pollination Control for Hybrid Seed Production", U.S. Pat. No. 5,530,192 
(use of antisense RNA to alter amino acid and fatty acid composition in 
plants). 
In conjunction with these genetic constructs, the present invention also 
includes methods for altering lignin biosynthesis in plants. Generally, 
such methods comprise introducing into the genome of a plant a genetic 
construct that includes all or part of the coniferin beta-glucosidase cDNA 
(either in sense or antisense orientation) or a sequence derived from this 
cDNA. Methods for introducing transformation vectors into plants are well 
known in the art and include electroporation of plant protoplasts, 
liposome-mediated transformation, polyethylene mediated transformation; 
transformation using viruses, micro-injection of plant cells, 
micro-projectile bombardment of plant cells, vacuum infiltration, and 
Agrobacterium tumeficiens (AT) mediated transformation. Methods 
particularly suited to the transformation of woody species are described 
in Ellis et al. (1993), Ellis et al. (1996), U.S. Pat. No. 5,122,466, 
"Ballistic Transformation of Conifer" and U.S. Pat. No. 4,795,855, 
"Transformation and Foreign Gene Expression with Woody Species". 
The invention also includes transformed plants having altered lignin 
compositions as a result of being transformed with a genetic construct as 
described above. Examples of plants that may be transformed in this manner 
include conifers, such as plants from the genera Picea, Pseudotsuga, 
Tsuga, Sequoia, Abies, Thuja, Libocedrus, Chamaecyparis and Laryx. Pines 
are expected to be a particularly suitable choice for genetic modification 
by the methods disclosed herein, including loblolly pine (Pinus taeda), 
slash pine (Pinus elliotii), longleaf pine (Pinus palustris), shortleaf 
pine (Pinus echinata), jack pine (Pinus banksiana), ponderosa pine (Pinus 
ponderosa), red pine (Pinus resinosa), Eastern white pine (Pinus strobus), 
Western white pine (Pinus monticola), sugar pine (Pinus lambertiana), 
lodgepole pine (Pinus contorta), Monterey pine (Pinus radiata), Afghan 
pine (Pinus eldarica), Scots pine (Pinus sylvestris) and Virginia pine 
(Pinus virginiana). Other tree species, including poplar, eucalyptus and 
aspen may also be transformed using the nucleotide sequences of this 
invention. However, the invention is not limited to trees: crop and forage 
plants such as maize, tobacco, alfalfa, wheat and grasses may also be 
transformed using the constructs provided by this invention in order to 
modify lignin content. In general, this invention can be applied to any 
plant species that can be transformed.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions and Abbreviations 
The following abbreviations are used herein: 
4-NPG: 4-nitrophenyl .beta.-glucoside 
2-NPG: 2-nitrophenyl .beta.-glucoside 
MUG: 4-methylumbelliferyl .beta.-glucoside 
VRA-G: 5,4-( .beta.-D-glucopyranosyloxy)-3- methoxyphenylmethylene 
-2-thioxothiazolidin-4-one-3-ethanoic acid. VRA-G is a substrate analog of 
coniferin synthesized by Biosynth International Inc., Skoke, Ill. 
EDC: 1-ethyl-3-(dimethylaminopropyl) carbodiimide 
: phenylalanine anunonia-lyase 
CAD: Cinnamyl alcohol dehydrogenase 
4CL: 4-coumarate: CoA ligase 
COMT: caffeic acid 3-o-methyltransferase 
PAGE: polyacrylamide gel electrophoresis 
CBG: coniferin .beta.-glucosidase 
Unless otherwise noted, technical terms are used according to conventional 
usage. Definitions of common terms in molecular biology may be found in 
Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 
0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular 
Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); 
and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a 
Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 
(ISBN 1-56081-569-8). 
In order to facilitate review of the various embodiments of the invention, 
the following definitions of terms are provided: 
Coniferin Beta-glucosidase Biological Activity 
The defining functional characteristic of the coniferin beta-glucosidase 
enzyme is its ability to hydrolyze coniferin to release coniferyl alcohol. 
This activity can be measured using the glucosidase assay described 
herein. Thus, a protein having coniferin beta-glucosidase biological 
activity is a protein that is able to hydrolyze coniferin to release 
coniferyl alcohol in the described assay. 
Coniferin Beta-glucosidase (CBG) Protein 
A protein having coniferin beta-glucosidase biological activity and sharing 
amino acid sequence identity with the amino acid sequence of the 
prototypical coniferin beta-glucosidase protein shown in Seq. I.D. No. 7. 
(the Pinus CBG protein). CBG proteins that are more distantly related to 
the prototypical CBG protein will share at least 50% amino acid sequence 
identity with the sequence shown in Seq. I.D. No. 7, as determined by the 
methods described below. More closely related CBG proteins may share at 
least 60%, 65%, 70%, 75% or 80% sequence identity with the Pinus CBG 
protein. CBG proteins that are most closely related to the Pinus protein 
will have CBG protein biological activity and share at least 85%, 90% or 
95% sequence identity with the Pinus protein. 
CBG gene/ CBG cDNA 
Nucleic acid molecules that encode a CBG protein. Nucleic acid molecules 
that encode the Pinus CBG protein are provided in Seq. I.D. No. 6 (Pinus 
CBG CDNA), and Seq. I.D. No. 8 (Pinus CBG ORF). The invention includes not 
only the nucleic acid molecules provided in Seq. I.D. Nos. 6 and 8, but 
also homologs and orthologs of these sequences, nucleic acid molecules 
that encode CBG proteins, and probes and primers that are derived from 
these sequences. 
Probes and Primers 
Nucleic acid probes and primers may readily be prepared based on the 
nucleic acids provided by this invention. A probe comprises an isolated 
nucleic acid attached to a detectable label or reporter molecule. Typical 
labels include radioactive isotopes, ligands, chemiluminescent agents, and 
enzymes. Methods for labeling and guidance in the choice of labels 
appropriate for various purposes are discussed, e.g., in Sambrook et al. 
(1989) and Ausubel et al. (1987). 
Primers are short nucleic acids, preferably DNA oligonucleotides 15 
nucleotides or more in length. Primers may be annealed to a complementary 
target DNA strand by nucleic acid hybridization to form a hybrid between 
the primer and the target DNA strand, and then extended along the target 
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for 
amplification of a nucleic acid sequence, e.g., by the polymerase chain 
reaction (PCR) or other nucleic-acid amplification methods known in the 
art. 
Methods for preparing and using probes and primers are described, for 
example, in Sambrook et al. (1989), Ausubel et al. (1987), and Innis et 
al., (1990). PCR primer pairs can be derived from a known sequence, for 
example, by using computer programs intended for that purpose such as 
Primer (Version 0.5, .COPYRGT.1991, Whitehead Institute for Biomedical 
Research, Cambridge, Mass. One of skill in the art will appreciate that 
the specificity of a particular probe or primer increases with its length. 
Thus, for example, a primer comprising 20 consecutive nucleotides of the 
Pinus CBG cDNA will anneal to a target sequence such as a CBG gene homolog 
from eucalyptus contained within a eucalyptus cDNA library with a higher 
specificity than a corresponding primer of only 15 nucleotides. Thus, in 
order to obtain greater specificity, probes and primers may be selected 
that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of 
the Pinus CBG cDNA or gene sequences. 
The invention thus includes isolated nucleic acid molecules that comprise 
specified lengths of the disclosed CBG cDNA sequence. Such molecules may 
comprise at least 20, 25, 30, 35, 40 or 50 consecutive nucleotides of 
these sequences and may be obtained from any region of the disclosed 
sequences. By way of example, the Pinus CBG cDNA sequences may be 
apportioned into halves or quarters based on sequence length, and the 
isolated nucleic acid molecule may be derived from the first or second 
halves of the molecule, or any of the four quarters. The Pinus CBG cDNA, 
shown in Seq. I.D. No. 6 may be used to illustrate this. The Pinus CBG 
cDNA is 1909 nucleotides in length and so may be hypothetically divided 
into halves (nucleotides 1-955 and 956-1909) or quarters (nucleotides 
1-477, 478-955, 956-1433 and 1434-1909). Nucleic acid molecules may be 
selected that comprise at least 20, 25, 30, 35, 40 or 50 consecutive 
nucleotides of any of these portions of the Pinus cDNA. Thus, one such 
nucleic acid molecule might comprise at least 25 consecutive nucleotides 
of the region comprising nucleotides 1-955 of the disclosed Pinus cDNA. 
Sequence Identity 
the similarity between two nucleic acid sequences, or two amino acid 
sequences is expressed in terms of the similarity between the sequences, 
otherwise referred to as sequence identity. Sequence identity is 
frequently measured in terms of percentage identity (or similarity or 
homlogy); the higher the percentage, the more similar the two sequences 
are. Homologs of the Pinus CBG protein will possess a relatively high 
degree of sequence identity when aligned using standard methods. 
Methods of alignment of sequences for comparison are well known in the art. 
Various programs and alignment algorithms are described in: Smith and 
Waterman (1981); Needleman and Wunsch (1970); Pearson and Lipman (1988); 
Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet et al. (1988); 
Huang et al. (1992); and Pearson et al. (1994). Altschul et al. (1994) 
presents a detailed consideration of sequence alignment methods and 
homology calculations. 
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) 
is available from several sources, including the National Center for 
Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for 
use in connection with the sequence analysis programs blastp, blastn, 
blastx, tblastn and tblastx. It can be accessed at 
htp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine 
sequence identity using this program is available at 
http://www.ncbi.nlm.nih.gov/BLAST/blast help.html. 
Homologs of the disclosed Pinus CBG protein are typically characterized by 
possession of at least 50% sequence identity counted over the full length 
alignment with the amino acid sequence of Pinus CBG using the NCBI Blast 
2.0, gapped blastp set to default parameters. Proteins with even greater 
similarity to the reference sequences will show increasing percentage 
identities when assessed by this method, such as at least 60%, at least 
65%, at least 70%, at least 75%, at least 80%, at least 90% or at least 
95% sequence identity. When less than the entire sequence is being 
compared for sequence identity, homologs will typically possess at least 
75% sequence identity over short windows of 10-20 amino acids, and may 
possess sequence identities of at least 85% or at least 90% or 95% 
depending on their similarity to the reference sequence. Methods for 
determining sequence identity over such short windows are described at 
http://www.ncbi.nlm. nih.gov/BLAST/blast FAQs.html. One of skill in the 
art will appreciate that these sequence identity ranges are provided for 
guidance only; it is entirely possible that strongly significant homologs 
could be obtained that fall outside of the ranges provided. The present 
invention provides not only the peptide homologs are described above, but 
also nucleic acid molecules that encode such homologs. 
