Note: Descriptions are shown in the official language in which they were submitted.
CA 02275885 2002-11-12
1
2
3
4
TRANSGENIC POTATOES HAVING REDUCED LEVELS OF
6 ALPHA GLUCAN L- OR H-TYPE TUBER PHOSPHORYLASE ACTIVITY
WITH REDUCED COLD-SWEETENING
8
9
11 FIELD OF THE INVENTION
12
13 The invention relates to the inhibition of the accumulation of sugars in
potatoes by
14 reducing the level of a glucan L-type tuber phosphorylase or a glucan H-
type tuber
phosphorylase enzyme activity in the potato plant.
16
17 BACKGROUND OF THE INVENTION
18
19 Plant stresses caused by a wide variety of factors including disease,
environment, and
2 0 storage of potato tubers (Solanum tuberosum) represent major determinants
of tuber quality.
21 Dormancy periods between harvesting and sprouting are critical to
maintaining quality
2 2 potatoes. Processing potatoes are usually stored between 7 and 12
°C. Cold storage at 2 to
2 3 6 ° C, versus storage at 7 to 12 ° C, provides the greatest
longevity by reducing respiration,
2 4 moisture loss, microbial infection, heating costs, and the need for
chemical sprout inhibitors
2 5 (Burton, 1989). However, low temperatures lead to cold-induced sweetening,
and the
2 6 resulting high sugar levels contribute to an unacceptable brown or black
color in the fried
2 7 product (Coffin et al., 1987, Weaver et al., 1978). The sugars that
accumulate are
2 8 predominantly glucose, fructose, and sucrose. It is primarily the glucose
and fructose
2 9 (reducing sugars) that react with free amino groups when heated during the
various cooking
3 0 processes such as frying via the Maillard reaction, resulting in the
formation of brown
31 pigments (Burton, 1989, Shallenberger et al., 1959). Sucrose produces a
black colouration
3 2 when fried due to caramelization and charring. The ideal reducing sugar
content is generally
33 accepted to be 0.1% of tuber fresh weight with 0.33% as the upper limit and
higher levels of
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 reducing sugars are sufficient to cause the formation of brown and black
pigments that
2 results in an unacceptable fried product (Davies and Viola, 1992). Although
the
3 accumulation of reducing sugars can be slowed in higher temperature (7 to
12°C) storage,
4 this increases microbial infection and the need to use sprout inhibitors.
Given the negative
environmental and health risks associated with chemical use, development of
pathogens
6 resistant to pesticides, and the fact that use of current sprout inhibitors
may soon be
7 prohibited, a need exists for potato varieties that can withstand stress and
long-term cold
8 storage without the use of chemicals, without the accumulation of reducing
sugars, and with
9 greater retention of starch.
Carbohydrate metabolism is a complex process in plant cells. Manipulation of a
11 number of different enzymatic processes may potentially affect the
accumulation of reducing
12 sugars during cold storage. For example, inhibition of starch breakdown
would reduce the
13 buildup of free sugar. Other methods may also serve to enhance the cold
storage properties of
14 potatoes through reduction of sugar content, including the resynthesis of
starch using reducing
sugars, removal of sugars through glycolysis and respiration, or conversion of
sugars into
16 other forms that would not participate in the Maillard reaction. However,
many of the
17 enzymatic processes are reversible, and the role of most of the enzymes
involved in
18 carbohydrate metabolism is poorly understood. The challenge remains to
identify an enzyme
19 that will deliver the desired result, achieve function at low temperatures,
and still retain the
2 0 product qualities desired by producers, processors, and consumers.
21 It has been suggested that phosphofructokinase (PFK) has an important role
in the
22 cold-induced sweetening process (Kruger and Hammond, 1988, ap Rees et al.,
1988, Dixon et
23 al., 1981, Claassen et al., 1991). ap Reese et al. (1988) suggested that
cold treatment had a
24 disproportionate effect on different pathways in carbohydrate metabolism in
that glycolysis
2 5 was more severely seduced due to the cold-sensitivity of PFK. The
reduction in PFK activity
2 6 would then lead to an increased availability of hexose-phosphates for
sucrose production. It
27 was disclosed in European Patent 0438904 (Burrell et al., July 31, 1991)
that increasing PFK
2 8 activity seduces sugar accumulation during storage by removing hexoses
through glycolysis
2 9 and further metabolism. A PFK enzyme from E coli was expressed in potato
tubers and the
3 0 report claimed to increase PFK activity and to reduce sucrose content in
tubers assayed at
31 harvest. However it has been shown that pyrophosphate:fructose 6-phosphate
2
CA 02275885 1999-06-29
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1 phosphotransferase (PFP) remains active at low temperatures (Claassen et
al., 1991 ). PFP
2 activity can supply fructose 6-phosphate for glycolysis just as PFK can,
since the two -
3 enzymes catalyse the same reaction. Therefore, the efficacy of this strategy
for improving
4 cold storage quality of potato tubers remains in doubt. Furthermore, removal
of sugars
through glycolysis and further metabolism would not be a preferred method of
enhancing
6 storage properties of potato tubers because of the resultant loss of
valuable dry matter through
7 respiration.
8 It has also been suggested that ADPglucose pyrophosphorylase (ADPGPP) has an
9 important role in the cold-induced sweetening process. It was disclosed in
International
Application WO 94/28149 (Barry, et al., filed May 18, 1994) that increasing
ADPGPP
11 activity reduces sugar accumulation during storage by re-synthesising
starch using reducing
12 sugars. An ADPGPP enzyme from E. coli was expressed in potato tubers under
the control of
13 a cold-induced promoter and the report claimed to increase ADPGPP activity
and lower
14 reducing sugar content in tubers assayed at harvest and after cold
temperature storage.
However, this strategy does not eliminate starch catabolism but instead
increases the rate of
16 starch resynthesis. Thus, catabolism of sugars through glycolysis and
respiration occurs and
17 re-incorporation into starch is limited. Up regulation of ADPGPP would not
be a preferred
18 method of enhancing storage properties of potato tubers because of the
resultant loss of
19 valuable dry matter through respiration. Again, a method involving the
reduction of
2 0 catabolism of starch would be preferable as dry matter would be retained.
21 The degradation of starch is believed to involve several enzymes including
a-amylase
22 (endoamylase), ~i-amylase (exoamylase), amyloglucosidase, and a-glucan
phosphorylase
23 (starch phosphorylase). By slowing starch catabolism, accumulation of
reducing sugars
2 4 should be prevented and the removal of sugars through glycolysis and
further metabolism
2 5 would be minimized.
2 6 Three different isozymes of a glucan phosphorylase have been described.
The tuber
2 7 L-type a 1,4 glucan phosphorylase (EC 2.4.1.1 ) isozyme (GLTP) (Nakano and
Fukui, 1986)
2 8 has a low affinity for highly branched glucans, such as glycogen, and is
localized in
2 9 amyoplasts. The monomer consists of 916 amino acids and sequence
comparisons with
3 0 phosphorylases from rabbit muscle and Escherichia coli revealed a high
level of homology,
31 51 % and 40% amino acids, respectively. The nucleotide sequence of the GLTP
gene and the
3
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1 amino acid sequence of the GLTP enzyme are shown in SEQ B7 NO: 1 and SEQ )D
NO. 2,
2 respectively. The H-type tuber a-glucan phosphorylase isozyme H (GHTP) (Mori
et al.,
3 199/) has a high affinity for glycogen and is localized in the cytoplasm.
The gene encodes
4 for 838 amino acids and shows 63% sequence homology with the tuber L-type
phosphorylase
but lacks the 78-residue insertion and 50-residue amino-terminal extension
found in the L-
6 type polypeptide. The nucleotide sequence of the GHTP gene and the amino
acid sequence of
7 the GHTP enzyme are shown in SEQ m NO: 3 and SEQ m NO: 4, respectively. A
third
8 isozyme has been reported (Sonnewald et al., 1995) that consists of 974
amino acids and is
9 highly homologous to the tuber L-type phosphorylase with 81 % identity over
most of the
polypeptide. However, the regions containing the transit peptide and insertion
sequence are
11 highly diverse. This isozyme is referred to as the leaf L-type
phosphorylase since the mRNA
12 accumulates equally in leaf and tuber, whereas the mRNA of the tuber L-type
phosphoryiase
13 accumulates strongly in potato tubers and only weakly in leaf tissues. The
tuber L-type
14 phosphorylase is mainly present in the tubers and the leaf L-type
phosphorylase is more
abundunt in the leaves (Sonnewald et al., 1995). The nucleotide sequence of
the leaf L-type
16 phosphorylase gene and the amino acid sequence of the leaf L-type
phosphorylase enzyme are
17 shown in SEQ m NO: 5 and SEQ ID NO: 6, respectively.
18 The role of the various starch degrading enzymes is not clear, however, and
19 considerable debate has occurred over conflicting results. For example,
reduced expression
2 0 of the leaf L-type phosphorylase (Sonnewald et al., 1995) had no
significant influence on
21 starch accumulation. Sonnewaid et al. (1995) reported that constitutive
expression of an
22 antisense RNA specific for the leaf L-type gene resulted in a strong
reduction of a glucan
23 phosphorylase L-type activity in leaf tissue, but had no effect in potato
tuber tissue. Since the
24 antisense repression of the a glucan phosphorylase activity had no
significant influence on
2 5 starch accumulation in leaves of transgenic potato plants, the authors
concluded that starch
2 6 breakdown was not catalysed by phosphorylases. Considering the high level
of sequence
27 homology between identified a glucan phosphorylase isozymes, a similar
negative response
2 8 would be expected with the H-type (GHTP) and L-type tuber (GLTP) isozymes.
2 9 In view of the foregoing, there remains a need for potato plants which
produce tubers
3 0 exhibiting reduced conversion of starches to sugars during propagation and
during storage at
31 ambient and reduced temperatures, particularly at temperatures below
7°C.
4
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1 SUMMARY OF THE INVENTION
2 -
3 The inventors have found that surprisingly, reduction of the level of a
glucan L-type
4 tuber phosphorylase (GLTP) or a glucan H-type tuber phosphorylase (GHTP)
enzyme activity
within the potato tuber results in a substantial reduction in the accumulation
of sugars in the
6 tuber during propagation and storage, relative to wildtype potatoes,
particularly at storage
7 temperatures below 10°C, and specifically at 4°C. It is
remarkable that, given the complexity
8 of carbohydrate metabolism in the tuber, reduction in the activity of a
single enzyme is
9 effective in reducing sugar accumulation in the tuber. The inventors'
discovery is even more
surprising in light of the previously discussed work of Sonnewald et al. (
1995) wherein it was
11 reported that reduced expression of the leaf L-type phosphorylase had no
significant influence
12 on starch accumulation in leaves of potato plants.
13 The present invention provides tremendous commercial advantages. Tubers in
which
14 cold-induced sweetening is inhibited or reduced may be stored at cooler
temperatures without
producing high levels of reducing sugars in the tuber which cause unacceptable
darkening of
16 fried potato products. Cold storage of tubers storage results in longer
storage life, prolonged
17 dormancy by limiting respiration and delaying sprouting, and lower
incidence of disease.
18 Reduction in GLTP or GHTP activity in potato plants and tubers can be
accomplished
19 by any of a number of known methods, including, without limitation,
antisense inhibition of
2 0 GLTP or GHTP mRNA, co-suppression, site-directed mutagenesis of wildtype
GLTP or
21 GHTP genes, chemical or protein inhibition, or plant breeding programs.
2 2 Thus, in broad terms, the invention provides modified potato plants having
a reduced
2 3 level of a glucan L-type tuber phosphorylase (GLTP) or a glucan H-type
tuber phosphorylase
24 (GHTP) activity in tubers produced by the plants, relative to that of
tubers produced by an
2 5 unmodified potato plant. In a preferred embodiment, the invention provides
a potato plant
2 6 transformed with an expression cassette having a plant promoter sequence
operably linked to
27 a DNA sequence which, when transcribed in the plant, inhibits expression of
an endogenous
2 8 GLTP gene or GHTP gene. As will be discussed in detail hereinafter, the
aforementioned
2 9 DNA sequence may be inserted in the expression cassette in either a sense
or antisense
3 0 orientation. Potato plants of the present invention could have reduced
activity levels of either
5
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 one of GLTP or GHTP independently, or could have reduced activity levels of
both GLTP
2 and GHTP.
3 As discussed above, the inventors have found that reduction of activity
levels of
4 GLTP or GHTP enzymes in potato plants results in potato tubers in which
sugar
accumulation, particularly over long storage periods at temperatures below
10°C, is reduced.
6 Therefore, the invention further extends to methods for reducing sugar
production in tubers
7 produced by a potato plant comprising reducing the level of activity of GLTP
or GHTP in the
8 potato plant. In a preferred embodiment, such methods involve introducing
into the potato
9 plant an expression cassette having a plant promoter sequence operably
linked to a DNA
sequence which, when transcribed in the plant, inhibits expression of an
endogenous GLTP
11 gene or GHTP gene. As above, the DNA sequence may be inserted in the
expression cassette
12 in either a sense or antisense orientation.
13 As described in detail in the examples herein, improvements in cold-storage
14 characteristics have been observed in the potato variety Desiree
transformed by the methods
of the present invention. A direct measure of improved cold-storage
characteristics is a
16 reduction in the level of GLTP or GHTP enzyme activity detected in potatoes
after harvest
17 and cold-storage. Transformed potato varieties have been developed wherein
the total a
18 glucan phosphorylase activity measured as pmol NADPH produced mg ' protein
' h-' in tubers
19 of plants stored at 4°C for 189 days is as much as 70% lower than
the total a glucan
2 0 phosphorylase activity in tubers of untransformed plants stored under the
same conditions.
21 Another relatively direct measure of improved cold-storage characteristics
is a
22 reduction in sweetening of potatoes observed after a period of cold-
storage. Transformed
2 3 potato varieties have been developed wherein the sum of the concentrations
of glucose and
2 4 fructose in tubers stored at 4 °C for 91 days is 39% lower than the
sum of the concentrations
2 5 of glucose and fructose in tubers of an untransformed plant stored under
the same conditions.
2 6 Yet another measure of improved cold-storage characteristics,
demonstrating a
27 practical advantage of the present invention, is a reduction in darkening
of a potato chip
2 8 during processing (cooking). As discussed hereinbefore, the accumulation
of sugars in
2 9 potatoes during cold-storage contributes to unacceptable darkening of the
fried product.
3 0 Reduced darkening upon frying can be quantified as a measure of the
reflectance, or chip
31 score, of the fried potato chip. Techniques for measuring chip scores are
discussed herein.
6
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1 Transformed potato varieties of the present invention have been developed
wherein the chip
2 score for tubers of plants stored at 4°C for 124 days was as much as
89% higher than the chip
3 scores for tubers of untransformed plants stored under the same conditions.
4 By reducing GLTP and/or GHTP activity in tubers of potato plants, thereby
inhibiting
sugar accumulation during cold-temperature storage, the present invention
allows for storage
6 of potatoes at cooler temperatures than would be possible with wildtype
potatoes of the same
7 cultivar. As discussed above, storage of potatoes at cooler temperatures
than those
8 traditionally used could result in increased storage life, increased
dormancy through reduced
9 respiration and sprouting, and reduced incidence of disease. It will be
apparent to those
skilled in the art that such additional benefits also constitute improved cold-
storage
11 characteristics and may be measured and quantified by known techniques.
12
13 BRIEF DESCRIPTION OF THE DRAWINGS
14
In drawings illustrating embodiments of the invention:
16 Figure I is a schematic diagram of the tuber L-type a glucan phosphorylase
antisense
17 sequence inserted into the pBIl2l transformation vector;
18 Figure 2 is a schematic diagram of the tuber H-type a glucan phosphorylase
antisense
19 sequence inserted into the pBI121 transformation vector;
2 0 Figure 3 shows the basic structure of the three isolated isoforms of
glucan
21 phosphorylase. The transit peptide (TS) and insertion sequence (IS) are
characteristic of the
2 2 L-type phosphorylases and are not found in the H-type phosphorylase. The
percentages
2 3 indicate the nucleic acid sequence homologies between the isoforms;
24 Figure 4 is a schematic diagram of carbohydrate interconversions in
potatoes
2 5 (Sowokinos 1990);
2 6 Figure 5 is a comparison of the amino acid sequences of the three isoforms
of
2 7 phosphorylase found in potato for the region targeted by the antisense
GLTP construct used in
2 8 the Examples herein. Highlighted amino acids are identical. The leaf L-
type a glucan
29 phosphorylase amino acid sequence is on top (amino acids 21 - 238 of SEQ U~
NO: 6), the
3 0 tuber L-type a glucan phosphorylase amino acid sequence is in the middle
(amino acids 49 -
7
CA 02275885 1999-06-29
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1 266 of SEQ m NO: 2), and tuber H-type a glucan phosphorylase amino acid
sequence is on
2 the bottom (amino acids 46 - 264 of SEQ m NO: 4);
3 Figure 6A and 6B are a comparison of the nucleotide sequences of the three
isoforms
4 of phosphorylase found in potato for the region targeted by the antisense
GLTP construct used
in the Examples herein. Highlighted nucleotides are identical. The leaf L-type
a giucan
6 phosphorylase nucleotide sequence is on top (nucleotides 389 - 1045 of SEQ m
NO: 5), the
7 tuber L-type a glucan phosphorylase nucleotide sequence is in the middle
(nucleotides 338 -
8 993 of SEQ ID NO: 1), and tuber H-type a glucan phosphorylase nucleotide
sequence is on
9 the bottom (nucleotides 147 - 805 of SEQ 1D NO: 3);
Figure 7 is a northern blot of polyadenylated RNA isolated from potato tubers
of wild
11 type and lines 3,4,5, and 9 transformed with the tuber L-type a glucan
phosphorylase. The
12 blot was probed with a radiolabelled probe specific for the tuber L-type a
glucan
13 phosphorylase;
I4 Figure 8 is a northern blot of total RNA isolated from potato tubers of
wild type and
lines 1 and 2 transformed with the H-type a-glucan phosphorylase. The blot was
probed with
16 a radio labelled probe specific for the H-type a-glucan phosphorylase;
17 Figure 9 shows the fried product obtained from (A) wild type and tuber L-
type a
18 glucan phosphorylase transformants (B) ATL1 (C) ATL3 (D} ATL4 (E) ATLS (F)
ATL9 field
19 grown tubers following 86 days storage at 4°C ("ATL" = antisense
tuber L-type
2 0 transformant);
21 Figure 10 shows the activity gel and western blot of L-type and H-type
isozymes of a
22 1,4 glucan phosphorylase extracted from wild type tubers and tubers
transformed with the
2 3 antisense construct for the L-type isoform; and
24 Figure 11 shows the activity gel and western blot of L-type and H-type
isozymes of a
1,4 glucan phosphorylase extracted from wild type tubers and transformed with
the antisense
2 6 construct for the H-type isoform..
27
2 8 DESCRIPTION OF THE PREFERRED EMBODIMENT
29
3 0 Potato plants having a reduced level of a glucan L-type tuber
phosphorylase (GLTP)
31 or a glucan H-type tuber phosphorylase (GHTP) activity in tubers produced
by the plants
8
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_ WO 98/35051 PCT/CA98/00055
1 relative to that of tubers produced by unmodified potato plants are
provided. In the _
2 exemplified case, reduction in a glucan phosphorylase activity is
accomplished by
3 transforming a potato plant with an expression cassette having a plant
promoter sequence
4 operably linked to a DNA sequence which, when transcribed in the plant,
inhibits expression
of an endogenous GLTP gene or GHTP gene. Although, in the exemplified case,
the DNA
6 sequence is inserted in the expression cassette in the antisense
orientation, a reduction in a
'1 glucan phosphorylase activity can be achieved with the DNA sequence
inserted in the
8 expression cassette in either a sense or antisense orientation.
9
1 Homology Dependent Silencing
11 The control of gene expression using sense or antisense gene fragments is
standard
12 laboratory practice and is well documented in the literature. Antisense and
sense suppression
13 are both gene sequence homology-dependent phenomena that may be described
as
14 "homology-dependent silencing" phenomena.
