U.S. patent application number 12/624484 was filed with the patent office on 2010-03-25 for plant galactinol synthase homologs.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Stephen M. Allen, Perry G. Caimi, Johan M. Stoop.
Application Number | 20100077510 12/624484 |
Document ID | / |
Family ID | 34972614 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100077510 |
Kind Code |
A1 |
Stoop; Johan M. ; et
al. |
March 25, 2010 |
PLANT GALACTINOL SYNTHASE HOMOLOGS
Abstract
Isolated nucleic acid fragments encoding galactinol synthase are
disclosed. Recombinant DNA construct(s) for use in altering
expression of endogenous genes encoding galactinol synthase are
also disclosed.
Inventors: |
Stoop; Johan M.; (Kennett
Square, PA) ; Allen; Stephen M.; (Wilmington, DE)
; Caimi; Perry G.; (Kennett Square, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
Wilmington
DE
|
Family ID: |
34972614 |
Appl. No.: |
12/624484 |
Filed: |
November 24, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12536516 |
Aug 6, 2009 |
|
|
|
12624484 |
|
|
|
|
12336578 |
Dec 17, 2008 |
|
|
|
12536516 |
|
|
|
|
11928675 |
Oct 30, 2007 |
7476778 |
|
|
12336578 |
|
|
|
|
11149403 |
Jun 8, 2005 |
7294756 |
|
|
11928675 |
|
|
|
|
60581851 |
Jun 22, 2004 |
|
|
|
Current U.S.
Class: |
800/284 ;
435/183; 435/320.1; 435/419; 435/468; 536/23.2; 800/278;
800/298 |
Current CPC
Class: |
C12N 15/8234 20130101;
C12N 9/1051 20130101; C12N 15/8245 20130101 |
Class at
Publication: |
800/284 ;
536/23.2; 435/320.1; 435/468; 435/419; 800/278; 800/298;
435/183 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 15/54 20060101 C12N015/54; C12N 15/82 20060101
C12N015/82; C12N 5/10 20060101 C12N005/10; C12N 9/00 20060101
C12N009/00 |
Claims
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide having galactinol synthase activity, wherein
the polypeptide has an amino acid sequence of at least 85%
identity, when compared to one of SEQ ID NO: 2 or 4 or 95% identity
when compared to one of SEQ ID NO:6, based on the Clustal V method
of alignment, (b) all or part of the isolated polynucleotide
comprising (a) for use in co-suppression or antisense suppression
of endogenous nucleic acid sequences encoding polypeptides having
galactinol synthase activity, or (c) a complement of the nucleotide
sequence of (a) or (b), wherein the complement and the nucleotide
sequence consist of the same number of nucleotides and are 100%
complementary.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:2 or
SEQ ID NO:4 have at least 90% identity based on the Clustal
alignment method.
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO:2 or
SEQ ID NO:4 have at least 95% identity based on the Clustal
alignment method.
4. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide comprises one of SEQ ID NO:2, SEQ ID NO:4 or SEQ
ID NO:6.
5. The polynucleotide of claim 1, wherein the nucleotide sequence
comprises one of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.
6. A vector comprising the polynucleotide of claim 1.
7. A recombinant DNA construct comprising the polynucleotide of
claim 1 operably linked to at least one regulatory sequence.
8. A method for transforming a cell, comprising transforming a cell
with the polynucleotide of claim 1.
9. A cell comprising the recombinant DNA construct of claim 7.
10. A method for producing a plant comprising transforming a plant
cell with the polynucleotide of claim 1 and regenerating a plant
from the transformed plant cell.
11. A plant comprising the recombinant DNA construct of claim
7.
12. A seed comprising the recombinant DNA construct of claim 7.
13. An isolated polypeptide having galactinol synthase activity,
wherein the polypeptide has an amino acid sequence of at least 85%
sequence identity when compared to one of SEQ ID NO: 2 or 4 or 95%
identity when compared to SEQ ID NO: 6, based on the Clustal V
method of alignment.
14. The polypeptide of claim 13, wherein the amino acid sequence of
the polypeptide has at least 90% sequence identity, based on the
Clustal V method of alignment, when compared to one of SEQ ID NO: 2
or 4.
15. The polypeptide of claim 13, wherein the amino acid sequence of
the polypeptide has at least 95% sequence identity, based on the
Clustal V method of alignment, when compared to one of SEQ ID NO: 2
or 4.
16. The polypeptide of claim 13, wherein the amino acid sequence of
the polypeptide comprises one of SEQ ID NO: 2, 4 or 6.
17. A method for isolating a polypeptide having galactinol synthase
activity comprising isolating the polypeptide from a cell or
culture medium of the cell, wherein the cell comprises a
recombinant DNA construct comprising the polynucleotide of claim 1
operably linked to at least on regulatory sequence.
18. A method for reducing the level of at least one raffinose
saccharide in soybean comprising: (d) constructing a recombinant
DNA construct comprising all or part of at least one isolated
polynucleotide comprising a nucleotide sequence encoding a
polypeptide having galactinol synthase activity for use in
co-suppression or antisense suppression of endogenous nucleic acid
sequences encoding polypeptides having galactinol synthase activity
operably linked to at least one regulatory sequence; and (e)
transforming a soybean cell with the recombinant DNA construct of
(a); and (f) regenerating soybean plants from the transformed cells
of step (c); and (g) screening seeds obtained from the plants of
(c) for an altered level of galactinol synthase in the transformed
soybean cell when compared to a corresponding nontransformed
soybean cell.
19. The method of claim 18, wherein the total raffinose saccharide
content in soybean seeds has been reduced by at least 27%.
20. The method of claim 18, wherein the total stachyose content in
soybean seeds has been reduced by at least 36%.
21. The method of claim 18, wherein the total raffinose saccharide
content in soybean seeds has been reduced by at least 71%.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/581,851, filed Jun. 22, 2004, the entire content
of which is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to isolated
polynucleotides comprising nucleic acid fragments encoding
galactinol synthase homologs in plants and seeds wherein all or
part of such isolated polynucleotides can be used to down-regulate
expression of endogenous genes encoding galactinol synthase.
BACKGROUND OF THE INVENTION
[0003] Raffinose saccharides are a group of D-galactose-containing
oligosaccharide derivatives of sucrose that are widely distributed
in plants. Raffinose saccharides are characterized by the general
formula:
[O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n-.alpha.-glucopyranosyl-(1-
.fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4 are
known respectively as sucrose, raffinose, stachyose, verbascose,
and ajugose. A set of galactosyltransferases is involved in the
biosynthesis of raffinose saccharides. Galactinol synthase (EC
2.4.1.123) catalyzes the synthesis of galactinol
(O-.alpha.-D-gal-actopyranosyl-[1.fwdarw.1]-L-myo-inositol) from
UDP-D-Gal and myo-inositol. Raffinose and stachyose are then
synthesized by addition of Gal units from galactinol to sucrose and
raffinose, respectively. These reversible reactions are mediated by
raffinose synthase (EC 2.4.1.82) and stachyose synthase (EC
2.4.1.67). Transfer of a further Gal residue from galactinol to
stachyose gives verbascose.
[0004] Extensive botanical surveys of the occurrence of raffinose
saccharides have been reported in the scientific literature [see
Dey (1985) in Biochemistry of Storage Carbohydrates in Green
Plants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp.
53-129]. Raffinose saccharides are thought to be second only to
sucrose with respect to abundance among the nonstructural
carbohydrates in the plant kingdom. In fact, raffinose saccharides
may be ubiquitous, at least among higher plants. Raffinose
saccharides accumulate in significant quantities in the edible
portion of many economically significant crop species. Examples
include soybean (Glycine max L. Merrill), sugar beet (Beta
vulgaris), cotton (Gossypium hirsutum L.), canola (Brassica sp.)
and all of the major edible leguminous crops including beans
(Phaseolus sp.), chick pea (Cicer arietinum), cowpea (Vigna
unguiculata), mung bean (Vigna radiata), peas (Pisum sativum),
lentil (Lens culinaris) and lupine (Lupinus sp.).
[0005] Although abundant in many species, raffinose saccharides are
an obstacle to the efficient utilization of some economically
important crop species. Raffinose saccharides are not digested
directly by animals, primarily because alpha-galactosidase is not
present in the intestinal mucosa [Gitzelmann et al. (1965)
Pediatrics 36:231-236; Rutloff et al. (1967) Nahrung 11:39-46].
However, microflora in the lower gut are readily able to ferment
the raffinose saccharides resulting in an acidification of the gut
and production of carbon dioxide, methane and hydrogen gases
[Murphy et al. (1972) J. Agr. Food. Chem. 20:813-817; Cristofaro et
al. (1974) in Sugars in Nutrition, H. L. Sipple and K. W. McNutt,
Eds. Academic Press, New York, Chap. 20, 313-335; Reddy et al.
(1980) J. Food Science 45:1161-1164]. The resulting flatulence can
severely limit the use of leguminous plants in animal, particularly
human, diets. It is unfortunate that the presence of raffinose
saccharides restricts the use of legumes in human diets because
many of these species are otherwise excellent sources of protein
and soluble fiber. Varieties of edible beans free of raffinose
saccharides would be more valuable for human diets and would more
fully use the desirable nutritional qualities of edible leguminous
plants.
[0006] The biosynthesis of raffinose saccharides has been well
characterized [see Dey (1985) in Biochemistry of Storage
Carbohydrates in Green Plants, P. M. Dey and R. A. Dixon, Eds.
Academic Press, London, pp. 53-129]. The committed reaction of
raffinose saccharide biosynthesis involves the synthesis of
galactinol from UDP-galactose and myo-inositol. The enzyme that
catalyzes this reaction is galactinol synthase (inositol
1-alpha-galactosyltransferase; EC 2.4.1.123). Synthesis of
raffinose and higher homologues in the raffinose saccharide family
from sucrose is thought to be catalyzed by distinct
galactosyltransferases (for example, raffinose synthase and
stachyose synthase). Studies in many species suggest that
galactinol synthase is the key enzyme controlling the flux of
reduced carbon into the biosynthesis of raffinose saccharides
[Handley et al. (1983) J. Amer. Soc. Hort. Sci. 108:600-605;
Saravitz, et al. (1987) Plant Physiol. 83:185-189].