An alternative indication that two nucleic acid molecules are closely 
related is that the two molecules hybridize to each other under stringent 
conditions. Stringent conditions are sequence dependent and are different 
under different environmental parameters. Generally, stringent conditions 
are selected to be about 5.degree. C. to 20.degree. C. lower than the 
thermal melting point (Tm) for the specific sequence at a defined ionic 
strength and pH. The T.sub.m is the temperature (under defined ionic 
strength and pH) at which 50% of the target sequence hybridizes to a 
perfectly matched probe. Conditions for nucleic acid hybridization and 
calculation of stringencies can be found in Sambrook et al. (1989) and 
Tijssen (1993). Nucleic acid molecules that hybridize under stringent 
conditions to the Pinus CBG sequences will typically hybridize to a probe 
based on either the entire Pinus CBG cDNA or selected portions of the cDNA 
under wash conditions of 0.2.times.SSC, 0.1% SDS at 65.degree. C. 
Nucleic acid sequences that do not show a high degree of identity may 
nevertheless encode similar amino acid sequences, due to the degeneracy of 
the genetic code. It is understood that changes in nucleic acid sequence 
can be made using this degeneracy to produce multiple nucleic acid 
sequence that all encode substantially the same protein. 
Specific Binding Agent 
An agent that binds substantially only to a defined target. Thus a CBG 
protein specific binding agent binds substantially only the CBG protein. 
As used herein, the term "CBG protein specific binding agent" includes 
anti-CBG protein antibodies and other agents that bind substantially only 
to the CBG protein. Anti-CBG protein antibodies may be produced using 
standard procedures described in a number of texts, including Harlow and 
Lane (1988). The determination that a particular agent binds substantially 
only to the CBG protein may readily be made by using or adapting routine 
procedures. One suitable in vitro assay makes use of the Western blotting 
procedure (described in many standard texts, including Harlow and Lane 
(1988)). Western blotting may be used to determine that a given CBG 
protein binding agent, such as an anti-CBG protein monoclonal antibody, 
binds substantially only to the CBG protein. 
Oligonucleotide 
A linear polynucleotide sequence of up to about 100 nucleotide bases in 
length. 
Vector 
A nucleic acid molecule as introduced into a host cell, thereby producing a 
transformed host cell. A vector may include nucleic acid sequences that 
permit it to replicate in a host cell, such as an origin of replication. A 
vector may also include one or more selectable marker genes and other 
genetic elements known in the art. 
Transformed 
A transformed cell is a cell into which has been introduced a nucleic acid 
molecule by molecular biology techniques. As used herein, the term 
transformation encompasses all techniques by which a nucleic-acid molecule 
might be introduced into such a cell, including transfection with viral 
vectors, transformation with plasmid vectors, and introduction of naked 
DNA by electroporation, lipofection, and particle gun acceleration. 
Isolated 
An "isolated" biological component (such as a nucleic acid or protein or 
organelle) has been substantially separated or purified away from other 
biological components in the cell of the organism in which the component 
naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and 
RNA, proteins and organelles. Nucleic acids and proteins that have been 
"isolated" include nucleic acids and proteins purified by standard 
purification methods. The term also embraces nucleic acids and proteins 
prepared by recombinant expression in a host cell as well as chemically 
synthesized nucleic acids. 
Purified 
The term purified does not require absolute purity; rather, it is intended 
as a relative term. Thus, for example, a purified CBG protein preparation 
is one in which the CBG protein is more enriched than the protein is in 
its natural environment within a cell. Generally, a preparation of CBG 
protein is purified such that the CBG represents at least 50% of the total 
protein content of the preparation. For particular applications, higher 
purity may be desired, such that preparations in which CBG represents at 
least 75% or at least 90% of the total protein content may be employed. 
Ortholog 
Two nucleotide or amino acid sequences are orthologs of each other if they 
share a common ancestral sequence and diverged when a species carrying 
that ancestral sequence split into two species. Orthologous sequences are 
also homologous sequences. 
Operably Linked 
A first nucleic acid sequence is operably linked with a second nucleic acid 
sequence when the first nucleic acid sequence is placed in a functional 
relationship with the second nucleic acid sequence. For instance, a 
promoter is operably linked to a coding sequence if the promoter affects 
the transcription or expression of the coding sequence. Generally, 
operably linked DNA sequences are contiguous and, where necessary to join 
two protein-coding regions, in the same reading frame. 
Recombinant 
A recombinant nucleic acid is one that has a sequence that is not naturally 
occurring or has a sequence that is made by an artificial combination of 
two otherwise separated segments of sequence. This artificial combination 
is often accomplished by chemical synthesis or, more commonly, by the 
artificial manipulation of isolated segments of nucleic acids, e.g., by 
genetic engineering techniques. 
cDNA (Complementary DNA) 
A piece of DNA lacking internal, non-coding segments (introns) and 
regulatory sequences that determine transcription. cDNA is synthesized in 
the laboratory by reverse transcription from messenger RNA extracted from 
cells. 
ORF (Open Reading Frame) 
A series of nucleotide triplets (codons) coding for amino acids without any 
termination codons. These sequences are usually translatable into a 
peptide. 
Transgenic Plant 
As used herein, this term refers to a plant that contains recombinant 
genetic material not normally found in plants of this type and which has 
been introduced into the plant in question (or into progenitors of the 
plant) by human manipulation. Thus, a plant that is grown from a plant 
cell into which recombinant DNA is introduced by transformation is a 
transgenic plant, as are all offspring of that plant that contain the 
introduced transgene (whether produced sexually or asexually). 
Sequence Listing 
The nucleic and amino acid sequences listed in the accompanying sequence 
listing are showed using standard letter abbreviations for nucleotide 
bases, and three letter code for amino acids. The letter "N" is used to 
indicate inosine in the nucleic acid sequences. Only one strand of each 
nucleic acid sequence is shown, but the complementary strand is understood 
as included by any reference to the displayed strand. 
Seq. I.D. No. 1 is primer N7A 
Seq. I.D. No. 2 is primer N7B 
Seq. I.D. No. 3 is primer N10 
Seq. I.D. No. 4 is primer CBG172 
Seq. I.D. No. 5 is primer CBG75 
Seq. I.D. No. 6 is the Pinus CBG cDNA 
Seq. I.D. No. 7 is the Pinus CBG protein 
Seq. I.D. No. 8 is the Pinus CBG ORF 
Seq. I.D. No. 9 is primer NT1 
Seq. I.D. No. 10 is primer CT1 
Seq. I.D. Nos. 11-14 are primers useful for amplification of the CBG cDNA 
sequence. 
Detailed aspects of the invention are provided in the following examples. 
EXAMPLE 1 
Identification of the Coniferin beta-glucosidase cDNA 
Actively differentiating Pinus contorta xylem was harvested as described by 
Dharmwardhana et al. (1995) and used to isolate total RNA as described by 
Lewinsohn et al. (1994). PolyA RNA isolated with an Oligotex mRNA 
isolation kit (Qiagen) was used to construct a cDNA library in the 
.lambda.ZAP-XR vector, employing Stratagene cDNA synthesis and GigapakIl 
Gold packaging kits. 
Coniferin beta-glucosidase enzyme was purified from Pinus contorta xylem 
tissue as described by Dharmwardhana et al. (1995). In order to determine 
the N-terminal amino acid sequence of the purified enzymne, it was run on 
native PAGE gels, stained for activity on the synthetic coniferin 
substrate VRA-G and the staining band excised and subjected to SDS-PAGE. 
The protein was then transferred to an Immobilon membrane for N-terminal 
amino acid sequencing using an Applied Biosystems 470A gas phase sequencer 
(Edman degradation). 
Gene-specific primers for PCR amplification of CBG sequence fragments were 
then designed based on the 15 N-terminal amino acid sequence obtained. 
Primers N7A and N7B were based on the first 7 N-terminal amino acid 
residues and were identical except at the third base from the 3' end where 
the degeneracy is split between the primers. 
N7A: 5' GCTCTAGAGCGAC(T)A(C)GIAAC(T)AAC(T)TTTCC 3' (Seq. I.D. No. 1) N7B: 
5' GCTCTAGAGCGAC(T)A(C)GIAAC(T)AAC(T)TTCCC 3' (Seq. I.D. No. 2) The 
amplification template used was the .lambda.ZAP-cDNA library described 
above. The initial PCR reactions contained 200-300 ng .lambda.ZAP-cDNA as 
template, 200 nM degenerate gene-specific primer N7A or N7B, 50 nM vector 
primer M13F or T7 (BRL), 200 M dNTP, and 1X reaction buffer (10 mM TrisHCl 
pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl) in a 50.mu.l volume. Prior to adding 
3 units Taq polymerase (Boehringer), the reaction mixture was heated to 
94.degree. C. for 2 min. The thermal cycling regime was as follows: 1-2 
cycles (94.degree. C./1 min., 48.degree.-52.degree. C./2 min., 72.degree. 
/2 min.);30 cycles (94.degree. C./45 sec., 55.degree./1 min., 72.degree. 
C./2 min.); 72.degree. C./10 min. extension. 
Amplification using primer N7B yielded 3-4 major bands, whereas 
amplification with N7B did not yield consistent product, suggesting a 
mismatch at the degenerate third base. To increase specificity and 
identify the desired amplification product, a 20 ng aliquot of reaction 
products from the initial PCR using N7B was reamplified using the 
partially nested gene-specific primer N10 [GAC(T)A(C)GIAAC(T)AAC(T) 
TTCCCIT(A)C(G)IGA(T)TT, Seq. I.D. No. 3] and vector primer T7 (30 cycles 
of 94.degree. C./45 sec., 55.degree./1 min., 72.degree. C./ 2 min. 
followed by 72.degree. C./10 min. final extension), yielding a 1.7 kb 
band. 
Following identification of the 1.7 kb band as the desired amplification 
product, the initial PCR reaction was repeated with less (0.9 mM) 
MgCl.sub.2 in the reaction buffer. The resulting 1.7 kb band was then 
isolated by gel purification (Qiagen) and cloned into EcoRV-digested 
T-tailed Bluescript II KS vector according to the T/A cloning protocol 
(Holton and Graham, 1991). Plasmid minipreps from several clones were used 
for restriction analysis of insert and for primer-directed sequencing of 
both strands using ABI AmpliTaq dye termination cycle sequencing. 
To amplify the 5' end of the CBG cDNA, .lambda.ZAP-cDNA from the library 
was again used as a template, this time in conjunction with a T3 vector 
primer and the gene-specific primer CBG172 (CACATATCTGTGATATTGGTCG, Seq. 
I.D. No. 4) based on the sequence of the 3' CBG amplification product. A 
second nested gene-specific primer CBG75 (CCATCTTCTCGGACTGCTC, Seq. I.D. 
No. 5) was used to re-amplify the former reaction products to confirm the 
authenticity of the PCR product. The cloning and sequencing of the 5' PCR 
product was conducted as described above. An exact sequence match in the 
overlapping regions of the 5' and and 3' end clones confirmed the 
authenticity of the 5' amplification product. 
EXAMPLE 2 
Analysis of the CBG cDNA Sequence 
The complete CBG cDNA sequence, shown in FIG. 1 and in Seq. I.D. No. 6, is 
1909 bp in length. The cDNA includes an open reading frame (Seq. I.D. No. 