A review of scientific research articles published during 1996 reveals several
hundred
16 reports of homology-dependent silencing in transgenic plants. The
mechanisms underlying
1~ homology-dependent silencing are not fully understood, but the
characteristics of the
18 phenomena have been studied in many plant genes and this body of work has
been
39 extensively reviewed (Meyer and Saedler 1996, Matzke and Matzke 1995,
Jorgensen 1995,
2 0 Weintraub 1990, Van der Krol et al. 1988) Homology-dependent silencing
appears to be a
21 general phenomenon that may be used to control the activity of many
endogenous genes.
22 Examples of genes exhibiting reduced expression after the introduction of
homologous
23 sequences include dihydroflavanol reductase (Van der Krol 1990),
polygalacturonidase
24 (Smith et al 1990), phytoene synthase (Fray and Grierson 1993),
pectinesterase (Seymour e~
al. 1993), phenylalanine ammonia-lyase (De Carvalho et al. 1992), ~3-1,3-
gIucanase (Hart et
2 6 al. 1992), chitinase (Dorlhac et al. 1994) nitrate reductase (Napoli et
al. 1990), and chalcone
2 7 synthase ( 14). Transformation of Russet Burbank potato plants with either
sense- or
2 8 antisense- constructs of the potato leafroll virus coat protein gene has
been reported to confer
2 9 resistance to potato leafroll virus infection (Kawchuk et al. 1991 ). The
transfer of a
3 0 homologous sense or antisense sequence usually generates transformants
with reduced
31 endogenous gene expression. As discussed in detail in the examples herein,
transformed
9
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1 potato plants exhibiting phenotypes indicating reduced GLTP or GHTP
expression can be
2 readily identified.
3 In the antisense suppression technique, a gene construct or expression
cassette is
4 assembled which, when inserted into a plant cell, results in expression of
an RNA which is of
complementary sequence to the mRNA produced by the target gene. It is
theorized that the
6 complementary RNA sequences form a duplex thereby inhibiting translation to
protein. The
7 theory underlying both sense and antisense inhibition has been discussed in
the literature,
8 including in Anti,rense Research and Applications (CRC Press, 1993) pp. /25-
I48. The
9 complementary sequence may be equivalent in length to the whole sequence of
the target
gene, but a fragment is usually sufficient and is more convenient to work
with. For instance,
11 Cannon et al. (1990) reveals that an antisense sequence as short as 41 base
pairs is sufficient
12 to achieve gene inhibition. United States Patent No. 5,585,545 (Bennett et
al., December 17,
13 1996) describes gene inhibition by an antisense sequence of only 20 base
pairs. There are
14 many examples in the patent literature of patents including descriptions
and claims to
methods for suppressing gene expression through the introduction of antisense
sequences to
16 an organism, including, for example, United States Patent No. 5,545,815
(Fischer et al.,
17 August 13, 1996) and United States Patent No. 5,387,757 (Bridges et al.,
February 7, 1995).
18 Sense-sequence homology-dependent silencing is conducted in a similar
manner to
19 antisense suppression, except that the nucleotide sequence is inserted in
the expression
2 0 cassette in the normal sense orientation. A number of patents, including
United States patents
21 5,034,323, 5,231,020 and 5,283,184, disclose the introduction of sense
sequences leading to
2 2 suppression of gene expression.
2 3 Both forms of homology-dependent silencing, sense- and antisense-
suppression, are
2 4 useful for accomplishing the down-regulation of GLTP or GHTP of the
present invention. It
2 5 is recognized in the art that both techniques are equally useful
strategies for gene suppression.
2 6 For instance, both US Patent No. 5,585,545 (Bennett et al., December 17,
1996) and US
27 Patent No. 5,451,514 (Boudet et al., September 15, 1995) claim methods for
inhibiting gene
2 8 expression or recombinant DNA sequences useful in methods for suppressing
gene
2 9 expression drawn to both sense- and antisense-suppression techniques.
31
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1 2 Alternate Techniques for Reducing GHTP and/or GLTP Activity in Tubers
2 Although homology-dependent silencing is a preferred technique for the down-
3 regulation of GLTP or GHTP in potato plants of the present invention, there
are several
4 commonly used alternative strategies available to reduce the activity of a
specific gene
product which will be understood by those skilled in the art to bear
application in the present
6 invention. Insertion of a related gene or promoter into a plant can induce
rapid turnover of
7 homologous endogenous transcripts, a process referred to as co-suppression
and believed to
8 have many similarities to the mechanism responsible for antisense RNA
inhibition
9 (Jorgensen, 1995; Brusslan and Tobin, 1995). Various regulatory sequences of
DNA can be
altered (promoters, polyadenylation signals, post-transcriptional processing
sites) or used to
11 alter the expression levels {enhancers and silencers) of a specific mRNA.
Another strategy to
12 reduce expression of a gene and its encoded protein is the use of ribozymes
designed to
13 specifically cleave the target mRNA rendering it incapable of producing a
fully functional
14 protein (Hasseloff and Gerlach, 1988). Identification of naturally
occurring alleles or the
development of genetically engineered alleles of an enzyme that have been
identified to be
16 important in determining a particular trait can alter activity levels and
be exploited by
17 classical breeding programs (Oritz and Huaman, 1994). Site-directed
mutagenesis is often
18 used to modify the activity of an identified gene product. The structural
coding sequence for a
19 phosphorylase enzyme can be mutagenized in E. toll or another suitable host
and screened for
2 0 reduced starch phosphorolysis. Alternatively, naturally occurring alleles
of the phosphorylase
21 with reduced affinity and/or specific activity may be identified.
Additionally, the activity of
22 a particular enzyme can be altered using various inhibitors. These
procedures are routinely
23 used and can be found in text books such as Sambrook et al. (1989).
24
2 5 3 Variants of GLTP and GHTP Enzymes and Sequences Used for Homology
Dependent
2 6 Silencing
2 7 As discussed in the background of the invention, and in greater detail by
Nakano et al.
2 8 ( 1986), Mori et al. ( I 991 ), and Sonnewald et al. ( 1990), there are
three known a glucan
2 9 phosphorylase isozymes that occur in potato plants. The present invention
relates to down-
3 0 regulation of the GLTP and/or GHTP isozymes. While it is believed that the
GLTP and
31 GHTP genes of all known commercial potato varieties are substantially
identical, it is
11
CA 02275885 1999-06-29
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1 expected that the principles and techniques of the present invention would
be effective in
2 potato plants having variant full length polynucleotide sequences or
subsequences which
3 encode polypeptides having the starch catabolizing enzymatic activity of the
described GLTP
4 and GHTP enzymes. The terms "GLTP" and "GHTP", as used herein and in the
claims, are
intended to cover the variants described above. The foregoing variants may
include GLTP
6 and GHTP nucleotide sequence variants that differ from those exemplified but
still encode
7 the same polypeptide due to codon degeneracy, as well as variants which
encode proteins
8 capable of recognition by antibodies raised against the GLTP and GHTP amino
acid
9 sequences set forth in SEQ 1D NO's. 2 and 4.
Similarly, those skilled in the art will recognize that homology dependent
silencing of
11 GLTP and/or GHTP in potato plants may be accomplished with sense or
antisense sequences
12 other than those exemplified. First, the region of the GLTP or GHTP cDNA
sequence from
13 which the antisense sequence is derived is not essential. Second, as
described hereinabove,
14 the length of the antisense sequence used may vary considerably. Further,
the sense or
antisense sequence need not be identical to that of the target GLTP or GHTP
gene to be
16 suppressed. As described in the Examples herein, the inventors have
observed that
17 transformation of potato plants with antisense DNA sequences derived from
the GHTP gene
18 not only substantially suppresses GHTP gene activity, but causes some
degree of suppression
19 of GLTP gene activity. The GHTP and GLTP genes antisense sequences have
56.8%
2 0 sequence identity. The sequence identity between the GLTP antisense
sequence and the
21 corresponding leaf type a glucan phosphorylase squence described by
Sonnewald et al.
22 (1990) is 71.3%. In the inventors' research to date, the same phenomenon of
cross-
23 downregulation has not been observed when potato plants are transformed
with antisense
24 DNA sequences derived from the GLTP gene. Nevertheless, these results
clearly indicate that
absolute sequence identity between the target endogenous a glucan
phosphorylase gene and
2 6 the recombinant DNA is not essential given that GLTP activity was
suppressed by an
27 antisense sequence having about 57% sequence identity with the target GLTP
sequence.
2 8 Thus, it will be understood by those skilled in the art that sense or
antisense sequences
29 other than those exemplified herein and other than those having absolute
sequence identity
3 0 with the target endogenous GLTP or GHTP gene will be effective to cause
suppression of the
31 endogenous GLTP or GHTP gene when introduced into potato plant cells.
Useful sense or
12
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 antisense sequences may differ from the exemplified antisense sequences or
from other
2 sequences derived from the endogenous GHTP or GLTP gene sequences by way of
3 conservative amino acid substitutions or differences in the percentage of
matched nucleotides
4 or amino acids over portions of the sequences which are aligned for
comparison purposes.
United States Patent 5,585,545 (Bennett et al., December 17, 1996) provides a
helpful
6 discussion regarding techniques for comparing sequence identity for
polynucleotides and
7 polypeptides, conservative amino acid substitutions, and hybridization
conditions indicative
8 of degrees of sequence identity. Relevant parts of that discussion are
summarized herein.
Percentage of sequence identity for polynucleotides and polypeptides may be
determined by comparing two optimally aligned sequences over a comparison
window,
11 wherein the portion of the polynucleotide or polypeptide sequence in the
comparison window
12 may include additions or deletions (i.e., gaps) as compared to the
reference sequence (which
13 does not comprise additions or deletions) for optimal alignment of the two
sequences. The
14 percentage is calculated by: (a) determining the number of positions at
which the identical
1 S nucleic acid base or amino acid residue occurs in both sequences to yield
the number of
16 matched positions; (b) dividing the number of matched positions by the
total number of
17 positions in the window of comparison; and, (c) multiplying the result by
100 to yield the
18 percentage of sequence identity. Optimal alignment of sequences for
comparison may be
19 conducted by computerized implementations of known algorithms (e.g., GAP,
BESTFTT,
2 0 FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer
21 Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available
from the
22 National Center for Biotechnology Information), or by inspection.
23 Polypeptides which are substantially similar share sequences as noted above
24 except that residue positions which are not identical may differ by
conservative amino acid
2 5 changes. Conservative amino acid substitutions refer to the
interchangeability of residues
2 6 having similar side chains. For example, a group of amino acids having
aliphatic side chains
27 is glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having
2 8 aliphatic-hydroxyl side chains is serine and threonine; a group of amino
acids having
2 9 amide-containing side chains is asparagine and glutamine; a group of amino
acids having
3 0 aromatic side chains is phenyialanine, tyrosine, and tryptophan; a group
of amino acids
13
CA 02275885 1999-06-29
-WO 98/35051 PCT/CA98/00055
1 having basic side chains is lysine, arginine, and histidine; and a group of
amino acids having
2 sulfur-containing side chains is cysteine and methionine.
3 Another indication that nucleotide sequences are substantially identical is
if two
4 molecules specifically hybridize to each other under stringent conditions.
Stringent
conditions are sequence dependent and will be different in different
circumstances.
6 Generally, stringent conditions are selected to be about 10°C lower
than the thermal melting
7 point (Tm) for the specific sequence at a defined ionic strength and pH. The
Tm is the
8 temperature (under defined ionic strength and pH) at which 50% of the target
sequence
9 hybridizes to a perfectly matched probe. The Tm of a hybrid, which is a
function of both the
length and the base composition of the probe, can be calculated as described
in Sambrook et
11 al. ( 1989). Typically, stringent conditions for a Southern blot protocol
involve washing at
12 65 °C with 0.2XSSC. For preferred oligonucleotide probes, washing
conditions are typically
13 about at 42°C in 6XSSC.
14
4 General Methods
16 Various methods are available to introduce and express foreign DNA
sequences in
17 plant cells. In brief, the steps involved in preparing antisense a glucan
phosphorylase cDNAs
18 and introducing them into a plant cell include: ( 1 ) isolating mRNA from
potato plants and
19 preparing cDNA from the mRNA; (2) screening the cDNA for the desired
sequences; (3)
2 0 linking a promoter to the desired cDNAs in the opposite orientation for
expression of the
21 phosphorylase genes; (4) transforming suitable host plant cells; and (5)
selecting and
22 regenerating cells which transcribe the inverted sequences.
23 In the exemplified case, DNA derived from potato GLTP and GHTP genes is
used to
2 4 create expression cassettes having a plant promoter sequence operably
linked to an antisense
2 5 DNA sequence which, when transcribed in the plant, inhibits expression of
an endogenous
2 6 GLTP gene or GHTP gene. Agrobacterium tumefaciens is used as a vehicle for
transmission
2 7 of the DNA to stem explants of potato plant shoots. A plant regenerated
from the
2 8 transformed explants transcribes the antisense DNA which inhibits activity
of the enzyme.
29 The recombinant DNA technology described herein involves standard
laboratory
3 0 techniques that are well known in the art and are described in standard
references such as
31 Sambrook et al. ( 1989). Generally, enzymatic reactions involving DNA
ligase, DNA
14
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 polymerase, restriction endonucleases and the like are performed according
to the
2 manufacturer's specifications. -
3
4 5 Preparation of GHTP and GLTP cDNA
cDNA is prepared from isolated potato tuber mRNA by reverse transcription. A
6 primer is annealed to the mRNA, providing a free 3' end that can be used for
extension by the
7 enzyme reverse transcriptase. The enzyme engages in the usual 5'-3'
elongation, as directed
8 by complementary base pairing with the mRNA template to form a hybrid
molecule,
9 consisting of a template RNA strand base-paired with the complementary cDNA
strand.
After degradation of the original mRNA, a DNA polymerase is used to synthesize
the
11 complementary DNA strand to convert the single-stranded cDNA into a duplex
DNA.
12 After DNA amplification, the double stranded cDNA is inserted into a vector
for
13 propagation in E. coli. Typically, identification of clones harbouring the
desired cDNA's
14 would be performed by either nucleic acid hybridization or immunological
detection of the
encoded protein, if an expression vector is used. In the exemplified case, the
matter is
16 simplified in that the DNA sequences of the GLTP and GHTP genes are known,
as are the
17 sequences of suitable primers (Brisson et al., 1990; Fukui et al., 1991).
The primers used
18 hybridize within the GLTP and GHTP genes. Thus, it is expected that the
amplified cDNA's
19 prepared represent portions of the GLTP and GHTP genes without further
analysis. E. coli
transformed with pUCl9 plasmids carrying the phosphorylase DNA insert were
detected by
21 color selection. Appropriate E. coli strains transformed with plasmids
which do not carry
22 inserts grow as blue colonies. Strains transformed with pBluescript
plasmids carrying inserts
23 grow as white colonies. Plasmids isolated from transformed E. coli were
sequenced to
24 confirm the sequence of the phosphorylase inserts.
2 6 6 Vector Construction
27 The cDNAs prepared can be inserted in the antisense or sense orientation
into
2 8 expression cassette in expression vectors for transformation of potato
plants to inhibit the
29 expression of the GLTP and/or GHTP genes in potato tubers.
3 0 As in the exemplified case, which involves antisense suppression, the
desired
31 recombinant vector will comprise an expression cassette designed for
initiating transcription
CA 02275885 1999-06-29
-WO 98/35051 PCT/CA98/00055
1 of the antisense cDNAs in plants. Additional sequences are included to allow
the vector to be
2 cloned in a bacterial or phage host.
3 The vector will preferably contain a prokaryote origin of replication having
a broad
4 host range. A selectable marker should also be included to allow selection
of bacterial cells
bearing the desired construct. Suitable prokaryotic selectable markers include
resistance to
6 antibiotics such ampicillin.
7 Other DNA sequences encoding additional functions may also be present in the
8 vector, as is known in the art. For instance, in the case of Agrobacterium
transformations,
9 T-DNA sequences will also be included for subsequent transfer to plant
chromosomes.
For expression in plants, the recombinant expression cassette will contain in
11 addition to the desired sequence, a plant promoter region, a transcription
initiation site (if
12 the sequence to be transcribed lacks one), and a transcription termination
sequence. Unique
13 restriction enzyme sites at the 5' and 3' ends of the cassette are
typically included to allow for
14 easy insertion into a pre-existing vector. Sequences controlling eukaryotic
gene
expression are well known in the art.
16 Transcription of DNA into mRNA is regulated by a region of DNA referred to
as the
17 promoter. The promoter region contains sequence of bases that signals RNA
polymerase to
18 associate with the DNA, and to initiate the transcription of mRNA using one
of the DNA
I9 strands as a template to make a corresponding complimentary strand of RNA.
Promoter
2 0 sequence elements include the TATA box consensus sequence (TATAAT), which
is usually
21 20 to 30 base pairs (bp) upstream (by convention -30 to -20 by relative to
the transcription
2 2 start site) of the transcription start site. In most instances the TATA
box is required for
2 3 accurate transcription initiation. The TATA box is the only upstream
promoter element that
24 has a relatively fixed location with respect to the staut point.
2 5 The CHAT box consensus sequence is centered at -75, but can function at
distances
2 6 that vary considerably from the start point and in either orientation.
2 7 Another common promoter element is the GC box at -90 which contains the
2 8 consensus sequence GGGCGG. It may occur in multiple copies and in either
orientation.
2 9 Other sequences conferring tissue specificity, response to environmental
signals, or
3 0 maximum efficiency of transcription may also be found in the promoter
region. Such
31 sequences are often found within 400 by of transcription initiation size,
but may extend as far
16
CA 02275885 1999-06-29
- WO 98135051 PCT/CA98100055
1 as 2000 by or more. In heterologous promoter/structural gene combinations,
the promoter is
2 preferably positioned about the same distance from the heterologous
transcription start site as
3 it is from the transcription start site in its natural setting. However,
some variation in this
4 distance can be accommodated without loss of promoter function.
The particular promoter used in the expression cassette is not critical to the
6 invention. Any of a number of promoters which direct transcription in plant
cells is suitable.
7 The promoter can be either constitutive or inducible.
8 A number of promoters which are active in plant cells have been described in
the
9 literature. These include the nopaline synthase (NOS) and octopine synthase
(OCS)
promoters (which are carried on tumour-inducing plasmids of Agrobacterium
ta~mefaciens),
11 the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S
and 355 and
12 the figwort mosaic virus 35S-promoters, the light-inducible promoter from
the small subunit
13 of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO, a very abundant plant
polypeptide),
14 and the chlorophyll alb binding protein gene promoter, etc. All of these
promoters have been
used to create various types of DNA constructs which have been expressed in
plants; see, e.g.,
16 PCT W08402913.
17 The CaMV 35S promoter used in the Examples herein, has been shown to be
highly
18 active and constitutively expressed in most tissues (Bevan et al., 1986). A
number of other
19 genes with tuber-specific or enhanced expression are known, including the
potato tuber
2 0 ADPGPP genes, large and small subunits (Muller et al., 1990). Other
promoters which are
21 contemplated to be useful in this invention include those that show
enhanced or specific
2 2 expression in potato tubers, that are promoters normally associated with
the expression of
2 3 starch biosynthetic or modification enzyme genes, or that show different
patterns of
2 4 expression, for example, or are expressed at different times during tuber
development.
2 5 Examples of these promoters include those for the genes for the granule-
bound and other
2 6 starch synthases, the branching enzymes (Blennow et al., 1991; WO 9214827;
WO 9211375),
27 disproportionating enzyme (Takaha et al., 1993) debranching enzymes,
amylases, starch
2 8 phosphorylases (Nakano et al., 1989; Mori et al., 1991 ), pectin esterases
(Ebbelaar et al.,
29 1993), the 40 kD glycoprotein; ubiquitin, aspartic proteinase inhibitor
(Stukerlj et al., 1990),
3 0 the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shahar et al.,
1992; GenBank
31 Accession Numbers M95196 and M95197), putative trypsin inhibitor and other
tuber cDNAs
17
CA 02275885 1999-06-29
w - WO 98/35051 PCT/CA98/00(!55
1 (Stiekema et al., 1988), and for amylases and sporamins (Yoshida et al.,
1992; Ohta et al.,
2 1991).
3 In addition to a promoter sequence, the expression cassette should also
contain a
4 transcription termination region downstream of the structural gene to
provide for efficient
termination. The termination region may be obtained from the same gene as the
promoter
6 sequence or may be obtained from different genes. In the exemplified case
the nopaline
7 synthase NOS 3' terminator sequence (Bevan et al. 1983) was used.
