Note: Descriptions are shown in the official language in which they were submitted.
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STARCH BRANCHING ENZYMES
TECHNICAL FIELD
The invention relates to the field of plant molecular biology, particularly to
enzymes of starch bio-synthesis.
BACKGROUND ART
The endosperm of wheat, barley, rye and triticale contain large A-type and
small B-type starch granules at maturityl. In wheat, the large A-type starch
granules are more than 10 ~m in diameter and lenticular in shape, while B-
type starch granules are less than 10 ~m in diameter and roughly spherical in
shape2. Because A- and B-type starch granules have significantly different
chemical compositions and functional properties3, wheat cultivars with
predominantly A- or B-type starch granules would be very useful to the food
and non-food industries.
A-type starch granules are produced in amyloplast at about four to five days-
post-anthesis (DPA), and their number increases until 12 to 14 DPA4.
Subsequently, the A-type starch granules grow in size to an eventual
diameter of from 10 ~m to more than 36 Vim. The number of A-type starch
granules per endosperm is constant from about 15 DPA to maturity.
B-type starch granules are actively initiated about 14-16 DPA. Both the
number and size of B-type starch granules increase until wheat grain
matures. The diameter of B-type starch granules is less than 10 ~mz.The
mechanisms controlling the initiation and size growth of A- and B-type
starch granules are unknown. Based on the current knowledge about starch
granule synthesis, several mechanisms could be proposed. The initiation and
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size growth of A- and B-type starch granules may be controlled by different
isoforms of starch synthases (SS), starch branching enzymes (SBE), and
debranching enzymes (DBE). These enzymes are involved in the biogenesis
of plant starch granules5. In the barley shrunken endosperm mutant (shx),
the size of A-type starch granules is reduced, giving the appearance of a
unimodal size distribution6. The soluble starch synthase I (SSS I) activity in
the shx endosperm is 86 % lower relative to the wild type, suggesting that
SSS-I may play a role in controlling the size growth of A-type starch
granules. However, there are no experimental results showing genetic
control of starch granule size distribution in wheats. 9.
Starch branching enzyme (a-1,4-glucan-6-glycosyltransferase; EC 2.4.1.18,
SBE) is a key enzyme in the starch biosynthesis pathway. The enzyme acts
on glucose polymers and catalyses excision and transfer of glucan chains to
the same or other glucan molecules. Translocated chains are attached to the
polymer through a-1,6-glucosidic bonds to form branches on the a-1,4-linked
glucose backbone. All of the reported SBE from plants to date can be divided
into two classes, SBEI and SBEII, based on their amino acid sequenceslo,
Most of the characterised plant SBEs are in the 80-100 kDa molecular mass
range and, like all enzymes of the a-amylase family, carry a (~3a)s barrel
domain with four highly conserved regions at the active sitell. Analysis of
plants with reduced SBEII activity and enzyme assays performed with
purified SBEI and SBEII proteins suggest that the two enzyme classes differ
in their enzymatic specificityl2 i3. The biochemical data suggest that SBEI
favours transfer of long glucan chains and acts primarily on amylose,
whereas SBEII produces shorter branches and prefers amylopectin as
substratel41s i6, However, the exact role of the different SBE classes in the
formation of the branched glucan polymers in planta is not clear. There is no
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previous evidence to suggest that there are SBEs specific to A- or B-type
starch granules.
DISCLOSURE OF THE INVENTION
In a first aspect, the invention provides a starch branching enzyme that is
bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
In a second aspect, the invention provides a DNA sequence encoding one of
the starch branching enzymes that is bound to A-type starch granules in
wheat, rye, barley or triticale endosperm.
In a third aspect, the invention provides a method for increasing the
concentration of A-type starch granules in endosperm of a wheat, rye, barley
or triticale plant by over-expressing in the plant a gene encoding a starch
branching enzyme that is bound to A-type starch granules.
In a fourth aspect, the invention provides a method for decreasing the
concentration of A-type starch granules in endosperm of a wheat, rye, barley
or triticale plant by suppressing the activity of a starch branching enzyme
that is bound to A-type starch granules.
In a fifth aspect, the invention provides a method for decreasing the
concentration of A-type starch granules in endosperm of a wheat, rye, barley
or triticale plant by suppressing the transcription and/ or translation of a
gene encoding a starch branching enzyme that is bound to A-type starch
granules in wheat, rye, barley or triticale endosperm.
In a sixth aspect, the invention provides a method of modifying starch
granule morphology in a plant expressing a gene encoding a starch
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branching enzyme that is bound to A-type starch granules in wheat, rye,
barley or triticale endosperm.
In a seventh aspect, the invention provides a method for analysing a plant to
determine the presence or absence of DNA encoding granule bound starch
branching enzyme, comprising the steps of:
providing a probe capable of hybridising with a DNA encoding a starch
branching enzyme that is bound to A-type starch granules in wheat, rye,
barley or triticale endosperm;
exposing the probe to sequences of DNA derived from the genome of the
plant; and
detecting whether hybridisation with the probe has occurred.
In an eight aspect, the invention provides a method for analysing a plant to
determine the presence or absence of transcripts encoding granule bound
starch branching enzyme, comprising the steps of:
providing a probe capable of hybridising with mRNA encoding a starch
branching enzyme that is bound to A-type starch granules in wheat, rye,
barley or triticale endosperm;
exposing the probe to RNA prepared from the plant or used in in situ
hybridisation analysis, and detecting whether hybridisation with the probe
has occurred;
providing specific primer for detection of transcripts encoding a granule
bound starch branching enzyme in wheat; where
detection is accomplished by RT-PCR analysis.
In a ninth aspect the invention provides an antibody raised to a starch
branching enzyme that is bound to A-type starch granules in wheat, rye,
barley or triticale endosperm.
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In a tenth aspect, the invention provides a method for analysing a plant to
determine the presence or absence of granule bound starch branching
enzyme, comprising the steps of:
exposing the proteins of the plant to an antibody raised to a starch branching
5 enzyme that is bound to A-type starch granules in wheat, rye, barley or
triticale endosperm; and
detecting whether the antibody has bound a starch branching enzyme that is
bound to A-type starch granules in wheat, rye, barley or triticale endosperm.
The invention also relates to a method of genetically transforming a plant so
that the plant expresses a starch branching enzyme that is bound to A-type
starch granules in wheat, barley, or triticale endosperm.
The invention further relates to a genetically modified plant expressing a
starch branching enzyme that is bound to A-type starch granules in wheat,
barley, or triticale endosperm.
The invention also relates to a genetically modified plant having within its
genome a hybrid gene, wherein the hybrid gene comprises a DNA sequence
encoding a starch branching enzyme that is bound to A-type starch granules
in wheat, barley or triticale endosperm, or a fragment thereof, fused to a
passenger-gene.
DETAILED DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic alignment of pABEI and pRN60 cDNA. Hatched
area of pABEI coding region (grey box) represents sequence encoding a
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putative transit peptide and horizontal arrows on the pRN60 cDNA show
location of imperfect direct repeats. The four black areas within the coding
region represent sequences encoding the highly conserved regions of
enzymes belonging to the a-amylase familyil. DNA fragments used as
probes in DNA and RNA hybridisations are indicated below.
Figure 2 shows RNA gel analysis of Sbe1 expression during wheat kernel
development.
A Analysis of total RNA (20 fig) prepared from developing kernels
harvested at different DPA. The blot was hybridised with probe 2 (Figure 1)
and estimated sizes of hybridising RNA species are shown to the left.
Migration of RNA size markers is indicated to the right.
B Same blot as above hybridised with a 25S rRNA DNA probe.
-15---Figure 3-shows isolation of cDNA corresponding-to- the 5-'-end- of
the°4.6 kb
Sbelc transcript.
A Schematic illustration of the 4.6 kb Sbelc transcript and product
obtained from 5'-RACE analysis. Start of pRN60 sequence and location of
PCR primers used in the 5'-RACE and RT-PCR reactions are indicated.
B Gel analysis of 5'-RACE products obtained in reactions with primers
indicated and poly(A)+ RNA prepared from 12-day-old wheat kernels.
Arrows indicate migration of product carrying the 5' end of the 4.6 kb Sbelc
cDNA. Migration of standard DNA fragments are indicated to the right.
C Gel analysis of RT-PCR products obtained from reactions with PCR
primers BE65 and BE38.
Figure 4 shows the nucleotide sequence and deduced amino acid sequence of
the 4.6 kb SBEIc transcript produced in the wheat endosperm. Possible
polyadenylation sequence is underlined and proposed transit peptide
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cleavage site is indicated by an vertical arrow. Shadowed regions represent
conserved sequences in enzymes belonging to the a-amylase familyll. Start
of pRN60 sequence and location of PCR primers used in the study are
indicated.
Figure 5 shows a schematic illustration of SBEIc precursor encoded by 4.6 kb
Sbelc transcript. DNA sequences corresponding to exons 1 to 14 on wheat
genomic Sbe1 1~ are indicated. Hatched areas indicate location of predicted
transit peptide and domains 1 and 2 encompass SBEI-like sequences. The
location of the four highly conserved regions on (~3a)8 barrels of amylolytic
enzymesll are indicated by black boxes and their sequences are shown
below. Highly conserved residues are indicated by asterisks and catalytic
residues present only on domain 2 are underlined. SBEIc is aligned with the
SBEI-like protein deduced from the wSBEI-D2 cDNAl$ and the wheat 87 kD
SBEIbI9 _ _ _ - _ . _ _ _ -- . _.
Figure 6 shows the expression analysis of Sbelc in Escherichia coli.
A Schematic illustration of the expression vector pQE-SBEIc carrying
sequences encoding mature SBEIc with histidine tag (black box) added at the
amino-terminal end.