8) that encodes a 513 amino acid protein (Seq. I.D. No. 7). The 5' and 
3'-untranslated regions of the cDNA contain 162 and 187 nucleotides, 
respectively. The 3'-untranslated region does not contain the conserved 
eukaryotic polyadenylation signal AAUAAA, as is the case for more than 50% 
of reported plant mRNA sequences (Wu et al., 1995). Instead, the CBG 
3'-untranslated region contains AAUAAA-like sequences like most plant 
mRNAs (Joshi, 1987). 
The 5'-UTR of the CBG cDNA carries a 9 bp AC-rich element (AACCAACAA) that 
is also present in Arabidopsis 1 and bean chalcone synthase (CHS15) 
genes, and has been proposed to be an elicitor-inducible hypersensitive 
site (Lawton et al., 1990; Ohl et al., 1990). This indirectly associates 
CBG with other phenylpropanoid metabolic genes/regulation, and is 
consistent with the induction of CBG activity in jackpine cell cultures by 
fungal elicitation (Campbell & Ellis, 1991). 
The deduced 513 amino acid protein has a molecular weight of 58.3 kD and a 
calculated isoelectric point of pH 4.9. The N-terminal amino acid sequence 
determined for the purified enzyme corresponds to amino acids 24 -40 in 
the deduced sequence. Met35 in the deduced sequence was identified as Thr 
during N-terminal amino acid sequencing. This mismatch could result from a 
misidentification during amino acid sequencing, or could represent a 
polymorphism. The nascent protein contains an N-terminal signal peptide 
with features characteristic of eukaryotic secretory signal sequences for 
ER targeting. The "weight matrix" method (von Heijne, 1986) predicts two 
possible cleavage sites for the signal peptide, one between residues Gly 
17 and Phe 18, and a second between Ala23 and Arg24. Since the N-terminal 
amino acid sequence of the mature protein begins at Arg24, the 
co-translational processing of the signal peptide appears to occur at the 
predicted second cleavage site. The protein contains two putative N- 
asparagine glycosylation sites at Asn223 and Asn447, consistent with the 
detection of oligosaccharide sidechains in the purified enzyme 
(Dharmawardhana et al., 1995). 
Nucleotide and amino acid sequence homology searches and comparisons were 
carried out using BLAST (Altschul et al., 1990) on Genbank, EMBL, PDB, 
SWISS-PROT and PIR databases. Further sequence analysis was performed 
using PC/GENE or GeneWorks (IntelliGenetics Inc.) software. The derived 
amino acid sequence of CBG, when compared to other glycohydrolase 
sequences in the databases, showed the strongest similarity to enzymes 
belonging to family 1 glycosyl hydrolases (Henrissat, 1991). The 
.beta.-glucosidases showing the highest similarity (30-50% identity) to 
CBG were from plant species Prunus, Hordeum, Trifolium, Manihot, Sorghum, 
Avena, and Costus. The dendrogram in FIG. 2 illustrates that among the 
plant .beta.-glucosidases, pine CBG is loosely clustered with cyanogenic 
.beta.-glucosidases from several species (FIG. 2: sequences 7 to 13). 
CBG contains several sequence elements that are highly conserved among many 
family 1 .beta.-glucosidases. Between residues 34 and 48 it carries the 
N-terminal signature sequence F,X,(FYWM),(GSTA),X,(GSTA),X,(GSTA),(GSTA), 
(FYN),X,E,X(GSTA) characteristic offamily 1 glycosyl hydrolases 
(Henrissat, 1991). Two of the five cysteine residues found in CBG (Cys 175 
and Cys225) are also conserved in these homologous .beta.-glucosidases, 
suggesting that they may be involved in forming important intramolecular 
disulfide bridges. 
Other conserved sequence elements include the sequence -ENG- at residues 
408-410 within the C-terminal signature, and the sequence -NEP- at 
residues 190-192. These sequence motifs are thought to be important for 
enzyme activity, and this region may be involved in binding of the 
pyranose ring during catalysis. The NEP motif of both Bacillus 
endo-.beta.-1-4-glucanase and CBG is flanked by hydrophobic amino acids; 
next to the signal peptide, it is the most hydrophobic region of the CBG 
enzyme. The hydrolytic mechanism of the family 1 .beta.-glucosidases is 
considered to be general acid catalysis (Sinnott, 1990) with Glu and Asp 
residues in conserved motifs serving as active site nucleophile and acid 
catalyst. Evidence from inhibitor and site-directed mutagenesis studies 
suggests the Glu408 within the conserved ENG motif is the active site 
nucleophile (Withers et al., 1990; Trimbur et al, 1992). A conserved 
aspartate residue (Asp427) located 19 residues downstream from the ENG 
motif of CBG appears to be analogous to Asp374 of Agrobacterium 
.beta.-glucosidase (cellobiase). This carboxylate side-chain may play the 
role of acid-base catalyst during hydrolysis of the glycosidic linkage 
(Trimbur et al., 1992). 
EXAMPLE 3 
Expression of CBG cDNA in E. coli 
To express CBG protein in E. coli, the full- length coding region for the 
mature protein (i.e. excluding the signal peptide) was amplified using the 
3' end clone (1A6) as the template with the N-terminal primer, NT1 (5' 
TAGCTAGCAGGCTGGACAGGAACAACTTC 3', Seq. I.D. No. 9) containing a 5' Nhe1 
site, and a C-terminal primer, CT1 (5' CTCGAGACAAGCAGTCTAAATGCT 3', Seq. 
I.D. No. 10) containing a Xho 1 site. The resulting 1.5 kb DNA fragment 
was ligated into Bluescript II KS by T/A cloning as described above. The 
structure of the junctions of this construct was confirmed by sequencing 
and it was then inserted as a Nhe1/Xho1 fragment into expression vector 
pET21a (Novagen). Because, the Nhe1 site was used to introduce the cDNA 
into the pET vector, three non-CBG amino acids (Met, Ala, Ser) were added 
to the N-terminus of the expressed protein. To avoid the expression of the 
vector His-tag at the 3' end, the native stop codon of CBG was included. 
The expressed protein was thus identical in sequence to the mature CBG 
expressed in planta, except for the additional tripeptide at the 
N-terminus. Following transformation into E.coli strain DH5.alpha. and 
verification of the plasmid integrity by restriction digestion, it was 
introduced into the expression host BL21 (DE3). 
To express CBG, the bacteria were grown to log phase (A.sub.600 =0.6-0.9) 
followed by an additional 2-3 h incubation at 29-37.degree. C. in the 
presence or absence of 0.4-1 mM IPTG. The expressed CBG in the soluble 
protein fraction was purified by preparative Q-Sepharose chromatography 
followed by QMA-Memsep (Millipore) chromatography. 
As noted above, the functional characteristic of the CBG enzyme is its 
ability to hydrolyze coniferin. This activity can be measured using the 
simple .beta.-glucosidase assay described by Dharwardhana et al. (1995), 
conducted as follows: enzyme preparations (10-50 .mu.l) and glucoside 
substrate (coniferin) (2 mM final concentration) in 0.2M MES, pH 5.5 
buffer in a final volume of 150 .mu.l are incubated at 30.degree. C. for 
30 min. The reaction is stopped by basification of the assay mixture with 
an equal volume of 0.5M CAPS buffer (Sigma Chemical Co., St. Louis, Mo., 
pH 10.5 and the activity measured by determining the absorbance of the 
released aglycone. The activity of the enzyme can be measured not only 
against coniferin, but also against related glucosides including 4-NPG, 
2-NPG, MUG and the synthetic coniferin analog VRA-G. For quantitative 
calculations, the following analysis wavelengths and values (mM.sup.-1 
.times.cm.sup.-1) were used: coniferyl alcohol, 325 nm, .epsilon.=7.0; 
sinapyl alcohol, 315 nm, .epsilon.=11.2; 2-nitrophenol, 420 nm, 
.epsilon.=4.55, 4-nitrophenol, 400 nm, .epsilon.=19.3; 4-methyl 
umbelliferone, 360 nm, .epsilon.=18.25; VRA-G, 490 nm, .epsilon.=38.6; 
salicyl alcohol, 295 nm, .epsilon.=3.3. 
Soluble proteins and insoluble proteins (inclusion bodies) prepared from 
induced and uninduced bacterial cells were assayed for coniferin 
hydrolysis activity by the method described above. Only the soluble 
protein fraction of induced cells displayed this activity. The activity in 
this fraction could be increased up to 2-fold by increasing the IPTG 
concentration from 0.4 -1.0 mM, and by reducing the growing temperature 
from 37.degree. C. to 29.degree. C. Activity staining of nondenaturing 
PAGE gels using the chromogenic coniferin analogue VRA-G revealed a 
.beta.-glucosidase-active protein band in induced cell extracts. This 
protein was purified by anion exchange chromatography using coniferin as 
the substrate for monitoring .beta.-glucosidase activity. The purified 
enzyme often migrated as a doublet on nondenaturing gels. Both protein 
bands in the doublet showed .beta.-glucosidase activity, as assayed by 
hydrolysis of VRA-G. This could be due to partial degradation, alternate 
forms of folding, or the synthesis of a truncated protein at the 5' end 
where CBG has a prokaryotic ribosome binding Shine-Dalgarno sequence 
(GAAGGAG). The latter would result in the synthesis of a polypeptide that 
is truncated at the N-terminus, as opposed to the full-length polypeptide 
initiated by ribosome binding to the standard ribosome binding site in the 
vector. As shown in Table 1 below, the CBG expressed in E. coli and the 
enzyme purified from the pine xylem showed almost identical substrate 
specificities. 
TABLE 1 
______________________________________ 
Substrate specificity of coniferin .beta.-glucosidase purified from 
pine xylem and E. coli- expressed CBG-cDNA. 100% activity 
represents 14pKat for native coniferin beta-glucosidase and 
22pKat for the recombinant enzyme. 
Relative activity 
Substrate Native CBG 
E. coli CBG 
______________________________________ 
coniferin 100 100 
syringin 51 65 
4-methyl umbelliferyl-.beta.-glucoside 18 20 
2-nitrophenyl-.beta.-glucoside 51 50 
4-nitrophenyl-.beta.-glucoside 30 35 
______________________________________ 
EXAMPLE 4 
Preferred Method for Making the CBG cDNA 
With the provision of the CBG cDNA sequence shown in Seq. I.D. No. 6, the 
polymerase chain reaction (PCR) may now be utilized in a preferred method 
for producing the CBG cDNA. PCR amplification of the CBG cDNA sequence may 
be accomplished either by direct PCR from an appropriate cDNA library or 
by Reverse-Transcription PCR (RT-PCR) using RNA extracted from plant cells 
as a template. Methods and conditions for both direct PCR and RT-PCR are 
known in the art and are described in Innis et al. (1990). Suitable plant 
cDNA libraries for direct PCR include the Pinus contorta library as 
described above. Other plant cDNA libraries may be used in order to 
amplify orthologous cDNAs of other species; for example, the Arabidopsis 
cDNA library described by Newman et al. (1994) may be used to amplify the 
Arabidopsis ortholog. 