8 Polyadenylation sequences are also commonly added to the vector construct if
the
9 mRNA encoded by the structural gene is to be efficiently translated (Alber
and Kawasaki,
1982). Polyadenylation is believed to have an effect on stabilizing mRNAs.
Polyadenylation
I 1 sequences include, but are not limited to the Agrobacterium octopine
synthase signal (Gielen
12 et al., 1984) or the nopaline synthase signal (Depicker et al., 1982).
13 The vector will also typically contain a selectable marker gene by which
transformed
14 plant cells can be identified in culture. Typically, the marker gene
encodes antibiotic
resistance. These markers include resistance to 6418, hygromycin, bleomycin,
kanamycin,
16 and gentamycin. In the exemplified case, the marker gene confers resistance
to kanamycin.
17 After transforming the plant cells, those cells containing the vector will
be identified by their
18 ability to grow in a medium containing the particular antibiotic.
19
2 0 7 Transformation of Plant Cells
21 Although in the exemplified case potato plant shoot stem explants were
transformed
2 2 via inoculation with Agrobacterium tumefaciens carrying the antisense
sequence linked to a
23 binary vector, direct transformation techniques which are known in the art
can also be used to
24 transfer the recombinant DNA. The vector can be microinjected directly into
plant cells.
2 5 Alternatively, nucleic acids may be introduced to the plant cell by high
velocity ballistic
2 6 penetration by small particles having the nucleic acid of interest
embedded within the matrix
27 of the particles or on the surface. Fusion of protoplasts with lipid-
surfaced bodies such as
2 8 minicells, cells or Iysosomes carrying the DNA of interest can be used.
The DNA may also
2 9 be introduced into plant cells by electroporation, wherein plant
protoplasts are electroporated
3 0 in the presence of plasmids carrying the expression cassette.
18
CA 02275885 1999-06-29
-WO 98135051 PCT/CA98/00055
1 In contrast to direct transformation methods, the exemplified case uses
vectored
2 transformation using Agrobacterium tumefaciens. Agrobacterium tumefaciens is
a Gram-
3 negative soil bacteria which causes a neoplastic disease known as crown gall
in
4 dicotyledonous plants. Induction of tumours is caused by tumour-inducing
plasmids known
as Ti plasmids. Ti plasmids direct the synthesis of opines in the infected
plant. The opines
6 are used as a source of carbon and/or nitrogen by the Agrobacteria.
7 The bacterium does not enter the plant cell, but transfers only part of the
Ti plasmid, a
8 portion called T-DNA, which is stably integrated into the plant genome,
where it expresses
9 the functions needed to synthesize opines and to transform the plant cell.
Vir (virulence)
genes on the Ti plasnlid, outside of the T-DNA region, are necessary for the
transfer of the T-
11 DNA. The vir region, however, is not transferred. In fact, the vir region,
although required
12 for T-DNA transfer, need not be physically linked to the T-DNA and may be
provided on a
13 separate plasmid.
14 The tumour-inducing portions of the T-DNA can be interrupted or deleted
without
loss of the transfer and integration functions, such that normal and healthy
transformed plant
16 cells may be produced which have lost all properties of tumour cells, but
still harbour and
17 express certain parts of T-DNA, particularly the T-DNA border regions.
Therefore, modified
18 Ti plasmids, in which the disease causing genes have been deleted, may be
used as vectors for
19 the transfer of the sense and antisense gene constructs of the present
invention into potato
2 0 plants (see generally Winnacker, 1987).
21 Transformation of plants cells with Agrobacterium and regeneration of whole
plants
22 typically involves either co-cultivation of Agrobacterium with cultured
isolated protoplasts or
23 transformation of intact cells or tissues with Agrobacterium. In the
exemplified case, stem
24 explants from potato shoot cultures are transformed~with Agrobacterium.
Alternatively, cauliflower mosaic virus (CaMV) may be used as a vector for
2 6 introducing sense or antisense DNA into plants of the Solanaceae family.
For instance,
27 United States Patent No. 4,407,956 (Howell, October 4, 1983) teaches the
use of cauliflower
2 8 mosaic virus DNA as a plant vehicle.
29
19
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 8 Selection and Regeneration of Transformed Plant Cells
2 After transformation, transformed plant cells or plants carrying the
antisense or sense
3 DNA must be identif ed. A selectable marker, such as antibiotic resistance,
is typically used.
4 In the exemplified case, transformed plant cells were selected by growing
the cells on growth
medium containing kanamycin. Other selectable markers will be apparent to
those skilled in
6 the art. For instance, the presence of opines can be used to identify
transformants if the plants
7 are transformed with Agrobacterium.
8 Expression of the foreign DNA can be confirmed by detection of RNA encoded
by the
9 inserted DNA using well known methods such as Northern blot hybridization.
The inserted
DNA sequence can itself be identified by Southern blot hybridization or the
polymerase chain
11 reaction, as well (See, generally, Sambrook et al. (1989)).
12 Generally, after it is determined that the transformed plant cells carry
the recombinant
13 DNA, whole plants are regenerated. In the exemplified case, stem and leaf
explants of potato
14 shoot cultures were inoculated with a culture of Agrobacterium tumefaciens
carrying the
desired antisense DNA and a kanamycin marker gene. Transformants were selected
on a
16 kanamycin-containing growth medium. After transfer to a suitable medium for
shoot
17 induction, shoots were transferred to a medium suitable for rooting. Plants
were then
18 transferred to soil and hardened off. The plants regenerated in culture
were transplanted and
19 grown to maturity under greenhouse conditions.
21 9 Analysis of GHTP and GLTP Activity Levels in Transformed Tubers
22 Following regeneration of potato plants transformed with antisense DNA
sequences
2 3 derived from the GHTP and GLTP genes, the biochemistry of transformed
tuber tissue was
2 4 analyzed several ways. The in vitro activity of a glucan phosphoryiase in
the phosphorolytic
2 5 direction was assayed according to the methods of Steup ( 1990) (Table 1
). The activity of the
2 6 enzyme in the synthetic direction and the amount of enzyme protein were
compared after
2 7 eiectrophoretic separation of the enzyme isoforms on a glycogen-
containing, polyacrylamide
2 8 gel (Figure 7). Starch synthesis by the tuber L-type and H-type isoforms
was determined by
2 9 iodine staining of the gel after incubation with glucose-1-phosphate and a
starch primer
3 0 (Steup, 1990). Western analysis was performed by blotting the protein from
an identical
31 unincubated native gel to nitrocellulose and probing with poIyclonal
antibodies specific for
CA 02275885 1999-06-29
1 tuber type L and type H glucan phosphorylase isc~forms. Le~.~els of reducing
;u:~ars (gla::ose~ a '
2 and fructose) in tuber tissues were quantified by HPLC (Tables 2. 3 and 4).
The extent of
3 Maillard reaction, which is proportional to the concentration of reducing
sugars in tubers was
4 examined by determining chip scores after frying (Table 5 and Figure 9).
6 10 Definitions . .
7 As used herein and in the claims, the term:
8 - "about three months", "about four months" and "about six months" refer,
respectively,
9 to periods of time of three months plus or minus two weeks, four months plus
or minus two
weeks, and six months plus or minus two weeks;
11 - "antisense orientation" refers to the orientation of nucleic acid
sequence from a
12 structural gene that is inserted in an expression cassette in an inverted
manner with respect to
13 its naturally occurring orientation. When the sequence is double stranded,
the strand that is
14 the template strand in the naturally occurring orientation becomes the
coding strand, and vice
versa;
16 - "chip score" of a tuber means the reflectance measurement recorded by an
Agtron
17 model E-15-FP Direct Reading Abridged Spectrophotometer (Agtron Inc. 109
Spice Island
18 Drive #100, Sparks Nevada 89431) of a center cut potato chip fried at 20~'F
in soybean oil
19 for approximately 3 minutes until bubbling stops;
2 0 - "cold storage" or "storage at reduced temperature" or variants thereof,
shall mean
21 holding at temperatures less than 10°C, that may be achieved by
refrigeration or ambient
2 2 temperatures;
2 3 - "endogenous", as it is used with reference to a glucan phosphorylase
genes of a potato
2 4 plant, shall mean a naturally occurring gene that was present in the
genome of the potato plant
2 5 prior to the introduction of an expression cassette carrying a DNA
sequence derived from an
2 6 a glucan phosphorylase gene;
27 - "expression" refers to the transcription and translation of a structural
Qene so that a
2 8 protein is synthesized;
2 9 - "heterologous sequence" or "heterologous expression cassette" is one
that originates
3 0 from a foreign species, or, if from the same species, is substantially
modified from its original
31 form;
21
AMENDED SHEET
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 - "improved cold-storage characteristics" includes, without limitation,
improvements in
2 chip score and reduction in sugar accumulation in tubers measured at harvest
or after a period
3 of storage below 10°C, and further includes improvements, advantages
and benefits which
4 may result from the storage of potatoes at cooler temperatures than those
traditionally used,
such as, without limitation, increased storage life of potatoes, increased
dormancy through
6 reduced respiration and sprouting of potatoes, and reduced incidence of
disease. Unless
7 further qualified by a specific measure or test, an improvement in a cold-
storage characteristic
8 refers to a difference in the described characteristic relative to that in a
control, wildtype or
9 unmodified potato plant;
- "modified" or variants thereof, when used to describe potato plants or
tubers, is used
11 to distinguish a potato plant or tuber that has been altered from its
naturally occurring state
12 through: the introduction of a nucleotide sequence from the same or a
different species,
13 whether in a sense or antisense orientation, whether by recombinant DNA
technology or by
14 traditional cross-breeding methods including the introduction of modified
structural or
regulatory sequences; modification of a native nucleotide sequence by site-
directed
16 mutagenesis or otherwise; or the treatment of the potato plant with
chemical or protein
17 inhibitors. An "unmodified" potato plant or tuber means a control, wildtype
or naturally
18 occurring potato plant or tuber that has not been modified as described
above;
19 - "nucleic acid sequence" or "nucleic acid segment" refer to a single or
double-stranded
2 0 polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. It
21 includes both self-replicating plasmids, infectious polymers of DNA or RNA
and non-
2 2 functional DNA or RNA;
23 - "operably linked" refers to functional linkage between a promoter and a
second
2 4 sequence, wherein the promoter sequence initiates transcription of RNA
corresponding to the
2 5 second sequence;
2 6 - "plant " includes whole plants, plant organs (e.g. leaves, stems, roots,
etc.) seeds and
2 7 plant cells;
28 - "promoter" refers to a region of DNA upstream from the structural gene
and involved
2 9 in recognition and binding RNA polymerase and other proteins that initiate
transcription. A
3 0 "plant promoter" is a promoter capable of initiating transcription in
plant cells;
22
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 - "reduced activity" or variants thereof, when used in reference to the
level of GLTP or _
2 GHTP enzyme activity in a potato tuber includes reduction of GLTP or GHTP
enzyme
3 activity resulting from reduced expression of the GLTP or GHTP gene product,
reduced
4 substrate affinity of the GLTP or GHTP enzyme, and reduced catalytic
activity of the GLTP
or GHTP enzyme;
- "reduced" or variants thereof, may be used herein with reference to, without
7 limitation, activity levels of GLTP or GHTP enzyme in potato tubers,
accumulation of sugars
8 in potato tubers and darkening of potato chips upon frying. Unless further
qualified by a
9 specific measure or test, reduced levels or reduced activity refers to a
demonstrable
statistically significant difference in the described characteristic relative
to that in a control,
11 wildtype or unmodified potato plant;
12 - "stress" or variants thereof, when used in relation to stresses
experienced by potato
13 plants and tubers, includes the effects of environment, fertility,
moisture, temperature,
14 handling, disease, atmosphere and aging that impact upon plant or tuber
quality and which
may be experienced by potato plants through all stages of their life cycle and
by tubers at all
16 stages of the growth and development cycle and during subsequent
harvesting, transport,
17 storage and processing;
18 - "stress resistance" or variants thereof, shall mean reduced effects of
temperature,
19 aging, disease, atmosphere, physical handling, moisture, chemical residues,
environment,
2 0 pests and other stresses;
21 - "suitable host" refers to a microorganism or cell that is compatible with
a recombinant
22 plasmid, DNA sequence or recombinant expression cassette and will permit
the plasmid to
2 3 replicate, to be incorporated into its genome, or to be expressed; and
24 - "uninterrupted" refers to a DNA sequence (e.g. cDNA) containing an open
reading
2 5 frame that lacks intervening, untranslated sequences.
26
2 7 EXAMPLE I
2 8 This example describes the reduction of GHTP and/or GLTP activity in
tubers of
2 9 potato plants by transforming potato plants with expression cassettes
containing DNA
3 0 sequences derived from the GLTP and GHTP gene sequences linked to the
promoter in the
31 antisense orientation.
23
CA 02275885 2002-11-12
1 A Isolation of Potato Tuber mRNA
2 Potato total RNA was purified at 4 ° C using autoclaved reagents from
1 g of tuber
3 tissue ground to a fine powder under liquid nitrogen with a mortar and
pestle. The powder
4 was transferred to a 30m1 corex tube and 3 volumes were added of 100 mM Tris-
Cl, pH 8.0,
100 mM NaCI, and 10 mM EDTA (10x TNE) containing 0.2% (w/v) SDS and 0.5% (v/v)
2-
6 mercaptoethanol. An equal volume of phenol-chloroform (1:1) was added and
the sample
7 gently vortexed before centrifugation at 4 °C in a SS34 rotor at
8,000 rpm for 5 min. The
8 organic phase was reextracted with 0.5 volume of l Ox THE containing 0.2%
(w/v)SDS and
9 0.5% (v/v) 2-mercaptoethanol and the combined aqueous phases were extracted
with
chloroform. Nucleic acids were precipitated from the aqueous phase with sodium
acetate and
11 absolute ethanol, pelleted by centrifugation, and resuspended in 3 ml of 1
x TNE. An equal
12 volume of 5 M LiCI was added and the sample stored at -20°C for at 4
h before centrifuging
13 at 8,000 rpm in a SS34 rotor at 4°C for 10 min. The RNA pellet was
washed with 70%
14 ethanol, dried, and resuspended in DEPC-treated water.
Poly (A+) RNA was isolated using oligo (dT) cellulose (Boehringer Mannheim)
16 column chromatography. Poly (A+) RNA was isolated from total RNA
resuspended in
17 RNAse free water. Columns were prepared using an autoclaved Bio-Rad Poly-
PrepT''~ 10 ml
18 column to which was added 50 mg of oligo (dT) cellulose suspended in 1 ml
of loading
19 buffer B which contains 20 mM Tris-Cl, pH 7.4, 0.1 M NaCI, 1 mM EDTA, and
0.1% (w/v)
2 0 SDS. The column was washed with 3 volumes of 0.1 M NaOH with 5 mM EDTA and
then
21 DEPC-treated water until the pH of effluent was less than 8, as determined
with pH paper.
2 2 The column was then washed with 5 volumes of loading buffer A containing
40 mM Tris-Cl,
2 3 pH 7.4, 1 M NaCI, 1 mM EDTA, and 0.1 % (w/v) SDS.
24 RNA samples were heated to 65 °C for 5 min at which time 400 ,u1 of
loading buffer
2 5 A, prewarmed to 65 °C, was added. The sample was mixed and allowed
to cool at room
2 6 temperature for 2 min before application to the column. Eluate was
collected, heated to 65 ° C
2 7 for 5 min, cooled to room temperature for 2 min, and reapplied to the
column. This was
2 8 followed by a 5 volume washing with loading buffer A followed by a 4
volume wash with
2 9 loading buffer B. Poly (A+) RNA was eluted with 3 volumes of 10 mM Tris-
Cl, pH 7.4, 1
3 0 mM EDTA, and 0.05% (w/v) SDS. Fractions were collected and those
containing RNA were
31 identified in an ethidium bromide plate assay, a petri dish with 1 %
agarose made with TAE
24
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 containing EtBr. RNA was precipitated, resuspended in 10 ~cl, and a 1 ~cl
aliquot quantitated
2 with a spectrophotometer.
3
4 B Isolation of GLTP and GHTP DNA Sequences
The nucleotide sequences utilized in the development of the antisense
construct were
6 randomly selected from the 5' sequence of GLTP (SEQ ID NO: 1 ) and GHTP (SEQ
ID NO:
7 3). DNA sequences used to develop the antisense constructs were obtained
using reverse
8 transcription-polymerase chain reaction. GLTP (SPL1 and SPL2)- and GHTP
(SPHI and
9 SPH2)-specific primers were designed according to the published sequences
(Brisson et al.
30 1990, Fukui et al. 1991) with minor modifications to facilitate restriction
with enzymes:
11 SPLI Primer: 5'ATTCGAAAAGCTCGAGATTTGCATAGA3' (SEQ ID NO: 7) (additional
12 CG creates Xho I site);
13 SPL2 Primer: 5'GTGTGCTCTCGAGCATTGAAAGC3' (SEQ m NO: 8) (changed C to G to
14 create Xho I site);
SPHI Primer: 5'GTTTATTTTCCATCGATGGAAGGTGGTG3' (SEQ ID NO: 9) (added
16 CGAT to create Cla I site);
17 SPH2 Primer: 5'ATAATATCCTGAATCGATGCACTGC3' (SEQ ID NO: 10) (changed G to
18 T to create Cla I site).
19 Reverse transcription was performed in a volume of 15 p1 containing I x PCR
buffer
2 0 ( 10 mM Tris-CI pH 8.2, 50 mM KCI, 0.001 % gelatin, 1.5 mM MgCI,), 670 p M
of each
21 dNTP, 6 pg of total potato tuber cv. Russet Burbank RNA, I mM each primer
(SPH1 and
22 SPL2, or SPH1 and SPH2) and 200 U of Maloney murine leukemia virus reverse
23 transcriptase (BRL). The reaction was set at 37°C for 30 minutes,
then heat-killed at 94°C for
24 5 minutes and snap cooled on ice. To the reverse transcription reaction was
added 2.5 U Taq
DNA polymerase (BRL) in 35 p1 of 1 x PCR buffer. DNA amplification was done in
a Perkin
26 Elmer 480 programmed for 30 cycles with a 1 min 94°C denaturation
step, a 1 min 56 °C
27 (SPL1 and SPL2) or 58°C (SPH1 and SPH2) annealing step, and a 2 min
72°C extension
2 8 step. PCR was completed with a final 10 min extension at 72°C.
29
CA 02275885 2002-11-12
1 C Construction of SP Vectors for Phosphorylase Inhibition
2 To express the antisense constructs in plant cells, it was necessary to fuse
the genes to
3 the proper plant regulatory regions. This was accomplished by cloning the
antisense DNA
4 into a plasmid vector that contained the needed sequences.
Amplified DNA was blunt ended and cloned into a pUC 19 vector at the SmaI
site.
6 The recombinant plasmid was transformed into sub-cloning efficiency E. coli
DHSa cells
7 (BRL). The transformed cells were plated on LB ( 15 g/1 Bactotyptone, 5 g/1
yeast extract, 10
8 g/1 NaCI, pH 7.3, and solidified with 1.5% agar) plates that contained
ampicillin at 100 ug/ml.
9 Selection of bacteria containing plasmids with inserted plant phosphorylase
sequence was
accomplished using color selection. The polylinker and T3 and T7 RNA
polymerase promoter
11 sequences are present in the N-terminal portion of the lacZ gene fragment.
pUC 19 plasmids
12 without inserts in the polylinker grow as blue colonies in appropriate
bacterial strains such as
13 DHSa. Color selection was made by spreading 100 p1 of 2% X-gal (prepared in
dimethyl
14 formamide) on LB plates containing 50 pg/ml ampicillin 30 minutes prior to
plating the
transformants. Colonies containing plasmids without inserts will be blue after
incubation for
16 12 to 18 hours at 37C and colonies with plasmids containing inserts will
remain white. An
17 isolated plasmid was sequenced to confirm the sequence of the phosphorylase
inserts.