[0007] Related galactinol synthase genes already known in the art
include sequences disclosed in WO 01/77306 and in U.S. Pat. No.
5,648,210, Kerr et al. (the contents of which are hereby
incorporated by reference), and Sprenger and Keller (2000) Plant J
21:249-258. Presumably related sequences are also disclosed in WO
98/50553.
[0008] There is a great deal of interest in identifying the genes
that encode proteins involved in raffinose saccharides in plants.
Specifically, the galactinol synthase gene may be used to alter
galactinol synthesis and modulate the level of raffinose
saccharides in plant cells. Accordingly, the availability of
nucleic acid sequences encoding all or a portion of a galactinol
synthase would facilitate studies to better understand raffinose
synthesis in plants, and provide genetic tools to alter raffinose
saccharide synthesis to enhance the nutritional qualities of many
edible leguminous plants.
SUMMARY OF THE INVENTION
[0009] In a first embodiment, the invention concerns an isolated
polynucleotide comprising: [0010] (a) a nucleotide sequence
encoding a polypeptide having galactinol synthase activity, wherein
the polypeptide has an amino acid sequence of at least 85%
identity, when compared to one of SEQ ID NO: 2 or 4 or 95% identity
when compared to one of SEQ ID NO:6, based on the Clustal V method
of alignment, [0011] (b) all or part of the isolated polynucleotide
comprising (a) for use in co-suppression or antisense suppression
of endogenous nucleic acid sequences encoding polypeptides having
galactinol synthase activity, or [0012] (c) a complement of the
nucleotide sequence of (a) or (b), wherein the complement and the
nucleotide sequence consist of the same number of nucleotides and
are 100% complementary.
[0013] In a second embodiment, the instant invention concerns a
recombinant DNA construct comprising any of the isolated
polynucleotides of the present invention operably linked to at
least one regulatory sequence, and a cell, a plant, and a seed
comprising the recombinant DNA construct.
[0014] In a third embodiment, the present invention includes a
vector comprising any of the isolated polynucleotides of the
present invention.
[0015] In a fourth embodiment, the present invention concerns a
method for transforming a cell comprising transforming a cell with
any of the isolated polynucleotides of the present invention. The
cell transformed by this method is also included. Advantageously,
the cell is eukaryotic, e.g., a yeast or plant cell, or
prokaryotic, e.g., a bacterium.
[0016] In a fifth embodiment, the present invention includes a
method for producing a transgenic plant comprising transforming a
plant cell with any of the isolated polynucleotides of the present
invention and regenerating a plant from the transformed plant cell.
The invention is also directed to the transgenic plant produced by
this method, and seed obtained from this transgenic plant.
[0017] In a sixth embodiment, the present invention concerns an
isolated polypeptide having galactinol synthase activity, wherein
the polypeptide has an amino acid sequence of at least 85%, 90%, or
95% identity, based on the Clustal V method of alignment, when
compared to one of SEQ ID NO: 2 or 4 and wherein the polypeptide
has an amino acid sequence of at least 95% identity, based on the
Clustal V method of alignment, when compared to SEQ ID NO:6.
[0018] In a seventh embodiment, the present invention concerns a
method for isolating a polypeptide having galactinol synthase
activity comprising isolating the polypeptide from a cell or
culture medium of the cell, wherein the cell comprises a
recombinant DNA construct comprising a polynucleotide of the
invention operably linked to at least one regulatory sequence.
[0019] In an eighth embodiment, this invention concerns a method
for selecting a transformed cell comprising: (a) transforming a
host cell with the recombinant DNA construct or an expression
cassette of the present invention; and (b) growing the transformed
host cell, preferably a plant cell, under conditions that allow
expression of the galactinol synthase polynucleotide in an amount
sufficient to complement a null mutant in order to provide a
positive selection means.
[0020] In a ninth embodiment, this invention relates to a method of
reducing the raffinose saccharide content of soybean seeds by at
least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% or any integer
percentage in between 30% to 100%.
[0021] In a tenth embodiment, this invention relates to a method of
reducing the total stachyose content by at least 36%, 40%, 50%,
60%, 70%, 80%, 90%; 95% or 100% or any integer percentage between
36% to 100%.
[0022] In another embodiment, this invention relates to a method of
reducing the level of at least one raffinose saccharide in
soybean.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING
[0023] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing, which form a part of this application.
[0024] FIG. 1 shows a comparison of the amino acid sequence
alignment between the galactinol synthase encoded by the nucleotide
sequences derived from soybean clones sdp3c.pk013.c9:fis,
srr3c.pk003.h12:fis and srr3c.pk001.i20:fis (SEQ ID NO:2, SEQ ID
NO:4 and SEQ ID NO:6, respectively) and the galactinol synthase
from Pisum sativum (NCBI GenBank Identifier (GI) No. 5541885; SEQ
ID NO:7), Arabidopsis thaliana (NCBI GenBank Identifier (GI) No.
15223567; SEQ ID NO:8) and Glycine max (NCBI GenBank Identifier
(GI) No. 32345694; SEQ ID NO:9). Amino acids which are conserved
among all sequences are indicated with an asterisk (*) below the
conserved residue. The program to maximize alignment of the
sequences uses dashes.
[0025] FIG. 2 shows vector pJMS08.
[0026] FIG. 3 shows vector pJMS10.
[0027] FIG. 4 shows a typical carbohydrate profile of transgenic
somatic embryos co-suppressing galactinol synthase.
[0028] FIG. 5 shows a typical carbohydrate profile of a wild type
soybean somatic embryo.
[0029] FIG. 6 shows vector pG4G.
[0030] FIG. 7 shows vector SH50.
[0031] FIG. 8 shows a TLC analysis of somatic embryos containing
the PM29 driven recombinant expression construct described in
Example 11.
[0032] FIG. 9 shows a TLC analysis of mature soybean seeds
containing the PM29 driven recombinant expression construct
described in Example 11.
[0033] FIG. 10 shows the carbohydrate profiles of a mutant and a
transgenic low raffinose saccharide soybean compared to wild type
soybean.
[0034] FIG. 11 shows vector pKR57.
[0035] FIG. 12 shows vector pKR63.
[0036] FIG. 13 shows vector pDS1.
[0037] FIG. 14 shows vector pKR72.
[0038] FIG. 15 shows vector pDS2.
[0039] FIG. 16 shows vector pDS3 (orientation 2).
[0040] FIG. 17 shows vector SH60.
[0041] FIG. 18 shows a TLC analysis of somatic embryos containing
the beta-conglycinin/KTI3 driven recombinant expression construct
described in Example 13.
[0042] SEQ ID NO:1 is the 1151 by sequence derived from clone
sdp3c.pk013.c9 (FIS) of the soybean nucleotide sequence containing
the ORF [nucleotides 71-1090 (Stop)] of the galactinol synthase 3
gene.
[0043] SEQ ID NO:2 is the 339 amino acid sequence encoded by the
ORF [nucleotides 71-1090 (Stop)] of SEQ ID NO: 1
[0044] SEQ ID NO:3 is the 1398 by sequence derived from clone
srr3c.pk003.h12 (FIS) of the soybean nucleotide sequence containing
the ORF [nucleotides 94-1089 (Stop)] of the galactinol synthase 4
gene.
[0045] SEQ ID NO:4 is the 331 amino acid sequence encoded by the
ORF [nucleotides 94-1089 (Stop)] of SEQ ID NO: 3.
[0046] SEQ ID NO:5 is the 1417 by sequence derived from clone
srr3c.pk001.120
[0047] (FIS) of the soybean nucleotide sequence containing the ORF
[nucleotides 213-1187 (Stop)] of the galactinol synthase 5
gene.
[0048] SEQ ID NO:6 is the 324 amino acid sequence encoded by the
ORF [nucleotides 213-1187 (Stop)] of SEQ ID NO: 5
[0049] SEQ ID NO:7 is the amino acid sequence of the galactinol
synthase from Pisum sativum (NCBI GenBank Identifier (GI) No.
5541885).
[0050] SEQ ID NO:8 is the amino acid sequence of the galactinol
synthase from Arabidopsis thaliana (NCBI GenBank Identifier (GI)
No. 15223567).
[0051] SEQ ID NO:9 is the amino acid sequence of the galactinol
synthase from Glycine max (NCBI GenBank Identifier (GI) No.
32345694).
[0052] SEQ ID NO:10 represents the 1406 by of the soybean
nucleotide sequence of the galactinol synthase 1 gene.
[0053] SEQ ID NO:11 represents the 1350 by of the soybean
nucleotide sequence galactinol synthase 2 gene.
[0054] SEQ ID NO:12 is the forward primer used to amplify part of
galactinol synthase 1 as described in Example 6.
[0055] SEQ ID NO:13 is the reverse primer used to amplify part of
galactinol synthase 1 as described in Example 6.
[0056] SEQ ID NO:14 is the 519 by sequence amplified from the
galactinol synthase 1 gene (SEQ ID NO:10) as described in Example
6.
[0057] SEQ ID NO:15 is the forward primer used to amplify part of
galactinol synthase 2 as described in Example 6.
[0058] SEQ ID NO:16 is the reverse primer used to amplify part of
galactinol synthase 2 as described in Example 6.
[0059] SEQ ID NO:17 is the 519 by sequence amplified from the
galactinol synthase 2 gene (SEQ ID NO:11) as described in Example
6.
[0060] SEQ ID NO:18 is the forward primer used to amplify part of
galactinol synthase 3 as described in Example 6.
[0061] SEQ ID NO:19 is the reverse primer used to amplify part of
galactinol synthase 3 as described in Example 6.
[0062] SEQ ID NO:20 is the 519 by sequence amplified from the
galactinol synthase 3 gene (SEQ ID NO:1) as described in Example
6.
[0063] SEQ ID NO:21 is the forward primer used to isolate and
amplify the soybean PM29 promoter as described in Example 10.
[0064] SEQ ID NO:22 is the reverse primer used to isolate and
amplify the soybean PM29 promoter as described in Example 10.