B Analysis of BE activity by iodine staining and phosphorylase a
stimulation assay. BE activities were determined from the BE-positive strain
DH5a and the BE-deficient strain KV832, transformed with plasmids
indicated. Construct pREP4-cm expresses the Lac repressor and pQE30 is a
cloning vector used for construction of pQE-SBEIc. The BE activity values
and standard errors determined by the phosphorylase a stimulation assay2o
are expressed as ~mol glucose-1-phosphate incorporated mg protein-1 min-1
and were determined from three separate experiments.
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C SDS-PAGE and immunoblot analysis of recombinant wheat SBEIc
produced in Escherichia coli. Total cell extracts of non-induced and IPTG-
induced cultures of the BE-deficient strain, KV832, harbouring pREP4-cm
and plasmid indicated were analysed. The immunoblot analysis was done
with antibodies prepared against wheat 87 kD SBEI. Migration of marker
proteins revealed by amido black staining is shown to the right.
Figure 7 shows an immunoblot analysis of starch granule-bound proteins.
A Analysis of starch granule-bound proteins by SDS-PAGE and silver
staining. Migration of marker proteins (St) is shown to the left.
B Immunoblot analysis of starch granule-bound proteins using
antibodies prepared against wheat 87 kD SBEI and SBEII. Migration of
marker proteins revealed by amido black staining is shown to the right.
Figure 8 shows SDS-PAGE analysis-of starch-granule proteins produced in
wheat endosperm.
A Analysis of granule-bound proteins produced in developing
endosperm of the hexaploid wheat cultivar, Fielder. Solid arrow indicates
migration of SBEIc isoforms and open arrow shows migration of 59 kD
GBSSII present in pericarp starch2l.
B SDS-PAGE analysis of granule-bound proteins extracted from mature
kernels of Triticum monococcum Tm 23 (lane 1), Triticum tauschii accession PI
511-380 (lane 2), Triticum turgidum ssp. durum cultivar Kyle (lane 3) and
Triticum aestivum cultivar Fielder (lane 4). Arrows indicates proteins
recognised by SBEI antibodies and with similar migration as SBEIc.
Figure 9 shows SDS-PAGE analysis of SGP extracted from wheat A- and B-
type starch granules. Each lane was loaded with protein extract from 5 mg A-
and B-type starch granules of five hexaploid and one tetraploid (Plenty)
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cultivar. Separated proteins were visualised by silver staining and migration
of protein molecular weight markers (Mr) is indicated to the right.
Figure 10 shows analysis of starch granule size distribution in wheat
endosperm.
A Light microscopic pictures (500x) of total starch granules harvested at
different stages of endosperm development of the hexaploid wheat cultivar
CDC Teal.
B Histogram of large-size (>10 Vim) and small-size (<10 Vim) granule size
distribution during wheat endosperm development.
Figure 11 shows SDS-PAGE analysis of SGP extracted from large-size (>10
Vim) and small-size (<10 Vim) starch granules of the hexaploid wheat cultivar
CDC Teal. Samples of SGP from 5 mg starch granules were from different
stages of wheat endosperm development as indicated. Gel-separated
proteins were visualised by silver staining and migration of protein
molecular weight marker (Mr) is indicated to the right.
Figure 12 shows immunoblot analysis of extracted SGP from wheat A- and
B-type starch granules. Each lane was loaded with SGP extracted from 2 mg
A- and B-type starch granules harvested from mature endosperm of the
hexaploid wheat cultivar CDC Teal. To the left is shown SGP separated by
SDS-PAGE and visualised by silver staining. To the right is shown
immunoblot analyses of gel-separated SGP using polyclonal antisera
prepared against different wheat starch biosynthetic enzymes as indicated.
Figure 13 shows sub-cellular localisation of SGP-140 and SGP-145 in
immature wheat kernels.
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SDS-PAGE analysis of SGP extracted from CDC Teal pericarp starch,
endosperm starch and soluble endosperm proteins were prepared from
different DPA of endosperm development as indicated. Samples of soluble
protein [280 (10 DPA), 250 (15 DPA) or 250 (20 DPA) fig] and starch granules
5 (5 mg) analysed were derived from the same amount of endosperm tissue.
Gel-separated proteins were visualised by silver staining (pericarp and
endosperm starch analysis) or Coomassie blue staining (soluble endosperm
analysis). Migration of molecular weight marker (Mr) is shown to the right.
Below is shown immunoreactive bands formed between gel-separated SGP-
10 140 and SGP-145 and wheat SBEI antibodies.
Figure 14 shows analysis of SGP in starches from various plant sources.
A SDS-PAGE analysis of SGP extracted from 5 mg starch of: A-type
starch granules from endosperm of triticale, wheat, barley and rye; total
-15 --starch from endosperm-of-canary-seed; rice and -maize; and potato
tubers.
Proteins were visualised by silver staining. Migration of molecular weight
marker (Mr) is shown to the right.
B Immunoblot analysis of gel-separated proteins shown above.
Immunoreactive bands obtained from interaction between wheat SBEI
polyclonal antibodies and SGP-140 and SGP-145 are indicated.
C SDS-PAGE analysis of extracted SGP from 5 mg A- and B-type
starches isolated from wheat, barley, rye and triticale endosperm. Proteins
were visualised by silver staining. Migration of molecular weight marker
(Mr) is shown to the right.
The inventors have characterised a cDNA encoding a novel form of SBEI in
wheat endosperm. The encoded polypeptide was found to be preferentially
associated with A-type starch granules.
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Isolation of a Partial SBEI cDNA Clone
During screening of a wheat (Triticum aestivum L. cv. Fielder) cDNA library
for Sbe1 clones using probe 1, the pRN60 clone was isolated (Figure 1). DNA
sequence analysis of pRN60 revealed a 2962 by insert that was 162 by longer
than a previously characterised full-length SBEI cDNA, pABEI, isolated from
the same library (Figure 1)19. The two cDNA clones matched almost
perfectly from the 3' end to 346 nucleotides from the 5' end of the pRN60
cDNA (99.8 % nucleotide identity and 100 % encoded amino acid identity), at
which point the two sequences diverged. In contrast to the pABEI cDNA, the
346 by 5' sequence of pRN60 cDNA did not seem to encode a transit peptide,
but instead matched sequences located further downstream on the same
cDNA. The unusual 5' sequence carried by pRN60 lacked stop codons in
frame with the downstream SBEI coding region, which suggested that the
isolated cDNA could be translated from the first base, and therefore, might
not represent a full-length transcript.
RNA Blot Analysis of Wheat Endosperm Reveals Two Sbe1 Transcripts
The existence of Sbe1 transcripts that were longer than those encoding the 87
to 88 kD SBEI isoforms was confirmed by an RNA gel blot analysis. This
analysis of wheat kernel RNA extracted at various time points during kernel
development showed that a transcript of about 5 kb, in addition to the
expected 2.8 Sbe1 mRNA, was recognised by the Sbe1-specific probe (Figure
2A). The signals from both the 5 and 2.8 kb transcripts were very weak in
samples of five-day-old kernels, in which the endosperm is very immature,
but were clearly seen in samples prepared from 10- to 25-day-old kernels. In
kernels younger than 10 days post anthesis (DPA), the 5 kb hybridisation
signals appeared stronger as compared to the signal from the 2.8 kb
transcript.
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Isolation of Full-length cDNA Corresponding to 4.6 kb Sbel Transcript
With the hypothesis that the pRN60 cDNA was a partial product of the about
kb Sbe1 transcript, the inventors isolated the 5' end of this mRNA species
using a 5'-RACE procedure. Gel analysis of products obtained from the final
5 PCR reaction revealed one major fragment of 1.9 kb and three minor
fragments (Figure 3B, lane AP2+BE39). No products were obtained from
control reactions employing only one primer (Figure 3B; lanes AP2 and
BE39). The different PCR products were analysed by DNA sequencing,
which showed that only the 1.9 kb fragment carried Sbe1-like sequences.
One of the 1.9 kb 5'-RACE products was found to correspond 100% to the 272
by region overlapping the 5' end of pRN60, and the composite cDNA
sequence obtained with this product and the pRN60 cDNA gave a 4563 by
long sequence. This assembled sequence was denoted Sbelc to distinguish it
from the inventors' previously characterised wheat Sbe1 clones, Sbelal~ and
-15 -Sbe1-b19-~_.__ - -_____ __- ____ _._. ___ _
The 5'-RACE analysis suggested that several variants of the 4.6 kb Sbelc
transcript were produced in the wheat endosperm. RT-PCR analysis using
the BE65/BE38 primer pair (Figure 3A) and endosperm RNA further
confirmed this observation. The 2.0 kb RT-PCR products generated from
three independent RT-PCR experiments (Figure 3C, lane BE65+BE38) were
found to be of at least three different variants, that differed slightly in
deduced amino acid sequence. One of the sequence variants matched exactly
to the corresponding sequence on Sbelc, and thus, independently confirmed
the 2.0 kb 5' sequence of Sbelc.
The 4563 by SBEI cDNA Encodes a Protein With Two SBEI-like Domains
DNA sequence analysis of the 4563 by Sbelc cDNA (Figure 4, and SEQ. ID
N0:1) revealed an open reading frame of 1425 codons that was initiated
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from the 5' end of the assembled sequence and terminated at nucleotides
4278-4280. The TAA stop codon was followed by a possible polyadenylation
signal sequence, AATAAA, located 19 by upstream of the polyadenylation
tail. Initiation of translation was assigned for the first ATG codon
(nucleotides 63-65), allowing translation of 1405 codons of the open reading
frame. Sequence analysis of the proposed amino-terminal region of SBEIc
revealed a 50% sequence identity to transit peptides predicted from wheat
Sbela and Sbelb. Thus, SBEIc appeared, like the 87 kD SBEI, to be imported
into plastids. Cleavage of the transit peptide was proposed to occur between
amino acids Ala6~ and Alabs of the deduced SBEIc sequence (Ile-Ala-
AIa.~Ala), as this site showed high resemblance to the consensus sequence for
transit peptide cleavage sites, Val/Ile-X-Ala/Cys~.Ala~. Processing of the
SBEIc precursor would leave a 1338 amino acid long mature protein with a
calculated molecular mass of 152 kD. The transit peptide cleavage site was
confirrried-by N-term'irial sequencing of SBEIc isoforms produced in the
wheat cultivar Teal (data presented further below in Table 1).