18 Sequences were determined using the ABI PrismT"~ Dye Terminator Cycle
Sequencing Core
19 Kit (Applied Biosystems, Foster City, CA), M13 universal and reverse
primers, and an ABI
2 0 automated DNA sequencer. The engineered plasmid was purified by the rapid
alkaline
21 extraction procedure from a 5 ml overnight culture (Birnboim and Doly,
1979). Orientation of
2 2 the SPL and SPH fragments in pUC 19 was determined by restriction enzyme
digestion. The
2 3 recombinant pUC 19 vectors and the binary vector pBI121 (Clonetech) were
restricted, run on
2 4 a agarose gel and the fragments purified by gel separation as described by
Thuring et al
(1975).
2 6 Ligation fused the antisense sequence to the binary vector pBI121. The
ligation
2 7 contained pBI121 vector that had been digested with BamHI and SacI, along
with the SPL or
2 8 SPH phosphorylase DNA product, that had been cut from the pUC 19 subclone
with BamHI
2 9 and SacI. Ligated DNA was transformed into SCE E. coli DHSa cells, and the
transformed
3 0 cells were plated on LB plates containing ampicillin. The nucleotide
sequences of the
31 antisense DNA SPL and SPH are nucleotides 338 to 993 of SEQ ID NO: 1 and
nucleotides
26
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 147 to 799 of SEQ m NO: 3, respectively. Selection of pBIl2l with
phosphorlylase inserts
2 was done with CAMV and NOS specific primers.
3 Samples 1 and 2 representing the tuber L-type and tuber H-type phosphorylase
DNA
4 fragments were picked from a plate after overnight growth. These samples
were inoculated
into 5 ml of LB media and grown overnight at 37°C. Plasmids were
isolated by the rapid
6 alkaline extraction procedure, and the DNA was electroporated into
Agrobactericcrn
7 tccmefaciens.
8 Constructs were engineered into the pBI121 vector that contains the CaMV 35S
9 promoter (Kay et al. 1987) and the NOS 3' terminator {Bevan et al. 1983)
sequence. The
pBI121 plasmid is made up of the following well characterized segments of DNA.
A 0.93 kb
11 fragment isolated from transposon Tn7 which encodes bacterial
spectinomycin/streptomycin
12 (Spc/Str) resistance and is a determinant for selection in E. coli and
Agrobacterium
13 tumefaciens (Fling et al., 1985). This is joined to a chimeric kanamycin
resistance gene
14 engineered for plant expression to allow selection of the transformed
tissue. The chimeric
gene consists of the 0.35 kb cauliflower mosaic virus 355 promoter (P-35S)
(Odell et aL,
16 1985), the 0.83 kb neomycin phosphotransferase type II gene (NPTII), and
the 026 kb 3' non-
17 translated region of the nopaline synthase gene (N05 3') (Fraley et al.,
1983). The next
18 segment is a 0.75 kb origin of replication from the RK2 plasmid (ori-V)
(Stalker et al., 1981).
19 It is joined to a 3. I kb SaII to PvccI segment of pBR322 which provides
the origin of
2 0 replication for maintenance in E. coli (ori-322) and the bom site for the
conjugational transfer
21 in the Agrobactericcm tumefaciens cells. Next is a 0.36 kb PvccI fragment
from the pTiT37
22 plasmid which contains the nopaline-type T-DNA right border region (Fraley
e2 al., 1985).
23 The antisense sequence was engineered for expression in the tuber by
placing the gene under
2 4 the control of a constitutive tissue non-specific promoter.
2 6 D Plant Transformation/Regeneration
27 The SPL and SPH vectors were transformed into the Desiree potato cultivar
according
2 8 to de Block ( 1988). To transform "Desiree" potatoes, sterile shoot
cultures of "Desiree" were
2 9 maintained in test tubes containing 8 ml of S 1 (Murashige and Skoog (MS)
medium
3 0 supplemented with 2°!o sucrose and 0.5 g/1 MES pH 5.7, solidified
with 6 g/1 Phytagar). When
31 plantlets reached approximately 5 cm in length, leaf pieces were excised
with a single cut
27
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 along the base and inoculated with a 1:10 dilution of an overnight culture
of A~robacterium
2 tumefaciens. The stem explants were co-cultured for 2 days at 20°C on
S I medium (De
3 Block 1988). Following co-culture, the explants were transferred to S4
medium (MS medium
4 without sucrose, supplemented with 0.5 g/1 MES pH 5.7, 200 mg/1 glutamine,
0.5 g/1 PVP, 20
g/1 mannitol, 20 g/1 glucose, 40 mg/1 adenine, 1 mg/1 trans zeatin, 0.1 mg/1
NAA, I g/1
6 carbenicillin, SO mg/1 kanamycin, solidified with 6 g/1 phytagar) for 1 week
and then 2 weeks
7 to induce callus formation.
8 After 3 weeks, the explants were transferred to S6 medium (S4 without NAA
and with
9 half the concentration (500 mg/1) of carbenicillin). After another two
weeks, the explants
were transferred to S8 medium {S6 with only 250 mg/1 carbenicillin and 0.01
mg/1 gibberellic
11 acid, GA3) to promote shoot formation. Shoots began to develop
approximately 2 weeks
12 after transfer to S8 shoot induction medium. These shoots were excised and
transferred to
13 vials of S 1 medium for rooting. After about 6 weeks of multiplication on
the rooting
14 medium. the plants were transferred to soil and are gradually hardened off.
Desiree plants regenerated in culture were transplanted in 1 gallon pots and
were
16 grown to maturity under greenhouse conditions. Tubers were harvested and
allowed to
17 suberize at room temperature for two days. All tubers greater than 2 cm in
length were
18 collected and stored at 4°C under high humidity.
19
2 0 E Field Trials
21 Untransformed controls, plants expressing the SPL construct, and plants
expressing
22 the SPH construct were propagated in field trials in a single replicate
randomized design. All
2 3 plants were grown side by side in the same field and exposed to similar
pesticide. fertilizer,
24 and irrigation regimes. Tubers were harvested and stored at 10°C for
2 weeks before
randomly selecting a fraction of the tubers from each line to be placed in
storage at 4°C.
26
27 F Sugar Analysis
28 Tubers were stored at 4°C and were not allowed to recondition at
room temperature
2 9 prior to sugar analysis. An intact longitudinal slice ( 1 cm thick, width
variable and equal to
3 0 the outside dimensions of the tuber) was cut from the central portion of
each tuber, thus
31 representing all of the tuber's tissues. At each harvest, the central
slices from four tubers per
28
CA 02275885 2002-11-12
1 clone (3 replicates) were collectively diced into 1-cm cubes and 15 g was
randomly selected
2 from the pooled tissue for analysis. Glucan phosphorylase (see below) and
sugars were
3 extracted with 15 mL of Tris buffer (50 mM, pH 7.0) containing 2 mM sodium
bisulfate, 2
4 mM EDTA. 0.5 mM PMSF and 10% (w/w) glycerol with a polytron homogenizer at
4°C.
The extracts were centrifuged at 4°C (30,000 g, 30 min) and reducing
sugars (glucose and
6 fructose) were measured on a 10-fold dilution of the supernatant using a
Spectra PhysicsT""
'7 high performance liquid chromatograph interfaced to a refractive index
detector. The
8 separation was performed at 80°C on a 30 x 0.78 cm AminexT"" HPX 87C
column (Biorad)
9 using 0.6 ml/min water as the mobile phase. Calibration of the instrument
was via authentic
standards of d-glucose and d-fructose.
11
12 H Analysis of a-Glucan Phosphorylase Activity
13 Tubers stored at 4°C were not allowed to warm prior to extraction
and analysis of a
14 glucan phosphorylase activity and isozymes. The in vitro activity of glucan
phosphorylase in
the phosphorolytic direction was assayed as described by Steup ( 1990).
Briefly, samples of
16 extracts obtained for sugar analysis (see above) were added to a reaction
medium which
1'7 coupled starch phosphorolysis to the reduction of NADP through the
sequential actions of
18 phosphoglucomutase and glucose-6-phosphate dehydrogenase. The rate of
reduction of
19 NADP during the reaction is stoichiometric with the rate of production of
glucose-1-
2 0 phosphate from the starch substrate. Reduction of NADP was followed at 340
nm in a Varian
21 Cary double-beam spectrophotometer. Protein levels in extracts were
determined according
2 2 to Bradford ( 1976).
23 Glucan phosphorylase activity gels were run essentially according to Steup
(1990).
24 Proteins were separated on native polyacrylamide gels (8.5 %) containing
1.5 % glycogen.
Following electrophoresis at 80 V for 15 h (4°C), the gels were
incubated (1-2 h) at 37°C in
2 6 0.1 M citrate-NaOH buffer (pH 6.0) containing 20 mM glucose-1-P and 0.05%
(w/v)
2 7 hydrolyzed potato starch. Gels were then rinsed and stained with an iodine
solution.
2 8 For Western blot analysis, proteins were electrophoresed on glycogen-
containing
2 9 polyacrylamide gels as described above. The proteins were electroblotted
to nitrocellulose
3 0 and blots were probed with polyclonal antibodies raised against GHTP and
GLTP.
29
CA 02275885 1999-06-29
1 Immunoblots were developed with alkaline pho~phatawe con;ugaced anti-ra>;bic
secon,iary
2 antibodies (Sigma).
3
4 H Chip Color Determination
Five transgenic potato lines expressing the GLTP antisense sequence, two
trans~enic
6 lines expressing the GHTP antisense sequence, non-transgenic Desiree control
lines, and two
7 control lines transformed with the pBI l2 ( vector T-DNA, were grown under
field conditions
8 in Alberta, Canada. Tubers were harvested and stored at 10°C and
4°C. Chip color was
9 determined for all potato lines by taking center cuts from representative
samples from each
line and frying at 205°F in soybean oil for approximately 3 minutes
until bubbling stops.
11
12 I Results
13 All tubers were harvested from plants of the same cultivar (Desiree), the
same age,
14 and grown side by side under identical growth conditions. Northern analysis
of tubers
showed a considerable reduction of endogenous GLTP transcript in transgenic
plants
16 expressing the homologous antisense transcript (Figure 7). Glucan
phosphorylase assays
17 showed that activities (pmol NADPH mg~~ protein h'') were reduced (Table 1)
at harvest and
18 for at least six months following harvest in transgenic plants expressing
the GLTP antisense
19 DNA. The results tabulated in Table 1 show that a glucan phosphorylase
activity in tubers
2 0 stored at 4°C for 189 days was reduced from approximately 16% to
70% in various
21 transformed potato varieties relative to the wildtype control strain.
Activity gels and western
2 2 blot analysis showed specific reduced expression of homologous enzymes and
lower
2 3 reduction of expression for heterologous enzymes (Figures 10 and 11 ).
This specificity for
24 homologous products may result from differences between the phosphorylases
(Figures 6A
2 5 and 6B).
2 6 Analysis of tubers at harvest (0 days) shows that those expressing the
antisense GLTP
27 transcript have up to 5-fold less reducing sugars than control tubers
(Table 2). Furthermore,
2 8 after 91 days storage at 4°C, transformed tubers contained 28-39%
lower reducing sugar
2 9 concentrations than the wildtype control strain. Concentrations of glucose
and fructose were
3 0 reduced significantly in tubers expressing the antisense GLTP transcript
(Tables 3 and 4).
31 These results suggest that reduced GLTP activity slows the catabolism of
starch into reducing
AMENDED SHEET
CA 02275885 1999-06-29
1 sugars in tubers, while in the control tubers the s~_vgars continue to
accurnulute. The
2 correlation between total phosphorylase activity and the concentration of
reducing sugars is
3 not direct, suggesting that certain isozymes of phosphorylase may play a
more important role
4 in the catabolism of starch, that specific levels of reduced expression of
particular
phosphorylase isozymes may be more optimum than others, and/or that there may
be
6 unidentified interactions involved in the lower reducing sugar levels.
7 Transgenic potato plants expressing the antisense GLTP or GHTP transcript
have
8 been grown under field conditions and their tubers stored at 4°C.
Chip color, which
9 correlated with sugar content, was determined prior to cold storage and
after 86 and 12=1 days
of cold storage. The chip color of tubers from all transgenic plants
expressing the antisense
11 GLTP transcript was significantly improved (lighter) relative to that of
control tubers (darker)
12 stored under identical conditions (Table ~ and Figure 9). Chip scores of
tubers from
13 "Desiree" potato plants expressing the GLTP transcript were improved by at
least 4.3 points
14 and 8.9 points as determined with an Agtron model E-15-FP Direct Reading
Abridged
1S Spectrophotometer (Agtron Inc. 1095 Spice Island Drive #100, Sparks Nevada
89431)
16 following storage at 10°C and 4°C, respectively, for 86 days.
Chip scores of GLTP
17 transformants measured after 124 days of storage at 4°C were
improved by 4490 to 89%
18 relative to wildtype (Table 5).
19 The Desiree cultivar is not a commercially desirable potato for chipping
due to its
2 0 high natural sugar content and propensity to sweeten rapidly in cold
storage. Nevertheless,
21 significant improvements in fried chip color were noted with the
transformed "Desiree"
22 potatoes. It is expected that superior color lightening would be achieved
if the methods of the
2 3 invention were applied to commercial processing potato varieties.
24 Analysis of tubers stored at 10°C and 4°C shows that those
expressing the antisense
2 S GHTP transcript sometimes provided chips that fried lighter than control
tubers, indicating a
2 6 lower buildup of reducing sugars (Table 5). Results showing heterologous
and homologous
27 reduction in phosphorylase activity (Figures 10 and 11) indicate that the
improvement may be
2 8 a result of reducing one or both tuber phosphorylases. However, these
results suggest that the
2 9 L-type phosphorylase plays a more important role in the catabolism of
starch into reducing
3 0 sugars.
31
I~I~r;C:'Jlv.__' ~~Iwl
CA 02275885 1999-06-29
Further, the results show that the diffErence ~u redwing sugar levels (Table
'~) and
2 chip scores (Table 5) between tubers wildtype plants and those expressing
tuber
31a
. _.
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 phosphorylase antisense RNA, are sustained during long-term storage. As
shown in Table 5,
2 the chip scores are approximately the same at 86 days and 124 days. No
further increases in
3 reducing sugar concentrations were evident after 49 and 91 days storage at
4°C (Table 2).
4 This equilibrium in sugar concentration was probably associated with the
kinetics of the tuber
phosphorylases. The capability of maintaining lower sugar levels has the
potential of
6 extending the period of storage by at least several months. Presently,
processing potatoes are
7 usually stored for a maximum of three to six months at 10°C to
12°C before the suear
8 accumulation reaches levels that reduce quality. Fresh product must be
imported until the
9 present season potatoes become available. Extending the storage period of
potatoes by many
months may reduce import costs.
11 Table 6 provides a summary of the percentage improvement in various
improved tuber
12 cold-storage characteristics of tubers of potato plants transformed with
antisense DNA
13 derived from the GLTP gene sequence (ATL3 - ATL9), and from the GHTP gene
sequence
14 (ATHI and ATH2) relative to untransformed control plants. It is apparent
from the results
summarized in Table 6 that substantial improvements in tuber cold-storage
characteristics
16 may be consistently obtained through the methods of the present invention.
Particularly
17 noteworthy are the percentage chip score improvements over wildtype
observed after storage
18 at 4°C for approximately four months (124 days). Relative chip score
improvements of up to
19 89% relative to wildtype were observed. Improved chip scores reflect the
commercial utility
2 0 of the invention. That is> by reducing cold-induced sweetening, tubers can
be stored at cooler
21 temperatures, without causing unacceptable darkening of fried potato
products.
22 The reduction in sugar accumulation of transformed potato lines relative to
wildtype,
23 both at harvest and after 91 day storage, also demonstrates significant
advantages of the
24 invention. Reduced sugar accumulation relates to the observed chip score
improvements, and
also reflects improved specific gravity of tubers, another important
commercial measure of
2 6 tuber quality.
27 Even at harvest, substantial improvements in chip score and reduced sugar
2 8 accumulation were noted for transformed lines relative to wildtype. Thus,
the benefits of the
29 invention are not limited to improvements that arise only after extended
periods of cold
3 0 storage, but that are present at the time of harvest. In this sense, the
invention is not limited
31 only to improvements in cold-storage characteristics but encompasses
improvements in tuber
32
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 quality characteristics such as chip score or sugar accumulation which are
present at the time
2 of harvest, resulting in earlier maturity.
3 Turning to specific improvements summarized in Table 6, it can be seen that
GLTP-
4 type transformants {ATL3 - ATL9) exhibited up to a 66%, 70% and 69%
reduction in a
glucan phosphorylase activity relative to wildtype, at harvest, and after
storage for 91 and 189
6 days, respectively. Most also exhibited improvements in excess of 10% and
30% relative to
7 wildtype at harvest and after storage for 91 and 189 days. After storage for
91 and 189 days,
8 the GHTP-type transformants (ATH1 and ATH2) exhibited, respectively, up to
28% and 39%
9 relative improvement over wildtype and generally showed at least 10%
improvement.
The GLTP-type transformants exhibited up to 80% and 39% reduction of sugar
11 accumulation relative to wildtype at harvest and at 91 days, respectively.
At harvest, all
12 GLTP-type transformants exhibited at least IO% and at least 30% relative
improvement. At
13 91 days, all GLTP-type transformants exhibited at least 10% and most
exhibited at least 30%
14 relative improvement.
The GLTP-type transformants exhibited up to 46%, 89% and 89% chip score
16 improvement relative to wildtype at harvest, and after storage for 86 days
and 124 days,
17 respectively. Almost all exhibited at least 10% and 30% relative
improvement at harvest, and
18 after storage for 86 and 124 days. At Least one of the GHTP-type
transformants exhibited at
19 least 5% and at least 10% improvement relative to wildtype at harvest, and
after storage for
2 0 86 and I24 days. After 124 days storage, at least one of the GHTP-type
transformants
21 exhibited up to 25% relative improvement in chip score.
22 The results clearly demonstrate that substantial improvements in tuber cold-
storage
23 characteristics may be readily obtained through the methods of the
invention. Results will
24 vary due to, among other things, the location within~the plant genome where
the recombinant
2 5 antisense or sense DNA is inserted, and the number of insertion events
that occur. It is
2 6 important to note that despite the variability in the results amongst the
various transformed
27 lines, there was little variation in the results amongst the samples within
a single transformed
2 8 potato line (see footnotes to Tables I to 5). Results are presented in
Table 6 for all potato
2 9 plant lines which were successfully transformed with the GHTP or GLTP
antisense DNA.
3 0 Therefore, all transformants show at least some improvement in one or more
cold-storage
31 characteristics such as increased chip score (lighter color on cooking) and
reduced sugar
33
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 accumulation, and most show very substantial improvements. Given the large
proportion of
2 positive transformants observed in the examples herein, it is expected that,
using the cold-
3 storage characteristic testing procedures described in the examples, potato
plants transformed
4 through the methods of the invention can be readily screened to identify
transformed lines
exhibiting significantly improved cold-storage characteristics. By applying
the techniques
6 disclosed herein to commercially important potato varieties, it will be
possible to readily
7 create and select transformants having significantly improved cold-storage
characteristics.
8 Those transformants showing the greatest relative improvements over wildtype
controls can
9 be used in the development of new commercial potato varieties.
34
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 Table 1
2 _
3 Effects of enzyme
an antisense
transcript
on glucan
phosphorylase
activity measured
in
4 extracts from
field grown
"Desiree"
tubers.
6 Glucan Phosphorylase Activity
7 Storage Period at 4C (days)
8
9
Clone 0 49 91 140 189
11
12 pmol NADPH mg'' protein
h-'
13 Wt~ 10.50 11.83 9.94 11.90 13
04
14 ATL3 4.90 4.86 4.49 4.73 .
4
gg
ATL4 11.45 7.17 8.09 11.32 ,
10
99
16 ATLS 3.58 3.56 2.97 4.59 .
4
79
17 ATL9 3.59 3.88 3.84 4.72 .
3
9g
18 ,
19
2 LSDo_osb 1.97 2.94 1.59 2.34 2
0 58
21 LSDo.o, 2.87 4.28 2.31 3.41 .
3
75
22 .