[0065] SEQ ID NO:23 is the 597 by sequence of the soybean PM29
promoter.
[0066] SEQ ID NO:24 is the forward primer used to re-amplify the
PM29 promoter as described in Example 11.
[0067] SEQ ID NO:25 is the reverse primer used to re-amplify the
PM29 promoter as described in Example 11.
[0068] SEQ ID NO:26 is the sequence of two copies of the
Eag1-ELVISLIVES sequence as described in Example 11.
[0069] SEQ ID NO:27 represents the sequence of the complementary
strand of SEQ ID NO: 26.
[0070] SEQ ID NO:28 represents the sequence of a truncated version
of the two copies of the ELVISLIVES (ELEL) linker.
[0071] SEQ ID NO:29 is the 8810 by sequence of vector SH50.
[0072] SEQ ID NO:30 is the 4479 by sequence of vector pKR57.
[0073] SEQ ID NO:31 is the 5010 by sequence of vector pKR63.
[0074] SEQ ID NO:32 is the 5414 by sequence of v pDS1.
[0075] SEQ ID NO:33 is the 7085 by sequence of vector pKR72.
[0076] SEQ ID NO:34 is the 5303 by sequence of vector pDS2.
[0077] SEQ ID NO:35 is the 8031 by sequence of vector pDS3
(orientation 2).
[0078] SEQ ID NO:36 is the 9616 by sequence of vector SH60.
[0079] SEQ ID NO:37 is the 1585 by sequence of the Not1 fragment of
vector pJMS10 (FIG. 3) described in Example 13.
[0080] The sequence descriptions and Sequence Listing attached
hereto comply with the rules governing nucleotide and/or amino acid
sequence disclosures in patent applications as set forth in 37
C.F.R. .sctn.1.821-1.825.
[0081] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res. 13:3021-3030 (1985) and in the
Biochemical J. 219 (No. 2):345-373 (1984) which are herein
incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0082] In the context of this disclosure, a number of terms shall
be utilized. The terms "polynucleotide," "polynucleotide sequence,"
"nucleic acid sequence," and "nucleic acid fragment"/"isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. An isolated polynucleotide of the present
invention may include at least 30 contiguous nucleotides,
preferably at least 40 contiguous nucleotides, most preferably at
least 60 contiguous nucleotides derived from SEQ ID NOs:1 or 3 or
5, or the complement of such sequences.
[0083] The term "isolated" refers to materials, such as nucleic
acid molecules and/or proteins, which are substantially free or
otherwise removed from components that normally accompany or
interact with the materials in a naturally occurring environment.
Isolated polynucleotides may be purified from a host cell in which
they naturally occur. Conventional nucleic acid purification
methods known to skilled artisans may be used to obtain isolated
polynucleotides. The term also embraces recombinant polynucleotides
and chemically synthesized polynucleotides.
[0084] The term "recombinant" means, for example, that a nucleic
acid sequence is made by an artificial combination of two otherwise
separated segments of sequence, e.g., by chemical synthesis or by
the manipulation of isolated nucleic acids by genetic engineering
techniques.
[0085] A "recombinant DNA construct" comprises any of the isolated
polynucleotides of the present invention operably linked to at
least one regulatory sequence.
[0086] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the polypeptide encoded by the
nucleotide sequence. "Substantially similar" also refers to nucleic
acid fragments wherein changes in one or more nucleotide bases does
not affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by gene silencing through for example
antisense or co-suppression technology. "Substantially similar"
also refers to modifications of the nucleic acid fragments of the
instant invention such as deletion or insertion of one or more
nucleotides that do not substantially affect the functional
properties of the resulting transcript vis-a-vis the ability to
mediate gene silencing or alteration of the functional properties
of the resulting protein molecule. It is therefore understood that
the invention encompasses more than the specific exemplary
nucleotide or amino acid sequences and includes functional
equivalents thereof. The terms "substantially similar" and
"corresponding substantially" are used interchangeably herein.
[0087] "Sequence identity" or "identity" in the context of nucleic
acid or polypeptide sequences refers to the nucleic acid bases or
amino acid residues in the two sequences that are the same when
aligned for maximum correspondence over a specified comparison
window.
[0088] Thus, "Percentage of sequence identity" refers to the valued
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the results by 100 to yield the percentage of sequence
identity. Useful examples of percent sequence identities include,
but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, or any integer percentage from 55% to 100%. These
identities can be determined using any of the programs described
herein.
[0089] The "Clustal V method of alignment" corresponds to the
alignment method labeled Clustal V (described by Higgins and Sharp
(1989) CABIOS. 5: 151-153) and found in the Megalign program of the
LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). The "default parameters" are the parameters preset by the
manufacturer of the program and for multiple alignments they
correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10, while for
pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5. After alignment of the sequences, using the
Clustal V program, it is possible to obtain a "percent identity" by
viewing the "sequence distances" table in the same program.
[0090] Substantially similar nucleic acid fragments may be selected
by screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant
invention, wherein one or more nucleotides are substituted, deleted
and/or inserted, for their ability to affect the level of the
polypeptide encoded by the unmodified nucleic acid fragment in a
plant or plant cell. For example, a substantially similar nucleic
acid fragment representing at least 30 contiguous nucleotides,
preferably at least 40 contiguous nucleotides, most preferably at
least 60 contiguous nucleotides derived from the instant nucleic
acid fragment can be constructed and introduced into a plant or
plant cell. The level of the polypeptide encoded by the unmodified
nucleic acid fragment present in a plant or plant cell exposed to
the substantially similar nucleic fragment can then be compared to
the level of the polypeptide in a plant or plant cell that is not
exposed to the substantially similar nucleic acid fragment.
[0091] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by using nucleic acid
fragments that do not share 100% sequence identity with the gene to
be suppressed. Moreover, alterations in a nucleic acid fragment,
which result in the production of a chemically equivalent amino
acid at a given site, but do not affect the functional properties
of the encoded polypeptide, are well known in the art. Thus, a
codon for the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
Consequently, an isolated polynucleotide comprising a nucleotide
sequence of at least 30 (preferably at least 40, most preferably at
least 60) contiguous nucleotides derived from a nucleotide sequence
of SEQ ID NOs:1, 3 or 5 and the complement of such nucleotide
sequences may be used to affect the expression and/or function of a
galactinol synthase in a host cell. A method of using an isolated
polynucleotide to affect the level of expression of a
polynucleotide in a host cell (eukaryotic, such as plant or yeast,
prokaryotic such as bacterial) may comprise the steps of:
constructing an isolated polynucleotide of the present invention or
an isolated recombinant DNA construct of the present invention;
introducing the isolated polynucleotide or the isolated recombinant
DNA construct into a host cell; measuring the level of a
polypeptide or enzyme activity in the host cell containing the
isolated polynucleotide; and comparing the level of a polypeptide
or enzyme activity in the host cell containing the isolated
polynucleotide with the level of a polypeptide or enzyme activity
in a host cell that does not contain isolated polynucleotide.
[0092] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridization, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times.SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 min, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C.
[0093] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least about 85% identical to the amino
acid sequences reported herein. More preferred nucleic acid
fragments encode amino acid sequences that are at least about 90%
identical to the amino acid sequences reported herein. Most
preferred are nucleic acid fragments that encode amino acid
sequences that are at least about 95% identical to the amino acid
sequences reported herein. Suitable nucleic acid fragments not only
have the above identities but typically encode a polypeptide having
at least 50 amino acids, preferably at least 100 amino acids, more
preferably at least 150 amino acids, still more preferably at least
200 amino acids, and most preferably at least 250 amino acids.
[0094] It is well understood by one skilled in the art that many
levels of sequence identity are useful in identifying related
polypeptide sequences. Useful examples of percent identities are
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer
percentage from 55% to 100%. Sequence alignments and percent
identity calculations were performed using the Megalign program of
the LASERGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.). Multiple alignment of the sequences was performed
using the Clustal method of alignment (Higgins and Sharp (1989)
CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP
LENGTH PENALTY=10). Default parameters for pairwise alignments
using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5.
[0095] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.
Biol. 215:403-410; see also the explanation of the BLAST algorithm
on the world wide web site for the National Center for
Biotechnology Information at the National Library of Medicine of
the National Institutes of Health). In general, a sequence of ten
or more contiguous amino acids or thirty or more contiguous
nucleotides is necessary in order to putatively identify a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene-specific oligonucleotide probes comprising 30 or more
contiguous nucleotides may be used in sequence-dependent methods of
gene identification (e.g., Southern hybridization) and isolation
(e.g., in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12 or more
nucleotides may be used as amplification primers in PCR in order to
obtain a particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence
comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid fragment
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0096] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequences set forth herein. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid fragment for improved
expression in a host cell, it is desirable to design the nucleic
acid fragment such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0097] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to a nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well-established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of the nucleotide sequence to
reflect the codon bias of the host cell. The skilled artisan
appreciates the likelihood of successful gene expression if codon
usage is biased towards those codons favored by the host.
Determination of preferred codons can be based on a survey of genes
derived from the host cell where sequence information is
available.
[0098] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Recombinant DNA construct"
refers to any gene that is not a native gene, comprising regulatory
and coding sequences that are not found together in nature.
Accordingly, a recombinant DNA construct may comprise regulatory
sequences and coding sequences that are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source, but arranged in a manner different than that found
in nature. "Endogenous gene" refers to a native gene in its natural
location in the genome of an organism. A "foreign-gene" refers to a
gene not normally found in the host organism, but that is
introduced into the host organism by gene transfer. Foreign genes
can comprise native genes inserted into a non-native organism,
recombinant DNA constructs, or recombinant DNA constructs. A
"transgene" is an isolated nucleic acid fragment or recombinant DNA
construct that has been introduced into the genome by a
transformation procedure.
[0099] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0100] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence, which can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or may be composed of different
elements derived from different promoters found in nature, or may
even comprise synthetic nucleotide segments. It is understood by
those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental conditions. Promoters which cause a nucleic acid
fragment to be expressed in most cell types at most times are
commonly referred to as "constitutive promoters". New promoters of
various types useful in plant cells are constantly being
discovered; numerous examples may be found in the compilation by
Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is
further recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, nucleic acid
fragments of different lengths may have identical promoter
activity.