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pat
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Analysis of the deduced mature SBEIc sequence disclosed the presence of
two SBEI-like sequences, domain 1 and 2, encompassing amino acids 1-561
and 570-1338, respectively, on the mature SBEIc (Figure 5). As already
mentioned, the sequence of the second domain was identical to that of the
5 mature protein encoded by the pABEI cDNA. The main difference between
the first domain and the second domain was the lack of a 21 and a 163 amino
acid long sequence on domain 1. These two sequences corresponded to exon
nine and exons 11 to 14, respectively, on wheat genomic DNA coding for the
87 kD SBEI (Figure 5). Further analysis of SBEIc showed that the first
10 domain including the transit peptide was very similar to the first 629
amino
acids (92% identical residues) of a 686 amino acid long SBEI-like protein,
wSBEI-D2, presumed to be produced in the wheat endospermls. The
proposed translational start codons coincided for wSBEI-D2 and SBEIc
cDNA, but no sequence corresponding to the 57 long carboxy-terminal
15 residues-of-wSBEI=D2-was present on-SBEIc. - -- - -
The first domain of SBEIc and the corresponding sequence on wSBEI-D2
differed from other characterised SBEI from plants at the four highly
conserved regions on enzymes belonging to the a-amylase family, which
include plant SBE11. It was especially notable that the Asp residues on
regions two and four and the Glu residue on region three, all proposed to be
directly involved in hydrolysis of a-1,4 glucan bondsll, were replaced by
non-equivalent residues (Figure 5).
Expression of Sbelc Complements a BE Mutation in Escherichia coli
To examine if the isolated cDNA encoded an active enzyme, a prokaryotic
expression vector, pQE-SBEIc, encoding a histidine-tagged mature SBEIc
(amino acids 1-1338) was constructed (Figure 6A) and tested for activity in a
Escherichia coli BE-deficient mutant, KV83223. Since high level expression of
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the His-tagged SBEIc was found to severely affect cell growth, a construct
expressing the Lac repressor (pREP4-cm) was also introduced into the cells to
control transcription from the strong T5 promoter. SDS-PAGE and
immunoblot analysis of extracts prepared from the transformed KV832 cells
confirmed that a polypeptide of-expected molecular mass (154 kD) was
produced at a very low level in non-induced cells, but was clearly seen in
cells induced with IPTG for two hours (Figure 6C, lane 4). The BE-mutant
carrying pREP4-cm and cloning vector pQE30 showed a blue/ grey colour
upon iodine staining, indicating low or no branching of the glucan polymers
(Figure 6B). Expression of pQE-SBEIc in KV832 cells harbouring pREP4-cm
resulted in a brown colour upon iodine staining, showing that the BE-mutant
had regained the ability to branch glucan molecules. The BE-positive strain,
DHSa, transformed with pREP4-cm and pQE30A gave a yellow/brown
colour upon treatment with iodine, as expected from a strain able to produce
-glycogen-hkE-polymers: The slight differences in iodine staining patterns of
cells producing plant and bacterial BE has been suggested to reflect
differences in enzyme specificity24. Production of BE activity from cells
expressing Sbelc was confirmed by the phosphorylase a assay2~, which
revealed a >90-fold higher level of BE activity in soluble cell extracts of
non-
induced KV832 cells harbouring pQE-SBEIc, as compared to KV832 cells
lacking this construct (Figure 6B). The BE-positive strain, DHSa, produced a
4.5 times lower level of BE activity than the complemented KV832 cells. The
BE activity in induced cells expressing Sbelc was not assessed, since most of
the produced SBEIc in these cells was deposited into inclusion bodies.
The 152 kD SBEI is Associated with Starch Granules of the Wheat
Endosperm
To test if the granule-bound protein of about 149 kD reported by Schofield
and Greenwell (19825 could correspond to SBEIc, the inventors analysed
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starch granule extracts by SDS-PAGE and immunoblotting. Silver-staining
of extracted and gel-separated proteins from granules of mature hexaploid
wheat kernels resolved seven clearly visible protein bands, of which one
band migrated as a 140 kD protein in the gel system used (Figure 7A).
An immunoblot analysis of the gel-separated proteins using polyclonal
antiserum prepared against the wheat 87 kD SBEIb confirmed that the 140
kD protein band was related to SBEI (Figure 7B, lane a-SBEI). The
immunoblot analysis also revealed an interaction with the 92 kD protein
band and several 62 to 67 kD protein bands of unknown identities. Since the
140 kD granule-bound protein corresponded reasonably well in mass to
SBEIc and no SBEI corresponding in mass with SBEIc was found by
immunoblot analysis of the soluble endosperm (data presented in Figure 13),
the inventors reasoned that SBEIc was incorporated into starch granules.
Further analysis of the granule-bound proteins using polyclonal antibodies
prepared against a 87 kD wheat SBEII, revealed only an interaction with the
92 kD protein band (Figure 7B, lane a.-SBEII), as previously reported by
Rahman et al. (1995)26. Thus, isoforms analogous to SBEIc and bound to
starch granules did not seem to exist for SBEII in wheat.
A gel analysis of granule-bound proteins extracted from developing kernels
at different stage after anthesis showed no presence of the 140 kD protein
band in starch prepared from kernels that were less than five days old. These
young kernel samples contained a substantial amount of pericarp starch, as
indicated by the presence of the 59 kD GBSSII21 (Figure 8A). The 140 kD
protein band appeared in total kernel starch between five and seven DPA
and its abundance was relatively constant from there on. Thus, the
accumulation of the large isoform of SBEI in the kernel starch coincided with
the accumulation of the 4.6 kb SBEI transcript during kernel maturation
(Figure 2).
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One or two proteins corresponding closely in migration with SBEIc were also
found associated with starch granules of Triticum monococcum, Triticum
tauschii and Triticum turgidum ssp. Durum (Figure 8B), and immunoblot
analysis confirmed that these proteins were recognised by SBEI antibodies.
The inventors concluded that SBEIc isofornis must be encoded by all three
genomes of hexaploid wheat.
The inventors have further demonstrated that SBEIc and its isoforms are
preferentially associated with A-type starch granules of wheat endosperm.
SBEIc Isoforms are Preferentially Associated with A-type Starch Granules
in Wheat Endosperm
The inventors compared starch granule proteins (SGPs) localised in A- and
B-type starch granules, by purifying the two granule fractions from wheat
-endosperm-of six wheat cu-1-tiva-r-s using a-method previously reported2~.
The
extracted SGPs were resolved by SDS-PAGE and visualised by silver
staining. To quantitatively compare the different polypeptides in A- and
B-type starch granules, the 60 kD GBSSI was used as an internal standard for
equal loading of proteins. The major SGP of 60, 80, 92,100,108 and 115 kD,
were present in similar concentrations in A- and B-type starch granules from
all the cultivars tested (Figure 9), and no difference was observed among
polypeptides with molecular masses lower than 60 kD. These results were
consistent with previous studies that reported almost identical polypeptide
profiles for wheat A- and B-type starch granules48 26 29
In addition to known SGPs, the inventors found that A-type starch granules
of all wheat cultivars tested contained a polypeptide co-migrating with SBEIc
of Fielder (Figure 9). A slightly larger polypeptide, with an apparent
molecular mass of 145 kD, was also present in A-type starch granules of all
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cultivars except Fielder (Figure 9). Analysis of B-type starch granules from
the six wheat cultivars showed a much lower abundance of the 140 and 145
kD polypeptides as compared to the A-type granules. In B-type granules of
the cultivar Fielder, only the 140 kD band was observed. Thus, the inventors
concluded that SGP-140 band, which includes SBEIc in Fielder, and SGP-145
are preferentially associated with A-type starch granules.
In developing wheat endosperm, A-type starch granules are initiated at
about four to 14 DPA, whereas B-type granules are formed after 14 DPA4 so
After initiation, both granule types continue to grow until maturity of the
endosperm3l. An image analysis of purified large-size and small-size starch
granule fractions from developing endosperm of the cultivar CDC Teal
showed that the growth of small starch granules formed before and after 15
DPA was significantly different (Figure 10). Prior to 15 DPA, the newly
-15 formed small starch granules grew-rapidly in size to become large-size
(>10
Vim) starch granules (Figure 10A). During the time period eight to 15 DPA,
large-size starch granules accounted for more than 70% of total endosperm
starch granules (Figure 10B). Small-size starch granules formed after 15 DPA
increased rapidly in number until maturity (from 25% to 94%), but they grew
very slowly and only reached diameters less than 10 Vim.
SGP-140 and SGP-145 are Preferentially Incorporated into A-type Starch
Granules Throughout Endosperm Development
The preferential incorporation of SGP-140 and SGP-145 into A-type granules
can be explained by synthesis of these polypeptides only during the first 15
DPA. To study this possibility, the inventors analysed the protein profiles of
large-size and small-size granules isolated at different DPA (Figure 11). The
large-size (>10 Vim) A-type starch granules were found to show no variation
in SGP-140 and SGP-145 concentration during development. Small-size
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starch granules (<10 ~m in diameter) formed before 15 DPA, which were of
the A-type, were also found to contain SGP-140 and SGP-145 at about the
same concentration as in large-size granules. On the other hand, small-size
B-type starch granules harvested after 15 DPA, showed very low presence of
5 SGP-140 and SGP-145. The analyses demonstrated no significant variation in
concentration of the other major granule-bound polypepHdes (60, 80, 92,100,
108 and 115 kD) for both small-size and large-size starch granules
throughout endosperm development. In the cultivar CDC Teal, most of the
A-type granule growth occurred after 15 DPA, when about 65% (w/w) of the
10 starch in A-type granules was synthesized. The constant levels of SGP-140
and SGP-145 in A-type granules strongly suggested that the two proteins
were continuously incorporated into A-type granules throughout endosperm
development.