2 Clone' 0.01
3
2 WT vs. ATL's 0.01
4
2 Days NS
5
2 Clone x Days 0.05
6
29
3 WT 1 I .49 8.90 12.66 13.66
0
31 ATH-1 10.40 9.69 10.79 10.10
32 ATH-2 6.46 6.40 6.56 g,3g
33
34
3 LSDo.osb 2.02 0.41 3.00 NS
5
3 LSDo.oi 4.78 0.95 NS NS
6
37
3 Clone' 0.01
8
3 WT vs. ATH's 0.01
9
4 Days 0.05
0
41 Clone x Days NS
42
43 aWT, wild type untransformed tubers. bLSD, least significant difference at
0.05 or 0.01 level
44 for each storage period. 'Sources of variation in factorial analysis.
dSignificance levels for
4 5 indicated sources of variation.
CA 02275885 1999-06-29
-WO 98/35051 PCT/CA98/00055
1 Table 2
2
3 Effects of
an antisense
GLTP transcript
on low temperature
induced sweetening
of field
4 grown "Desiree"tubers.
6 Reducing Sugars (glucose + fructose)
7 Storage Period at 4C (days)
8
9 Clone 0 49 91
11 mg g' fresh weight
12 Wta 5.63 31.8 28.0
13 ATL3 1.88 I7.3 17.3
14 ATL~ 1.11 14.3 20.1
ATLS 1.51 18.3 17.0
16 ATL9 I.36 17.3 I8.5
17
18
19 WT vs. ATL'sb 0.01 0.01 0.05
21 Clone' 0.01 d
2 Days 0.01
2
2 Clone x Days NS
3
24
2 6 aWT, wild type untransformed tubers. bOrthogonal comparisons for ANOVA's
at each
27 storage period. 'sources of variation in factorial analysis, dSignificance
levels for indicated
2 8 sources of variation.
36
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
Table 3
2 -
3 Effects of an
antisense GLTP
transcript on
Iow temperature
induced fructose
accumulation
of
4 field grown "Desiree"tubers.
6 Fructose
7 Storage Period at 4C
(days)
8
9 Clone 0 49 91
11 mg g' fresh weight
12 Wta 3.53 15.10 12.20
13 ATL3 1.21 8.40 8.79
14 ATL4 0.79 7.22 8.56
ATLS 0.61 10.00 8.09
16 ATL9 0.54 8.38 8.72
17
18
19 WT vs. ATL's b 0.01 0.01 NS
21
2 Clone' 0.01 d
2
2 Days 0.01
3
2 Clone x Days NS
4
2 6 aWT, wild type untransformed tubers. bOrthogonal comparisons for ANOVA's
at each
27 storage period. 'Sources of variation in factorial analysis. Significance
levels for indicated
2 8 sources of variation.
37
CA 02275885 1999-06-29
- WO 98/35051 PCT/CA98/00055
1 Table 4
2
'
3 Effects of an glucose accumulation
antisense GLTP of
transcript on
low temperature
induced
4 field grown "Desiree"tubers.
6 Glucose
7 Storage Period at 4C
(days)
8
9 Clone 0 49 91
11 mg g' ' fresh weight
12 Wt~ 2.10 16.60 15.90
13 ATL3 0.68 8.94 8.49
14 ATL4 0.3 2 7.07 11.06
ATLS 1.05 8.33 8.91
16 ATL9 0.83 8.87 9.78
17
18
19 WT vs. ATL's 0.01 0.01 0.05
21 Clone' 0.01 d
22 Days 0.01
2 Clone x Days NS
3
24
aWT, wild type untransformed tubers. °Orthogonal comparisons for
ANOVA's at each
2 6 storage period. 'sources of variation in factorial analysis. dSignificance
levels for indicated
27 sources of variation.
38
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 Table S
2 _
3 Average chip color of field growntubers. The chip color rating
"Desiree" was assigned using
4 an Agtron meter similar to that
used by industry to measure
color of fried potatoes. In
this
index, the higher the number hip product but color does not
the lighter the c represent a linear
6 relationship to the index.
7
8 Storage Temperature , Period, and Agtron Readinga
9
Harvest 86 days 86 days 124 days
11
12 lOC 4C 4C
13
14
Wt~ 26 25.3 1 S.4 17
1
16 .
17 ATL3' 2S 37.4 26.7 30.8
18 ATL4 3S 43.7 29.1 32
3
19 ATLS 36 29.6 .
24.7 24
6
2 ATL9 38 38.7 .
0 24.3 26
6
21 .
2 ATH l d 26 49.7 17.5 21
2 4
2 ATH2 29 31.2 .
3 15.6 15
9
24 .
GMP1 31 15.7 15
7
2 GMP2 35 .
6 16.7 16
6
27 .
28
aAgtron Inc. 1095 Spice Island Drive #100, Sparks Nevada 89431. Agtron model E-
15-FP (Direct Reading Abridged Spectrophotometer). Measures ratio of
reflectance in two
spectral modes, near infrared and green. Results represent the measurement of
6 to 8 chips
from 3 randomly selected tubers approximately 3 to 4 cm in diameter.
bWT, negative control, wild type untransformed tubers.
'ATL, tubers transformed with the tuber L-type« glucan phosphorylase.
BATH, tubers transformed with the tuber H-type« glucan phosphorylase.
eGMP, negative control, tubers transformed with pBI121 T-DNA.
39
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 Table 6
2 Summary of Results -
3
4
Sample % Reduction % Reduction % Chip
of of Score
a
giucan Sugar Improvement
phosphorylase relative
to
activity Accumulation wildtype
relative
to
wildtype relative
to
wildtype
at 91 at 91 at 86
189 124
harvest harvest harvest
days days days
days days
6 ATL 3 53 55 63 67 38 -4 73 80
7 ATL 4 -9 19 16 80 28 35 89 89
8 ATL 5 66 70 63 73 39 38 60 44
9 ATL 9 66 61 69 76 34 46 58 56
ATH 1 n/a -9 26 n/a n/a 0 14 25
11 ATH 2 n/a 28 39 n/a n/a 12 1 -7
12
13
CA 02275885 1999-06-29
WO 98/35051 PCT/CA98/00055
1 REFERENCES
2 Alber and Kawasaki ( I 982) Mol. And Appl. Genet. 1:419-434.
3 ap Rees et al. (1988) Symp. Soc. Exp. Biol. 42:377-393.
4 Bevan et al. (/983) Nature (London) 304:184-187.
Bevan et al. ( 1986) Nucleic Acids Res. 14 ( 11 ):4625-4638.
6 Birnboim et al. (1979) Nucleic Acids Res. 7:1513-1523.
7 Blennow et al. ( 1991 ) Phytochemistry 30:437-444.
8 Bradford, M.M. 1976. A rapid and sensitive method for quantification of
microgram
9 quantities of protein utilizing the principle of protein dye binding. Anal.
Biochem. 72:
243-254.
11 Brisson et al. ( 1989) The Plant Cell 1:559-566.
12 Brusslan and Tobin ( 1995) Plant Molecular Biology 27:809-813.
13 Burton, W.G. ( 1989) The Potato. Longman Scientific and Technical,
14 Cannon et al. (1990) Plant Molecular Biology 15:39-47
Claassen et al. (1991) Plant Physiol. 95:1243-1249.
16 Coffin et al. (1987) J. Food Sci. 52:639-645.
17 Davies and Viola (1992) Postharvest News and Information 3:97-100.
18 De Block, M. (1988) Theoretical and Applied Genetics 76:767-774.
19 De Carvalho et al. (1992) EMBO J. 11:2595-2602.
2 0 Depicker et al. ( 1982) Mol. And Appl. Genet. 1:561-573.
21 Dixon et al. ( 1981 ) Phytochemistry 20:969-972.
2 2 Dorlhac et al. 1994 Mol. Gen. Genetic. 243:613-621.
23 Ebbelaar et al. (1993) Int. Symp. on Gen. Manip. of Plant Metabolism and
Growth, 29-31
2 4 March, Norwich UK Abstract #9.
Ecker and Davis (1986) Proc. Nati. Acad. Sci. 83: 5373-5376.
2 6 FIing et al. (1985) Nucleic Acids Research 13 no. 19, 7095-7106.
2 7 Fraley, et al. ( 1983) Proc Natl Acad Sci USA 80, 4803-4807.
2 8 Fraley et al. ( 1985) Bio/Technology 3, 629-635.
29 Fray and Grierson 1993 Plant Mol. Biol. 22:589-602.
3 0 Gielen et al. ( 1984) EMBO J. 3:835-846.
31 Hasseloff, J. And W.L. Gerlach ( 1988) Nature 334:585-591.
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1 Hart et al. ( 1992) Mol. Gen. Genetic. 235:179-i 88.
2 Jorgensen. R.A. ( 1995) Science 268: 686-691.
3 Kawchuk et al. ( 1990) Molecular Piant-Microbe Interactions 3:301-307.
4 Kawchuk et al. ( 1991 ) Mol. Plant Microbe-Inter. 4:247-253.
Kay et al. (1987) Science 236:1299-1302.
6 Kruger, N.J. and Hammond, J.B.W. (1988) Plant Physiol. 86:645-648.
7 Laemmli, U.K. (1970) Nature (London) 227:680-685.
8 Lin et al. (1988) Plant Physiol. 86:1131-1135.
9 Loiselle et al. ( 1990) American Potato Journal 67:633-646.
Lynch et al. ( 1992) Can,. 3. Plant Sci. 72: 535-543.
11 Matzke and Matzke ( 1995) Plant Physiol. 107:679.
12 Meyer and Saedler (1996) Annu. Rev. Plant Physiol. 47:23-48.
13 Mori et al. {1991) J. Biol. Chem. 266:18446-18453.
14 Muller, et al. (1990) Mol. Gen. Genet. 224:136-146.
Nakano et al. (1989) J. Biochem. 106:691-695.
16 Nakano, K. and Fukui, T. ( 1986) J. Biol. Chem. 266:8230-8256.
17 Napoli et al. ( 1990) Plant Cell 2:279-289.
18 Odell, et al. ( 1985) Nature 313, 810-812.
19 Ohta et al. ( 1991 ) Mol. Gen. Genet. 225:369-378.
Ortiz, R. and Huaman, Z. (1994) In:Potato Genetics. Bradshaw, J.E. and Mackay
G.R. (eds.)
21 Sambrook et al. ( 1989) Molecular Cloning, A Laboratory Manual. 2nd Ed.
Cold Spring
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23 Seymour et al. 1993 Plant Mol. Biol. 23:1-9.
24 Shahar et al. (1992) Plant Cell 4:135-147.
Shallenberger et al. (1959) Agric. and Food Chem. 7:274-277.
2 6 Smith et al. ( 1988) Nature 334:724-726.
2 7 Smith et al. ( 1990) Mol. Gen. Genet. 244:447-481.
2 8 Sonnewald et al. ( 1995) Plant Molecular Biology 27:567-576.
29 Sowokinos, J. (1990) In: The molecular and Cellular Biology of the Potato.
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31 Stalker et al. (1981) Mol Gen Genet 181, 8-12.
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CA 02275885 2002-11-12
1 Steup, M. (1990) Starch Degrading Enzymes in "Methods in Plant Biochemistry"
Vol 3.
2 P.M. Dey and J.B. Harborne, eds. Academic Press, London
3 Stiekema et al. (1988) Plant Mol. Biol. 11:255-269.
4 Stukerlj et al. (1990) Nucl. Acids Res. 18:46050.
S Takaha et al., (1993) J. Biol. Chem. 26 8:1391-1396.
6 Thuring et al. (1975) Analytical Biochemistry 66:213-220.
Van der Krol et al (1988) Gene 72:45-50.
8 Van der Krol (1990) Plant Cell 2:291-299.
9 Weaver et al. (1978) Am. Pot. J. 55:83-93.
Weintraub (1990) Scientific American 1:34-40.
11 Winnacker, Ernst L. (1987) From Genes to Clones. VCH Verlagsgesellschaft
mbH, Federal
12 Replublic of Germany
13 Yoshida et al. (1992) Geneg 10:255-259.
14
All publications mentioned in this specification are indicative of the level
of skill in
16 the art to which this invention pertains. Although the foregoing invention
has been described
17 in some detail by way of illustration and example for purposes of clarity
of understanding, it
18 will be obvious that certain changes and modifications may be practised
within the scope of
19 the appended claims.
43
CA 02275885 1999-08-31
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Her Majesty the Queen in Right of Canada as Represented
by the Department of Agriculture and Agri-Food Canada
(ii) TITLE OF INVENTION: Potatoes Having Improved Quality
Characteristics and Methods for Their Production
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: McKay-Carey & Company
(B) STREET: 2125 Commerce Place, 10155-102nd Street
(C) CITY: Edmonton
(D) STATE: Alberta
(E) COUNTRY: Canada
(F) ZIP: T5J 4G8
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE: 10-FEB-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/036,946
(B) FILING DATE: 10-FEB-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/868,786
(B) FILING DATE: 04-JUN-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McKay-Carey, Mary Jane
(B) REGISTRATION NUMBER: 3790
(C) REFERENCE/DOCKET NUMBER: 24002W00
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (403) 424-0222
(B) TELEFAX: (403) 421-0834
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3101 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
44
CA 02275885 1999-08-31
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 44..2944
(D) OTHER INFORMATION: /product= "potato alpha-glucan
L-type tuber phosphorylase"
(ix) FEATURE:
(A) NAME/KEY: mat-peptide
(B) LOCATION: 194..2941
(ix) FEATURE:
(A) NAME/KEY: sig-peptide
(B) LOCATION: 44..193
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
ATCACTCTCA TTCGAAAAGC TAGATTTGCA TAGAGAGCAC AAA ATG GCG ACT GCA 55
Met Ala Thr Ala
-50
AAT GGA GCA CAC TTG TTC AAC CAT TAC AGC TCC AAT TCC AGA TTC ATC 103
Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn Ser Arg Phe Ile
-45 -40 -35
CAT TTC ACT TCT AGA AAC ACA AGC TCC AAA TTG TTC CTT ACC AAA ACC 151
His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe Leu Thr Lys Thr
-30 -25 -20 -15
TCC CAT TTT CGG AGA CCC AAA CGC TGT TTC CAT GTC AAC AAT ACC TTG 199
Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val Asn Asn Thr Leu
-10 -5 1
AGT GAG AAA ATT CAC CAT CCC ATT ACT GAA CAA GGT GGT GAG AGC GAC 247
Ser Glu Lys Ile His His Pro Ile Thr Glu Gln Gly Gly Glu Ser Asp
10 15
CTG AGT TCT TTT GCT CCT GAT GCC GCA TCT ATT ACC TCA AGT ATC AAA 295
Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser Ile Thr Ser Ser Ile Lys
20 25 30
TAC CAT GCA GAA TTC ACA CCT GTA TTC TCT CCT GAA AGG TTT GAG CTC 343
Tyr His Ala Glu Phe Thr Pro Val Phe Ser Pro Glu Arg Phe Glu Leu
35 40 45 50
CA 02275885 1999-08-31
CCT AAG GCA TTC TTT GCA ACA GCT CAA AGT GTT CGT GAT TCG CTC CTT 391
Pro Lys Ala Phe Phe Ala Thr Ala Gln Ser Val Arg Asp Ser Leu Leu
55 60 65
ATT AAT TGG AAT GCT ACG TAT GAT ATT TAT GAA AAG CTG AAC ATG AAG 439
Ile Asn Trp Asn Ala Thr Tyr Asp Ile Tyr Glu Lys Leu Asn Met Lys
70 75 80
CAA GCG TAC TAT CTA TCC ATG GAA TTT CTG CAG GGT AGA GCA TTG TTA 487
Gln Ala Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly Arg Ala Leu Leu
85 90 95
AAT GCA ATT GGT AAT CTG GAG CTT ACT GGT GCA TTT GCG GAA GCT TTG 535
Asn Ala Ile Gly Asn Leu Glu Leu Thr Gly Ala Phe Ala Glu Ala Leu
100 105 110
AAA AAC CTT GGC CAC AAT CTA GAA AAT GTG GCT TCT CAG GAA CCA GAT 583
Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser Gln Glu Pro Asp
115 120 125 130
GCT GCT CTT GGA AAT GGG GGT TTG GGA CGG CTT GCT TCC TGT TTT CTG 631
Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu
135 140 145
GAC TCT TTG GCA ACA CTA AAC TAC CCA GCA TGG GGC TAT GGA CTT AGG 679
Asp Ser Leu Ala Thr Leu Asn Tyr Pro Ala Trp Gly Tyr Gly Leu Arg
150 155 160
TAC AAG TAT GGT TTA TTT AAG CAA CGG ATT ACA AAA GAT GGT CAG GAG 727
Tyr Lys Tyr Gly Leu Phe Lys Gln Arg Ile Thr Lys Asp Gly Gln Glu
165 170 175
GAG GTG GCT GAA GAT TGG CTT GAA ATT GGC AGT CCA TGG GAA GTT GTG 775
Glu Val Ala Glu Asp Trp Leu Glu Ile Gly Ser Pro Trp Glu Val Val
180 185 190
AGG AAT GAT GTT TCA TAT CCT ATC AAA TTC TAT GGA AAA GTC TCT ACA 823
Arg Asn Asp Val Ser Tyr Pro Ile Lys Phe Tyr Gly Lys Val Ser Thr