[0101] "Convergent promoters" refers to promoters that are situated
on either side of the isolated nucleic acid fragment of interest
such that the direction of transcription from each promoter is
opposing each other. Any promoter useful in plant transgene
expression can be used. The promoters can be the same or different.
The promoters are convergent with the isolated nucleic acid
fragment being situated between the convergent promoters. It is
important that the promoters have similar spatial and temporal
activity, i.e., similar spatial and temporal patterns of
expression, so that double-stranded RNA is produced in plants or
plant organs by the recombinant construct that is stably integrated
into the genome of the plant or plant organ. This has been
described in U.S. provisional application 60/578,404, filed Jun. 9,
2004 which is filed simultaneously herewith. Also, this is
described in U.S. provisional application 60/625,835, filed Nov. 8,
2004.
[0102] "Translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).
[0103] "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0104] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into polypeptides by the cell. "cDNA" refers to DNA that
is complementary to and derived from an mRNA template. The cDNA can
be single-stranded or converted to double stranded form using, for
example, the Klenow fragment of DNA polymerase I. "Sense-RNA"
refers to an RNA transcript that includes the mRNA and so can be
translated into a polypeptide by the cell. "Antisense RNA" refers
to an RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by
reference). The complementarity of an antisense RNA may be with any
part of the specific nucleotide sequence, i.e., at the 5'
non-coding sequence, 3' non-coding sequence, introns, or the coding
sequence. "Functional RNA" refers to sense RNA, antisense RNA,
ribozyme RNA, or other RNA that may not be translated but yet has
an effect on cellular processes.
[0105] Cosuppression technology constitutes the subject matter of
U.S. Pat. No. 5,231,020, which issued to Jorgensen et al. on Jul.
27, 1999. The phenomenon observed by Napoli et al. in petunia was
referred to as "cosuppression" since expression of both the
endogenous gene and the introduced transgene were suppressed (for
reviews see Vaucheret et al., Plant J. 16:651-659 (1998); and Gura,
Nature 404:804-808 (2000)).
[0106] Co-suppression constructs in plants previously have been
designed by focusing on overexpression of a nucleic acid sequence
having homology to an endogenous mRNA, in the sense orientation,
which results in the reduction of all RNA having homology to the
overexpressed sequence (see Vaucheret et al. (1998) Plant J
16:651-659; and Gura (2000) Nature 404:804-808). The overall
efficiency of this phenomenon is low, and the extent of the RNA
reduction is widely variable. Recent work has described the use of
"hairpin" structures that incorporate all, or part, of an mRNA
encoding sequence in a complementary orientation that results in a
potential "stem-loop" structure for the expressed RNA (PCT
Publication WO 99/53050 published on Oct. 21, 1999). This increases
the frequency of co-suppression in the recovered transgenic plants.
Another variation describes the use of plant viral sequences to
direct the suppression, or "silencing", of proximal mRNA encoding
sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998).
Both of these co-suppressing phenomena have not been elucidated
mechanistically, although recent genetic evidence has begun to
unravel this complex situation (Elmayan et al. (1998) Plant Cell
10:1747-1757).
[0107] In addition to cosuppression, antisense technology has also
been used to block the function of specific genes in cells.
Antisense RNA is complementary to the normally expressed RNA, and
presumably inhibits gene expression by interacting with the normal
RNA strand. The mechanisms by which the expression of a specific
gene are inhibited by either antisense or sense RNA are on their
way to being understood. However, the frequencies of obtaining the
desired phenotype in a transgenic plant may vary with the design of
the construct, the gene, the strength and specificity of its
promoter, the method of transformation and the complexity of
transgene insertion events (Baulcombe, Curr. Biol. 12(3):R82-84
(2002); Tang et al., Genes Dev. 17(1):49-63 (2003); Yu et al.,
Plant Cell. Rep. 22(3):167-174 (2003)). Cosuppression and antisense
inhibition are also referred to as "gene silencing",
"post-transcriptional gene silencing" (PTGS), RNA interference or
RNAi. See for example U.S. Pat. No. 6,506,559.
[0108] MicroRNAs (miRNA) are small regulatory RNSs that control
gene expression. miRNAs bind to regions of target RNAs and inhibit
their translation and, thus, interfere with production of the
polypeptide encoded by the target RNA. miRNAs can be designed to be
complementary to any region of the target sequence RNA including
the 3' untranslated region, coding region, etc. miRNAs are
processed from highly structured RNA precursors that are processed
by the action of a ribonuclease III termed DICER. While the exact
mechanism of action of miRNAs is unknown, it appears that they
function to regulate expression of the target gene. See, e.g., U.S.
Patent Publication No. 2004/0268441 A1 which was published on Dec.
30, 2004.
[0109] The term "operably linked" refers to the association of two
or more nucleic acid fragments on a single polynucleotide so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0110] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0111] A "protein" or "polypeptide" is a chain of amino acids
arranged in a specific order determined by the coding sequence in a
polynucleotide encoding the polypeptide. Each protein or
polypeptide has a unique function.
[0112] "Altered levels" or "altered expression" refers to the
production of gene product(s) in transgenic organisms in amounts or
proportions that differ from that of normal or non-transformed
organisms.
[0113] "Mature protein" or the term "mature" when used in
describing a protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or pro-peptides present
in the primary translation product have been removed. "Precursor
protein" or the term "precursor" when used in describing a protein
refers to the primary product of translation of mRNA; i.e., with
pre- and pro-peptides still present. Pre- and pro-peptides may be
but are not limited to intracellular localization signals.
[0114] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference). Thus, isolated polynucleotides of the present invention
can be incorporated into recombinant constructs, typically DNA
constructs, capable of introduction into and replication in a host
cell. Such a construct can be a vector that includes a replication
system and sequences that are capable of transcription and
translation of a polypeptide-encoding sequence in a given host
cell. A number of vectors suitable for stable transfection of plant
cells or for the establishment of transgenic plants have been
described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory
Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for
Plant Molecular Biology, Academic Press, 1989; and Flevin et al.,
Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990.
Typically, plant expression vectors include, for example, one or
more cloned plant genes under the transcriptional control of 5' and
3' regulatory sequences and a dominant selectable marker. Such
plant expression vectors also can contain a promoter regulatory
region (e.g., a regulatory region controlling inducible or
constitutive, environmentally- or developmentally-regulated, or
cell- or tissue-specific expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0115] "Stable transformation" refers to the transfer of a nucleic
acid fragment into a genome of a host organism, including both
nuclear and organellar genomes, resulting in genetically stable
inheritance. The term "transformation" as used herein refers to
both stable transformation and transient transformation.
[0116] The terms "recombinant construct", "expression construct"
and "recombinant expression construct" are used interchangeably
herein. These terms refer to a functional unit of genetic material
that can be inserted into the genome of a cell using standard
methodology well known to one skilled in the art.
[0117] The term "vector" refers to a vehicle used for gene cloning
to insert a foreign nucleic acid fragment into the genome of a host
cell.
[0118] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Maniatis").
[0119] "Motifs" or "subsequences" refer to short regions of
conserved sequences of nucleic acids or amino acids that comprise
part of a longer sequence. For example, it is expected that such
conserved subsequences would be important for function, and could
be used to identify new homologues in plants. It is expected that
some or all elements may be found in a homologue. Also, it is
expected that one or two of the conserved amino acids in any given
motif may differ in a true homologue.
[0120] "PCR" or "polymerase chain reaction" is well known by those
skilled in the art as a technique used for the amplification of
specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).
[0121] The present invention concerns an isolated polynucleotide
comprising: [0122] (a) a nucleotide sequence encoding a polypeptide
having galactinol synthase activity, wherein the polypeptide has an
amino acid sequence of at least 85% identity, when compared to one
of SEQ ID NO: 2 or 4 or 95% identity when compared to one of SEQ ID
NO:6, based on the Clustal V method of alignment, [0123] (b) all or
part of the isolated polynucleotide comprising (a) for use in
co-suppression or antisense suppression of endogenous nucleic acid
sequences encoding polypeptides having galactinol synthase
activity, or [0124] (c) a complement of the nucleotide sequence of
(a) or (b), wherein the complement and the nucleotide sequence
consist of the same number of nucleotides and are 100%
complementary.
[0125] This invention also includes the isolated complement of such
polynucleotides, wherein the complement and the polynucleotide
consist of the same number of nucleotides, and the nucleotide
sequence of the complement and the polynucleotide have 100%
complementarity.
[0126] Nucleic acid fragments encoding at least a portion of
several galactinol synthases have been isolated and identified by
comparison of random plant cDNA sequences to public databases
containing nucleotide and protein sequences using the BLAST
algorithms well known to those skilled in the art. The nucleic acid
fragments of the instant invention may be used to isolate cDNAs and
genes encoding homologous proteins from the same or other plant
species. Isolation of homologous genes using sequence-dependent
protocols is well known in the art. Examples of sequence-dependent
protocols include, but are not limited to, methods of nucleic acid
hybridization, and methods of DNA and RNA amplification as
exemplified by various uses of nucleic acid amplification
technologies (e.g., polymerase chain reaction, ligase chain
reaction).
[0127] For example, genes encoding other galactinol synthases,
either as cDNAs or genomic DNAs, could be isolated directly by
using all or a portion of the instant nucleic acid fragments as DNA
hybridization probes to screen libraries from any desired plant
employing methodology well known to those skilled in the art.
Specific oligonucleotide probes based upon the instant nucleic acid
sequences can be designed and synthesized by methods known in the
art (Maniatis). Moreover, an entire sequence can be used directly
to synthesize DNA probes by methods known to the skilled artisan
such as random primer DNA labeling, nick translation, end-labeling
techniques, or RNA probes using available in vitro transcription
systems. In addition, specific primers can be designed and used to
amplify a part or all of the instant sequences. The resulting
amplification products can be labeled directly during amplification
reactions or labeled after amplification reactions, and used as
probes to isolate full length cDNA or genomic fragments under
conditions of appropriate stringency.