15 The-ratio of total SG-P=140=plus-SGP-145 in-A=type granules versus total
SGP-140 plus SGP-145 in B-type granules is preferably at least about 4, more
preferable at least about 5, most preferably at least about 10.
Both SGP-140 and SGP-145 are Related to SBEI
20 To confirm the identity SGP-140 as an SBEI isoform in the cultivar CDC Teal
and to identify SGP-145, immunoblots of SGP from A- and B-type starch
granules were reacted with polyclonal antibodies raised against wheat SBEI,
SBEII, SSI, SSII and GBSSI, respectively (Figure 12). The major polypepHdes
of 60 kD (GBSSI), 80 kD (SSI), 92 kD (SBEII) and 100 to 115 kD (SSII), were
recognised by their respective antibodies, as expected, with no difference in
intensity between A-type and B-type granules. Among the five antibodies
tested, only the wheat SBEI antibodies reacted with SGP-140 and were also
found to recognise SGP-145. A weaker interaction between the SBEI
antibodies and a protein co-migrating with SBEII and proteins of
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approximately 63 kD were also seen. Similar to the analysis of SGP-140 and
SGP-145 by SDS-PAGE, the immunoreactive bands were strong in A-type,
but weak in B-type starch granules.
The inventors have further corifirnied thafSGP-140 and SGP-145 protein
bands of the wheat cultivar CDC Teal have very similar N-terminal
sequences as SBEIc. Direct amino acid sequencing of the protein bands
purified from SDS-PAGE gels suggested variation in amino acid sequence as
indicated in Table I. This is likely due to presence of several polypeptides
that differ slightly in sequence within the same protein band. Presence of
several isoforms of SBEIc was also suggested by reverse transcription PCR
analysis of transcripts produced in the cultivar Fielder. Alignment of the
determined N-terminal sequences of the SGP-140 and SGP-145 with those
predicted for SBEIc and wSBEI-D2 revealed striking similarities, thus
-suggesting that all four-polypeptides-were closely=related (-Table I). A
lower
level of similarity was noted to the predicted N-terminal sequence for the
wheat 87 kD SBEIbI9 isoform. Since the molecular masses of SGP-140 and
SGP-145 were reasonably close to that of SBEIc (152 kD) predicted from Sbelc
cDNA, the inventors concluded that SGP-140 and SGP-145 bands contain
isoforms of SBEIc.
SGP-140 and SGP-145 are Only Located to Starch Granules in the Wheat
Endosperm
To localise SGP-140 and SGP-145 in the developing kernels, SGP from
pericarp and endosperm starch granules, and the soluble endosperm fraction
were prepared from developing wheat kernels, and analysed by SDS-PAGE
and immunoblotting (Figure 13). The results of these analyses confirmed
that SGP-140 and SGP-145 were present within the endosperm starch
granules, but could not be found in the endosperm soluble fraction. Nor
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were SGP-140 and SGP-145 observed in pericarp starch granules harvested
from 5 to 10 DPA, but could be seen as two very faint bands in pericarp
granules of 15 DPA. Since pericarp from kernels older than 15 DPA was
rather difficult to separate from the endosperm, it is possible that the two
faint bands seen in 15 DPA pericarp sample originated from some
endosperm starch granules mixed with the pericarp starch granules.
SGP-140 and/or SGP-145 Homologues Exist in Plant Species Known to
Produce A- and B-type Starch Granules
The inventors' study included starches from plants with bimodal (rye, barley
and triticale) and unimodal (rice, maize, potato, canary seed) starch granule
size distributions. SDS-PAGE analysis of extracted SGP from triticale, barley
and rye revealed one (barley and rye) or two protein bands (triHcale) with
similar relative mobility as SGP-140 and SGP-145 of wheat (Figure 14A).
~'hese protein bands-were-also=found -to react w-ith-SBEI antibodies (Figure
14B), and thus appeared to be SGP-140 and SGP-145 homologues. Analysis
of canary seed, rice, maize and potato SGP did not reveal presence of any
polypepddes similar in size to SGP-140 and SGP-145 and reacting with SBEI
antibodies (Figures 14A and 14B). Thus, it appeared that proteins similar to
SGP-140 and SGP-145 were only present in cereal starches with bimodal
granule size distribution.
To determine if the SGP-140 and SGP-145 counterparts in triticale, barley and
rye were, like in wheat, preferentially associated with A-type starch
granules,
the A- and B-type starch granules from these cereals were analysed. Similar
to wheat endosperm starch, the SGP-140 and SGP-145 homologues were
abundant in A-type starch granules, but very scarce in B-type starch granules
(Figure 14C).
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The results show that SGP-140 and SGP-145 are preferentially found on both
small-size and large-size A-type granules (Figure 11). No reduction was
noted in SGP-140 and SGP-145 concentrations in large granules harvested
after 15 DPA (Figure 11), a developmental stage when most of the A-type
granule starch is being produced. This suggests that SGP-140 and SGP-145
are continuously targeted to A-type granules, even when B-type granules are
produced. Since SGP-140 and SGP-145 did not accumulate in the soluble
phase of the endosperm, these proteins must be actively produced both
before and after 15 DPA. This was also indicated by RNA analysis of SGP-
140 gene expression during kernel development, which showed only a small
reduction in transcript levels after 15 DPA, as compared to before 15 DPA
(Figure 2).
The inventors demonstrated that the 140 kD protein band revealed by SDS-
PAGE analysis of Fielder wheat starch granules contains a novel 152 kD
isoform of SBEI in plants. SBEIc encoded by the isolated cDNA differed from
previously characterised SBEI isoforms by its high molecular mass and by
the presence of two domains of SBEI-like sequences. Domain 1 differs from
domain 2 by the lack of a 21 amino acid long peptide and a 163 residue long
(~17 kD) C-terminal sequence (Figure 5).
The inventors study showed that the 152 kD SBEIc represents a granule-
bound form of SBEI.
The analysis of SBEI transcripts produced in the developing wheat
endosperm of the cultivar Fielder suggested that there are at least three
different forms of SBEIc transcripts produced. These variants would encode
proteins of very similar molecular masses (<1 kD difference), and thus,
cannot be distinguished as separate bands on one-dimensional SDS-PAGE
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gels. Our analysis of starch granules of Triticum species suggested that
variants of SBEIc also exist in both diploid (Triticum monococcum, Triticum
tauschii) and tetraploid (Triticum turgidum ssp. durum) wheat (Figure 8B). For
the tetraploid wheat cultivar Kyle, two separate protein bands were
distinguished, and apparently, the difference between the SBEIc isoforms in
this cultivar is more distinguishable on SDS-PAGE gels than those of the
hexaploid wheat cultivar Fielder.
INDUSTRIAL APPLICABILITY
SEBIc is a novel starch branching enzyme. It can be used in vitro to
synthesise or modify starch. Modified starches find use in the food and
beverage industries as a thickener and sweetener, as well as in industrial
uses, such as the production of stiffening agents for laundering, sizing for
-_ -paper and as thickening agents and_adhesives 3z 33. _-_
The Sbelc sequence, or fragments thereof, or complementary sequences to
any of these can be used to screen plant genomes to locate genes that are
homologous (i.e. which encode similar activities).
Expression of SBEIc in a plant can be expected to result in modification of
starch granule morphology and size distribution in seed endosperm. The
Sbelc gene may be expressed in a plant already having a copy of this gene, in
which case the expression SBEIc can be expected to increase. Increase in
SBEIc expression may result in increase in A-type starch granule
concentration, and/or in increase in starch granule size. Cultivars having
increased A-type granules would be desirable, for example, in the
production of gluten, as A-type granules are more easily separated from the
protein of the endosperm. Wheat starch with elevated A-granule content has
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applications in the manufacture of biodegradable plastic film and carbonless
copy paper.
In addition to the sequence of Sbelc, listed in SEQ ID NO:1, the invention
5 also relates to homologous variants of SEQ ID N0:1, including DNA
sequences from plants encoding proteins with two SBEI-like domains, as
illustrated in Figure 5, and deduced amino acid sequences of 25% or greater
identity, and 40% or greater similarity, isolated and/or characterised and/or
designed by known methods using the sequence information of SEQ ID
10 N0:1 or SEQ ID NO: 2, and to parts of reduced length that are able to
function as inhibitors of gene expression by use in an anti-sense, co-
suppression [Transwitch~ gene suppression technology; U.S. patent no.
5,231,020, July 27,1993; for reviews see Iyer et al. (2000)35, Baulcombe
(1996)36
and Vaucheret et al. (1998)3] or other gene silencing technologies. It will be
15 -appreciated by-per-sons-skilled-in-the-ar-t-that small changes in the
identities
of nucleotides in a specific gene sequence may result in reduced or enhanced
effectiveness of the genes and that, in some applications (e.g. anti-sense or
co-
suppression), partial sequences often work as effectively as full length
versions. The ways in which the gene sequence can be varied or shortened
20 are well known to persons skilled in the art, as are ways of testing the
effectiveness of the altered genes. All such variations of the genes are
therefore claimed as part of the present invention.