195 200 205 210
GGA TCA GAT GGA AAG AGG TAT TGG ATT GGT GGA GAG GAT ATA AAG GCA 871
Gly Ser Asp Gly Lys Arg Tyr Trp Ile Gly Gly Glu Asp Ile Lys Ala
215 220 225
GTT GCG TAT GAT GTT CCC ATA CCA GGG TAT AAG ACC AGA ACC ACA ATC 919
Val Ala Tyr Asp Val Pro Ile Pro Gly Tyr Lys Thr Arg Thr Thr Ile
230 235 240
AGC CTT CGA CTG TGG TCT ACA CAG GTT CCA TCA GCG GAT TTT GAT TTA 967
Ser Leu Arg Leu Trp Ser Thr Gln Val Pro Ser Ala Asp Phe Asp Leu
245 250 255
TCT GCT TTC AAT GCT GGA GAG CAC ACC AAA GCA TGT GAA GCC CAA GCA 1015
Ser Ala Phe Asn Ala Gly Glu His Thr Lys Ala Cys Glu Ala Gln Ala
260 265 270
46
CA 02275885 1999-08-31
AAC GCT GAG AAG ATA TGT TAC ATA CTC TAC CCT GGG GAT GAA TCA GAG 1063
Asn Ala Glu Lys Ile Cys Tyr Ile Leu Tyr Pro Gly Asp Glu Ser Glu
275 280 285 290
GAG GGA AAG ATC CTT CGG TTG AAG CAA CAA TAT ACC TTA TGC TCG GCT 1111
Glu Gly Lys Ile Leu Arg Leu Lys Gln Gln Tyr Thr Leu Cys Ser Ala
295 300 305
TCT CTC CAA GAT ATT ATT TCT CGA TTT GAG AGG AGA TCA GGT GAT CGT 1159
Ser Leu Gln Asp Ile Ile Ser Arg Phe Glu Arg Arg Ser Gly Asp Arg
310 315 320
ATT AAG TGG GAA GAG TTT CCT GAA AAA GTT GCT GTG CAG ATG AAT GAC 1207
Ile Lys Trp Glu Glu Phe Pro Glu Lys Val Ala Val Gln Met Asn Asp
325 330 335
ACT CAC CCT ACA CTT TGT ATC CCT GAG CTG ATG AGA ATA TTG ATA GAT 1255
Thr His Pro Thr Leu Cys Ile Pro Glu Leu Met Arg Ile Leu Ile Asp
340 345 350
CTG AAG GGC TTG AAT TGG AAT GAA GCT TGG AAT ATT ACT CAA AGA ACT 1303
Leu Lys Gly Leu Asn Trp Asn Glu Ala Trp Asn Ile Thr Gln Arg Thr
355 360 365 370
GTG GCC TAC ACA AAC CAT ACT GTT TTG CCT GAG GCA CTG GAG AAA TGG 1351
Val Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu Lys Trp
375 380 385
AGT TAT GAA TTG ATG CAG AAA CTC CTT CCC AGA CAT GTC GAA ATC ATT 1399
Ser Tyr Glu Leu Met Gln Lys Leu Leu Pro Arg His Val Glu Ile Ile
390 395 400
GAG GCG ATT GAC GAG GAG CTG GTA CAT GAA ATT GTA TTA AAA TAT GGT 1447
Glu Ala Ile Asp Glu Glu Leu Val His Glu Ile Val Leu Lys Tyr Gly
405 410 415
TCA ATG GAT CTG AAC AAA TTG GAG GAA AAG TTG ACT ACA ATG AGA ATC 1495
Ser Met Asp Leu Asn Lys Leu Glu Glu Lys Leu Thr Thr Met Arg Ile
420 425 430
TTA GAA AAT TTT GAT CTT CCC AGT TCT GTT GCT GAA TTA TTT ATT AAG 1543
Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu Leu Phe Ile Lys
435 440 445 450
CCT GAA ATC TCA GTT GAT GAT GAT ACT GAA ACA GTA GAA GTC CAT GAC 1591
Pro Glu Ile Ser Val Asp Asp Asp Thr Glu Thr Val Glu Val His Asp
455 460 465
AAA GTT GAA GCT TCC GAT AAA GTT GTG ACT AAT GAT GAA GAT GAC ACT 1639
Lys Val Glu Ala Ser Asp Lys Val Val Thr Asn Asp Glu Asp Asp Thr
470 475 480
GGT AAG AAA ACT AGT GTG AAG ATA GAA GCA GCT GCA GAA AAA GAC ATT 1687
Gly Lys Lys Thr Ser Val Lys Ile Glu Ala Ala Ala Glu Lys Asp Ile
485 490 495
47
CA 02275885 1999-08-31
GAC AAG AAA ACT CCC GTG AGT CCG GAA CCA GCT GTT ATA CCA CCT AAG 1735
Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val Ile Pro Pro Lys
500 505 510
AAG GTA CGC ATG GCC AAC TTG TGT GTT GTG GGC GGC CAT GCT GTT AAT 1783
Lys Val Arg Met Ala Asn Leu Cys Val Val Gly Gly His Ala Val Asn
515 520 525 530
GGA GTT GCT GAG ATC CAT AGT GAA ATT GTG AAG GAG GAG GTT TTC AAT 1831
Gly Val Ala Glu Ile His Ser Glu Ile Val Lys Glu Glu Val Phe Asn
535 540 545
GAC TTC TAT GAG CTC TGG CCG GAA AAG TTC CAA AAC AAA ACA AAT GGA 1879
Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gln Asn Lys Thr Asn Gly
550 555 560
GTG ACT CCA AGA AGA TGG ATT CGT TTC TGC AAT CCT CCT CTT AGT GCC 1927
Val Thr Pro Arg Arg Trp Ile Arg Phe Cys Asn Pro Pro Leu Ser Ala
565 570 575
ATC ATA ACT AAG TGG ACT GGT ACA GAG GAT TGG GTC CTG AAA ACT GAA 1975
Ile Ile Thr Lys Trp Thr Gly Thr Glu Asp Trp Val Leu Lys Thr Glu
580 585 590
AAG TTG GCA GAA TTG CAG AAG TTT GCT GAT AAT GAA GAT CTT CAA AAT 2023
Lys Leu Ala Glu Leu Gln Lys Phe Ala Asp Asn Glu Asp Leu Gln Asn
595 600 605 610
GAG TGG AGG GAA GCA AAA AGG AGC AAC AAG ATT AAA GTT GTC TCC TTT 2071
Glu Trp Arg Glu Ala Lys Arg Ser Asn Lys Ile Lys Val Val Ser Phe
615 620 625
CTC AAA GAA AAG ACA GGG TAT TCT GTT GTC CCA GAT GCA ATG TTT GAT 2119
Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp Ala Met Phe Asp
630 635 640
ATT CAG GTA AAA CGC ATT CAT GAG TAC AAG CGA CAA CTG TTA AAT ATC 2167
Ile Gln Val Lys Arg Ile His Glu Tyr Lys Arg Gln Leu Leu Asn Ile
645 650 655
TTC GGC ATC GTT TAT CGG TAT AAG AAG ATG AAA GAA ATG ACA GCT GCA 2215
Phe Gly Ile Val Tyr Arg Tyr Lys Lys Met Lys Glu Met Thr Ala Ala
660 665 670
GAA AGA AAG ACT AAC TTC GTT CCT CGA GTA TGC ATA TTT GGG GGA AAA 2263
Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys Ile Phe Gly Gly Lys
675 680 685 690
GCT TTT GCC ACA TAT GTG CAA GCC AAG AGG ATT GTA AAA TTT ATC ACA 2311
Ala Phe Ala Thr Tyr Val Gln Ala Lys Arg Ile Val Lys Phe Ile Thr
695 700 705
GAT GTT GGT GCT ACT ATA AAT CAT GAT CCA GAA ATC GGT GAT CTG TTG 2359
Asp Val Gly Ala Thr Ile Asn His Asp Pro Glu Ile Gly Asp Leu Leu
710 715 720
48
CA 02275885 1999-08-31
AAG GTA GTC TTT GTG CCA GAT TAC AAT GTC AGT GTT GCT GAA TTG CTA 2407
Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val Ala Glu Leu Leu
725 730 735
ATT CCT GCT AGC GAT CTA TCA GAA CAT ATC AGT ACG GCT GGA ATG GAG 2455
Ile Pro Ala Ser Asp Leu Ser Glu His Ile Ser Thr Ala Gly Met Glu
740 745 750
GCC AGT GGA ACC AGT AAT ATG AAG TTT GCA ATG AAT GGT TGT ATC CAA 2503
Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Met Asn Gly Cys Ile Gln
755 760 765 770
ATT GGT ACA TTG GAT GGC GCT AAT GTT GAA ATA AGG GAA GAG GTT GGA 2551
Ile Gly Thr Leu Asp Gly Ala Asn Val Glu Ile Arg Glu Glu Val Gly
775 780 785
GAA GAA AAC TTC TTT CTC TTT GGT GCT CAA GCT CAT GAA ATT GCA GGG 2599
Glu Glu Asn Phe Phe Leu Phe Gly Ala Gln Ala His Glu Ile Ala Gly
790 795 800
CTT AGA AAA GAA AGA GCT GAC GGA AAG TTT GTA CCT GAT GAA CGT TTT 2647
Leu Arg Lys Glu Arg Ala Asp Gly Lys Phe Val Pro Asp Glu Arg Phe
805 810 815
GAA GAG GTG AAG GAA TTT GTT AGA AGC GGT GCT TTT GGC TCT TAT AAC 2695
Glu Glu Val Lys Glu Phe Val Arg Ser Gly Ala Phe Gly Ser Tyr Asn
820 825 830
TAT GAT GAC CTA ATT GGA TCG TTG GAA GGA AAT GAA GGT TTT GGC CGT 2743
Tyr Asp Asp Leu Ile Gly Ser Leu Glu Gly Asn Glu Gly Phe Gly Arg
835 840 845 850
GCT GAC TAT TTC CTT GTG GGC AAG GAC TTC CCC AGT TAC ATA GAA TGC 2791
Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser Tyr Ile Glu Cys
855 860 865
CAA GAG AAA GTT GAT GAG GCA TAT CGC GAC CAG AAA AGG TGG ACA ACG 2839
Gln Glu Lys Val Asp Glu Ala Tyr Arg Asp Gln Lys Arg Trp Thr Thr
870 875 880
ATG TCA ATC TTG AAT ACA GCG GGA TCG TAC AAG TTC AGC AGT GAC AGA 2887
Met Ser Ile Leu Asn Thr Ala Gly Ser Tyr Lys Phe Ser Ser Asp Arg
885 890 895
ACA ATC CAT GAA TAT GCC AAA GAC ATT TGG AAC ATT GAA GCT GTG GAA 2935
Thr Ile His Glu Tyr Ala Lys Asp Ile Trp Asn Ile Glu Ala Val Glu
900 905 910
ATA GCA TAA GAGGGGGAAG TGAATGAAAA ATAACAAAGG CACAGTAAGT 2984
Ile Ala
915
AGTTTCTCTT TTTATCATGT GATGAAGGTA TATAATGTAT GTGTAAGAGG ATGATGTTAT 3044
TACCACATAA TAAGAGATGA AGAGTCTCAT TTTGCTTCAA AAAAAAA 3101
49
CA 02275885 1999-08-31
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 966 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Thr Ala Asn Gly Ala His Leu Phe Asn His Tyr Ser Ser Asn
-50 -45 -40 -35
Ser Arg Phe Ile His Phe Thr Ser Arg Asn Thr Ser Ser Lys Leu Phe
-30 -25 -20
Leu Thr Lys Thr Ser His Phe Arg Arg Pro Lys Arg Cys Phe His Val
-15 -10 -5
Asn Asn Thr Leu Ser Glu Lys Ile His His Pro Ile Thr Glu Gln Gly
1 5 10
Gly Glu Ser Asp Leu Ser Ser Phe Ala Pro Asp Ala Ala Ser Ile Thr
15 20 25 30
Ser Ser Ile Lys Tyr His Ala Glu Phe Thr Pro Val Phe Ser Pro Glu
35 40 45
Arg Phe Glu Leu Pro Lys Ala Phe Phe Ala Thr Ala Gln Ser Val Arg
50 55 60
Asp Ser Leu Leu Ile Asn Trp Asn Ala Thr Tyr Asp Ile Tyr Glu Lys
65 70 75
Leu Asn Met Lys Gln Ala Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly
80 85 90
Arg Ala Leu Leu Asn Ala Ile Gly Asn Leu Glu Leu Thr Gly Ala Phe
95 100 105 110
Ala Glu Ala Leu Lys Asn Leu Gly His Asn Leu Glu Asn Val Ala Ser
115 120 125
Gln Glu Pro Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala
130 135 140
Ser Cys Phe Leu Asp Ser Leu Ala Thr Leu Asn Tyr Pro Ala Trp Gly
145 150 155
Tyr Gly Leu Arg Tyr Lys Tyr Gly Leu Phe Lys Gln Arg Ile Thr Lys
160 165 170
Asp Gly Gln Glu Glu Val Ala Glu Asp Trp Leu Glu Ile Gly Ser Pro
175 180 185 190
CA 02275885 1999-08-31
Trp Glu Val Val Arg Asn Asp Val Ser Tyr Pro Ile Lys Phe Tyr Gly
195 200 205
Lys Val Ser Thr Gly Ser Asp Gly Lys Arg Tyr Trp Ile Gly Gly Glu
210 215 220
Asp Ile Lys Ala Val Ala Tyr Asp Val Pro Ile Pro Gly Tyr Lys Thr
225 230 235
Arg Thr Thr Ile Ser Leu Arg Leu Trp Ser Thr Gln Val Pro Ser Ala
240 245 250
Asp Phe Asp Leu Ser Ala Phe Asn Ala Gly Glu His Thr Lys Ala Cys
255 260 265 270
Glu Ala Gln Ala Asn Ala Glu Lys Ile Cys Tyr Ile Leu Tyr Pro Gly
275 280 285
Asp Glu Ser Glu Glu Gly Lys Ile Leu Arg Leu Lys Gln Gln Tyr Thr
290 295 300
Leu Cys Ser Ala Ser Leu Gln Asp Ile Ile Ser Arg Phe Glu Arg Arg
305 310 315
Ser Gly Asp Arg Ile Lys Trp Glu Glu Phe Pro Glu Lys Val Ala Val
320 325 330
Gln Met Asn Asp Thr His Pro Thr Leu Cys Ile Pro Glu Leu Met Arg
335 340 345 350
Ile Leu Ile Asp Leu Lys Gly Leu Asn Trp Asn Glu Ala Trp Asn Ile
355 360 365
Thr Gln Arg Thr Val Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala
370 375 380
Leu Glu Lys Trp Ser Tyr Glu Leu Met Gln Lys Leu Leu Pro Arg His
385 390 395
Val Glu Ile Ile Glu Ala Ile Asp Glu Glu Leu Val His Glu Ile Val
400 405 410
Leu Lys Tyr Gly Ser Met Asp Leu Asn Lys Leu Glu Glu Lys Leu Thr
415 420 425 430
Thr Met Arg Ile Leu Glu Asn Phe Asp Leu Pro Ser Ser Val Ala Glu
435 440 445
Leu Phe Ile Lys Pro Glu Ile Ser Val Asp Asp Asp Thr Glu Thr Val
450 455 460
Glu Val His Asp Lys Val Glu Ala Ser Asp Lys Val Val Thr Asn Asp
465 470 475
Glu Asp Asp Thr Gly Lys Lys Thr Ser Val Lys Ile Glu Ala Ala Ala
480 485 490
51
CA 02275885 1999-08-31
Glu Lys Asp Ile Asp Lys Lys Thr Pro Val Ser Pro Glu Pro Ala Val
495 500 505 510
Ile Pro Pro Lys Lys Val Arg Met Ala Asn Leu Cys Val Val Gly Gly
515 520 525
His Ala Val Asn Gly Val Ala Glu Ile His Ser Glu Ile Val Lys Glu
530 535 540
Glu Val Phe Asn Asp Phe Tyr Glu Leu Trp Pro Glu Lys Phe Gln Asn
545 550 555
Lys Thr Asn Gly Val Thr Pro Arg Arg Trp Ile Arg Phe Cys Asn Pro
560 565 570
Pro Leu Ser Ala Ile Ile Thr Lys Trp Thr Gly Thr Glu Asp Trp Val
575 580 585 590
Leu Lys Thr Glu Lys Leu Ala Glu Leu Gln Lys Phe Ala Asp Asn Glu
595 600 605
Asp Leu Gln Asn Glu Trp Arg Glu Ala Lys Arg Ser Asn Lys Ile Lys
610 615 620
Val Val Ser Phe Leu Lys Glu Lys Thr Gly Tyr Ser Val Val Pro Asp
625 630 635
Ala Met Phe Asp Ile Gln Val Lys Arg Ile His Glu Tyr Lys Arg Gln
640 645 650
Leu Leu Asn Ile Phe Gly Ile Val Tyr Arg Tyr Lys Lys Met Lys Glu
655 660 665 670
Met Thr Ala Ala Glu Arg Lys Thr Asn Phe Val Pro Arg Val Cys Ile
675 680 685
Phe Gly Gly Lys Ala Phe Ala Thr Tyr Val Gln Ala Lys Arg Ile Val
690 695 700
Lys Phe Ile Thr Asp Val Gly Ala Thr Ile Asn His Asp Pro Glu Ile
705 710 715
Gly Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val
720 725 730
Ala Glu Leu Leu Ile Pro Ala Ser Asp Leu Ser Glu His Ile Ser Thr
735 740 745 750
Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Met Asn
755 760 765
Gly Cys Ile Gln Ile Gly Thr Leu Asp Gly Ala Asn Val Glu Ile Arg
770 775 780
Glu Glu Val Gly Glu Glu Asn Phe Phe Leu Phe Gly Ala Gln Ala His
785 790 795
52
CA 02275885 1999-08-31
Glu Ile Ala Gly Leu Arg Lys Glu Arg Ala Asp Gly Lys Phe Val Pro
800 805 810
Asp Glu Arg Phe Glu Glu Val Lys Glu Phe Val Arg Ser Gly Ala Phe
815 820 825 830
Gly Ser Tyr Asn Tyr Asp Asp Leu Ile Gly Ser Leu Glu Gly Asn Glu
835 840 845
Gly Phe Gly Arg Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Ser
850 855 860
Tyr Ile Glu Cys Gln Glu Lys Val Asp Glu Ala Tyr Arg Asp Gln Lys
865 870 875
Arg Trp Thr Thr Met Ser Ile Leu Asn Thr Ala Gly Ser Tyr Lys Phe
880 885 890
Ser Ser Asp Arg Thr Ile His Glu Tyr Ala Lys Asp Ile Trp Asn Ile
895 900 905 910
Glu Ala Val Glu Ile Ala
915
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2655 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 12..2528
(D) OTHER INFORMATION: /product= "potato alpha-glucan
H-type tuber phosphorylase"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 12..2525
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GTTTATTTTC C ATG GAA GGT GGT GCA AAA TCG AAT GAT GTA TCA GCA GCA 50
Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala
1 5 10
53
CA 02275885 1999-08-31
CCT ATT GCT CAA CCA CTT TCT GAA GAC CCT ACT GAC ATT GCA TCT AAT 98
Pro Ile Ala Gln Pro Leu Ser Glu Asp Pro Thr Asp Ile Ala Ser Asn
15 20 25
ATC AAG TAT CAT GCT CAA TAT ACT CCT CAT TTT TCT CCT TTC AAG TTT 146
Ile Lys Tyr His Ala Gln Tyr Thr Pro His Phe Ser Pro Phe Lys Phe
30 35 40 45
GAG CCA CTA CAA GCA TAC TAT GCT GCT ACT GCT GAC AGT GTT CGT GAT 194
Glu Pro Leu Gln Ala Tyr Tyr Ala Ala Thr Ala Asp Ser Val Arg Asp
50 55 60
CGC TTG ATC AAA CAA TGG AAT GAC ACC TAT CTT CAT TAT GAC AAA GTT 242
Arg Leu Ile Lys Gln Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val
65 70 75
AAT CCA AAG CAA ACA TAC TAC TTA TCA ATG GAG TAT CTC CAG GGG CGA 290
Asn Pro Lys Gln Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gln Gly Arg
80 85 90
GCT TTG ACA AAT GCA GTT GGA AAC TTA GAC ATC CAC AAT GCA TAT GCT 338
Ala Leu Thr Asn Ala Val Gly Asn Leu Asp Ile His Asn Ala Tyr Ala
95 100 105
GAT GCT TTA AAC AAA CTG GGT CAG CAG CTT GAG GAG GTC GTT GAG CAG 386
Asp Ala Leu Asn Lys Leu Gly Gln Gln Leu Glu Glu Val Val Glu Gln
110 115 120 125
GAA AAA GAT GCA GCA TTA GGA AAT GGT GGT TTA GGA AGG CTC GCT TCA 434