The selection of PCR primers will be made according to the portions of the 
cDNA which are to be amplified. Primers may be chosen to amplify small 
segments of the cDNA or the entire cDNA molecule. Variations in 
amplification conditions may be required to accommodate primers of 
differing lengths; such considerations are well known in the art and are 
discussed in Innis et al. (1990), Sambrook et al. (1989), and Ausubel et 
al (1992). By way of example only, the entire CBG cDNA molecule as shown 
in Seq. I.D. No. 6 may be amplified using the following combination of 
primers: 
5' GGATTTGGACCTGAAAATATCAAT 3' (Seq. I.D. No. 11) 
5' CAATGTTCTTACCCTGCAGTTCCC 3' (Seq. I.D. No. 12) 
The open reading frame portion of the cDNA may be amplified using the 
following primer pair: 
5' ATGGAGGTGTCTGTGTTGATGTGGGTA 3' (Seq. I.D. No. 13) 
5' AATGCTGCTGCTGCTTCTAATACTTCC 3' (Seq. I.D. No. 14) 
These primers are illustrative only; it will be appreciated by one skilled 
in the art that many different primers may be derived from the provided 
cDNA sequence in order to amplify particular regions of this cDNA. 
Suitable amplification conditions include those described above for the 
original isolation of the CBG cDNA. As is well known in the art, 
amplification conditions may need be varied in order to amplify 
orthologous genes where the sequence identity is not 100%; in such cases, 
the use of nested primers, as described above may be beneficial. 
Resequencing of PCR products obtained by these amplification procedures is 
recommended; this will facilitate confirmation of the CBG cDNA sequence 
and will also provide information on natural variation on this sequence in 
different ecotypes, cultivars and plant populations. 
Oligonucleotides which are derived from the CBG cDNA sequence and which are 
suitable for use as PCR primers to amplify the CBG cDNA are encompassed 
within the scope of the present invention. Preferably, such 
oligonucleotide primers will comprise a sequence of 15-20 consecutive 
nucleotides of the CBG cDNA. To enhance amplification specificity, primers 
of 20-30 nucleotides or more in length may also be used. 
EXAMPLE 5 
Use of the CBG CDNA to Produce Plants with Modified Lignin Content 
Once a gene (or cDNA) encoding a protein involved in the determination of a 
particular plant characteristic has been isolated, standard techniques may 
be used to express the cDNA in transgenic plants in order to modify that 
particular plant characteristic. The basic approach is to clone the cDNA 
into a transformation vector, such that it is operably linked to control 
sequences (e.g., a promoter) which direct expression of the cDNA in plant 
cells. The transformation vector is then introduced into plant cells by 
one of a number of techniques (e.g., electroporation) and progeny plants 
containing the introduced cDNA are selected. Preferably all or part of the 
transformation vector will stably integrate into the genome of the plant 
cell. That part of the transformation vector which integrates into the 
plant cell and which contains the introduced cDNA and associated sequences 
for controlling expression (the introduced "transgene") may be referred to 
as the recombinant expression cassette. 
Selection of progeny plants containing the introduced transgene may be made 
based upon the detection of an altered phenotype. Such a phenotype may 
result directly from the cDNA cloned into the transformation vector or may 
be manifested as enhanced resistance to a chemical agent (such as an 
antibiotic) as a result of the inclusion of a dominant selectable marker 
gene incorporated into the transformation vector. 
The choice of (a) control sequences and (b) how the cDNA (or selected 
portions of the cDNA) are arranged in the transformation vector relative 
to the control sequences determine, in part, how the plant characteristic 
affected by the introduced cDNA is modified. For example, the control 
sequences may be tissue specific, such that the cDNA is only expressed in 
particular tissues of the plant (e.g., vascular systems) and so the 
affected characteristic will be modified only in those tissues. The cDNA 
sequence may be arranged relative to the control sequence such that the 
cDNA transcript is expressed normally, or in an antisense orientation. 
Expression of an antisense RNA that is the reverse complement of the 
cloned cDNA will result in a reduction of the targeted gene product (the 
targeted gene product being the protein encoded by the plant gene from 
which the introduced cDNA was derived). Over-expression of the introduced 
cDNA, resulting from a plus-sense orientation of the cDNA relative to the 
control sequences in the vector, may lead to an increase in the level of 
the gene product, or may result in a reduction in the level of the gene 
product due to co-suppression (also termed "sense suppression") of that 
gene product. 
Successful examples of the modification of plant characteristics by 
transformation with cloned cDNA sequences are replete in the technical and 
scientific literature. Selected examples, which serve to illustrate the 
level knowledge in this field of technology include: 
U.S. Pat. No. 5,451,514 to Boudet (modification of lignin synthesis using 
antisense RNA and co-suppression); 
U.S. Pat. No. 5,443,974 to Hitz (modification of saturated and unsaturated 
fatty acid levels using antisense RNA and co-suppression); 
U.S. Pat. No. 5,530,192 to Murase (modification of amino acid and fatty 
acid composition using antisense RNA); 
U.S. Pat. No. 5,455,167 to Voelker (modification of medium chain fatty 
acids) 
U.S. Pat. No. 5,231,020 to Jorgensen (modification of flavonoids using 
co-suppression); and 
U.S. Pat. No. 5,583,021 to Dougherty (modification of virus resistance by 
expression of plus-sense RNA) 
These examples include descriptions of transformation vector selection, 
transformation techniques and the construction of constructs designed to 
over-express the introduced cDNA, untranslatable RNA forms or antisense 
RNA. In light of the foregoing and the provision herein of the CBG cDNA, 
it is thus apparent that one of skill in the art will be able to introduce 
this cDNA, or derivative forms of the cDNA (e.g., antisense forms), into 
plants in order to produce plants having modified lignin content. Example 
6 below provides an exemplary illustration of how an antisense form of the 
CBG cDNA may be introduced into conifers using ballistic transformation, 
in order to produce conifers having altered lignin content. 
a. Plant Types 
Lignins are found in all plant types, and thus DNA molecules according to 
the present invention (e.g., the CBG cDNA, homologs of the CBG cDNA and 
antisense forms) may be introduced into any plant type in order to modify 
the lignin composition of the plant. Thus, the sequences of the present 
invention may be used to modify lignin composition in any higher plants 
including monocotyledonous plants such as lily, corn, rice, wheat and 
barley as well as dicotyledonous plants, such as tomato, potato, soy bean, 
cotton, tobacco, sunflower, safflower and brasicca. As noted above, the 
present invention is expected to be particularly useful in woody species 
such as species belonging to the genera Picea, Pseudotsuga, Tsuga, 
Sequoia, Abies, Thuja, Libocedrus, Chamaecyparis and Laryx. Pines are 
expected to be a particularly suitable choice for genetic modification by 
the methods disclosed herein, including lodgepole pine (Pinus contorta), 
the species from which the CBG cDNA was cloned. 
b. Vector Construction, Choice of Promoters 
A number of recombinant vectors suitable for stable transfection of plant 
cells or for the establishment of transgenic plants have been described 
including those described in Pouwels et al., (1987), Weissbach and 
Weissbach, (1989), and Gelvin et al., (1990). Typically, plant 
transformation vectors include one or more cloned plant genes (or cDNAs) 
under the transcriptional control of 5' and 3' regulatory sequences and a 
dominant selectable marker. Such plant transformation vectors typically 
also contain a promoter regulatory region (e.g., a regulatory region 
controlling inducible or constitutive, environmentally-or 
developmentally-regulated, or cell- or tissue-specific expression), a 
transcription initiation start site, a ribosome binding site, an RNA 
processing signal, a transcription termination site, and/or a 
polyadenylation signal. 
Examples of constitutive plant promoters which may be useful for expressing 
the CBG cDNA include: the cauliflower mosaic virus (CaMV) 35S promoter, 
which confers constitutive, high-level expression in most plant tissues 
(see, e.g., Odel et al., 1985; Dekeyser et al., 1990; Terada and 
Shimamoto, 1990); the nopaline synthase promoter (An et al., 1988); and 
the octopine synthase promoter (Fromm et al., 1989). 
A variety of plant gene promoters that are regulated in response to 
environmental, hormonal, chemical, and/or developmental signals, also can 
be used for expression of the CBG cDNA in plant cells, including promoters 
regulated by: (a) heat (Callis et al., 1988); (b) light (e.g., the pea 
rbcS-3A promoter, Kuhlemeier et al., 1989, the maize rbcS promoter, 
Schaffner and Sheen, 1991, and the chlorophyll a/b-binding protein 
promoter); (c) hormones, such as abscisic acid (Marcotte et al., 1989); 
(d) wounding (e.g., wunI, Siebertz et al., 1989); and (e) chemicals such 
as methyl jasminate or salicylic acid. It may also be advantageous to 
employ tissue-specific promoters, such as those described by Roshal et 
al., (1987), Schernthaner et al., (1988), and Bustos et al., (1989). 
Plant transformation vectors may also include RNA processing signals, for 
example, introns, which may be positioned upstream or downstream of the 
CBG cDNA sequence in the transgene. In addition, the expression vectors 
may also include additional regulatory sequences from the 3'-untranslated 
region of plant genes, e.g., a 3' terminator region to increase mRNA 
stability of the mRNA, such as the PI-II terminator region of potato or 
the octopine or nopaline synthase 3' terminator regions. 
Finally, as noted above, plant transformation vectors may also include 
dominant selectable marker genes to allow for the ready selection of 
transformants. Such genes include those encoding antibiotic resistance 
genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, 
streptomycin or spectinomycin) and herbicide resistance genes (e.g., 
phosphinothricin acetyltransferase). 
C. Arrangement of CBG cDNA in Vector 
As noted above, the particular arrangement of the CBG cDNA in the 
transformation vector will be selected according to the expression of the 
cDNA desired. 
Sense Expression 
Where enhanced lignin synthesis is desired, the CBG cDNA may be operably 
linked to a constitutive high-level promoter such as the CaMV 35S 
promoter. As noted below, modification of lignin synthesis may also be 
achieved by introducing into a plant a transformation vector containing a 
variant form of the CBG cDNA, for example a form which varies from the 
exact nucleotide sequence of the CBG cDNA, but which encodes a protein 
that retains the functional characteristic of the CBG protein, i.e. 
coniferin hydrolysis activity. 
Sense Suppression 
Constructs in which the CBG cDNA (or variants thereon) are over-expressed 
may also be used to obtain co-suppression of the endogenous CBG gene in 
the manner described in U.S. Pat. No. 5,231,021 to Jorgensen. Such 
co-suppression (also termed sense suppression) does not require that the 
entire CBG cDNA be introduced into the plant cells, nor does it require 
that the introduced sequence be exactly identical to the CBG cDNA. 
However, as with antisense suppression, the suppressive efficiency will be 
enhanced as (1) the introduced sequence is lengthened and (2) the sequence 
similarity between the introduced sequence and the endogenous CBG geneis 
increased. Sense-suppression is believed to be modulated, in part, by the 
position on the plant genome into which the introduced sequence 
integrates. 