[0128] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998-9002) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments
can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA
86:5673-5677; Loh et al. (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165). Consequently, a polynucleotide comprising a nucleotide
sequence of at least 30 (preferably at least 40, most preferably at
least 60) contiguous nucleotides derived from a nucleotide sequence
selected from the group consisting of SEQ ID NOs:1, 3, or 5 and the
complement of such nucleotide sequences may be used in such methods
to obtain a nucleic acid fragment encoding a substantial portion of
an amino acid sequence of a galactinol synthase polypeptide.
[0129] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).
[0130] In another embodiment, this invention concerns viruses and
host cells comprising either the recombinant DNA constructs of the
invention as described herein or isolated polynucleotides of the
invention as described herein. Examples of host cells which can be
used to practice the invention include, but are not limited to,
yeast, bacteria, and plants.
[0131] Plant tissue includes differentiated and undifferentiated
tissues or plants, including but not limited to, roots, stems,
shoots, leaves, pollen, seeds, tumor tissue, and various forms of
cells and culture such as single cells, protoplasm, embryos, and
callus tissue. The plant tissue may in plant or in organ, tissue or
cell culture.
[0132] The term "plant organ" refers to plant tissue or group of
tissues that constitute a morphologically and functionally distinct
part of a plant. The term "genome" refers to the following: 1. The
entire complement of genetic material (genes and non-coding
sequences) is present in each cell of an organism, or virus or
organelle. 2. A complete set of chromosomes inherited as a
(haploid) unit from one parent. The term "stably integrated" refers
to the transfer of a nucleic acid fragment into the genome of a
host organism or cell resulting in genetically stable
inheritance.
[0133] As was noted above, the nucleic acid fragments of the
instant invention may be used to create transgenic plants in which
the disclosed polypeptides are present at higher or lower levels
than normal or in cell types or developmental stages in which they
are not normally found. This would have the effect of altering the
level of galactinol synthase, galactinol, and raffinose saccharides
in those cells.
[0134] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a recombinant DNA construct
in which the coding region is operably linked to a promoter capable
of directing expression of a gene in the desired tissues at the
desired stage of development. The recombinant DNA construct may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
recombinant DNA construct may also comprise one or more introns in
order to facilitate gene expression of the recombinant DNA
construct.
[0135] Plasmid vectors comprising the instant isolated
polynucleotides) (or recombinant DNA constructs) may be
constructed. The choice of plasmid vector is dependent upon the
method that will be used to transform host plants. The skilled
artisan is well aware of the genetic elements that must be present
on the plasmid vector in order to successfully transform, select
and propagate host cells containing the recombinant DNA construct
or recombinant DNA construct. The skilled artisan will also
recognize that different independent transformation events will
result in different levels and patterns of expression (Jones et al.
(1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.
Genetics 218:78-86), and thus that multiple events must be screened
in order to obtain lines displaying the desired expression level
and pattern. Such screening may be accomplished by Southern
analysis of DNA, Northern analysis of mRNA expression, Western
analysis of protein expression, or phenotypic analysis.
[0136] It may also be desirable to reduce or eliminate expression
of genes encoding the instant polypeptides in plants for some
applications. In order to accomplish this, a recombinant DNA
construct designed for co-suppression of the instant polypeptide
can be constructed by linking a gene or gene fragment encoding that
polypeptide to plant promoter sequences. Alternatively, a
recombinant DNA construct designed to express antisense RNA for all
or part of the instant nucleic acid fragment can be constructed by
linking the gene or gene fragment in reverse orientation to plant
promoter sequences. Either the co-suppression or antisense
recombinant DNA constructs could be introduced into plants via
transformation wherein expression of the corresponding endogenous
genes are reduced or eliminated.
[0137] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
a specific phenotype to the reproductive tissues of the plant by
the use of tissue specific promoters may confer agronomic
advantages relative to conventional mutations which may have an
effect in all tissues in which a mutant gene is ordinarily
expressed.
[0138] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different
recombinant DNA constructs utilizing different regulatory elements
known to the skilled artisan. Once transgenic plants are obtained
by one of the methods described above, it will be necessary to
screen individual transgenics for those that most effectively
display the desired phenotype. Accordingly, the skilled artisan
will develop methods for screening large numbers of transformants.
The nature of these screens will generally be chosen on practical
grounds. For example, one can screen by looking for changes in gene
expression by using antibodies specific for the protein encoded by
the gene being suppressed, or one could establish assays that
specifically measure enzyme activity. A preferred method will be
one which allows large numbers of samples to be processed rapidly,
since it will be expected that a large number of transformants will
be negative for the desired phenotype.
[0139] In still another embodiment, the present invention concerns
a galactinol synthase polypeptide having an amino acid sequence
comprising at least 85% identical, based on the Clustal method of
alignment, to a polypeptide of SEQ ID NO:2 or 4 or at least 95%
identical to a polypeptide of SEQ ID NO:6. The instant polypeptides
(or portions thereof) may be produced in heterologous host cells,
particularly in the cells of microbial hosts, and can be used to
prepare antibodies to these proteins by methods well known to those
skilled in the art. The antibodies are useful for detecting the
polypeptides of the instant invention in situ in cells or in vitro
in cell extracts. Preferred heterologous host cells for production
of the instant polypeptides are microbial hosts. Microbial
expression systems and expression vectors containing regulatory
sequences that direct high level expression of foreign proteins are
well known to those skilled in the art. Any of these could be used
to construct a recombinant DNA construct for production of the
instant polypeptides. This recombinant DNA construct could then be
introduced into appropriate microorganisms via transformation to
provide high level expression of the encoded galactinol synthase.
An example of a vector for high level expression of the instant
polypeptides in a bacterial host is provided (Example 5).
[0140] All or a substantial portion of the polynucleotides of the
instant invention may also be used as probes for genetically and
physically mapping the genes that they are a part of, and used as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J. Hum. Genet. 32:314-331).
[0141] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe
genetic mapping of specific cDNA clones using the methodology
outlined above or variations thereof. For example, F2 intercross
populations, backcross populations, randomly mated populations,
near isogenic lines, and other sets of individuals may be used for
mapping. Such methodologies are well known to those skilled in the
art.
[0142] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0143] Nucleic acid probes derived from the instant nucleic acid
sequences may be used in direct fluorescence in situ hybridization
(FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although
current methods of FISH mapping favor use of large clones (several
hundred kb; see Laan et al. (1995) Genome Res. 5:13-20),
improvements in sensitivity may allow performance of FISH mapping
using shorter probes.
[0144] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0145] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl. Acad. Sci. USA
86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci. USA
92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter
approach may be accomplished in two ways. First, short segments of
the instant nucleic acid fragments may be used in polymerase chain
reaction protocols in conjunction with a mutation tag sequence
primer on DNAs prepared from a population of plants in which
Mutator transposons or some other mutation-causing DNA element has
been introduced (see Bensen, supra). The amplification of a
specific DNA fragment with these primers indicates the insertion of
the mutation tag element in or near the plant gene encoding the
instant polypeptide. Alternatively, the instant nucleic acid
fragment may be used as a hybridization probe against PCR
amplification products generated from the mutation population using
the mutation tag sequence primer in conjunction with an arbitrary
genomic site primer, such as that for a restriction enzyme
site-anchored synthetic adaptor. With either method, a plant
containing a mutation in the endogenous gene encoding the instant
polypeptide can be identified and obtained. This mutant plant can
then be used to determine or confirm the natural function of the
instant polypeptides disclosed herein.
[0146] This invention also relates to a method of reducing the
raffinose saccharide content of soybean seeds by at least 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95% or 100% or any integer percentage in
between 30% to 100%.
[0147] Raffinose saccharides are a group of D-galactose-containing
oligosaccharide derivatives of sucrose that are widely distributed
in plants. Raffinose saccharides are characterized by the following
general formula:
[O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n-.alpha.-glucopyr-
anosyl-(1.fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4
are known respectively as sucrose, raffinose, stachyose, verbascose
and ajugose.
[0148] More specifically, this invention concerns method for
reducing the level of at least one raffinose saccharide in soybean
comprising: [0149] (a) constructing a recombinant DNA construct
comprising all or part of at least one isolated polynucleotide
comprising a nucleotide sequence encoding a polypeptide having
galactinol synthase activity for use in co-suppression or antisense
suppression of endogenous nucleic acid sequences encoding
polypeptides having galactinol synthase activity operably linked to
at least one regulatory sequence; and [0150] (b) transforming a
soybean cell with the recombinant DNA construct of (a); and [0151]
(c) regenerating soybean plants from the transformed cells of step
(c); and screening seeds obtained from the plants of (c) for an
altered level of galactinol synthase in the transformed soybean
cell when compared to a corresponding nontransformed soybean
cell.
[0152] The regeneration, development, and cultivation of plants
from single plant protoplast transformants or from various
transformed explants is well known in the art (Weissbach and
Weissbach, In: Methods for Plant Molecular Biology, (Eds.),
Academic Press, Inc. San Diego, Calif., (1988)). This regeneration
and growth process typically includes the steps of selection of
transformed cells, culturing those individualized cells through the
usual stages of embryonic development through the rooted plantlet
stage. Transgenic embryos and seeds are similarly regenerated. The
resulting transgenic rooted shoots are thereafter planted in an
appropriate plant growth medium such as soil. Preferably, the
regenerated plants are self-pollinated to provide homozygous
transgenic plants. Otherwise, pollen obtained from the regenerated
plants is crossed to seed-grown plants of agronomically important
lines. Conversely, pollen from plants of these important lines is
used to pollinate regenerated plants. A transgenic plant of the
present invention containing a desired polypeptide is cultivated
using methods well known to one skilled in the art.
[0153] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0154] Methods for transforming dicots, primarily using
Agrobacterium tumefaciens, and obtaining transgenic plants have
been published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No.
5,159,135, U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No.
5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al. (1988)
BiolTechnology 6:923, Christou et al. (1988) Plant Physiol.
87:671-674); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et
al. (1996) Plant Cell Rep. 15:653-657, McKently et al. (1995) Plant
Cell Rep. 14:699-703); papaya and pea (Grant et al. (1995) Plant
Cell Rep. 15:254-258).