Other preferred degrees of identity to the indicated sequences are at least
25 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%; and other preferred degrees of
similarity are at least 50%, 60%, 70%, 80%, 90% and 95%. To assess sequence
homology, a computer program known as MegAlign~, DNASTAR~ of
DNASTAR Inc.,1228 South Park Street, Madison, WI 53715, USA, may be
used. This program is based on the Clustal V algorithm38. For each gap
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introduced in the alignment, the program deducts a penalty from the score.
A higher gap penalty suppresses gapping; a lower value promotes it. The
program also assesses penalties based on the length of the gap. The more
residues the gap spans, the greater the penalty. The program deducts these
penalties from the overall score of the alignment.
The expression "homologous variant", when referring to a DNA sequence,
encompasses all DNA sequences encoding a protein having the same
functionality as the recited sequence, as well as those having two SBEI-like
domains, illustrated in Figure 5. The same expression, when referring to an
amino acid sequence, encompasses all amino acid sequences having the same
functionality as the given sequence.
Suppression of transcription and/or translation of Sbelc, for example, by
15- casing anti-sense approaches, would be expected to reduce the
concentration
of A-type starch granules. Reduction in A-type granules is desirable if the
starch is going to be used as face powder, as a laundry-stiffening agent, a
fat
replacement or in the production of degradable plastic films39 40.
For the purposes of breeding cultivars having enhanced A-type starch
granule concentration, probes based on the sequence of Sbelc (SEQ ID NO:
1) or complementary sequences may be used to screen the genome of existing
cultivars to find those cultivars having within their genome homologues
(particularly alleles) of Sbelc, encoding SBEs that are preferentially bound
to
A-type starch granules. Such cultivars can be chosen for crossbreeding with
one-another, resulting in progeny strains having a high level of SBEIc or
homologue expression. Alternatively, cultivars having a low level of
Sbelc-like sequences within their genome can be expected to have a low level
of A-type starch granules. Such culdvars could be chosen for crossbreeding
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with one-another, resulting in progeny strains having a low level of SBEIc
expression, and a reduced content of A-type starch granules.
Similarly, strains expressing SBEIc or homologous variants can be found
using antibodies raised to SBEIc (polyclonal or monoclonal) to screen cereal
varieties to find those having SBEIc or variants. Antibodies to SBEIc can be
produced by known methods4i 42 4s 44.
The invention also relates to a method of genetically transforming a plant so
that the plant expresses a starch branching enzyme that is bound to A-type
starch granules in wheat, barley, or triticale endosperm.
The invention further relates to a genetically modified plant expressing a
starch branching enzyme that is bound to A-type starch granules in wheat,
-barley, or triticale endosperm: _- -- ~ ---~ - --
The invention also relates to a genetically modified plant having within its
genome a hybrid gene, wherein the hybrid gene comprises a DNA sequence
encoding a starch branching enzyme that is bound to A-type starch granules
in wheat, barley or triticale endosperm, or a fragment thereof, fused to a
passenger-gene. The protein encoded by the hybrid gene is preferably
targeted to starch granules. The passenger-gene preferably encodes a
vaccine, an antibody, a pigment, a preservative, a fragrant or flavour
inducing agent, a receptor, or an enzyme involved in lipid, carbohydrate or
protein synthesis, degradation or modification.
Genetically modified plants expressing SBEIc activity would be expected to
have altered starch granule morphology. In plants having unimodal starch
granule deposition in the wild type, the transformant could be expected to be
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either bimodal (i.e. large and small starch granules), or unimodal, but with
an increase in starch granule size.
Methods for transforming plants are known in the art [see, for example,
Potrykus (1991)45; Vasil (1994)46; Walden and Wingender (1995)4'; Songstad et
al.
(1995)4$; Bechtold et al. (1993)49; Katavic et al. (1994)5°; DeBlock et
al. (1989)5';
Moloney et al. (1989)52; Sanford et al. (1987)53; Nehra et al. (1994)54;
Becker et al.
(1994)55; Rhodes et al. (1988)56; Shimamoto et al. (1989)5'; Meyer, (1995)5$;
Datla et al.
( 1997)s9~
BEST MODES FOR CARRYING OUT THE INVENTION
Abbreviations:
DTT: diothiothreitol
EDTA: ethylene diamxnine tetraacetate
IPTG: isopropyl ~i-D-thiogalactopyranoside
5'-RACE: 5'-rapid amplification of cDNA ends
RT-PCR: reverse transcription -polymerase chain reaction
SDS: sodium dodecyl sulfate
Tris: tris(hydroxymethyl)aminomethane
SCREENING OF A WHEAT CDNA LIBRARY
Approximately 200,000 plaques of a cDNA library, constructed from wheat
poly(A)+ RNA isolated from 12-day-old wheat kernels6~, were screened for
Sbe1 clones by plaque hybridisation6l. Probe 1 used in the library screening
consisted of an 828 by Reverse Transcription-PCR (RT-PCR) product,
obtained from a reaction using 12 day old wheat kernel RNA and the
Sbe1-specific primers BE11 and BE12 (Figures 1 and 4). The primers were
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based on sequences of previously characterised Sbe1 clones from wheatl~ is,
Ten of the positive clones were plaque-purified and their inserts were excised
in vivo from the Uru-ZAP XRTM vector (Stratagene). The clone with the
longest insert was denoted pRN60 and chosen for further characterisation.
DNA SEQUENCE ANALYSIS
Templates for sequencing were prepared by subcloning DNA fragments into
the pBluescript II SK+ vector (Stratagene). DNA sequencing reactions were
performed by the dye terminator cycle sequencing technique and analysed
on an automated DNA Sequencer (Applied Biosystems, Foster City, CA). All
reported sequences were determined on both strands and from overlapping
templates. Nucleotide sequences were assembled and analysed using the
LasergeneTM software (DNASTAR Inc.). Pair-wise alignments of DNA and
protein sequences were calculated by the Clustal method using a ktuple
value 1, gap penalty value 3 and window size 5.
ISOLATION OF RNA AND RNA GEL BLOT ANALYSIS
Total RNA was isolated from 12-day-old wheat kernels using a hot-phenol
method as described62. RNA gel blot analysis was performed with 20 ~g total
RNA fractionated on a 1 % agarose-2.2 M formaldehyde gel, transferred to a
HybondTM (Amersham) membrane, hybridised with probe 2 (nucleotides
1993 to 4209 of Sbelc; Figure 1) and washed as described by Nair et al.
(1997)6. To assure that about the same amount of RNA was loaded onto
each lane, the hybridised blot was stripped and rehybridised with a 25S
ribosomal DNA probe as described6~. Probes were radio-labelled using the
RediprimeTM random primer labelling kit from Amersham.
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5'-RA CE
5'-RACE was performed with poly(A)*RNA extracted from 12-day-old wheat
endosperm following the protocol supplied with the MarathonTM cDNA
Amplification Kit from Clontech. The first strand synthesis was primed with
5 the Sbe1-specific BE19 primer (Figures 3 arid 4). After synthesis of the
second
strand, the double-stranded cDNA was ligated to the Marathon cDNA
Adapter (Clontech), followed by a first round PCR amplification performed
with the adapter primer AP1 (5'-CCATCCTAATACGACTCACTATAGGGC-
3'; Clontech) and the Sbe1-specific primer BE25 (Figures 3 and 4). The
10 reaction was initiated by a denaturation step at 94°C for 3 min
followed, by
25 cycles of 94°C 30 sec, 62°C 20 sec and 68°C 3 min and
a final 10 min
extension at 68°C. Products derived from the 4.8 kb Sbe1 transcripts
were
separated from shorter products derived from the 2.8 kb Sbe1 mRNA by
agarose gel electrophoresis. Products of 1.9 to 2.7 kb were gel-purified and
15 used as a template in a nested amplification employing nested adapter
primer AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3'; Clontech) and the
gene-specific primer BE39 (Figures 3 and 4). The amplification conditions
were 94°C 3 min, 30 cycles of 94°C 30 sec, 65°C 20 sec
and 68°C 3 min,
followed by a final extension at 68°C for 10 min. Amplified fragments
were
20 separated by agarose gel electrophoresis, isolated, cloned and analysed by
DNA sequencing.
RT-PCR
First strand cDNA, used as a template in the RT-PCR reactions, was
25 synthesised from 1.0 ~g total RNA isolated from 12-day-old wheat
endosperm. The RNA was primed with oligo(dT)i2-is and reverse-
transcribed in a total volume of 20 ~1 using SuperscriptTM II (Gibco-BRL).
PCR reactions (25 ~1) were performed with a 0.5 ~1 aliquot of the first-strand
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31
cDNA using the Long Expand TemplateTM PCR System (Boehringer
Mannheim) and the primer pair BE65/BE38 (Figures 3 and 4). Reactions
were initiated by a denaturation step at 94°C for 3 min, followed by 30
cycles
of 94°C 30 sec, 65°C 20 sec, 68°C 2 min 30 sec and a
final 10 min extension at
68°C. Amplified fragments were fractionated by agarose gel
electrophoresis,
isolated, cloned and analysed by DNA sequencing.
-CONS~TR.UC.TIO _N_O~XPRESSIO_ _N VECT9R_S ___.
Assembly of the pQE-SBEIc plasmid (Figure 6A) was initiated by PCR
amplification (30 cycles of 94°C, 65°C 20 sec, 68°C 2 min
30 sec) of Sbelc
nucleotides 265-1879, using the BE63/BE39 primer pair (Figure 4). This
reaction introduced a NcoI recognition site at the start of the sequence
encoding fhe mature ~BEIc: Thereafter, the NcoI~lza~I-fragment carrying
---~~Sbelc nucl_e_ofides 265=1732-was isolated from the amplified product,
filled-in
and inserted into a filled-in BamHI site of the His-tag expression vector
pQE30 (Qiagen). Construction of pQE-SBEIc was completed by insertion of a
2.2 kb EcoRV-XhoI fragment (Sbelc nucleotides 1623 to 4563 with XhoI site
added at the end) into the EcoRV and SaII sites.