Antisense Expression 
In contrast, a reduction of lignin synthesis may be obtained by introducing 
antisense constructs based on the CBG cDNA sequence into plants. For 
antisense suppression, the CBG EDNA is arranged in reverse orientation 
relative to the promoter sequence in the transformation vector. The 
introduced sequence need not be the full length CBG cDNA, and need not be 
exactly homologous to the CBG cDNA. Generally, however, where the 
introduced sequence is of shorter length, a higher degree of homology to 
the native CBG sequence will be needed for effective antisense 
suppression. Preferably, the introduced antisense sequence in the vector 
will be at least 30 nucleotides in length, and improved antisense 
suppression will typically be observed as the length of the antisense 
sequence increases. Preferably, the length of the antisense sequence in 
the vector will be greater than 100 nucleotides. Transcription of an 
antisense construct as described results in the production of RNA 
molecules that are the reverse complement of mRNA molecules transcribed 
from the endogenous CBG gene in the plant cell. Although the exact 
mechanism by which antisense RNA molecules interfere with gene expression 
has not been elucidated, it is believed that antisense RNA molecules bind 
to the endogenous MRNA molecules and thereby inhibit translation of the 
endogenous mRNA. 
Suppression of endogenous CBG gene expression can also be achieved using 
ribozymes. Ribozymes are synthetic RNA molecules that possess highly 
specific endoribonuclease activity. The production and use of ribozymes 
are disclosed in U.S. Pat. No. 4,987,071 to Cech and U.S. Pat. No. 
5,543,508 to Haselhoff. The inclusion of ribozyme sequences within 
antisense RNAs may be used to confer RNA cleaving activity on the 
antisense RNA, such that endogenous mRNA molecules that bind to the 
antisense RNA are cleaved, which in turn leads to an enhanced antisense 
inhibition of endogenous gene expression. 
Untranslatable RNA 
Suppression of native gene expression may be achieved by transforming the 
plant with a sequence that is homologous to the target gene, but which is 
rendered untranslatable by a genetic modification such as the introduction 
of a premature stop codon. This approach is described in U.S. Pat. No. 
5,583,021. The introduced CBG sequence is preferably 50-100 nucleotides in 
length, although longer sequences, such as 100-250 nucleotides are 
preferred. The introduced sequence is engineered to encode an 
untranslatable RNA; the introduction of a premature stop codon early on in 
the coding region is a preferred way of achieving this. The sequence need 
not be perfectly homologous to the target CBG sequence, but at least 80%, 
and preferably 85% sequence homology will likely be more effective than 
lower homologies. 
d. Transformation and Regeneration Techniques 
Transformation and regeneration of both monocotyledonous and dicotyledonous 
plant cells are now routine, and the selection of the most appropriate 
transformation and regeneration techniques will be determined by the 
practitioner. The choice of method will vary with the type of plant to be 
transformed; those skilled in the art will recognize the suitability of 
particular methods for given plant types. Suitable methods may include, 
but are not limited to: electroporation of plant protoplasts; 
liposome-mediated transformation; polyethylene mediated transformation; 
transformation using viruses; micro-injection of plant cells; 
micro-projectile bombardment of plant cells; vacuum infiltration; and 
Agrobacterium tumeficiens (AT) mediated transformation. Typical procedures 
for transforming and regenerating plants are described in the patent 
documents listed at the beginning of this section. In addition, methods 
for transforming woody species are described in Ellis et al. (1993), Ellis 
et al. (1996), U.S. Pat. No. 5,122,466, "Ballistic Transformation of 
Conifer" and U.S. Pat. No. 4,795,855, "Transformation and Foreign Gene 
Expression with Woody Species". 
e. Selection of Transformed Plants 
Following transformation and regeneration of plants with the transformation 
vector, transformed plants are preferably selected using a dominant 
selectable marker incorporated into the transformation vector. Typically, 
such a marker will confer antibiotic resistance on the seedlings of 
transformed plants, and selection of transformants can be accomplished by 
exposing the seedlings to appropriate concentrations of the antibiotic. 
After transformed plants are selected and grown to maturity, they can be 
assayed to determine whether coniferin beta-glucosidase synthesis has been 
altered as a result of the introduced transgene. This can be done in 
several ways, including by extracting and quantifying the enzyme activity 
as described in Example 6. In addition, lignification may be determined 
histochemically, and lignin content may be quantified, as described in 
Example 6. Also, antisense or sense suppression of the endogenous CBG gene 
may be detected by analyzing mRNA expression on Northern blots. 
EXAMPLE 6 
Introduction Of Antisense CBG cDNA Sequence Into White Spruce (Picea 
Glauca) 
By way of example, the following methodology may be used to produce white 
spruce trees having an altered lignin content. The CBG cDNA is operably 
linked, but in reverse orientation, to the enhanced cauliflower mosaic 
virus (CaMV) 35S promoter in place of the BT gene in plasmid pTVBT41100 
(Ellis et al., 1993). (Many other plants tranaformation vectors have been 
described and would be suitable for introducing CBG-based constructs into 
plants. Vector pBACGGUS shown in FIG. 3 is one such alternative vector 
that may be used). Somatic embryos of Picea glauca are differentiated from 
embryogenic white spruce callus line and cultured as described by Ellis et 
al. (1993). Plasmid DNA is adhered to 1-3 M gold particles (0.5 g DNA /mg 
gold) by calcium chloride and spermidine precipitation. Gold particles 
containing the DNA are then loaded on to carrier sheets at a rate of 0.05 
mg/cm.sup.2 and these particles are then introduced into somatic embryos 
as described by Ellis et al. (1991). Transformed embryos are selected 
using kanamycin. Regeneration of transgenic plants (via the production of 
embryogenic callus) is achieved using the culture conditions described by 
Ellis et al. (1993). 
In order to determine coniferin beta-glucosidase activity in the transgenic 
plants, the enzyme is extracted as described in Example 1 above, and the 
activity is assayed using the .beta.-glucosidase assay described in 
Example 3 above. Plants transformed with the same vector without the CBG 
cDNA insert should preferably be used as controls. In situ localization of 
the enzyme activity can be determined using VRA-G as described by 
Dharmawardhana et al. (1995). Lignin in the stem sections is detected 
histochemically by Basic Fuchsin-induced fluorescence and imaging on a 
confocal laser scanning microscope as described by Dharmawardhana et al. 
(1992). In order to determine the effect of introducing the antisense 
construct into the plant on lignin content, standard methods are used to 
quantify lignin in the transformed plant (and control plants). Standard 
methods of quantifying lignin include the thioglycolic acid procedure as 
described by Whitmore (1978) and the acetyl bromide procedure as described 
by Liyama and Wallis (1990). 
EXAMPLE 7 
CBG Genes in Other Plant Species 
Orthologs of the CBG gene may be isolated from a number of plant species, 
particularly woody species such as plants from the genera Picea, 
Pseudotsuga, Tsuga, Sequoia, Abies, Thuja, Libocedrus, Chamaecyparis and 
Laryx. With the provision herein of the prototypical CBG protein from 
Pinus and the cDNA sequence that encodes this protein, the cloning by 
standard methods of cDNAs and genes that encode CBG protein orthologs in 
other plant species is now enabled. As described above, orthologs of the 
disclosed Pinus CBG protein have CBG protein biological activity and are 
typically characterized by possession of at least 50% sequence identity 
counted over the full length alignment with the amino acid sequence of 
Pinus CBG using the NCBI Blast 2.0, gapped blastp set to default 
parameters. Proteins with even greater similarity to the reference 
sequences will show increasing percentage identities when assessed by this 
method, such as at least 60%, at least 65%, at least 70%, at least 75%, at 
least 80%, at least 90% or at least 95% sequence identity. 
Both conventional hybridization and PCR amplification procedures may be 
utilized to clone sequences encoding CBG protein orthologs. Common to both 
of these techniques is the hybridization of probes or primers derived from 
the Pinus CBG cDNA sequence to a target nucleotide preparation, which may 
be, in the case of conventional hybridization approaches, a cDNA or 
genomic library or, in the case of PCR amplification, a cDNA or genomic 
library, or an mRNA preparation. 
Direct PCR-amplification may be performed on cDNA or genomic libraries 
prepared from the plant species in question, or RT-PCR may be performed 
using MRNA extracted from the plant cells using standard methods. PCR 
primers will comprise at least 15 consecutive nucleotides of the Pinus CBG 
cDNA. One of skill in the art will appreciate that sequence differences 
between the Pinus CBG cDNA or gene and the target nucleic acid to be 
amplified may result in lower amplification efficiencies. To compensate 
for this, longer PCR primers or lower annealing temperatures may be used 
during the amplification cycle. Where lower annealing temperatures are 
used, sequential rounds of amplification using nested primer pairs may be 
necessary to enhance specificity. 
For conventional hybridization techniques the hybridization probe is 
preferably conjugated with a detectable label such as a radioactive label, 
and the probe is preferably of at least 20 nucleotides in length. As is 
well known in the art, increasing the length of hybridization probes tends 
to give enhanced specificity. The labeled probe derived from the Pinus 
cDNA sequence may be hybridized to a plant cDNA or genomic library and the 
hybridization signal detected using means known in the art. The 
hybridizing colony or plaque (depending on the type of library used) is 
then purified and the cloned sequence contained in that colony or plaque 
isolated and characterized. 
Homologs and orthologs of the Pinus CBG cDNA sequence include molecules 
that hybridize under stringent conditions to the disclosed prototypical 
CBG cDNA, or fragments thereof. Stringent hybridization conditions are 
hybridization at 65.degree. C. in 6.times.SSC, 5.times.Denhardt's 
solution, 0.5% SDS and 100 .mu.g sheared salmon testes DNA, followed by 
15-30 minute sequential washes at 65.degree. C. in 2.times.SSC, 0.1% SDS, 
followed by 1.times.SSC, 0.1% SDS and finally 0.2.times.SSC, 0.1% SDS. 
Lower stringency hybridization conditions (to detect less closely related 
homologs) are performed as described above but at 50.degree. C. (both 
hybridization and wash conditions); however, depending on the strength of 
the detected signal, the wash steps may be terminated after the first 
2.times.SSC, 0.1% SDS wash. 
Orthologs of the Pinus CBG may alternatively be obtained by immunoscreening 
of an expression library. With the provision herein of the disclosed Pinus 
CBG nucleic acid sequences, the enzyme may be expressed and purified in a 
heterologous expression system (e.g., E coli) and used to raise antibodies 
(monoclonal or polyclonal) specific for the Pinus CBG protein. Antibodies 
may also be raised against synthetic peptides derived from the Pinus CBG 
amino acid sequence presented herein. Methods of raising antibodies are 
well known in the art and are described in Harlow and Lane (1988). Such 
antibodies can then be used to screen an expression cDNA library produced 
from the plant from which it is desired to clone the CBG ortholog, using 
routine methods. The selected cDNAs can be confirmed by sequencing and 
enzyme activity. 