Glu Lys Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser
130 135 140
TGC TTT CTT GAT TCC ATG GCC ACA TTG AAC CTT CCA GCA TGG GGT TAT 482
Cys Phe Leu Asp Ser Met Ala Thr Leu Asn Leu Pro Ala Trp Gly Tyr
145 150 155
GGC TTG AGG TAC AGA TAT GGA CTT TTT AAG CAG CTT ATC ACA AAG GCT 530
Gly Leu Arg Tyr Arg Tyr Gly Leu Phe Lys Gln Leu Ile Thr Lys Ala
160 165 170
GGG CAA GAA GAA GTT CCT GAA GAT TGG TTG GAG AAA TTT AGT CCC TGG 578
Gly Gln Glu Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp
175 180 185
GAA ATT GTA AGG CAT GAT GTT GTC TTT CCT ATC AGG TTT TTT GGT CAT 626
Glu Ile Val Arg His Asp Val Val Phe Pro Ile Arg Phe Phe Gly His
190 195 200 205
GTT GAA GTC CTC CCT TCT GGC TCG CGA AAA TGG GTT GGT GGA GAG GTC 674
Val Glu Val Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Glu Val
210 215 220
CTA CAG GCT CTT GCA TAT GAT GTG CCA ATT CCA GGA TAC AGA ACT AAA 722
Leu Gln Ala Leu Ala Tyr Asp Val Pro Ile Pro Gly Tyr Arg Thr Lys
225 230 235
54
CA 02275885 1999-08-31
AAC ACT AAT AGT CTT CGT CTC TGG GAA GCC AAA GCA AGC TCT GAG GAT 770
Asn Thr Asn Ser Leu Arg Leu Trp Glu Ala Lys Ala Ser Ser Glu Asp
240 245 250
TTC AAC TTG TTT CTG TTT AAT GAT GGA CAG TAT GAT GCT GCT GCA CAG 818
Phe Asn Leu Phe Leu Phe Asn Asp Gly Gln Tyr Asp Ala Ala Ala Gln
255 260 265
CTT CAT TCT AGG GCT CAG CAG ATT TGT GCT GTT CTC TAC CCT GGG GAT 866
Leu His Ser Arg Ala Gln Gln Ile Cys Ala Val Leu Tyr Pro Gly Asp
270 275 280 285
GCT ACA GAG AAT GGA AAA CTC TTA CGG CTA AAG CAA CAA TTT TTT CTG 914
Ala Thr Glu Asn Gly Lys Leu Leu Arg Leu Lys Gln Gln Phe Phe Leu
290 295 300
TGC AGT GCA TCG CTT CAG GAT ATT ATT GCC AGA TTC AAA GAG AGA GAA 962
Cys Ser Ala Ser Leu Gln Asp Ile Ile Ala Arg Phe Lys Glu Arg Glu
305 310 315
GAT GGA AAG GGT TCT CAC CAG TGG TCT GAA TTC CCC AAG AAG GTT GCG 1010
Asp Gly Lys Gly Ser His Gln Trp Ser Glu Phe Pro Lys Lys Val Ala
320 325 330
ATA CAA CTA AAT GAC ACA CAT CCA ACT CTT ACG ATT CCA GAG CTG ATG 1058
Ile Gln Leu Asn Asp Thr His Pro Thr Leu Thr Ile Pro Glu Leu Met
335 340 345
CGG TTG CTA ATG GAT GAT GAA GGA CTT GGG TGG GAT GAA TCT TGG AAT 1106
Arg Leu Leu Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn
350 355 360 365
ATC ACT ACT AGG ACA ATT GCC TAT ACG AAT CAT ACA GTC CTA CCT GAA 1154
Ile Thr Thr Arg Thr Ile Ala Tyr Thr Asn His Thr Val Leu Pro Glu
370 375 380
GCA CTT GAA AAA TGG TCT CAG GCA GTC ATG TGG AAG CTC CTT CCT AGA 1202
Ala Leu Glu Lys Trp Ser Gln Ala Val Met Trp Lys Leu Leu Pro Arg
385 390 395
CAT ATG GAA ATC ATT GAA GAA ATT GAC AAA CGG TTT GTT GCT ACA ATA 1250
His Met Glu Ile Ile Glu Glu Ile Asp Lys Arg Phe Val Ala Thr Ile
400 405 410
ATG TCA GAA AGA CCT GAT CTT GAG AAT AAG ATG CCT AGC ATG CGC ATT 1298
Met Ser Glu Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg Ile
415 420 425
TTG GAT CAC AAC GCC ACA AAA CCT GTT GTG CAT ATG GCT AAC TTG TGT 1346
Leu Asp His Asn Ala Thr Lys Pro Val Val His Met Ala Asn Leu Cys
430 435 440 445
GTT GTC TCT TCA CAT ACG GTA AAT GGT GTT GCC CAG CTG CAT AGT GAC 1394
Val Val Ser Ser His Thr Val Asn Gly Val Ala Gln Leu His Ser Asp
450 455 460
CA 02275885 1999-08-31
ATC CTG AAG GCT GAG TTA TTT GCT GAT TAT GTC TCT GTA TGG CCC ACC 1442
Ile Leu Lys Ala Glu Leu Phe Ala Asp Tyr Val Ser Val Trp Pro Thr
465 470 475
AAG TTC CAG AAT AAG ACC AAT GGT ATA ACT CCT CGT AGG TGG ATC CGA 1490
Lys Phe Gln Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg Trp Ile Arg
480 485 490
TTT TGT AGT CCT GAG CTG AGT CAT ATA ATT ACC AAG TGG TTA AAA ACA 1538
Phe Cys Ser Pro Glu Leu Ser His Ile Ile Thr Lys Trp Leu Lys Thr
495 500 505
GAT CAA TGG GTG ACG AAC CTC GAA CTG CTT GCT AAT CTT CGG GAG TTT 1586
Asp Gln Trp Val Thr Asn Leu Glu Leu Leu Ala Asn Leu Arg Glu Phe
510 515 520 525
GCT GAT AAT TCG GAG CTC CAT GCT GAA TGG GAA TCA GCC AAG ATG GCC 1634
Ala Asp Asn Ser Glu Leu His Ala Glu Trp Glu Ser Ala Lys Met Ala
530 535 540
AAC AAG CAG CGT TTG GCA CAG TAT ATA CTG CAT GTG ACA GGT GTG AGC 1682
Asn Lys Gln Arg Leu Ala Gln Tyr Ile Leu His Val Thr Gly Val Ser
545 550 555
ATC GAT CCA AAT TCC CTT TTT GAC ATA CAA GTC AAA CGT ATC CAT GAA 1730
Ile Asp Pro Asn Ser Leu Phe Asp Ile Gln Val Lys Arg Ile His Glu
560 565 570
TAC AAA AGG CAG CTT CTA AAT ATT CTG GGC GTC ATC TAT AGA TAC AAG 1778
Tyr Lys Arg Gln Leu Leu Asn Ile Leu Gly Val Ile Tyr Arg Tyr Lys
575 580 585
AAG CTT AAG GGA ATG AGC CCT GAA GAA AGG AAA AAT ACA ACT CCT CGC 1826
Lys Leu Lys Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg
590 595 600 605
ACA GTC ATG ATT GGA GGA AAA GCA TTT GCA ACA TAC ACA AAT GCA AAA 1874
Thr Val Met Ile Gly Gly Lys Ala Phe Ala Thr Tyr Thr Asn Ala Lys
610 615 620
CGA ATT GTC AAG CTC GTG ACT GAT GTT GGC GAC GTT GTC AAT AGT GAC 1922
Arg Ile Val Lys Leu Val Thr Asp Val Gly Asp Val Val Asn Ser Asp
625 630 635
CCT GAC GTC AAT GAC TAT TTG AAG GTG GTT TTT GTT CCC AAC TAC AAT 1970
Pro Asp Val Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn
640 645 650
GTA TCT GTG GCA GAG ATG CTT ATT CCG GGA AGT GAG CTA TCA CAA CAC 2018
Val Ser Val Ala Glu Met Leu Ile Pro Gly Ser Glu Leu Ser Gln His
655 660 665
ATC AGT ACT GCA GGC ATG GAA GCA AGT GGA ACA AGC AAC ATG AAA TTT 2066
Ile Ser Thr Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe
670 675 680 685
56
CA 02275885 1999-08-31
GCC CTT AAT GGA TGC CTT ATC ATT GGG ACA CTA GAT GGG GCC AAT GTG 2114
Ala Leu Asn Gly Cys Leu Ile Ile Gly Thr Leu Asp Gly Ala Asn Val
690 695 700
GAA ATT AGG GAG GAA ATT GGA GAA GAT AAC TTC TTT CTT TTT GGT GCA 2162
Glu Ile Arg Glu Glu Ile Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala
705 710 715
ACA GCT GAT GAA GTT CCT CAA CTG CGC AAA GAT CGA GAG AAT GGA CTG 2210
Thr Ala Asp Glu Val Pro Gln Leu Arg Lys Asp Arg Glu Asn Gly Leu
720 725 730
TTC AAA CCT GAT CCT CGG TTT GAA GAG GCA AAA CAA TTT ATT AGG TCT 2258
Phe Lys Pro Asp Pro Arg Phe Glu Glu Ala Lys Gln Phe Ile Arg Ser
735 740 745
GGA GCA TTT GGG ACG TAT GAT TAT AAT CCC CTC CTT GAA TCA CTG GAA 2306
Gly Ala Phe Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu
750 755 760 765
GGG AAC TCG GGA TAT GGT CGT GGA GAC TAT TTT CTT GTT GGT CAT GAT 2354
Gly Asn Ser Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp
770 775 780
TTT CCG AGC TAC ATG GAT GCT CAG GCA AGG GTT GAT GAA GCT TAC AAG 2402
Phe Pro Ser Tyr Met Asp Ala Gln Ala Arg Val Asp Glu Ala Tyr Lys
785 790 795
GAC AGG AAA AGA TGG ATA AAG ATG TCT ATA CTG AGC ACT AGT GGG AGT 2450
Asp Arg Lys Arg Trp Ile Lys Met Ser Ile Leu Ser Thr Ser Gly Ser
800 805 810
GGC AAA TTT AGT AGT GAC CGT ACA ATT TCT CAA TAT GCA AAA GAG ATC 2498
Gly Lys Phe Ser Ser Asp Arg Thr Ile Ser Gln Tyr Ala Lys Glu Ile
815 820 825
TGG AAC ATT GCC GAG TGT CGC GTG CCT TGA GCACACTTCT GAACCTGGTA 2548
Trp Asn Ile Ala Glu Cys Arg Val Pro
830 835
TCTAATAAGG ATCTAATGTT CATTGTTTAC TAGCATATGA ATAATGTAAG TTCAAGCACA 2608
ACATGCTTTC TTATTTCCTA CTGCTCTCAA GAAGCAGTTA TTTGTTG 2655
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 838 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
57
CA 02275885 1999-08-31
Met Glu Gly Gly Ala Lys Ser Asn Asp Val Ser Ala Ala Pro Ile Ala
1 5 10 15
Gln Pro Leu Ser Glu Asp Pro Thr Asp Ile Ala Ser Asn Ile Lys Tyr
20 25 30
His Ala Gln Tyr Thr Pro His Phe Ser Pro Phe Lys Phe Glu Pro Leu
35 40 45
Gln Ala Tyr Tyr Ala Ala Thr Ala Asp Ser Val Arg Asp Arg Leu Ile
50 55 60
Lys Gln Trp Asn Asp Thr Tyr Leu His Tyr Asp Lys Val Asn Pro Lys
65 70 75 80
Gln Thr Tyr Tyr Leu Ser Met Glu Tyr Leu Gln Gly Arg Ala Leu Thr
85 90 95
Asn Ala Val Gly Asn Leu Asp Ile His Asn Ala Tyr Ala Asp Ala Leu
100 105 110
Asn Lys Leu Gly Gln Gln Leu Glu Glu Val Val Glu Gln Glu Lys Asp
115 120 125
Ala Ala Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu
130 135 140
Asp Ser Met Ala Thr Leu Asn Leu Pro Ala Trp Gly Tyr Gly Leu Arg
145 150 155 160
Tyr Arg Tyr Gly Leu Phe Lys Gln Leu Ile Thr Lys Ala Gly Gln Glu
165 170 175
Glu Val Pro Glu Asp Trp Leu Glu Lys Phe Ser Pro Trp Glu Ile Val
180 185 190
Arg His Asp Val Val Phe Pro Ile Arg Phe Phe Gly His Val Glu Val
195 200 205
Leu Pro Ser Gly Ser Arg Lys Trp Val Gly Gly Glu Val Leu Gln Ala
210 215 220
Leu Ala Tyr Asp Val Pro Ile Pro Gly Tyr Arg Thr Lys Asn Thr Asn
225 230 235 240
Ser Leu Arg Leu Trp Glu Ala Lys Ala Ser Ser Glu Asp Phe Asn Leu
245 250 255
Phe Leu Phe Asn Asp Gly Gln Tyr Asp Ala Ala Ala Gln Leu His Ser
260 265 270
Arg Ala Gln Gln Ile Cys Ala Val Leu Tyr Pro Gly Asp Ala Thr Glu
275 280 285
Asn Gly Lys Leu Leu Arg Leu Lys Gln Gln Phe Phe Leu Cys Ser Ala
290 295 300
58
CA 02275885 1999-08-31
Ser Leu Gln Asp Ile Ile Ala Arg Phe Lys Glu Arg Glu Asp Gly Lys
305 310 315 320
Gly Ser His Gln Trp Ser Glu Phe Pro Lys Lys Val Ala Ile Gln Leu
325 330 335
Asn Asp Thr His Pro Thr Leu Thr Ile Pro Glu Leu Met Arg Leu Leu
340 345 350
Met Asp Asp Glu Gly Leu Gly Trp Asp Glu Ser Trp Asn Ile Thr Thr
355 360 365
Arg Thr Ile Ala Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu
370 375 380
Lys Trp Ser Gln Ala Val Met Trp Lys Leu Leu Pro Arg His Met Glu
385 390 395 400
Ile Ile Glu Glu Ile Asp Lys Arg Phe Val Ala Thr Ile Met Ser Glu
405 410 415
Arg Pro Asp Leu Glu Asn Lys Met Pro Ser Met Arg Ile Leu Asp His
420 425 430
Asn Ala Thr Lys Pro Val Val His Met Ala Asn Leu Cys Val Val Ser
435 440 445
Ser His Thr Val Asn Gly Val Ala Gln Leu His Ser Asp Ile Leu Lys
450 455 460
Ala Glu Leu Phe Ala Asp Tyr Val Ser Val Trp Pro Thr Lys Phe Gln
465 470 475 480
Asn Lys Thr Asn Gly Ile Thr Pro Arg Arg Trp Ile Arg Phe Cys Ser
485 490 495
Pro Glu Leu Ser His Ile Ile Thr Lys Trp Leu Lys Thr Asp Gln Trp
500 505 510
Val Thr Asn Leu Glu Leu Leu Ala Asn Leu Arg Glu Phe Ala Asp Asn
515 520 525
Ser Glu Leu His Ala Glu Trp Glu Ser Ala Lys Met Ala Asn Lys Gln
530 535 540
Arg Leu Ala Gln Tyr Ile Leu His Val Thr Gly Val Ser Ile Asp Pro
545 550 555 560
Asn Ser Leu Phe Asp Ile Gln Val Lys Arg Ile His Glu Tyr Lys Arg
565 570 575
Gln Leu Leu Asn Ile Leu Gly Val Ile Tyr Arg Tyr Lys Lys Leu Lys
580 585 590
Gly Met Ser Pro Glu Glu Arg Lys Asn Thr Thr Pro Arg Thr Val Met
595 600 605
59
CA 02275885 1999-08-31
Ile Gly Gly Lys Ala Phe Ala Thr Tyr Thr Asn Ala Lys Arg Ile Val
610 615 620
Lys Leu Val Thr Asp Val Gly Asp Val Val Asn Ser Asp Pro Asp Val
625 630 635 640
Asn Asp Tyr Leu Lys Val Val Phe Val Pro Asn Tyr Asn Val Ser Val
645 650 655
Ala Glu Met Leu Ile Pro Gly Ser Glu Leu Ser Gln His Ile Ser Thr
660 665 670
Ala Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ala Leu Asn
675 680 685
Gly Cys Leu Ile Ile Gly Thr Leu Asp Gly Ala Asn Val Glu Ile Arg
690 695 700
Glu Glu Ile Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala Thr Ala Asp
705 710 715 720
Glu Val Pro Gln Leu Arg Lys Asp Arg Glu Asn Gly Leu Phe Lys Pro
725 730 735
Asp Pro Arg Phe Glu Glu Ala Lys Gln Phe Ile Arg Ser Gly Ala Phe
740 745 750
Gly Thr Tyr Asp Tyr Asn Pro Leu Leu Glu Ser Leu Glu Gly Asn Ser
755 760 765
Gly Tyr Gly Arg Gly Asp Tyr Phe Leu Val Gly His Asp Phe Pro Ser
770 775 780
Tyr Met Asp Ala Gln Ala Arg Val Asp Glu Ala Tyr Lys Asp Arg Lys
785 790 795 800
Arg Trp Ile Lys Met Ser Ile Leu Ser Thr Ser Gly Ser Gly Lys Phe
805 810 815
Ser Ser Asp Arg Thr Ile Ser Gln Tyr Ala Lys Glu Ile Trp Asn Ile
820 825 830
Ala Glu Cys Arg Val Pro
835
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3171 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
CA 02275885 1999-08-31
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 87..3011
(D) OTHER INFORMATION: /product= "potato alpha-glucan
L-type leaf phosphorylase"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 330..3008
(ix) FEATURE:
(A) NAME/KEY: sig~eptide
(B) LOCATION: 87..329
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TTTTTTTTTT CAACATGCAC AACAATTATT TTGATTAAAT TTTGTATCTA AAAATTTAGC 60
ATTTTGAAAT TCAGTTCAGA GACATC ATG GCA ACT TTT GCT GTC TCT GGA TTG 113
Met Ala Thr Phe Ala Val Ser Gly Leu
-81 -80 -75
AAC TCA ATT TCA AGT ATT TCT AGT TTT AAT AAC AAT TTC AGA AGC AAA 161
Asn Ser Ile Ser Ser Ile Ser Ser Phe Asn Asn Asn Phe Arg Ser Lys
-70 -65 -60
AAC TCA AAC ATT TTG TTG AGT AGA AGG AGG ATT TTA TTG TTC AGT TTT 209
Asn Ser Asn Ile Leu Leu Ser Arg Arg Arg Ile Leu Leu Phe Ser Phe
-55 -50 -45
AGA AGA AGA AGA AGA AGT TTC TCT GTT AGC AGT GTT GCT AGT GAT CAA 257
Arg Arg Arg Arg Arg Ser Phe Ser Val Ser Ser Val Ala Ser Asp Gln
-40 -35 -30 -25
AAGCAGAAGACA AAGGAT TCTTCC TCTGATGAA GGATTT ACATTA GAT 305
LysGlnLysThr LysAsp SerSer SerAspGlu GlyPhe ThrLeu Asp
-20 -15 -10
GTTTTTCAGCCG GACTCC ACGTCT GTTTTATCA AGTATA AAGTAT CAC 353
ValPheGlnPro AspSer ThrSer ValLeuSer SerIle LysTyr His
-5 1 5
GCTGAGTTCACA CCATCA TTTTCT CCTGAGAAG TTTGAA CTTCCC AAG 401
AlaGluPheThr ProSer PheSer ProGluLys PheGlu LeuPro Lys
15 20
GCATACTATGCA ACTGCA GAGAGT GTTCGAGAT ACGCTC ATTATA AAT 449
AlaTyrTyrAla ThrAla GluSer ValArgAsp ThrLeu IleIle Asn
25 30 35 40
61
CA 02275885 1999-08-31
TGG AAT GCC ACA TAC GAA TTC TAT GAA AAG ATG AAT GTA AAG CAG GCA 497
Trp Asn Ala Thr Tyr Glu Phe Tyr Glu Lys Met Asn Val Lys Gln Ala
45 50 55
TAT TAC TTG TCT ATG GAA TTT CTT CAG GGA AGA GCT TTA CTC AAT GCT 545
Tyr Tyr Leu Ser Met Glu Phe Leu Gln Gly Arg Ala Leu Leu Asn Ala
60 65 70
ATT GGT AAC TTG GGG CTA ACC GGA CCT TAT GCA GAT GCT TTA ACT AAG 593
Ile Gly Asn Leu Gly Leu Thr Gly Pro Tyr Ala Asp Ala Leu Thr Lys
75 80 85
CTC GGA TAC AGT TTA GAG GAT GTA GCC AGG CAG GAA CCG GAT GCA GCT 641
Leu Gly Tyr Ser Leu Glu Asp Val Ala Arg Gln Glu Pro Asp Ala Ala
90 95 100
TTA GGT AAT GGA GGT TTA GGA AGA CTT GCT TCT TGC TTT CTG GAC TCA 689
Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ser Cys Phe Leu Asp Ser
105 110 115 120
ATG GCG ACA CTA AAC TAC CCT GCA TGG GGC TAT GGA CTT AGA TAC CAA 737
Met Ala Thr Leu Asn Tyr Pro Ala Trp Gly Tyr Gly Leu Arg Tyr Gln
125 130 135
TAT GGC CTT TTC AAA CAG CTT ATT ACA AAA GAT GGA CAG GAG GAA GTT 785
Tyr Gly Leu Phe Lys Gln Leu Ile Thr Lys Asp Gly Gln Glu Glu Val
140 145 150
GCT GAA AAT TGG CTC GAG ATG GGA AAT CCA TGG GAA ATT GTG AGG AAT 833
Ala Glu Asn Trp Leu Glu Met Gly Asn Pro Trp Glu Ile Val Arg Asn
155 160 165
GAT ATT TCG TAT CCC GTA AAA TTC TAT GGG AAG GTC ATT GAA GGA GCT 881
Asp Ile Ser Tyr Pro Val Lys Phe Tyr Gly Lys Val Ile Glu Gly Ala
170 175 180
GAT GGG AGG AAG GAA TGG GCT GGC GGA GAA GAT ATA ACT GCT GTT GCC 929
Asp Gly Arg Lys Glu Trp Ala Gly Gly Glu Asp Ile Thr Ala Val Ala
185 190 195 200
TAT GAT GTC CCA ATA CCA GGA TAT AAA ACA AAA ACA ACG ATT AAC CTT 977
Tyr Asp Val Pro Ile Pro Gly Tyr Lys Thr Lys Thr Thr Ile Asn Leu
205 210 215
CGA TTG TGG ACA ACA AAG CTA GCT GCA GAA GCT TTT GAT TTA TAT GCT 1025
Arg Leu Trp Thr Thr Lys Leu Ala Ala Glu Ala Phe Asp Leu Tyr Ala
220 225 230
TTT AAC AAT GGA GAC CAT GCC AAA GCA TAT GAG GCC CAG AAA AAG GCT 1073
Phe Asn Asn Gly Asp His Ala Lys Ala Tyr Glu Ala Gln Lys Lys Ala
235 240 245
GAA AAG ATT TGC TAT GTC TTA TAT CCA GGT GAC GAA TCG CTT GAA GGA 1121
Glu Lys Ile Cys Tyr Val Leu Tyr Pro Gly Asp Glu Ser Leu Glu Gly
250 255 260
62
CA 02275885 1999-08-31
AAG ACG CTT AGG TTA AAG CAG CAA TAC ACA CTA TGT TCT GCT TCT CTT 1169
Lys Thr Leu Arg Leu Lys Gln Gln Tyr Thr Leu Cys Ser Ala Ser Leu
265 270 275 280
CAG GAC ATT ATT GCA CGG TTC GAG AAG AGA TCA GGG AAT GCA GTA AAC 1217
Gln Asp Ile Ile Ala Arg Phe Glu Lys Arg Ser Gly Asn Ala Val Asn
285 290 295
TGG GAT CAG TTC CCC GAA AAG GTT GCA GTA CAG ATG AAT GAC ACT CAT 1265
Trp Asp Gln Phe Pro Glu Lys Val Ala Val Gln Met Asn Asp Thr His
300 305 310
CCA ACA CTT TGT ATA CCA GAA CTT TTA AGG ATA TTG ATG GAT GTT AAA 1313
Pro Thr Leu Cys Ile Pro Glu Leu Leu Arg Ile Leu Met Asp Val Lys
315 320 325
GGT TTG AGC TGG AAG CAG GCA TGG GAA ATT ACT CAA AGA ACG GTC GCA 1361
Gly Leu Ser Trp Lys Gln Ala Trp Glu Ile Thr Gln Arg Thr Val Ala
330 335 340
TAC ACT AAC CAC ACT GTT CTA CCT GAG GCT CTT GAG AAA TGG AGC TTC 1409
Tyr Thr Asn His Thr Val Leu Pro Glu Ala Leu Glu Lys Trp Ser Phe
345 350 355 360
ACA CTT CTT GGT GAA CTG CTT CCT CGG CAC GTG GAG ATC ATA GCA ATG 1457
Thr Leu Leu Gly Glu Leu Leu Pro Arg His Val Glu Ile Ile Ala Met
365 370 375
ATA GAT GAG GAG CTC TTG CAT ACT ATA CTT GCT GAA TAT GGT ACT GAA 1505
Ile Asp Glu Glu Leu Leu His Thr Ile Leu Ala Glu Tyr Gly Thr Glu
380 385 390
GAT CTT GAC TTG TTG CAA GAA AAG CTA AAC CAA ATG AGG ATT CTG GAT 1553
Asp Leu Asp Leu Leu Gln Glu Lys Leu Asn Gln Met Arg Ile Leu Asp
395 400 405
AAT GTT GAA ATA CCA AGT TCT GTT TTG GAG TTG CTT ATA AAA GCC GAA 1601
Asn Val Glu Ile Pro Ser Ser Val Leu Glu Leu Leu Ile Lys Ala Glu
410 415 420
GAA AGT GCT GCT GAT GTC GAA AAG GCA GCA GAT GAA GAA CAA GAA GAA 1649
Glu Ser Ala Ala Asp Val Glu Lys Ala Ala Asp Glu Glu Gln Glu Glu
425 430 435 440
GAA GGT AAG GAT GAC AGT AAA GAT GAG GAA ACT GAG GCT GTA AAG GCA 1697
Glu Gly Lys Asp Asp Ser Lys Asp Glu Glu Thr Glu Ala Val Lys Ala
445 450 455
GAA ACT ACG AAC GAA GAG GAG GAA ACT GAG GTT AAG AAG GTT GAG GTG 1745
Glu Thr Thr Asn Glu Glu Glu Glu Thr Glu Val Lys Lys Val Glu Val
460 465 470
GAG GAT AGT CAA GCA AAA ATA AAA CGT ATA TTC GGG CCA CAT CCA AAT 1793
Glu Asp Ser Gln Ala Lys Ile Lys Arg Ile Phe Gly Pro His Pro Asn
475 480 485
63
CA 02275885 1999-08-31
AAA CCA CAG GTG GTT CAC ATG GCA AAT CTA TGT GTA GTT AGC GGG CAT 1841
Lys Pro Gln Val Val His Met Ala Asn Leu Cys Val Val Ser Gly His
490 495 500
GCA GTT AAC GGT GTT GCT GAG ATT CAT AGT GAA ATA GTT AAG GAT GAA 1889
Ala Val Asn Gly Val Ala Glu Ile His Ser Glu Ile Val Lys Asp Glu
505 510 515 520
GTT TTC AAT GAA TTT TAC AAG TTA TGG CCA GAG AAA TTC CAA AAC AAG 1937
Val Phe Asn Glu Phe Tyr Lys Leu Trp Pro Glu Lys Phe Gln Asn Lys
525 530 535
ACA AAT GGT GTG ACA CCA AGA AGA TGG CTA AGT TTC TGT AAT CCA GAG 1985
Thr Asn Gly Val Thr Pro Arg Arg Trp Leu Ser Phe Cys Asn Pro Glu
540 545 550
TTG AGT GAA ATT ATA ACC AAG TGG ACA GGA TCT GAT GAT TGG TTA GTA 2033
Leu Ser Glu Ile Ile Thr Lys Trp Thr Gly Ser Asp Asp Trp Leu Val
555 560 565
AAC ACT GAA AAA TTG GCA GAG CTT CGA AAG TTT GCT GAT AAC GAA GAA 2081
Asn Thr Glu Lys Leu Ala Glu Leu Arg Lys Phe Ala Asp Asn Glu Glu
570 575 580
CTC CAG TCT GAG TGG AGG AAG GCA AAA GGA AAT AAC AAA ATG AAG ATT 2129
Leu Gln Ser Glu Trp Arg Lys Ala Lys Gly Asn Asn Lys Met Lys Ile
585 590 595 600
GTC TCT CTC ATT AAA GAA AAA ACA GGA TAC GTG GTC AGT CCC GAT GCA 2177
Val Ser Leu Ile Lys Glu Lys Thr Gly Tyr Val Val Ser Pro Asp Ala
605 610 615
ATG TTT GAT GTT CAG ATC AAG CGC ATC CAT GAG TAT AAA AGG CAG CTA 2225
Met Phe Asp Val Gln Ile Lys Arg Ile His Glu Tyr Lys Arg Gln Leu
620 625 630
TTA AAT ATA TTT GGA ATC GTT TAT CGC TAT AAG AAG ATG AAA GAA ATG 2273
Leu Asn Ile Phe Gly Ile Val Tyr Arg Tyr Lys Lys Met Lys Glu Met
635 640 645
AGC CCT GAA GAA CGA AAA GAA AAG TTT GTC CCT CGA GTT TGC ATA TTT 2321
Ser Pro Glu Glu Arg Lys Glu Lys Phe Val Pro Arg Val Cys Ile Phe
650 655 660
GGA GGA AAA GCA TTT GCT ACA TAT GTT CAG GCC AAG AGA ATT GTA AAA 2369
Gly Gly Lys Ala Phe Ala Thr Tyr Val Gln Ala Lys Arg Ile Val Lys
665 670 675 680
TTT ATC ACT GAT GTA GGG GAA ACA GTC AAC CAT GAT CCC GAG ATT GGT 2417
Phe Ile Thr Asp Val Gly Glu Thr Val Asn His Asp Pro Glu Ile Gly
685 690 695
GAT CTT TTG AAG GTT GTA TTT GTT CCT GAT TAC AAT GTC AGT GTA GCA 2465
Asp Leu Leu Lys Val Val Phe Val Pro Asp Tyr Asn Val Ser Val Ala
700 705 710
64
CA 02275885 1999-08-31
GAA GTG CTA ATT CCT GGT AGT GAG TTG TCC CAG CAT ATT AGT ACT GCT 2513
Glu Val Leu Ile Pro Gly Ser Glu Leu Ser Gln His Ile Ser Thr Ala
715 720 725
GGT ATG GAG GCT AGT GGA ACC AGC AAC ATG AAA TTT TCA ATG AAT GGC 2561
Gly Met Glu Ala Ser Gly Thr Ser Asn Met Lys Phe Ser Met Asn Gly
730 735 740
TGC CTC CTC ATC GGG ACA TTA GAT GGT GCC AAT GTT GAG ATA AGA GAG 2609
Cys Leu Leu Ile Gly Thr Leu Asp Gly Ala Asn Val Glu Ile Arg Glu
745 750 755 760
GAA GTT GGA GAG GAC AAT TTC TTT CTT TTC GGA GCT CAG GCT CAT GAA 2657
Glu Val Gly Glu Asp Asn Phe Phe Leu Phe Gly Ala Gln Ala His Glu
765 770 775
ATT GCT GGC CTA CGA AAG GAA AGA GCC GAG GGA AAG TTT GTC CCG GAC 2705
Ile Ala Gly Leu Arg Lys Glu Arg Ala Glu Gly Lys Phe Val Pro Asp
780 785 790
CCA AGA TTT GAA GAA GTA AAG GCG TTC ATT AGG ACA GGC GTC TTT GGC 2753
Pro Arg Phe Glu Glu Val Lys Ala Phe Ile Arg Thr Gly Val Phe Gly
795 800 805
ACC TAC AAC TAT GAA GAA CTC ATG GGA TCC TTG GAA GGA AAC GAA GGC 2801
Thr Tyr Asn Tyr Glu Glu Leu Met Gly Ser Leu Glu Gly Asn Glu Gly
810 815 820
TAT GGT CGT GCT GAC TAT TTT CTT GTA GGA AAG GAT TTC CCC GAT TAT 2849
Tyr Gly Arg Ala Asp Tyr Phe Leu Val Gly Lys Asp Phe Pro Asp Tyr
825 830 835 840
ATA GAG TGC CAA GAT AAA GTT GAT GAA GCA TAT CGA GAC CAG AAG AAA 2897
Ile Glu Cys Gln Asp Lys Val Asp Glu Ala Tyr Arg Asp Gln Lys Lys
845 850 855
TGG ACC AAA ATG TCG ATC TTA AAC ACA GCT GGA TCG TTC AAA TTT AGC 2945
Trp Thr Lys Met Ser Ile Leu Asn Thr Ala Gly Ser Phe Lys Phe Ser
860 865 870
AGT GAT CGA ACA ATT CAT CAA TAT GCA AGA GAT ATA TGG AGA ATT GAA 2993
Ser Asp Arg Thr Ile His Gln Tyr Ala Arg Asp Ile Trp Arg Ile Glu
875 880 885
CCT GTT GAA TTA CCT TAA AAGTTAGCCA GTTAAAGGAT GAAAGCCAAT 3041
Pro Val Glu Leu Pro
890
TTTTTCCCCC TGAGGTTCTC CCATACTGTT TATTAGTACA TATATTGTCA ATTGTTGCTA 3101
CTGAAATGAT AGAAGTTTTG AATATTTACT GTCAATAAAA TACAGTTGAT TCCATTTGAA 3161
3171
(2) INFORMATION FOR SEQ ID N0:6:
CA 02275885 1999-08-31
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 974 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Ala Thr Phe Ala Val Ser Gly Leu Asn Ser Ile Ser Ser Ile Ser
-81 -80 -75 -70
Ser Phe Asn Asn Asn Phe Arg Ser Lys Asn Ser Asn Ile Leu Leu Ser
-65 -60 -55 -50
Arg Arg Arg Ile Leu Leu Phe Ser Phe Arg Arg Arg Arg Arg Ser Phe
-45 -40 -35
Ser Val Ser Ser Val Ala Ser Asp Gln Lys Gln Lys Thr Lys Asp Ser
-30 -25 -20
Ser Ser Asp Glu Gly Phe Thr Leu Asp Val Phe Gln Pro Asp Ser Thr
-15 -10 -5
Ser Val Leu Ser Ser Ile Lys Tyr His Ala Glu Phe Thr Pro Ser Phe
1 5 10 15
Ser Pro Glu Lys Phe Glu Leu Pro Lys Ala Tyr Tyr Ala Thr Ala Glu
20 25 30
Ser Val Arg Asp Thr Leu Ile Ile Asn Trp Asn Ala Thr Tyr Glu Phe
35 40 45
Tyr Glu Lys Met Asn Val Lys Gln Ala Tyr Tyr Leu Ser Met Glu Phe
50 55 60
Leu Gln Gly Arg Ala Leu Leu Asn Ala Ile Gly Asn Leu Gly Leu Thr
65 70 75
Gly Pro Tyr Ala Asp Ala Leu Thr Lys Leu Gly Tyr Ser Leu Glu Asp
80 85 90 95
Val Ala Arg Gln Glu Pro Asp Ala Ala Leu Gly Asn Gly Gly Leu Gly
100 105 110
Arg Leu Ala Ser Cys Phe Leu Asp Ser Met Ala Thr Leu Asn Tyr Pro
115 120 125
Ala Trp Gly Tyr Gly Leu Arg Tyr Gln Tyr Gly Leu Phe Lys Gln Leu
130 135 140
Ile Thr Lys Asp Gly Gln Glu Glu Val Ala Glu Asn Trp Leu Glu Met
145 150 155
Gly Asn Pro Trp Glu Ile Val Arg Asn Asp Ile Ser Tyr Pro Val Lys
160 165 170 175
66
CA 02275885 1999-08-31
Phe Tyr Gly Lys Val Ile Glu Gly Ala Asp Gly Arg Lys Glu Trp Ala
180 185 190
Gly Gly Glu Asp Ile Thr Ala Val Ala Tyr Asp Val Pro Ile Pro Gly
195 200 205
Tyr Lys Thr Lys Thr Thr Ile Asn Leu Arg Leu Trp Thr Thr Lys Leu
210 215 220
Ala Ala Glu Ala Phe Asp Leu Tyr Ala Phe Asn Asn Gly Asp His Ala
225 230 235
Lys Ala Tyr Glu Ala Gln Lys Lys Ala Glu Lys Ile Cys Tyr Val Leu
240 245 250 255
Tyr Pro Gly Asp Glu Ser Leu Glu Gly Lys Thr Leu Arg Leu Lys Gln
260 265 270
Gln Tyr Thr Leu Cys Ser Ala Ser Leu Gln Asp Ile Ile Ala Arg Phe
275 280 285
Glu Lys Arg Ser Gly Asn Ala Val Asn Trp Asp Gln Phe Pro Glu Lys
290 295 300
Val Ala Val Gln Met Asn Asp Thr His Pro Thr Leu Cys Ile Pro Glu
305 310 315
Leu Leu Arg Ile Leu Met Asp Val Lys Gly Leu Ser Trp Lys Gln Ala
320 325 330 335
Trp Glu Ile Thr Gln Arg Thr Val Ala Tyr Thr Asn His Thr Val Leu
340 345 350
Pro Glu Ala Leu Glu Lys Trp Ser Phe Thr Leu Leu Gly Glu Leu Leu
355 360 365
Pro Arg His Val Glu Ile Ile Ala Met Ile Asp Glu Glu Leu Leu His
370 375 380
Thr Ile Leu Ala Glu Tyr Gly Thr Glu Asp Leu Asp Leu Leu Gln Glu
385 390 395
Lys Leu Asn Gln Met Arg Ile Leu Asp Asn Val Glu Ile Pro Ser Ser
400 405 410 415
Val Leu Glu Leu Leu Ile Lys Ala Glu Glu Ser Ala Ala Asp Val Glu
420 425 430
Lys Ala Ala Asp Glu Glu Gln Glu Glu Glu Gly Lys Asp Asp Ser Lys
435 440 445
Asp Glu Glu Thr Glu Ala Val Lys Ala Glu Thr Thr Asn Glu Glu Glu
450 455 460
Glu Thr Glu Val Lys Lys Val Glu Val Glu Asp Ser Gln Ala Lys Ile
465 470 475
67
CA 02275885 1999-08-31
Lys Arg Ile Phe Gly Pro His Pro Asn Lys Pro Gln Val Val His Met
480 485 490 495
Ala Asn Leu Cys Val Val Ser Gly His Ala Val Asn Gly Val Ala Glu
500 505 510
Ile His Ser Glu Ile Val Lys Asp Glu Val Phe Asn Glu Phe Tyr Lys
515 520 525
Leu Trp Pro Glu Lys Phe Gln Asn Lys Thr Asn Gly Val Thr Pro Arg
530 535 540
Arg Trp Leu Ser Phe Cys Asn Pro Glu Leu Ser Glu Ile Ile Thr Lys
545 550 555
Trp Thr Gly Ser Asp Asp Trp Leu Val Asn Thr Glu Lys Leu Ala Glu
560 565 570 575
Leu Arg Lys Phe Ala Asp Asn Glu Glu Leu Gln Ser Glu Trp Arg Lys
580 585 590
Ala Lys Gly Asn Asn Lys Met Lys Ile Val Ser Leu Ile Lys Glu Lys
595 600 605
Thr Gly Tyr Val Val Ser Pro Asp Ala Met Phe Asp Val Gln Ile Lys
610 615 620
Arg Ile His Glu Tyr Lys Arg Gln Leu Leu Asn Ile Phe Gly Ile Val
625 630 635
Tyr Arg Tyr Lys Lys Met Lys Glu Met Ser Pro Glu Glu Arg Lys Glu
640 645 650 655
Lys Phe Val Pro Arg Val Cys Ile Phe Gly Gly Lys Ala Phe Ala Thr
660 665 670
Tyr Val Gln Ala Lys Arg Ile Val Lys Phe Ile Thr Asp Val Gly Glu
675 680 685
Thr Val Asn His Asp Pro Glu Ile Gly Asp Leu Leu Lys Val Val Phe
690 695 700
Val Pro Asp Tyr Asn Val Ser Val Ala Glu Val Leu Ile Pro Gly Ser
705 710 715
Glu Leu Ser Gln His Ile Ser Thr Ala Gly Met Glu Ala Ser Gly Thr
720 725 730 735
Ser Asn Met Lys Phe Ser Met Asn Gly Cys Leu Leu Ile Gly Thr Leu
740 745 750
Asp Gly Ala Asn Val Glu Ile Arg Glu Glu Val Gly Glu Asp Asn Phe
755 760 765
Phe Leu Phe Gly Ala Gln Ala His Glu Ile Ala Gly Leu Arg Lys Glu
770 775 780
68
CA 02275885 1999-08-31
Arg Ala Glu Gly Lys Phe Val Pro Asp Pro Arg Phe Glu Glu Val Lys
785 790 795
Ala Phe Ile Arg Thr Gly Val Phe Gly Thr Tyr Asn Tyr Glu Glu Leu
800 805 810 815
Met Gly Ser Leu Glu Gly Asn Glu Gly Tyr Gly Arg Ala Asp Tyr Phe
820 825 830
Leu Val Gly Lys Asp Phe Pro Asp Tyr Ile Glu Cys Gln Asp Lys Val
835 840 845
Asp Glu Ala Tyr Arg Asp Gln Lys Lys Trp Thr Lys Met Ser Ile Leu
850 855 860
Asn Thr Ala Gly Ser Phe Lys Phe Ser Ser Asp Arg Thr Ile His Gln
865 870 875
Tyr Ala Arg Asp Ile Trp Arg Ile Glu Pro Val Glu Leu Pro
880 885 890
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..27
(D) OTHER INFORMATION: /function= "primer"
/label= SPL1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ATTCGAAAAG CTCGAGATTT GCATAGA 27
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
69
CA 02275885 1999-08-31
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 1..27
(D) OTHER INFORMATION: /function= "primer"
/label= SPL2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GTTTATTTTC CATCGATGGA AGGTGGT 27
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 1..23
(D) OTHER INFORMATION: /function= "primer"
/label= SPH1
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GTGTGCTCTC GAGCATTGAA AGC 23
(2) INFORMATION FOR SEQ ID N0:10:
CA 02275885 1999-08-31
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..25
(D) OTHER INFORMATION: /function= "primer"
/label= SPH2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
ATAATATCCT GAATCGATGC ACTGC 25
71