Construct pREP4-cm, encoding the Lac repressor, was derived from pREP4
(Qiagen) by replacing the NPTII gene carried on a CIaI-SmaI fragment, with
the chloroamphenicol resistance gene isolated as a PvuII-BstBI fragment
from the pACYC184 vector.
Construction of pKKABEI, encoding the mature 87 kD wheat SBEI, was
initiated by inserting nucleotides 221-923 (NcoI-KpnI fragment) of pABEI
cDNAl9 into NcoI-KpnI sites of the bacterial expression vector pKK388-1
(Clontech). Then nucleotides 923-2729, isolated as a KpnI fragment, were
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introduced to give pKKABEI. The SBEII expression vector, pQRN33,
encoding the mature wheat SBEII was obtained by two cloning steps. First
the pRN336° nucleotides 317-1442 carried by a HaeIII fragment were
inserted
into a filled-in BamHI site of the His-tag expression vector pQE31 (Qiagen).
The resulting construct was restricted with KpnI and SmaI, followed by
introduction of nucleotides 1245-2632 located on a KpnI-PvuII fragment, to
give pQRN33.
ANALYSIS OF BE ACTIVITY PRODUCED IN ESCHERICHIA COLI
The BE-deficient Escherichia coli strain KV832~ carrying pREP4-cm was
transformed with pQE-SBEIc or the cloning vector pQE30. Plasmids pREP4-
cm and pQE30 were also introduced into the BE-positive Escherichia coli
strain DHSa. The bacterial cultures were grown at 37°C, in liquid YT
_medium6l containing_1.0% glucose,100 ~g/ml carbenicillin and 25 ~g/ml
chloramphenicol, to an ODboo = 0.6, and induced for two hours by addition of
IPTG to 1 mM final concentration. Production of SBEIc was verified by SDS-
PAGE gel analysis of cell lysates prepared from non-induced and induced
cultures.
Visualisation of BE activity in bacterial cells grown on solid media was done
by iodine staining of colonies as described by Kossman et al. (1991)24. The BE
activity levels in cells from non-induced cultures was determined by the
phosphorylase a stimulation assay2° performed at 30°C for 30 min
using two
and five ~g of soluble protein extract. The cell extracts were prepared from
cells of 1 ml culture that were lysed by sonication in 0.25 ml extraction
buffer
(50 mM Tris-HCl pH 7.5, 2 mM EDTA, 5 mM DTT,1 mM
phenylmethylsulfonyl fluoride) and centrifuged at 15,000 x g for 20 min.
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Determination of protein concentration in the soluble extracts was done
using the dye-binding assay (Bio-Rad).
LARGE SCALE PRODUCTION OF WHEAT SBE IN ESCHERICHIA COLI
A culture of KV832 cells transformed with pKKABEI was grown at
37°C in
LB medium containing 100 ~g mL-1 ampicillin. At OD6oo = 0.6, IPTG was
added to a final concentration of 0.5 mM and the culture was grown at
25°C
-for-14-h: Cells-were-harvested-by-centrifugation and SBEI was purified
according to Guan et al. (1994)63. The final protein extract was loaded onto a
10% preparative SDS-PAGE gel and the 87 kD SBEI band was isolated by
electroelution (Model 422 Electro-eluterTM, Bio-Rad). The protein eluate was
concentrated using a Centriplus-30TM column (Amicon) before
-~ imrriurusation.
The SBEII expression vector, pQRN33, was introduced into the Escherichia coli
strain, MI5, carrying pREP4 and grown at 22°C in medium containing 25
g/ 1
tryptone,15 g/ 1 yeast extract, 5 g/ 1 NaCI,1 % glucose,100 fig/ ml ampicillin
and 25 ~g/ml kanamycin. Cells were grown to OD6oo=0.7, IPTG was added
to give a 1 mM final concentration and the cells were grown for an additional
14 h. Harvested cells were lysed under denaturing conditions and the His-
tagged SBEI was purified using the QIAexpress purification system (Qiagen).
The guanidine hydrochloride denaturation buffer, column washing buffers
and elution buffer were all supplemented with 10 mM ~i-mercaptoethanol
and 0.25% Tween 20. The homogeneity of the column fractions used for
immunisation was verified by SDS-PAGE.
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PREPARATION OF SBEI AND SBE11 ANTIBODIES
About 100 ~g purified SBEI, or 250 ~g His-tagged SBEII in 500 ~1 phosphate-
buffered saline, was emulsified with an equal volume of Freund's complete
adjuvant (Difco) and injected intradermally into cereal-starved rabbits. The
injection was repeated twice at two-weeks intervals using about 50 ~g
antigen and an equal volume of Freund's incomplete adjuvant (Difco). The
antiserum was collected two weeks after the final injection.
ISOLATION OF TOTAL STARCH
Starch granules were extracted from mature and developing wheat kernels
according to procedure described by Zhao and Sharp (1996)x, with the
exception of the steeping step, which was only done with mature seeds.
ISOLATION OF A-TYPE AND B-TYPE STARCH GRANULES
Starch granules were isolated from mature endosperm of five hexaploid
wheat cultivars (Triticum aestivum L. cv. CDC Teal, McKenzie, AC Karma,
AC Crystal, and Fielder), one tetraploid wheat (Triticum turgidum L. cv.
Plenty) cultivar, barley (Hordeum vulgare L.), rye (Secale cereale L.),
triticale (X
Triticosecale Wittmack), rice (Oryza sativa L.), maize (Zea mays L.), canary
seed
(Phalaris canariensis L.) and potato (Solanum tuberosum L.) tubers as
described
by Peng et al. (1999)2. Pericarp and developing endosperm tissues were
manually dissected from wheat (Triticum aestivum L. cv. CDC Teal) kernels
and immediately placed in extraction buffer B [50 mM Tris-HCI, pH 7.5,10
mM EDTA, 5 mM DTT,10% glycerol, 0.1% (w/v) polyvinyl pyrrolidone]
held at 40°C. The pericarp fraction was washed three times with
extraction
buffer B to remove endosperm starch granules. The endosperm and pericarp
fractions were homogenised with a mortar and pestle in three volumes of
extraction buffer B and filtered through four layers of MiraclothTM
(Calbiochem) to remove cell debris. The crude starch granule fraction was
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pelleted by centrifugation at 15,000 x g for 30 min and further purified as
described2~. The endosperm starch granules were separated into large-size
(diameter >10 Vim) and small-size (diameter <10 Vim) fractions and studied by
image analysis as described2~.
5 PREPARATION OF ENDOSPERM SOLUBLE FRACTIONS
The supernatant remaining from centrifugation of the homogenised
endosperm (see above) constituted the endosperm soluble fraction. Protein
concentration in the extract was determined using a dye-binding assay from
Bio-Rad. For each endosperm fraction, the total amount of extracted soluble
10 protein was determined.
SDS-PAGE ANALYSIS OF STARCH GRANULES
Extracted total starch (10 mg) was resuspended in 150 ~l of sample buffer --
(62.5 mM Tris-HCl pH 8.0,10% SDS,10% glycerol, 5% (3-mercaptoethanol
and 0.005% bromophenol blue), boiled for 7 min, cooled on ice for 5 min and
15 centrifuged at 15,000 x g for 20 min. Extracted A-type and B-type starch
granules ( 50 mg) were suspended in 350 ~l extraction buffer A [62.5 mM
Tris-HCI, pH 6.8,10% (w/v) SDS, 5% (v/v) (3-mercaptoethanol], boiled for 15
min, cooled to room temperature, and centrifuged at 15,000 x g for 20 min.
SDS-PAGE analysis of total and size fractionated starch granules was done
20 on 10% resolving gels (30:0.135) and proteins were visualized by Coomassie
blue staining and/or silver staining (BIO-RAD).
IMMUNOBLOTTING
Total starch granule proteins separated by SDS-PAGE were transferred by
vertical electroblotting6l onto ImmobilonTM nitrocellulose membranes
25 (Millipore) at 1.4 V/cm for 2.5 h using buffer 3 described by Bolt and
Mahoney (1997)65. The filters were blocked for 2 h in blocking buffer [5%
w/v non-fat dry milk, 0.1% Tween 20 in phosphate-buffered saline6l] and
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36
subsequently incubated for 1 h with primary antibodies in blocking buffer
(1:1000 dilution). Blots were washed for 1 h in blocking buffer, followed by
incubation with alkaline phosphatase-conjugated goat anti-rabbit antibodies
(Stratagene) in blocking buffer (1:5000 dilution). Thereafter, the membranes
were washed with blocking buffer for 1 h and with 50 mM Tris-HCl pH 7.5,
150 mM NaCI for 45 min. Gel-separated proteins extracted from A-type and
B-type granules were electrophoretically transferred at 40°C onto
PVDF
membranes (Millipore) using transfer buffer [25 mM Tris-HCI, pH 8.3,192
mM Glycine and 20% methanolJ. Membranes were incubated, for 1 h in TBS
buffer [20 mM Tris-HCI, pH 7.5,150 mM NaCIJ containing 3% (w/v) bovine
serum albumin, to block nonspecific binding sites. Antibodies, at a dilution
of 1:4000 in TBS buffer, were then added to the blot and incubated for 4 h at
room temperature. Following three washes in TBS buffer containing 0.05%
TweenTM 20 and one wash in TBS buffer, membranes were incubated with
alkaline phosphatase-conjugated goat anti-rabbit IgG (Stratagene) at a
dilution of 1:5000 for 1 h. Membranes were washed three times in TBS buffer
containing 0.05% Tween 20, once in TBS buffer, and equilibrated in 20 mM
Tris-HCI, pH 9.5,100 mM NaCI, 5 mM MgCl2. Immunoreactive bands were
detected with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-
indolyl phosphate (Stratagene).