EXAMPLE 8 
CBG Sequence Variants 
With the provision of the Pinus CBG protein and CBG cDNA sequences herein, 
the creation of variants of these sequences is now enabled. Variant CBG 
proteins include proteins that differ in amino acid sequence from the 
Pinus CBG sequence disclosed but which retain CBG protein biological 
activity. Such proteins may be produced by manipulating the nucleotide 
sequence of the Pinus CBG cDNA using standard procedures such as 
site-directed mutagenesis or the polymerase chain reaction. The simplest 
modifications involve the substitution of one or more amino acids for 
amino acids having similar biochemical properties. These so-called 
conservative substitutions are likely to have minimal impact on the 
activity of the resultant protein. Table 2 shows amino acids which may be 
substituted for an original amino acid in a protein and which are regarded 
as conservative substitutions. 
TABLE 2 
______________________________________ 
Original Residue Conservative Substitutions 
______________________________________ 
Ala ser 
Arg lys 
Asn gln; his 
Asp glu 
Cys ser 
Gln asn 
Glu asp 
Gly pro 
His asn; gln 
Ile leu; val 
Leu ile; val 
Lys arg; gln; glu 
Met leu; ile 
Phe met; leu; tyr 
Ser thr 
Thr ser 
Trp tyr 
Tyr trp; phe 
Val ile; leu 
______________________________________ 
More substantial changes in enzymatic function or other features may be 
obtained by selecting substitutions that are less conservative than those 
in Table 1, i.e., selecting residues that differ more significantly in 
their effect on maintaining (a) the structure of the polypeptide backbone 
in the area of the substitution, for example, as a sheet or helical 
conformation, (b) the charge or hydrophobicity of the molecule at the 
target site, or (c) the bulk of the side chain. The substitutions which in 
general are expected to produce the greatest changes in protein properties 
will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, 
is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, 
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted 
for (or by) any other residue; (c) a residue having an electropositive 
side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) 
an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue 
having a bulky side chain, e.g., phenylalanine, is substituted for (or by) 
one not having a side chain, e.g., glycine. The effects of these amino 
acid substitutions or deletions or additions may be assessed for CBG 
protein derivatives by analyzing the ability of the derivative proteins to 
hydrolyze coniferin in the assay described. 
Variant CBG cDNA or genes may be produced by standard DNA mutagenesis 
techniques, for example, M13 primer mutagenesis. Details of these 
techniques are provided in Sambrook et al. (1989), Ch. 15. By the use of 
such techniques, variants may be created which differ in minor ways from 
the Pinus CBG cDNA sequences disclosed, yet which still encode a protein 
having CBG protein biological activity. DNA molecules and nucleotide 
sequences which are derivatives of those specifically disclosed herein and 
which differ from those disclosed by the deletion, addition or 
substitution of nucleotides while still encoding a protein that has CBG 
protein biological activity are comprehended by this invention. In their 
simplest form, such variants may differ from the disclosed sequences by 
alteration of the coding region to fit the codon usage bias of the 
particular organism into which the molecule is to be introduced. 
Alternatively, the coding region may be altered by taking advantage of the 
degeneracy of the genetic code to alter the coding sequence in such a way 
that, while the nucleotide sequence is substantially altered, it 
nevertheless encodes a protein having an amino acid sequence identical or 
substantially similar to the disclosed Pinus CBG protein sequence. For 
example, the 23rd amino acid residue of the Pinus CBG protein is alanine. 
This is encoded in the Pinus CBG open reading frame (ORF) by the 
nucleotide codon triplet GCT. Because of the degeneracy of the genetic 
code, three other nucleotide codon triplets--GCA, GCC and GCG--also code 
for alanine. Thus, the nucleotide sequence of the Pinus CBG ORF could be 
changed at this position to any of these three codons without affecting 
the amino acid composition of the encoded protein or the characteristics 
of the protein. Based upon the degeneracy of the genetic code, variant DNA 
molecules may be derived from the cDNA sequence disclosed herein using 
standard DNA mutagenesis techniques as described above, or by synthesis of 
DNA sequences. Thus, this invention also encompasses nucleic acid 
sequences which encode a CBG protein but which vary from the disclosed 
nucleic acid sequences by virtue of the degeneracy of the genetic code. 
Variants of the CBG protein may also be defined in terms of their sequence 
identity with the prototype CBG protein shown in Seq. I.D. No. 7. As 
described above, CBG proteins have CBG biological activity and share at 
least 60% sequence identity with the Pinus CBG protein. Nucleic acid 
sequences that encode such proteins may readily be determined simply by 
applying the genetic code to the amino acid sequence of a CBG protein, and 
such nucleic acid molecules may readily be produced by assembling 
oligonucleotides corresponding to portions of the sequence. 
The Pinus CBG gene or cDNA, and orthologs of these sequences from other 
plants, may be incorporated into transformation vectors and introduced 
into plants to produce plants having modified lignin content, as described 
above. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - &lt;160&gt; NUMBER OF SEQ ID NOS: 14 
- - &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
N7A 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: modified.sub.-- base 
&lt;222&gt; LOCATION: (16) 
&lt;223&gt; OTHER INFORMATION: The letter 'n' in - #this sequence (position 
16) 
is an inosine (i) 
- - &lt;400&gt; SEQUENCE: 1 
- - gctctagagc gaymgnaaya aytttcc - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
N7B 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: modified.sub.-- base 
&lt;222&gt; LOCATION: (16) 
&lt;223&gt; OTHER INFORMATION: The letter 'n' in - #this sequence (position 
16) 
is an inosine (i) 
- - &lt;400&gt; SEQUENCE: 2 
- - gctctagagc gaymgnaaya ayttccc - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 3 
&lt;211&gt; LENGTH: 25 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
N10 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: modified.sub.-- base 
&lt;222&gt; LOCATION: (6,18,21) 
&lt;223&gt; OTHER INFORMATION: The letter 'n' in - #this sequence (positions 
6,18,and 21) are inosine (i) 
- - &lt;400&gt; SEQUENCE: 3 
- - gaymgnaaya ayttcccnws ngwtt - # - # 
25 
- - - - &lt;210&gt; SEQ ID NO 4 
&lt;211&gt; LENGTH: 22 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
CBG172 
- - &lt;400&gt; SEQUENCE: 4 
- - cacatatctg tgatattggt cg - # - # 
22 
- - - - &lt;210&gt; SEQ ID NO 5 
&lt;211&gt; LENGTH: 19 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
CBG75 
- - &lt;400&gt; SEQUENCE: 5 
- - ccatcttctc ggactgctc - # - # 
- # 19 
- - - - &lt;210&gt; SEQ ID NO 6 
&lt;211&gt; LENGTH: 1909 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Pinus contorta 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: CDS 
&lt;222&gt; LOCATION: (183)..(1724) 
- - &lt;400&gt; SEQUENCE: 6 
- - ggatttggac ctgaaaatat caatttcaaa gcaattccag agggataacg tg - 
#ggatcctt 60 
- - accattacca acaacccacc attccgccct gccgacctca ggcatatttt ga - 
#ttctattt 120 
- - aaccattaat tcatctgggc agttgtgatt ctgtataatt cgatcgctcc gt - 
#tttagcag 180 
- - ac atg gag gtg tct gtg ttg atg tgg gta ctg - # ctc ttc tat tcc tta 
227 
Met Glu Val Ser Val Leu Met Trp Val - #Leu Leu Phe Tyr Ser Leu 
1 - # 5 - # 10 - # 15 
- - tta ggt ttt caa gtg acg aca gct agg ctg ga - #c agg aac aac ttc ccc 
275 