[0155] Transformation of monocotyledons using electroporation,
particle bombardment, and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) (1987)
84:5354); barley (Wan and Lemaux (1994) Plant Physiol. 104:37); Zea
mays (Rhodes et al. (1988) Science 240:204, Gordon-Kamm et al.
(1990) Plant Cell 2:603-618, Fromm et al. (1990) BiolTechnology
8:833; Koziel et al. (1993) BiolTechnology 11: 194, Armstrong et
al. (1995) Crop Science 35:550-557); oat (Somers et al. (1992)
BiolTechnology 10: 15 89); orchard grass (Horn et al. (1988) Plant
Cell Rep. 7:469); rice (Toriyama et al. (1986) Theor. Appl. Genet.
205:34; Part et al. (1996) Plant Mol. Biol. 32:1135-1148; Abedinia
et al. (1997) Aust. J. Plant Physiol. 24:133-141; Zhang and Wu
(1988) Theor. Appl. Genet. 76:835; Zhang et al. (1988) Plant Cell
Rep. 7:379; Battraw and Hall (1992) Plant Sci. 86:191-202; Christou
et al. (1991) Bio/Technology 9:957); rye (De la Pena et al. (1987)
Nature 325:274); sugarcane (Bower and Birch (1992) Plant J. 2:409);
tall fescue (Wang et al. (1992) BiolTechnology 10:691), and wheat
(Vasil et al. (1992) Bio/Technology 10:667; U.S. Pat. No.
5,631,152).
[0156] Assays for gene expression based on the transient expression
of cloned nucleic acid constructs have been developed by
introducing the nucleic acid molecules into plant cells by
polyethylene glycol treatment, electroporation, or particle
bombardment (Marcotte et al., Nature 335:454-457 (1988); Marcotte
et al., Plant Cell 1:523-532 (1989); McCarty et al., Cell
66:895-905 (1991); Hattori et al., Genes Dev. 6:609-618 (1992);
Goff et al., EMBO J. 9:2517-2522 (1990)).
[0157] Transient expression systems may be used to functionally
dissect isolated nucleic acid fragment constructs (see generally,
Maliga et al., Methods in Plant Molecular Biology, Cold Spring
Harbor Press (1995)). It is understood that any of the nucleic acid
molecules of the present invention can be introduced into a plant
cell in a permanent or transient manner in combination with other
genetic elements such as vectors, promoters, enhancers etc.
[0158] In addition to the above discussed procedures the standard
resource materials which describe specific conditions and
procedures for the construction, manipulation and isolation of
macromolecules (e.g., DNA molecules, plasmids, etc.), generation of
recombinant organisms and screening and isolating of clones (see
for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in
Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et
al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, N.Y.
(1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold
Spring Harbor, N.Y. (1998); Plant Molecular Biology: A Laboratory
Manual, eds. Clark, Springer, New York (1997)) are well known.
EXAMPLES
[0159] The present invention is further defined in the following
Examples, in which parts and percentages are by weight and degrees
are Celsius, unless otherwise stated. It should be understood that
these Examples, while indicating preferred embodiments of the
invention, are given by way of illustration only. From the above
discussion and these Examples, one skilled in the art can ascertain
the essential characteristics of this invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0160] The disclosure of each reference set forth herein is
incorporated herein by reference in its entirety.
Example 1
Composition of cDNA Libraries
Isolation and Sequencing of cDNA Clones
[0161] A cDNA library representing mRNAs from soybean (Glycine max)
tissue was prepared. The characteristics of the library are
described below.
TABLE-US-00001 TABLE 1 cDNA Libraries from Soybean Library Tissue
Clone sdp3c Soybean (Glycine max [L.]) developing sdp3c.pk013.c9
pods 8-9 mm srr3c Soybean (Glycine max [L.], Bell) roots
srr3c.pk003.h12:fis control for src3c. srr3c.pk001.i20:fis
[0162] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
[0163] Full-insert sequence (FIS) data is generated utilizing a
modified transposition protocol. Clones identified for FIS are
recovered from archived glycerol stocks as single colonies, and
plasmid DNAs are isolated via alkaline lysis. Isolated DNA
templates are reacted with vector primed M13 forward and reverse
oligonucleotides in a PCR-based sequencing reaction and loaded onto
automated sequencers. Confirmation of clone identification is
performed by sequence alignment to the original EST sequence from
which the FIS request is made.
[0164] Confirmed templates are transposed via the Primer Island
transposition kit (PE Applied Biosystems, Foster City, Calif.)
which is based upon the Saccharomyces cerevisiae Ty1 transposable
element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772).
The in vitro transposition system places unique binding sites
randomly throughout a population of large DNA molecules. The
transposed DNA is then used to transform DH10B electro-competent
cells (Gibco BRL/Life Technologies, Rockville, Md.) via
electroporation. The transposable element contains an additional
selectable marker (named DHFR; Fling and Richards (1983) Nucleic
Acids Res. 11:5147-5158), allowing for dual selection on agar
plates of only those subclones containing the integrated
transposon. Multiple subclones are randomly selected from each
transposition reaction, plasmid DNAs are prepared via alkaline
lysis, and templates are sequenced (ABI Prism dye-terminator
ReadyReaction mix) outward from the transposition event site,
utilizing unique primers specific to the binding sites within the
transposon.
[0165] Sequence data is collected (ABI Prism Collections) and
assembled using Phred/Phrap (P. Green, University of Washington,
Seattle). Phrep/Phrap is a public domain software program which
re-reads the ABI sequence data, re-calls the bases, assigns quality
values, and writes the base calls and quality values into editable
output files. The Phrap sequence assembly program uses these
quality values to increase the accuracy of the assembled sequence
contigs. Assemblies are viewed by the Consed sequence editor (D.
Gordon, University of Washington, Seattle).
[0166] In some of the clones the cDNA fragment corresponds to a
portion of the 3'-terminus of the gene and does not cover the
entire open reading frame. In order to obtain the upstream
information one of two different protocols are used. The first of
these methods results in the production of a fragment of DNA
containing a portion of the desired gene sequence while the second
method results in the production of a fragment containing the
entire open reading frame. Both of these methods use two rounds of
PCR amplification to obtain fragments from one or more libraries.
The libraries some times are chosen based on previous knowledge
that the specific gene should be found in a certain tissue and some
times are randomly-chosen. Reactions to obtain the same gene may be
performed on several libraries in parallel or on a pool of
libraries. Library pools are normally prepared using from 3 to 5
different libraries and normalized to a uniform dilution. In the
first round of amplification both methods use a vector-specific
(forward) primer corresponding to a portion of the vector located
at the 5'-terminus of the clone coupled with a gene-specific
(reverse) primer. The first method uses a sequence that is
complementary to a portion of the already known gene sequence while
the second method uses a gene-specific primer complementary to a
portion of the 3'-untranslated region (also referred to as UTR). In
the second round of amplification a nested set of primers is used
for both methods. The resulting DNA fragment is ligated into a
pBluescript vector using a commercial kit and following the
manufacturer's protocol. This kit is selected from many available
from several vendors including Invitrogen (Carlsbad, Calif.),
Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.).
The plasmid DNA is isolated by alkaline lysis method and submitted
for sequencing and assembly using Phred/Phrap, as above.
Example 2
Identification of cDNA Clones
[0167] cDNA clones encoding galactinol synthase were identified by
conducting BLAST (Basic Local Alignment Search Tool; Altschul et
al. (1993) J. Mol. Biol. 215:403-410;) searches for similarity to
sequences contained in the BLAST "nr" database (comprising all
non-redundant GenBank CDS translations, sequences derived from the
3-dimensional structure Brookhaven Protein Data Bank, the last
major release of the SWISS-PROT protein sequence database, EMBL,
and DDBJ databases). The cDNA sequences obtained in Example 1 were
analyzed for similarity to all publicly available DNA sequences
contained in the "nr" database using the BLASTN algorithm provided
by the National Center for Biotechnology Information (NCBI). The
DNA sequences were translated in all reading frames and compared
for similarity to all publicly available protein sequences
contained in the "nr" database using the BLASTX algorithm (Gish and
States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
[0168] ESTs submitted for analysis are compared to the GenBank
database as described above. ESTs that contain sequences more 5- or
3-prime can be found by using the BLAST algorithm (Altschul et al
(1997) Nucleic Acids Res. 25:3389-3402.) against the Du Pont
proprietary database comparing nucleotide sequences that share
common or overlapping regions of sequence homology. Where common or
overlapping sequences exist between two or more nucleic acid
fragments, the sequences can be assembled into a single contiguous
nucleotide sequence, thus extending the original fragment in either
the 5 or 3 prime direction. Once the most 5-prime EST is
identified, its complete sequence can be determined by Full Insert
Sequencing as described in Example 1. Homologous genes belonging to
different species can be found by comparing the amino acid sequence
of a known gene (from either a proprietary source or a public
database) against an EST database using the tBLASTn algorithm. The
tBLAST algorithm searches an amino acid query against a nucleotide
database that is translated in all 6 reading frames. This search
allows for differences in nucleotide codon usage between different
species, and for codon degeneracy.
Example 3
Characterization of cDNA Clones Encoding Galactinol Synthase
[0169] The BLASTX search using the EST sequences from the clones
listed in Table 2 revealed similarity of the polypeptides encoded
by the cDNAs to galactinol synthase from Pisum sativum (NCBI
GenBank Identifier (GI) No. 5541885, SEQ ID NO:7), Arabidopsis
thaliana (NCBI GenBank Identifier (GI) No. 15223567, SEQ ID NO:8)
and Glycine max (NCBI GenBank Identifier (GI) No. 32345694, SEQ ID
NO:9). Shown in Table 2 are the BLAST results for the sequences
encoding an entire protein ("CGS") derived from the entire cDNA
inserts comprising the indicated cDNA clones ("fis"):
TABLE-US-00002 TABLE 2 BLAST Results for Sequences Encoding
Polypeptides Homologous to Galactinol Synthase BLAST pLog Score
Clone Status (NCBI) sdp3c.pk013.c9:fis (SEQ ID NO: 2) CGS 149.57
(GI: 5541885) srr3c.pk003.h12:fis (SEQ ID NO: 4) CGS 135.89 (GI:
15223567) srr3c.pk001.i20:fis (SEQ ID NO: 6) CGS 166.70 (GI:
32345694)
[0170] The sequence of the entire cDNA insert in the clones listed
in Table 2 was determined. The data in Table 3 represent a
calculation of the percent identity of the amino acid sequences set
forth in SEQ ID Nos: 2, 4, and 6 and the sequences of Pisum sativum
(SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8) and Glycine max
(SEQ ID NO: 9).