N-TERMINAL SEQUENCING OF SGP-140 AND SGP-145
SGP were extracted from 10 g A-type starch granules of CDC Teal and
resolved on preparative SDS-PAGE gels. The migration of SGP-140 and SGP-
145 was determined by silver staining a slice of the gel. The proteins were
eluted from the unstained part of the gel using an electro-eluter (Model 422
Electro-EluterTM, BIO-RAD) and elution buffer (25 mM Tris,192 mM glycine,
0.1% SDS). The eluate was dialysed for 8 h against 21 of dialysis buffer (50
mM Tris-acetate, pH 6.8, 5 mM DTT), with one buffer change. The dialysed
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37
solution was concentrated to 500 ~l through ultrafiltration (Amicon 100), and
200 ~1 of the concentrate was loaded on a preparative SDS-PAGE gel. Gel-
separated proteins were blotted on a PVDF membrane, as described above.
SGP-140 and SGP-145 were identified by amido black staining and subjected
to N-terminal sequencing using a gas-phase protein sequencer (Applied
Biosystem Model 476A).
NUCLEOTIDE AND AMINO ACID SEQUENCES
SEQ ID N0:1 is the DNA sequence of Sbelc
SEQ ID NO: 2 is the amino acid sequence of SBEIc
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SEQUENCE LISTING
<110> National Research Council of Canada
<120> Starch Branching Enzymes
<130> 45187pt
<140>
<141>
<150> US60/162,144
<151> 1999-10-29
<160> 2
<170> PatentIn Ver. 2.1
<210> 1
<211> 4563
<212> DNA
<213> Triticum aestivum
<400> 1
gggactttcg tccgccacca aggctgacag ctccaccgcc ctcggttgcg ccgtcgacga 60
cgatgctttg cctcagctcc tctctcctgc cgcgcccgtc tgccgctgct gaccggccgg 120
ctcccgggat catcgcgggc ggcggcggca agcggctgag cgtggtgccg gctgtcccgt 180
ttttacttcg ctggtcgtgg ccacggaagg ccaagagcag gtcttctgtt tccgtgactg 240
cacgaggaaa caaaattgcg gcagcaaatg gatatggttc tgaccacctt cccatgtatg 300
atctggaacc aaagttggct gaattcaaag accacttcaa ctatacgatg aaaaggtacc 360
ttgaacagaa acttttgatt gagaaacatg agggaggcct agaggaattc tctaaaggct 420
atttgaagtt tgggatcaac acggagcatg gtgcatctct gtacagggaa tgggcccctg 480
cagcagagga agcacaacta gttggtgact tcaacaactg gaatggttct ggccacaaga 540
tgacgaagga taactttggc gtttggtcaa tcaggatttc ccatgtcaat gggaaacctg 600
ccatccctca caattccaag gttaaatttc gatttaggca tgatggagta tgggttgaac 660
ggattccagc atggattcgt tatgcaactg ttactgcctc tgaatctgga gctccatatg 720
atggtgttca ctgggatcca ccaactagtg aaaggtatgt atttaaccat cctcgacctc 780
caaagcctga tgttccacgt atctatgagg ctcatgtggg ggtgagtggt ggaaagcttg 840
aagcaggcac acacagggaa tttgcagaca atgtgttacc gcgcttaagg gcaactacat 900
acaacacggt tcagttgatg ggaatcatgg aacattctga cgctgcttct tttgggtatt 960
atgtgacgaa tttcttcgca gttagcagca gatcaggcac accagacgac ctcaaatatc 1020
ttattgacaa ggcacatagt cttggattgt gtgttctgat ggatgttgtc cacagccatg 1080
cgagcaataa tgtgatagat ggtcccaatg gctatgatgt tggacaaagt gcacacgaat 1140
cctatttcta cacaggagac aggggctata ataagatgtg gaatggccgc atgttcaact 1200
atgccaattg ggaggtccta agattcctgc tttccaattt gagatattgg atggacgaat 1260
tcatgtttga tggcttccga tttgttgggg ttacatcgat gctatataat caaaatggta 1320
tcaatatgtc attcactgga aattacaaag agtattttgg tttggatacc aatgtagatg 1380
cagttgttta tatgatgctc gcgaaccatt taatgcacaa actctaccca gaagcaattg 1440
ttgtggccgt agatgtttca ggcatgccag ttctttgttg gccagttgat gaaggtggat 1500
tagggtttga ctatcgccag gctatgacta ttcccgatag atggattgaa tacttggaga 1560
acaaaggtga tcaacagtgg tcaatgagta gtgtaatatc acaaactttg actaacaggc 1620
gatatccgga aaagttcatt gcgtatgctg agaggcaaaa tcattctatt attggcagca 1680
agactatggc atttctcttg atgggatggg aaacgtattc cggtatgtcg gccatggagc 1740
ctgattcacc tacaatagat cgtggcattg cacttcaaaa gatgattcat ttcatcagga 1800
tggcctttgg aggtgatagc tacttaaaat ttatgggtaa tgagtacatg aatgcatttg 1860
atcaagcagt ggacacgccc agcgataaat gttccttcct atcatcatca aagcagactg 1920
ccagcgacat gaatgaggaa gaaaaggcca agagcaagtt ctctgttccc gtgtctgcgc 1980
caagagacta caccatggca acagctgaag atggtgttgg cgaccttccg atatacgatc 2040
CA 02389390 2002-04-29
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2
tggatccgaa gtttgccggc ttcaaggaac acttcagtta taggatgaaa aagtaccttg 2100
accagaaaca ttcgattgag aagcacgagg gaggccttga agagttctct aaaggctatt 2160
tgaagtttgg gatcaacaca gaaaatgacg caactgtgta ccgggaatgg gcccctgcag 2220
caatggatgc acaacttatt ggtgacttca acaactggaa tggctctggg cacaggatga 2280
caaaggataa ttatggtgtt tggtcaatca ggatttccca tgtcaatggg aaacctgcca 2340
tcccccataa ttccaaggtt aaatttcgat ttcaccgtgg agatggacta tgggtcgatc 2400
gggttcctgc atggattcgt tatgcaactt ttgatgcctc taaatttgga gctccatatg 2460
acggtgttca ctgggatcca ccttctggtg aaaggtatgt gtttaagcat cctcggcctc 2520
gaaagcctga cgctccacgt atttacgagg ctcatgtggg gatgagtggt gaaaagcctg 2580
aagtaagcac atacagagaa tttgcagaca atgtgttacc gcgcataaag gcaaacaact 2640
acaacacagt tcagctgatg gcaatcatgg aacattcata ttatgcttct tttgggtacc 2700
atgtgacgaa tttcttcgca gttagcagca gatcaggaac gccagaggac ctcaaatatc 2760
ttgttgacaa ggcacatagt ttagggttgc gtgttctgat ggatgttgtc catagccatg 2820
cgagcagtaa taagacagat ggtcttaatg gctatgatgt tgggcaaaac acacaggagt 2880
cctatttcca cacaggagaa aggggctatc ataaactgtg ggatagccgc ctgttcaact 2940
atgccaattg ggaggtctta cgatttcttc tttctaatct gagatattgg atggacgaat 3000
tcatgtttga tggcttccga tttgatgggg taacatccat gctatataat caccatggta 3060
tcaatatgtc attcgctgga agttacaagg aatattttgg tttggatact gatgtagatg 3120
cagttgttta cctgatgctt gcgaaccatt taatgcacaa actcttgcca gaagcaactg 3180
ttgttgcaga agatgtttca ggcatgccag tgctttgtcg gtcagttgat gaaggtggag 3240
tagggtttga ctatcgcctg gctatggcta ttcctgatag atggatcgac tacttgaaga 3300
acaaagatga ccttgaatgg tcaatgagtg gaatagcaca tactctgacc aacaggagat 3360
atacggaaaa gtgcattgca tatgctgaga gccatgatca gtctattgtt ggcgacaaga 3420
ctatggcatt tctcttgatg gacaaggaaa tgtatactgg catgtcagac ttgcagcctg 3480
cttcgcctac aattgatcgt ggaattgcac ttcaaaagat gattcacttc atcaccatgg 3540
cccttggagg tgatggctac ttgaatttta tgggtaatga gtttggccac ccagaatgga 3600
ttgactttcc aagagaaggc aacaactgga gttatgataa atgcagacgc cagtggagcc 3660
tcgcagacat tgatcaccta cgatacaagt acatgaacgc atttgatcaa gcaatgaatg 3720
cgctcgacga caaattttcc ttcctatcat catcaaagca gattgtcagc gacatgaatg 3780
aggaaaagaa gattattgta tttgaacgtg gagatctggt cttcgtcttc aattttcatc 3840
ccagtaaaac ttatgatggt tacaaagtcg gatgtgactt gcctgggaag tacaaggtag 3900
ctctggactc tgatgctctg atgtttggtg gacatggaag agtggcccat gacaacgatc 3960
actttacgtc acctgaagga gtaccaggag tacctgaaac aaacttcaac aaccgcccta 4020
actcattcaa aatcctgtct ccatcccgca cttgtgtggc ttactatcgc gtcgaggaga 4080
aagcggaaaa gcccaaggat gaaggagctg cttcttgggg gaaaactgct ctcgggtaca 4140
tcgatgttga agccactggc gtcaaagacg cagcagatgg tgaggcgact tctggttccg 4200
aaaaggcgtc tacaggaggt gactccagca agaagggaat taactttgtc tttctgtcac 4260
ccgacaaaga caacaaataa gcaccatatc aacgcttgat caggaccgtg tgccgacgtc 4320
cttgtaatac tcctgctatt gctagtagta gcaatactgt caaactgtgc agacttgaga 4380
ttctggcttg gactttgctg aggttaccta ctatatagaa agataaataa gcggtgatgg 4440
tgcgggtcga gtccagctat atgtgccaaa tatgcgccat cccgagtcct ctgtcataaa 4500
gaaagtttcg ggcttccatc ccagaataaa aacagttgtc tgtttgccca aaaaaaaaaa 4560
aaa 4563
<210> 2
<211> 1405
<212> PRT
<213> Triticum aestivum
<400> 2
Met Leu Cys Leu Ser Ser Ser Leu Leu Pro Arg Pro Ser Ala Ala Ala
1 5 10 15
Asp Arg Pro Ala Pro Gly Ile Ile Ala Gly Gly Gly Gly Lys Arg Leu
20 25 30
CA 02389390 2002-04-29
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3.