Leu Gly Phe Gln Val Thr Thr Ala Arg Leu As - #p Arg Asn Asn Phe Pro 
20 - # 25 - # 30 
- - tca gat ttc atg ttc ggc aca gcc tct tca gc - #g tat cag tat gaa gga 
323 
Ser Asp Phe Met Phe Gly Thr Ala Ser Ser Al - #a Tyr Gln Tyr Glu Gly 
35 - # 40 - # 45 
- - gca gtc cga gaa gat ggc aag ggt cct agc ac - #a tgg gac gcc tta aca 
371 
Ala Val Arg Glu Asp Gly Lys Gly Pro Ser Th - #r Trp Asp Ala Leu Thr 
50 - # 55 - # 60 
- - cat atg cct ggt aga ata aaa gat agc agc aa - #t gga gac gtg gca gtc 
419 
His Met Pro Gly Arg Ile Lys Asp Ser Ser As - #n Gly Asp Val Ala Val 
65 - # 70 - # 75 
- - gac caa tat cac aga tat atg gaa gat atc ga - #g ctt atg gct tca ctt 
467 
Asp Gln Tyr His Arg Tyr Met Glu Asp Ile Gl - #u Leu Met Ala Ser Leu 
80 - # 85 - # 90 - # 95 
- - gga cta gat gcc tat aga ttc tcc ata tcc tg - #g tct cga atc ctt cca 
515 
Gly Leu Asp Ala Tyr Arg Phe Ser Ile Ser Tr - #p Ser Arg Ile Leu Pro 
100 - # 105 - # 110 
- - gaa gga aga ggt gaa att aac atg gct ggg at - #t gaa tat tac aat aat 
563 
Glu Gly Arg Gly Glu Ile Asn Met Ala Gly Il - #e Glu Tyr Tyr Asn Asn 
115 - # 120 - # 125 
- - ctg att gac gct ctt ctg caa aat ggg atc ca - #g ccg ttc gtg aca ttg 
611 
Leu Ile Asp Ala Leu Leu Gln Asn Gly Ile Gl - #n Pro Phe Val Thr Leu 
130 - # 135 - # 140 
- - ttc cat ttc gat ctt ccc aaa gca ctt gaa ga - #c tcc tat ggg gga tgg 
659 
Phe His Phe Asp Leu Pro Lys Ala Leu Glu As - #p Ser Tyr Gly Gly Trp 
145 - # 150 - # 155 
- - ctg agt cct caa ata att aac gac ttc gaa gc - #c tat gca gag att tgc 
707 
Leu Ser Pro Gln Ile Ile Asn Asp Phe Glu Al - #a Tyr Ala Glu Ile Cys 
160 1 - #65 1 - #70 1 - 
#75 
- - ttc cgg gca ttc ggt gac cgt gtc aaa tat tg - #g gcg aca gtg aac 
gag 755 
Phe Arg Ala Phe Gly Asp Arg Val Lys Tyr Tr - #p Ala Thr Val Asn Glu 
180 - # 185 - # 190 
- - cca aat ctg ttt gtg ccg ttg gga tac acc gt - #c gga ata ttt cca ccg 
803 
Pro Asn Leu Phe Val Pro Leu Gly Tyr Thr Va - #l Gly Ile Phe Pro Pro 
195 - # 200 - # 205 
- - acg agg tgt gct gcc cct cac gcc aat cct tt - #g tgc atg aca ggg aat 
851 
Thr Arg Cys Ala Ala Pro His Ala Asn Pro Le - #u Cys Met Thr Gly Asn 
210 - # 215 - # 220 
- - tgc tcg tca gca gag cca tat cta gct gca ca - #t cac gtt ttg ctc gcc 
899 
Cys Ser Ser Ala Glu Pro Tyr Leu Ala Ala Hi - #s His Val Leu Leu Ala 
225 - # 230 - # 235 
- - cac gca tct gca gtg gag aaa tat agg gag aa - #a tat cag aaa att caa 
947 
His Ala Ser Ala Val Glu Lys Tyr Arg Glu Ly - #s Tyr Gln Lys Ile Gln 
240 2 - #45 2 - #50 2 - 
#55 
- - gga gga tct ata ggg tta gtt ata agc gcg cc - #a tgg tac gaa ccc 
ttg 995 
Gly Gly Ser Ile Gly Leu Val Ile Ser Ala Pr - #o Trp Tyr Glu Pro Leu 
260 - # 265 - # 270 
- - gaa aat tct cca gaa gag aga tca gct gtt ga - #t aga att tta tcc ttc 
1043 
Glu Asn Ser Pro Glu Glu Arg Ser Ala Val As - #p Arg Ile Leu Ser Phe 
275 - # 280 - # 285 
- - aat ctc cga tgg ttt ttg gat cca att gtt tt - #t gga gat tat cca caa 
1091 
Asn Leu Arg Trp Phe Leu Asp Pro Ile Val Ph - #e Gly Asp Tyr Pro Gln 
290 - # 295 - # 300 
- - gaa atg cgt gaa aga tta gga tcg cgc tta cc - #c tcc ata tcc tcg gaa 
1139 
Glu Met Arg Glu Arg Leu Gly Ser Arg Leu Pr - #o Ser Ile Ser Ser Glu 
305 - # 310 - # 315 
- - cta tct gcg aaa ctt cgg gga tcg ttc gac ta - #t atg ggt att aat cac 
1187 
Leu Ser Ala Lys Leu Arg Gly Ser Phe Asp Ty - #r Met Gly Ile Asn His 
320 3 - #25 3 - #30 3 - 
#35 
- - tat aca acc tta tat gca aca agc act cct cc - #c ctt tcc ccc gac 
cac 1235 
Tyr Thr Thr Leu Tyr Ala Thr Ser Thr Pro Pr - #o Leu Ser Pro Asp His 
340 - # 345 - # 350 
- - acg caa tat cta tat cca gac tct agg gtt ta - #t ctg act gga gag cgc 
1283 
Thr Gln Tyr Leu Tyr Pro Asp Ser Arg Val Ty - #r Leu Thr Gly Glu Arg 
355 - # 360 - # 365 
- - cac gga gtc tcc atc gga gaa cgg aca ggg at - #g gac ggt ttg ttt gtg 
1331 
His Gly Val Ser Ile Gly Glu Arg Thr Gly Me - #t Asp Gly Leu Phe Val 
370 - # 375 - # 380 
- - gta cct cat gga att caa aaa ata gtg gag ta - #t gta aaa gaa ttc tat 
1379 
Val Pro His Gly Ile Gln Lys Ile Val Glu Ty - #r Val Lys Glu Phe Tyr 
385 - # 390 - # 395 
- - gac aac ccg act att att atc gca gag aac gg - #t tat cca gag tct gag 
1427 
Asp Asn Pro Thr Ile Ile Ile Ala Glu Asn Gl - #y Tyr Pro Glu Ser Glu 
400 4 - #05 4 - #10 4 - 
#15 
- - gaa tcc tcg tcg act ctg caa gaa aat cta aa - #c gat gtg agg aga 
ata 1475 
Glu Ser Ser Ser Thr Leu Gln Glu Asn Leu As - #n Asp Val Arg Arg Ile 
420 - # 425 - # 430 
- - agg ttt cat gga gat tgt ttg agt tat ctc ag - #t gca gca atc aaa aat 
1523 
Arg Phe His Gly Asp Cys Leu Ser Tyr Leu Se - #r Ala Ala Ile Lys Asn 
435 - # 440 - # 445 
- - ggc tca gat gtt cga ggg tac ttt gtg tgg tc - #a ctt ctg gat aat ttt 
1571 
Gly Ser Asp Val Arg Gly Tyr Phe Val Trp Se - #r Leu Leu Asp Asn Phe 
450 - # 455 - # 460 
- - gag tgg gca ttt ggg tat acc att aga ttt gg - #t ctt tat cac gtg gat 
1619 
Glu Trp Ala Phe Gly Tyr Thr Ile Arg Phe Gl - #y Leu Tyr His Val Asp 
465 - # 470 - # 475 
- - ttc att tct gat caa aag aga tat ccc aag ct - #c tcg gct caa tgg ttc 
1667 
Phe Ile Ser Asp Gln Lys Arg Tyr Pro Lys Le - #u Ser Ala Gln Trp Phe 
480 4 - #85 4 - #90 4 - 
#95 
- - aga caa ttt ctt cag cac gac gat cag gga ag - #t att aga agc agc 
agc 1715 
Arg Gln Phe Leu Gln His Asp Asp Gln Gly Se - #r Ile Arg Ser Ser Ser 
500 - # 505 - # 510 
- - agc att tag actgcgttgt ctatttgcta atcaaagcgc acacattcc - #t 
1764 
Ser Ile 
- - gcaactctac ccaaaatcct gcaagcaaat atgttgtgtt cggatctatc ca - 
#ccgtgaga 1824 
- - cacattacaa agaaatcatc aatctattcc aaaatgcaga aaaccccatt ca - 
#gatgttct 1884 
- - agggaactgc agggtaagaa cattg - # - # 
1909 
- - - - &lt;210&gt; SEQ ID NO 7 
&lt;211&gt; LENGTH: 513 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Pinus contorta 
- - &lt;400&gt; SEQUENCE: 7 
- - Met Glu Val Ser Val Leu Met Trp Val Leu Le - #u Phe Tyr Ser Leu Leu 
1 5 - # 10 - # 15 
- - Gly Phe Gln Val Thr Thr Ala Arg Leu Asp Ar - #g Asn Asn Phe Pro Ser 
20 - # 25 - # 30 
- - Asp Phe Met Phe Gly Thr Ala Ser Ser Ala Ty - #r Gln Tyr Glu Gly Ala 
35 - # 40 - # 45 
- - Val Arg Glu Asp Gly Lys Gly Pro Ser Thr Tr - #p Asp Ala Leu Thr His 
50 - # 55 - # 60 
- - Met Pro Gly Arg Ile Lys Asp Ser Ser Asn Gl - #y Asp Val Ala Val Asp 
65 - # 70 - # 75 - # 80 
- - Gln Tyr His Arg Tyr Met Glu Asp Ile Glu Le - #u Met Ala Ser Leu Gly 
85 - # 90 - # 95 
- - Leu Asp Ala Tyr Arg Phe Ser Ile Ser Trp Se - #r Arg Ile Leu Pro Glu 
100 - # 105 - # 110 
- - Gly Arg Gly Glu Ile Asn Met Ala Gly Ile Gl - #u Tyr Tyr Asn Asn Leu 
115 - # 120 - # 125 
- - Ile Asp Ala Leu Leu Gln Asn Gly Ile Gln Pr - #o Phe Val Thr Leu Phe 
130 - # 135 - # 140 
- - His Phe Asp Leu Pro Lys Ala Leu Glu Asp Se - #r Tyr Gly Gly Trp Leu 
145 1 - #50 1 - #55 1 - 
#60 
- - Ser Pro Gln Ile Ile Asn Asp Phe Glu Ala Ty - #r Ala Glu Ile Cys 
Phe 
165 - # 170 - # 175 
- - Arg Ala Phe Gly Asp Arg Val Lys Tyr Trp Al - #a Thr Val Asn Glu Pro 
180 - # 185 - # 190 
- - Asn Leu Phe Val Pro Leu Gly Tyr Thr Val Gl - #y Ile Phe Pro Pro Thr 
195 - # 200 - # 205 
- - Arg Cys Ala Ala Pro His Ala Asn Pro Leu Cy - #s Met Thr Gly Asn Cys 
210 - # 215 - # 220 
- - Ser Ser Ala Glu Pro Tyr Leu Ala Ala His Hi - #s Val Leu Leu Ala His 
225 2 - #30 2 - #35 2 - 
#40 
- - Ala Ser Ala Val Glu Lys Tyr Arg Glu Lys Ty - #r Gln Lys Ile Gln 
Gly 
245 - # 250 - # 255 
- - Gly Ser Ile Gly Leu Val Ile Ser Ala Pro Tr - #p Tyr Glu Pro Leu Glu 
260 - # 265 - # 270 
- - Asn Ser Pro Glu Glu Arg Ser Ala Val Asp Ar - #g Ile Leu Ser Phe Asn 
275 - # 280 - # 285 
- - Leu Arg Trp Phe Leu Asp Pro Ile Val Phe Gl - #y Asp Tyr Pro Gln Glu 
290 - # 295 - # 300 
- - Met Arg Glu Arg Leu Gly Ser Arg Leu Pro Se - #r Ile Ser Ser Glu Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - Ser Ala Lys Leu Arg Gly Ser Phe Asp Tyr Me - #t Gly Ile Asn His 
Tyr 
325 - # 330 - # 335 
- - Thr Thr Leu Tyr Ala Thr Ser Thr Pro Pro Le - #u Ser Pro Asp His Thr 
340 - # 345 - # 350 
- - Gln Tyr Leu Tyr Pro Asp Ser Arg Val Tyr Le - #u Thr Gly Glu Arg His 
355 - # 360 - # 365 
- - Gly Val Ser Ile Gly Glu Arg Thr Gly Met As - #p Gly Leu Phe Val Val 
370 - # 375 - # 380 