TABLE-US-00003 TABLE 3 Percent Identity of Amino Acid Sequences
Deduced From the Nucleotide Sequences of cDNA Clones Encoding
Polypeptides Homologous to Galactinol Synthase Percent Identity to
Clone SEQ ID NO: (Accession No.) sdp3c.pk013.c9:fis 2 82 (GI:
5541885) srr3c.pk003.h12:fis 4 75 (GI: 15223567)
srr3c.pk001.i20:fis 6 92 (GI: 32345694)
[0171] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a galactinol synthase.
These sequences represent new soybean sequences encoding galactinol
synthase.
[0172] The expression pattern of galactinol synthase 3, 4 and 5
during soybean seed development was analyzed via Lynx MPSS Brenner
et al (2000) Proc Natl Acad Sci USA 97:1665-70) and is shown in
Table 4.
TABLE-US-00004 TABLE 4* 15 20 30 40 45 50 55 Clone Designation DAF
DAF DAF DAF DAF DAF DAF mature sdp3c.pk013.c9 -- -- -- 6 143 796
1979 1604 srr3c.pk003.h12 -- -- -- -- 21 151 152 133
srr3c.pk001.i20 -- -- 13 -- 45 79 365 329 *Lynx MPSS profiles
(expressed as adjusted PPM) of galactinol synthase 3
(sdp3c.pk013.c9), galactinol synthase 4 (srr3c.pk003.h12) and
galactinol synthase 5 (srr3c.pk001.i20) during soybean seed
development (DAF = days after flowering, mature = mature seed).
[0173] The results shown in Table 4 demonstrate that expression of
all three galactinol synthases are only detectable during the later
stages of seed development (45 DAF to mature seed). Galactinol
synthase 4 is very lowly expressed compared to galactinol synthase
3 and 5, which show high and intermediate expression levels during
late seed development. The pattern of expression between galactinol
synthase 3 and 5 also differs: whereas galactinol synthase 3
expression levels increase during the course of late seed
development, reaching a plateau in the mature seed, galactinol
synthase 5 expression appears to be prominent mainly at 55 DAF and
in mature seed.
Example 4
Expression of Recombinant DNA Constructs in Dicot Cells
[0174] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the alpha
subunit of the seed storage protein phaseolin from the bean
Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem.
261:9228-9238) can be used for expression of the instant
polypeptides in transformed soybean. The phaseolin cassette
includes about 500 nucleotides upstream (5') from the translation
initiation codon and about 1650 nucleotides downstream (3') from
the translation stop codon of phaseolin. Between the 5' and 3'
regions are the unique restriction endonuclease sites Nco I (which
includes the ATG translation initiation codon), Sma I, Kpn I and
Xba I. The entire cassette is flanked by Hind III sites.
[0175] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0176] Soybean embryos may then be transformed with the expression
vector comprising sequences encoding the instant polypeptides. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0177] Soybean embryogenic suspension cultures can be maintained in
35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 mL of liquid medium.
[0178] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein et al.
(1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0179] A selectable marker gene which can be used to facilitate
soybean transformation is a recombinant DNA construct composed of
the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985)
Nature 313:810-812), the hygromycin phosphotransferase gene from
plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188)
and the 3' region of the nopaline synthase gene from the T-DNA of
the Ti plasmid of Agrobacterium tumefaciens. The seed expression
cassette comprising the phaseolin 5' region, the fragment encoding
the instant polypeptide and the phaseolin 3' region can be isolated
as a restriction fragment. This fragment can then be inserted into
a unique restriction site of the vector carrying the marker
gene.
[0180] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0181] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0182] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 5
Expression of Recombinant DNA Constructs in Microbial Cells
[0183] The cDNAs encoding the instant polypeptides can be inserted
into the T7 E. coli expression vector pBT430. This vector is a
derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135)
which employs the bacteriophage T7 RNA polymerase/T7 promoter
system. Plasmid pBT430 was constructed by first destroying the EcoR
I and Hind III sites in pET-3a at their original positions. An
oligonucleotide adaptor containing EcoR I and Hind III sites was
inserted at the BamH I site of pET-3a. This created pET-3aM with
additional unique cloning sites for insertion of genes into the
expression vector. Then, the Nde I site at the position of
translation initiation was converted to an Nco I site using
oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM
in this region, 5'-CATATGG, was converted to 5'-CCCATGG in
pBT430.
[0184] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% low melting agarose gel.
Buffer and agarose contain 10 .mu.g/mL ethidium bromide for
visualization of the DNA fragment. The fragment can then be
purified from the agarose gel by digestion with GELase (Epicentre
Technologies, Madison, Wis.) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20 L
of water. Appropriate oligonucleotide adapters may be ligated to
the fragment using T4 DNA ligase (New England Biolabs (NEB),
Beverly, Mass.). The fragment containing the ligated adapters can
be purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the instant polypeptide are then screened for the
correct orientation with respect to the T7 promoter by restriction
enzyme analysis.
[0185] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree. C. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
[0186] Crude, partially purified or purified enzyme, either alone
or as a fusion protein, may be utilized in assays for the
evaluation of enzymatic activity of the instant polypeptides
disclosed herein. Assays may be conducted under well-known
experimental conditions which permit optimal enzymatic activity.
Assays for galactinol synthase activity are presented by Odegard
and Lumen (1995) Plant Physiol. 109: 505-511.
Example 6
Construction of Chimeric Vectors for Seed-Targeted Co-Suppression
of Galactinol Synthase in Transgenic Glycine max
[0187] Vectors designed for the seed-specific co-suppression of
galactinol synthase 1 galactinol synthase 2 and galactinol synthase
3 in soybean were assembled as described below.
Amplification of Partial Galactinol Synthase Polynucleotides
[0188] Polynucleotide fragments encoding parts of the galactinol
synthase 1 (GAS1 (SEQ ID NO:6 of U.S. Pat. Nos. 5,773,699 and
5,648,210), galactinol synthase 2 (GAS2) in clone ses4d.pk0017.b8
(WO 01/77306) and galactinol synthase 3 (GAS3) in clone
sdp3c.pk013.c9 were amplified by standard PCR methods using Pfu
Turbo DNA polymerase (Stratagene, La Jolla, Calif.) and the
following primer sets. The GAS1 oligonucleotide primers were
designed to add a Not I restriction endonuclease site at the 5' end
and a XhoI site to the 3' end (SEQ ID NO:12 and SEQ ID NO:13,
respectively). The DNA sequence comprising the 519 by
polynucleotide from soybean GAS1 is shown in SEQ ID NO:14.
[0189] The GAS2 oligonucleotide primers were designed to add a XhoI
restriction endonuclease site at the 5' end and a PstI site to the
3' end (SEQ ID NO:15 and SEQ ID NO:16 respectively). The DNA
sequence comprising the 519 by polynucleotide from soybean GAS2 is
shown in SEQ ID NO:17.
[0190] The GAS3 oligonucleotide primers were designed to add a PstI
restriction endonuclease site at the 5' end and a NotI site to the
3' end (SEQ ID NO:18 and 19, respectively). The DNA sequence
comprising the 519 by polynucleotide from soybean GAS3 is shown in
SEQ ID NO:20.
Assembly of Vectors for the Co-Suppression of Galactinol
Synthase
[0191] Preparation of pJMS08: The polynucleotide products for GAS1,
GAS2 and GAS3 obtained from the amplifications described above were
digested with Not I, Xho1 and PSt1 and assembled into vector pJMS08
(FIG. 2) by the following steps. First, the plasmid KS151 [US
patent publication 2003/0036197A1] was digested with Not I. Then,
the isolated DNA fragments containing partial sequences of GAS1,
GAS2 and GAS3 were inserted into Not I-digested plasmid KS151 to
obtain plasmid pJMS08 (FIG. 2).
[0192] Plasmid KS151 also comprises nucleotides encoding HPT under
the control of the T7 promoter and termination signals and the 35S
promoter and Nos 3' terminator (US patent publication
2003/0036197A1). The KTi3 promoter and 3' transcription terminator
region have been described by Jofuku et al. ((1989) Plant Cell
1:1079-1093). The KTI3 promoter directs strong embryo-specific
expression of transgenes.
[0193] Preparation of pJMS10: The polynucleotide products for GAS1,
GAS2 and GAS3 obtained from the amplifications described above were
digested with Not I, Xho1 and PSt1 and assembled into vector pJMS10
(FIG. 3) by the following steps. From plasmid KS123 (prepared
according to US Application No. 2004/0073975 A1, which published on
Apr. 15, 2004) the HindIII cassette containing the beta-conglycinin
promoter-phaseolin terminator was removed creating the plasmid
KS120. To the unique BamHI site of plasmid KS120 a lea
promoter-phaseolin terminator was inserted as a BamHI fragment
creating plasmid KS127. The Lea promoter (Lee et al (1992) Plant
Physiol. 100:2121-2122; Genbank Accession no. M97285) was amplified
from genomic A2872 soybean DNA and a phaseolin 3' end was added as
described in US patent publication 2003/0036197 A1. To KS127 an EL
linker was added to a unique Not1 site as described in US patent
publication 2003/0036197 A1, creating plasmid KS139. To KS139 an EL
linker was added to a unique Not1 site as described in US patent
publication 2003/0036197 A1, creating plasmid KS147. Plasmid KS147
also comprises nucleotides encoding HPT under the control of the T7
promoter and termination signals and the 35S promoter and Nos 3'.
Then, the isolated DNA fragments containing partial sequences of
GAS1, GAS2 and GAS3 were inserted into the Not I-digested plasmid
KS147 to obtain plasmid pJMS10 (FIG. 3).