Ser Val Val Pro Ala Val Pro Phe Leu Leu Arg Trp Ser Trp Pro Arg
35 40 45
Lys Ala Lys Ser Arg Ser Ser Val Ser Val Thr Ala Arg Gly Asn Lys
50 55 60
Ile Ala Ala Ala Asn Gly Tyr Gly Ser Asp His Leu Pro Met Tyr Asp
65 70 75 80
Leu Glu Pro Lys Leu Ala Glu Phe Lys Asp His Phe Asn Tyr Thr Met
85 90 95
Lys Arg Tyr Leu Glu Gln Lys Leu Leu Ile Glu Lys His Glu Gly Gly
100 105 110
Leu Glu Glu Phe Ser Lys Gly Tyr Leu Lys Phe Gly Ile Asn Thr Glu
115 120 125
His Gly Ala Ser Leu Tyr Arg Glu Trp Ala Pro Ala Ala Glu Glu Ala
130 135 140
Gln Leu Val Gly Asp Phe Asn Asn Trp Asn Gly Ser Gly His Lys Met
145 150 155 160
Thr Lys Asp Asn Phe Gly Val Trp Ser Ile Arg Ile Ser His Val Asn
165 170 175
Gly Lys Pro Ala Ile Pro His Asn Ser Lys Val Lys Phe Arg Phe Arg
180 185 190
His Asp Gly Val Trp Val Glu Arg Ile Pro Ala Trp Ile Arg Tyr Ala
195 200 205
Thr Val Thr Ala Ser Glu Ser Gly Ala Pro Tyr Asp Gly Val His Trp
210 215 220
Asp Pro Pro Thr Ser Glu Arg Tyr Val Phe Asn His Pro Arg Pro Pro
225 230 235 240
Lys Pro Asp Val Pro Arg Ile Tyr Glu Ala His Val Gly Val Ser Gly
245 250 255
Gly Lys Leu Glu Ala Gly Thr His Arg Glu Phe Ala Asp Asn Val Leu
260 265 270
Pro Arg Leu Arg Ala Thr Thr Tyr Asn Thr Val Gln Leu Met Gly Ile
275 280 285
Met Glu His Ser Asp Ala Ala Ser Phe Gly Tyr Tyr Val Thr Asn Phe
290 295 300
Phe Ala Val Ser Ser Arg Ser Gly Thr Pro Asp Asp Leu Lys Tyr Leu
305 310 315 320
Ile Asp Lys Ala His Ser Leu Gly Leu Cys Val Leu Met Asp Val Val
325 330 335
CA 02389390 2002-04-29
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4
His Ser His Ala Ser Asn Asn Val Ile Asp Gly Pro Asn Gly Tyr Asp
340 345 350
Val Gly Gln Ser Ala His Glu Ser Tyr Phe Tyr Thr Gly Asp Arg Gly
355 360 365
Tyr Asn Lys Met Trp Asn Gly Arg Met Phe Asn Tyr Ala Asn Trp Glu
370 375 380
Val Leu Arg Phe Leu Leu Ser Asn Leu Arg Tyr Trp Met Asp Glu Phe
385 390 395 400
Met Phe Asp Gly Phe Arg Phe Val Gly Val Thr Ser Met Leu Tyr Asn
405 410 415
Gln Asn Gly Ile Asn Met Ser Phe Thr Gly Asn Tyr Lys Glu Tyr Phe
420 425 430
Gly Leu Asp Thr Asn Val Asp Ala Val Val Tyr Met Met Leu Ala Asn
435 440 445
His Leu Met His Lys Leu Tyr Pro Glu Ala Ile Val Val Ala Val Asp
450 455 460
Val Ser Gly Met Pro Val Leu Cys Trp Pro Val Asp Glu Gly Gly Leu
465- - 470 - 475 480
Gly Phe Asp Tyr Arg Gln Ala Met Thr Ile Pro Asp Arg Trp Ile Glu
485 490 495
Tyr Leu Glu Asn Lys Gly Asp Gln Gln Trp Ser Met Ser Ser Val Ile
500 505 510
Ser Gln Thr Leu Thr Asn Arg Arg Tyr Pro Glu Lys Phe Ile Ala Tyr
515 520 525
Ala Glu Arg Gln Asn His Ser Ile Ile Gly Ser Lys Thr Met Ala Phe
530 535 540
Leu Leu Met Gly Trp Glu Thr Tyr Ser Gly Met Ser Ala Met Glu Pro
545 ~ 550 555 560
Asp Ser Pro Thr Ile Asp Arg Gly Ile Ala Leu Gln Lys Met Ile His
565 570 575
Phe Ile Arg Met Ala Phe Gly Gly Asp Ser Tyr Leu Lys Phe Met Gly
580 585 590
Asn Glu Tyr Met Asn Ala Phe Asp Gln Ala Val Asp Thr Pro Ser Asp
595 600 605
Lys Cys Ser Phe Leu Ser Ser Ser Lys Gln Thr Ala Ser Asp Met Asn
610 615 620
Glu Glu Glu Lys Ala Lys Ser Lys Phe Ser Val Pro Val Ser Ala Pro
625 630 635 640
CA 02389390 2002-04-29
WO 01/32886 PCT/CA00/01276
6
Gly Glu Arg Gly Tyr His Lys Leu Trp Asp Ser Arg Leu Phe Asn Tyr
945 950 955 960
Ala Asn Trp Glu Val Leu Arg Phe Leu Leu Ser Asn,Leu Arg Tyr Trp
965 970 975
Met Asp Glu Phe Met Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser
980 985 990
Met Leu Tyr Asn His His Gly Ile Asn Met Ser Phe Ala Gly Ser Tyr
995 1000 1005
Lys Glu Tyr Phe Gly Leu Asp Thr Asp Val Asp Ala Val Val Tyr Leu
1010 1015 1020
Met Leu Ala Asn His Leu Met His Lys Leu Leu Pro Glu Ala Thr Val
1025 1030 1035 1040
Val Ala Glu Asp Val Ser Gly Met Pro Val Leu Cys Arg Ser Val Asp
1045 1050 1055
Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu Ala Met Ala Ile Pro Asp
1060 1065 1070
Arg Trp Ile Asp Tyr Leu Lys Asn Lys Asp Asp Leu Glu Trp Ser Met
1075 1080 1085
Ser Gly Ile Ala His Thr Leu Thr Asn Arg Arg Tyr Thr Glu Lys Cys
1090 1095 1100
Ile Ala Tyr Ala Glu Ser His Asp Gln Ser Ile Val Gly Asp Lys Thr
1105 1110 1115 1120
Met Ala Phe Leu Leu Met Asp Lys Glu Met Tyr Thr Gly Met Ser Asp
1125 1130 1135
Leu Gln Pro Ala Ser Pro Thr Ile Asp Arg Gly Ile Ala Leu Gln Lys
1140 1145 1150
Met Ile His Phe Ile Thr Met Ala Leu Gly Gly Asp Gly Tyr Leu Asn
1155 1160 1165
Phe Met Gly Asn Glu Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg
1170 1175 1180
Glu Gly Asn Asn Trp Ser Tyr Asp Lys Cys Arg Arg Gln Trp Ser Leu
1185 1190 1195 1200
Ala Asp Ile Asp His Leu Arg Tyr Lys Tyr Met Asn Ala Phe Asp Gln
1205 1210 1215
Ala Met Asn Ala Leu Asp Asp Lys Phe Ser Phe Leu Ser Ser Ser Lys
1220 1225 1230
Gln Ile Val Ser Asp Met Asn Glu Glu Lys Lys Ile Ile Val Phe Glu
1235 1240 1245
CA 02389390 2002-04-29
WO 01/32886 PCT/CA00/01276
7
Arg Gly Asp Leu Val Phe Val Phe Asn Phe His Pro Ser Lys Thr Tyr
1250 1255 1260
Asp Gly Tyr Lys Val Gly Cys Asp Leu Pro Gly Lys Tyr Lys Val Ala
1265 1270 1275 1280
Leu Asp Ser Asp Ala Leu Met Phe Gly Gly His Gly Arg Val Ala His
1285 1290 1295
Asp Asn Asp His Phe Thr Ser Pro Glu Gly Val Pro Gly Val Pro Glu
1300 1305 1310
Thr Asn Phe Asn Asn Arg Pro Asn Ser Phe Lys Ile Leu Ser Pro Ser
1315 1320 1325
Arg Thr Cys Val Ala Tyr Tyr Arg Val Glu Glu Lys Ala Glu Lys Pro
1330 1335 1340
Lys Asp Glu Gly Ala Ala Ser Trp Gly Lys Thr Ala Leu Gly Tyr Ile
1345 1350 1355 1360
Asp Val Glu Ala Thr Gly Val Lys Asp Ala Ala Asp Gly Glu Ala Thr
1365 1370 1375
Ser Gly Ser Glu Lys Ala Ser Thr Gly Gly Asp Ser Ser Lys Lys Gly
1380 1385 1390
Ile Asn Phe Val Phe Leu Ser Pro Asp Lys Asp Asn Lys
1395 1400 1405