- - Pro His Gly Ile Gln Lys Ile Val Glu Tyr Va - #l Lys Glu Phe Tyr Asp 
385 3 - #90 3 - #95 4 - 
#00 
- - Asn Pro Thr Ile Ile Ile Ala Glu Asn Gly Ty - #r Pro Glu Ser Glu 
Glu 
405 - # 410 - # 415 
- - Ser Ser Ser Thr Leu Gln Glu Asn Leu Asn As - #p Val Arg Arg Ile Arg 
420 - # 425 - # 430 
- - Phe His Gly Asp Cys Leu Ser Tyr Leu Ser Al - #a Ala Ile Lys Asn Gly 
435 - # 440 - # 445 
- - Ser Asp Val Arg Gly Tyr Phe Val Trp Ser Le - #u Leu Asp Asn Phe Glu 
450 - # 455 - # 460 
- - Trp Ala Phe Gly Tyr Thr Ile Arg Phe Gly Le - #u Tyr His Val Asp Phe 
465 4 - #70 4 - #75 4 - 
#80 
- - Ile Ser Asp Gln Lys Arg Tyr Pro Lys Leu Se - #r Ala Gln Trp Phe 
Arg 
485 - # 490 - # 495 
- - Gln Phe Leu Gln His Asp Asp Gln Gly Ser Il - #e Arg Ser Ser Ser Ser 
500 - # 505 - # 510 
- - Ile 
- - - - &lt;210&gt; SEQ ID NO 8 
&lt;211&gt; LENGTH: 1542 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Pinus contorta 
&lt;220&gt; FEATURE: 
&lt;221&gt; NAME/KEY: CDS 
&lt;222&gt; LOCATION: (1)..(1542) 
- - &lt;400&gt; SEQUENCE: 8 
- - atg gag gtg tct gtg ttg atg tgg gta ctg ct - #c ttc tat tcc tta tta 
48 
Met Glu Val Ser Val Leu Met Trp Val Leu Le - #u Phe Tyr Ser Leu Leu 
1 5 - # 10 - # 15 
- - ggt ttt caa gtg acg aca gct agg ctg gac ag - #g aac aac ttc ccc tca 
96 
Gly Phe Gln Val Thr Thr Ala Arg Leu Asp Ar - #g Asn Asn Phe Pro Ser 
20 - # 25 - # 30 
- - gat ttc atg ttc ggc aca gcc tct tca gcg ta - #t cag tat gaa gga gca 
144 
Asp Phe Met Phe Gly Thr Ala Ser Ser Ala Ty - #r Gln Tyr Glu Gly Ala 
35 - # 40 - # 45 
- - gtc cga gaa gat ggc aag ggt cct agc aca tg - #g gac gcc tta aca cat 
192 
Val Arg Glu Asp Gly Lys Gly Pro Ser Thr Tr - #p Asp Ala Leu Thr His 
50 - # 55 - # 60 
- - atg cct ggt aga ata aaa gat agc agc aat gg - #a gac gtg gca gtc gac 
240 
Met Pro Gly Arg Ile Lys Asp Ser Ser Asn Gl - #y Asp Val Ala Val Asp 
65 - # 70 - # 75 - # 80 
- - caa tat cac aga tat atg gaa gat atc gag ct - #t atg gct tca ctt gga 
288 
Gln Tyr His Arg Tyr Met Glu Asp Ile Glu Le - #u Met Ala Ser Leu Gly 
85 - # 90 - # 95 
- - cta gat gcc tat aga ttc tcc ata tcc tgg tc - #t cga atc ctt cca gaa 
336 
Leu Asp Ala Tyr Arg Phe Ser Ile Ser Trp Se - #r Arg Ile Leu Pro Glu 
100 - # 105 - # 110 
- - gga aga ggt gaa att aac atg gct ggg att ga - #a tat tac aat aat ctg 
384 
Gly Arg Gly Glu Ile Asn Met Ala Gly Ile Gl - #u Tyr Tyr Asn Asn Leu 
115 - # 120 - # 125 
- - att gac gct ctt ctg caa aat ggg atc cag cc - #g ttc gtg aca ttg ttc 
432 
Ile Asp Ala Leu Leu Gln Asn Gly Ile Gln Pr - #o Phe Val Thr Leu Phe 
130 - # 135 - # 140 
- - cat ttc gat ctt ccc aaa gca ctt gaa gac tc - #c tat ggg gga tgg ctg 
480 
His Phe Asp Leu Pro Lys Ala Leu Glu Asp Se - #r Tyr Gly Gly Trp Leu 
145 1 - #50 1 - #55 1 - 
#60 
- - agt cct caa ata att aac gac ttc gaa gcc ta - #t gca gag att tgc 
ttc 528 
Ser Pro Gln Ile Ile Asn Asp Phe Glu Ala Ty - #r Ala Glu Ile Cys Phe 
165 - # 170 - # 175 
- - cgg gca ttc ggt gac cgt gtc aaa tat tgg gc - #g aca gtg aac gag cca 
576 
Arg Ala Phe Gly Asp Arg Val Lys Tyr Trp Al - #a Thr Val Asn Glu Pro 
180 - # 185 - # 190 
- - aat ctg ttt gtg ccg ttg gga tac acc gtc gg - #a ata ttt cca ccg acg 
624 
Asn Leu Phe Val Pro Leu Gly Tyr Thr Val Gl - #y Ile Phe Pro Pro Thr 
195 - # 200 - # 205 
- - agg tgt gct gcc cct cac gcc aat cct ttg tg - #c atg aca ggg aat tgc 
672 
Arg Cys Ala Ala Pro His Ala Asn Pro Leu Cy - #s Met Thr Gly Asn Cys 
210 - # 215 - # 220 
- - tcg tca gca gag cca tat cta gct gca cat ca - #c gtt ttg ctc gcc cac 
720 
Ser Ser Ala Glu Pro Tyr Leu Ala Ala His Hi - #s Val Leu Leu Ala His 
225 2 - #30 2 - #35 2 - 
#40 
- - gca tct gca gtg gag aaa tat agg gag aaa ta - #t cag aaa att caa 
gga 768 
Ala Ser Ala Val Glu Lys Tyr Arg Glu Lys Ty - #r Gln Lys Ile Gln Gly 
245 - # 250 - # 255 
- - gga tct ata ggg tta gtt ata agc gcg cca tg - #g tac gaa ccc ttg gaa 
816 
Gly Ser Ile Gly Leu Val Ile Ser Ala Pro Tr - #p Tyr Glu Pro Leu Glu 
260 - # 265 - # 270 
- - aat tct cca gaa gag aga tca gct gtt gat ag - #a att tta tcc ttc aat 
864 
Asn Ser Pro Glu Glu Arg Ser Ala Val Asp Ar - #g Ile Leu Ser Phe Asn 
275 - # 280 - # 285 
- - ctc cga tgg ttt ttg gat cca att gtt ttt gg - #a gat tat cca caa gaa 
912 
Leu Arg Trp Phe Leu Asp Pro Ile Val Phe Gl - #y Asp Tyr Pro Gln Glu 
290 - # 295 - # 300 
- - atg cgt gaa aga tta gga tcg cgc tta ccc tc - #c ata tcc tcg gaa cta 
960 
Met Arg Glu Arg Leu Gly Ser Arg Leu Pro Se - #r Ile Ser Ser Glu Leu 
305 3 - #10 3 - #15 3 - 
#20 
- - tct gcg aaa ctt cgg gga tcg ttc gac tat at - #g ggt att aat cac 
tat 1008 
Ser Ala Lys Leu Arg Gly Ser Phe Asp Tyr Me - #t Gly Ile Asn His Tyr 
325 - # 330 - # 335 
- - aca acc tta tat gca aca agc act cct ccc ct - #t tcc ccc gac cac acg 
1056 
Thr Thr Leu Tyr Ala Thr Ser Thr Pro Pro Le - #u Ser Pro Asp His Thr 
340 - # 345 - # 350 
- - caa tat cta tat cca gac tct agg gtt tat ct - #g act gga gag cgc cac 
1104 
Gln Tyr Leu Tyr Pro Asp Ser Arg Val Tyr Le - #u Thr Gly Glu Arg His 
355 - # 360 - # 365 
- - gga gtc tcc atc gga gaa cgg aca ggg atg ga - #c ggt ttg ttt gtg gta 
1152 
Gly Val Ser Ile Gly Glu Arg Thr Gly Met As - #p Gly Leu Phe Val Val 
370 - # 375 - # 380 
- - cct cat gga att caa aaa ata gtg gag tat gt - #a aaa gaa ttc tat gac 
1200 
Pro His Gly Ile Gln Lys Ile Val Glu Tyr Va - #l Lys Glu Phe Tyr Asp 
385 3 - #90 3 - #95 4 - 
#00 
- - aac ccg act att att atc gca gag aac ggt ta - #t cca gag tct gag 
gaa 1248 
Asn Pro Thr Ile Ile Ile Ala Glu Asn Gly Ty - #r Pro Glu Ser Glu Glu 
405 - # 410 - # 415 
- - tcc tcg tcg act ctg caa gaa aat cta aac ga - #t gtg agg aga ata agg 
1296 
Ser Ser Ser Thr Leu Gln Glu Asn Leu Asn As - #p Val Arg Arg Ile Arg 
420 - # 425 - # 430 
- - ttt cat gga gat tgt ttg agt tat ctc agt gc - #a gca atc aaa aat ggc 
1344 
Phe His Gly Asp Cys Leu Ser Tyr Leu Ser Al - #a Ala Ile Lys Asn Gly 
435 - # 440 - # 445 
- - tca gat gtt cga ggg tac ttt gtg tgg tca ct - #t ctg gat aat ttt gag 
1392 
Ser Asp Val Arg Gly Tyr Phe Val Trp Ser Le - #u Leu Asp Asn Phe Glu 
450 - # 455 - # 460 
- - tgg gca ttt ggg tat acc att aga ttt ggt ct - #t tat cac gtg gat ttc 
1440 
Trp Ala Phe Gly Tyr Thr Ile Arg Phe Gly Le - #u Tyr His Val Asp Phe 
465 4 - #70 4 - #75 4 - 
#80 
- - att tct gat caa aag aga tat ccc aag ctc tc - #g gct caa tgg ttc 
aga 1488 
Ile Ser Asp Gln Lys Arg Tyr Pro Lys Leu Se - #r Ala Gln Trp Phe Arg 
485 - # 490 - # 495 
- - caa ttt ctt cag cac gac gat cag gga agt at - #t aga agc agc agc agc 
1536 
Gln Phe Leu Gln His Asp Asp Gln Gly Ser Il - #e Arg Ser Ser Ser Ser 
500 - # 505 - # 510 
- - att tag - # - # - 
# 1542 
Ile 
- - - - &lt;210&gt; SEQ ID NO 9 
&lt;211&gt; LENGTH: 29 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
NT1 
- - &lt;400&gt; SEQUENCE: 9 
- - tagctagcag gctggacagg aacaacttc - # - # 
29 
- - - - &lt;210&gt; SEQ ID NO 10 
&lt;211&gt; LENGTH: 24 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
CT1 
- - &lt;400&gt; SEQUENCE: 10 
- - ctcgagacaa gcagtctaaa tgct - # - # 
24 
- - - - &lt;210&gt; SEQ ID NO 11 
&lt;211&gt; LENGTH: 24 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
for 
amplification of the CBG cDNA seq - #uence 
- - &lt;400&gt; SEQUENCE: 11 
- - ggatttggac ctgaaaatat caat - # - # 
24 
- - - - &lt;210&gt; SEQ ID NO 12 
&lt;211&gt; LENGTH: 24 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
for 
the amplification of the CBG cDNA - # sequence 
- - &lt;400&gt; SEQUENCE: 12 
- - caatgttctt accctgcagt tccc - # - # 
24 
- - - - &lt;210&gt; SEQ ID NO 13 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
for 
the amplification of the CBG cDNA - # sequence 
- - &lt;400&gt; SEQUENCE: 13 
- - atggaggtgt ctgtgttgat gtgggta - # - # 
27 
- - - - &lt;210&gt; SEQ ID NO 14 
&lt;211&gt; LENGTH: 27 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Artificial Sequence 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: Description of Artificial - #Sequence:primer 
for 
the amplification of the CBG cDNA - # sequence 
- - &lt;400&gt; SEQUENCE: 14 
- - aatgctgctg ctgcttctaa tacttcc - # - # 
27 
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