Example 7
Construction of Chimeric Vectors for Seed-Targeted Co-Suppression
of Galactinol Synthase in Transgenic Glycine max
[0194] Vectors designed for the seed-specific co-suppression of
galactinol synthase 3, galactinol synthase 4 and galactinol
synthase 5 in soybean can be assembled as described below.
Amplification of Partial Galactinol Synthase Polynucleotides
[0195] Polynucleotide fragments encoding parts of the galactinol
synthase 4 (SEQ ID NO: 3), galactinol synthase 5 (SEQ ID NO:5) and
galactinol synthase 3 (SEQ ID NO:1) are amplified by standard PCR
methods using Pfu Turbo DNA polymerase (Stratagene, La Jolla,
Calif.). Appropriate primer sets are chosen, giving polynucleotide
fragments of similar length as described for GAS1, 2 and 3 (Example
6), which is well within the routine skill in the art.
Assembly of Vectors for the Co-Suppression of Galactinol
Synthase
[0196] The assembly of vectors containing GAS 3, 4, and 5 for the
co-suppression of galactinol synthase is performed essentially as
described for GAS 1, 2 and 3 in Example 6.
[0197] Transformation into soybean somatic embryos and carbohydrate
analysis will be performed as described below for GAS 1, 2, and
3.
[0198] It is expected, that a similar reduction in Raffinose
Saccharides in soybean seeds will be observed using GAS 3, 4, 5 as
the one observed with GAS1, 2, and 3.
Example 8
Transformation of Soybean Somatic Embryos with Galactinol Synthase
Co-Suppression Vectors
[0199] To study the possibility of reducing Raffinose Family
Oligosaccharides (RFOs), soybean somatic embryos were transformed
with the seed-specific expression vectors pJMS08 (FIG. 2) or pJMS10
(FIG. 3) by the method of particle gun bombardment (Klein, T. M. et
al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050).
Soybean somatic embryos from the Jack cultivar were induced as
follows. Cotyledons (3 mm in length) were dissected from surface
sterilized, immature seeds and were cultured for an additional 6-10
weeks in the light at 26.degree. C. on a Murashige and Skoog media
containing 7 g/L agar and supplemented with 10 mg/mL 2,4-D.
Globular stage somatic embryos, which produced secondary embryos,
were then excised and placed into flasks containing liquid MS
medium supplemented with 2,4-D (10 mg/mL) and cultured in the light
on a rotary shaker. After repeated selection for clusters of
somatic embryos that multiplied as early, globular staged embryos,
the soybean embryogenic suspension cultures were maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
fluorescent lights on a 16:8 hour day/night schedule. Cultures were
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0200] Soybean embryogenic suspension cultures were then
transformed by the method of particle gun bombardment using a
DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit). To 50
.mu.L of a 60 mg/.mu.L 1 mm gold particle suspension were added (in
order): 5 .mu.L of 1 mg/.mu.L DNA (pJMS01 plus pJMS02, pRM02 plus
pRM03, pRM01, or pRM04), 20 .mu.L of 0.1 M spermidine, and 50 .mu.L
of 2.5 M CaCl.sub.2. The particle preparation was then agitated for
three minutes, spun in a microfuge for 10 seconds and the
supernatant removed. The DNA-coated particles were then washed once
in 400 .mu.L 70% ethanol and resuspended in 40 .mu.L of anhydrous
ethanol. The DNA/particle suspension was sonicated three times for
one second each. Five .mu.L of the DNA-coated gold particles was
then loaded on each macro carrier disk.
[0201] Approximately 300-400 mg of a two-week-old suspension
culture was placed in an empty 60.times.15-mm Petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5 to 10 plates of tissue
were bombarded. Membrane rupture pressure was set at 1100 psi and
the chamber was evacuated to a vacuum of 28 inches mercury. The
tissue was placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
was divided in half and placed back into liquid and cultured as
described above.
[0202] Five to seven days post bombardment, the liquid media was
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media was refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue was observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
was removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line was treated as an independent transformation event.
These suspensions were then subcultured and maintained as clusters
of immature embryos.
[0203] These immature soybean embryos were dried-down (by
transferring them into an empty small petridish that was seated on
top of a 10 cm petridish containing some agar gel to allow slow dry
down) to mimic the last stages of soybean seed development.
[0204] Dried-down embryos are capable of producing plants when
transferred to soil or soil-less media. Storage products produced
by embryos at this stage are similar in composition to storage
products produced by zygotic embryos at a similar stage of
development and most importantly the storage product profile is
predictive of plants derived from a somatic embryo line (WO
94/11516, published May 26, 1994)).
Example 9
Carbohydrate Analysis of Transgenic Soybean Somatic Embryos
[0205] The carbohydrate composition of transgenic somatic embryos
identified in Example 6 as containing the pJMS08 or pJMS10
cassettes was measured by high performance anion exchange
chromatography/pulsed amperometric detection (HPAE/PAD). Fresh
individual somatic embryos from transgenic lines were rapidly
washed in water, dried on a paper towel, and transferred into 1.5
mL microcentrifuge tubes. Ethanol (80%) was added to the tubes and
the samples were heated to 70.degree. C. for 15 minutes. The
samples were centrifuged at 14,000 rpm for 5 minutes at 4.degree.
C. and the supernatant collected. The pellet was re-extracted two
additional times with 80% ethanol at 70.degree. C. The supernatants
were combined, dried down in a speedvac, and the pellet
re-suspended in water.
[0206] For HPAE analysis, the extracts were filtered through a 0.2
.mu.m Nylon-66 filter (Rainin, Emeryville, Calif.) and analyzed by
HPAE/PAD using a DX500 anion exchange analyzer (Dionex, Sunnyvale,
Calif.) equipped with a 250.times.4 mm CarboPac PA1 anion exchange
column and a 25.times.4 mm CarboPac PA guard column. Soluble
carbohydrates were separated with a 25 minute linear gradient of
0.5 to 170 mM NaAc in 150 mM NaOH at a flow rate of 1.0 mL/min.
Soluble sugars were identified by comparison to standards (glucose,
fructose, sucrose, raffinose, stachyose, and verbascose) using
HPAE/PAD.
[0207] FIG. 4 and Table 5 show a typical carbohydrate profile
resulting from HPAE/PAD analysis of transgenic soybean somatic
embryos co-suppressing galactinol synthase. A clear reduction in
RFOs (raffinose, stachyose and vebascose) can be observed as
compared to FIG. 5 and wild type values in Table 5.
[0208] FIG. 5 shows a typical carbohydrate profile resulting from
HPAE/PAD analysis of a soybean somatic embryo showing a wild type
carbohydrate phenotype.
[0209] The results for two different events showing cosuppression
experiments of the three isoforms of Galactinol Synthases 1, 2, and
3 are shown in Table 5 above. For each event, 6 seeds were
analyzed. The results are expressed in .mu.mol/g dwt (sugar unit),
where the dry weight calculation was based on 7% moisture content
of seed.
TABLE-US-00005 TABLE 5 Strach/ Pheno- % Event seed wt(g) gol sucr
raff stach verb totRSA raff type reduction 1231- 1 0.14 1.37 179.99
18.35 11.90 0.14 43.94 0.65 Low 48.45 1-1-1 RFO 1231- 2 0.14 1.12
120.71 19.84 32.33 0.00 85.62 1.63 WT 1-1-1 1231- 3 0.19 0.68
159.38 18.06 20.57 0.00 59.88 1.14 Low 29.77 1-1-1 RFO 1231- 4 0.20
0.83 122.36 17.84 31.62 0.99 84.88 1.77 WT 1-1-1 1231- 5 0.19 0.78
148.20 16.14 19.20 0.00 55.32 1.19 Low 35.10 1-1-1 RFO 1231- 6 0.14
0.57 161.80 15.75 12.98 0.00 42.28 0.82 Low 50.41 1-1-1 RFO Mean
0.17 0.97 121.49 18.84 31.97 0.49 85.24 1.70 0.00 WT Mean 0.16 0.85
162.34 17.07 16.16 0.03 50.35 0.95 40.93 Low RFO 1231- 1 0.13 2.12
187.39 21.03 13.56 0.41 51.51 0.64 Low 52.67 1-1-3 RFO 1231- 2 0.13
2.15 186.36 25.69 16.09 0.43 61.30 0.63 Low 43.68 1-1-3 RFO 1231- 3
0.13 2.09 193.18 25.31 33.42 0.00 94.25 1.32 WT 1-1-3 1231- 4 0.15
0.81 231.88 20.46 8.90 0.21 39.72 0.44 Low 63.51 1-1-3 RFO 1231- 5
0.14 1.28 236.19 19.82 13.43 0.19 48.54 0.68 Low 55.40 1-1-3 RFO
1231- 6 0.15 1.63 164.68 17.05 43.41 1.11 108.84 2.55 WT 1-1-3 Mean
0.14 1.86 178.93 21.18 38.41 0.56 101.55 0.00 WT Mean 0.14 1.59
210.46 21.75 12.98 0.31 50.27 50.50 Low RFO TotRSA (total raffinose
saccharides) refers to the .alpha.-galactose content present in the
sum of galactinol (gol, 1 mol .alpha.-galactose/mole), raffinose
(raff, 1 mol .alpha.-galactose/mole), stachyose (stach, 2 mol
.alpha.-galactose/mole) and verbascose (verb, 3 mol
.alpha.-galactose/mole). Sucrose is sucr. % Reduction indicates the
change in total RFOs compared to the wild type.
Example 10
Isolation of Soybean PM29 Promoter
[0210] The promoter of a soybean seed maturation protein was
isolated using a polymerase chain reaction (PCR) based approach.
Soybean genomic DNA was digested to completion with a DNA
restriction enzyme that generates blunt ends (DraI, EcoRV, PvuII or
StuI, for example) according to standard protocols. The Universal
GenomeWalker.TM. kit from Clonetech.TM. (Product User Manual No.
PT3042-1) was used to ligate adaptors to the ends of the genomic
DNA fragments. Nested primers are also supplied in the Universal
GenomeWalker.TM. kit that are specific for the adaptor sequence
(AP1 and AP2, for the first and second adaptor primer,
respectively). Two gene specific primers (GSP1 and GSP2) were
designed for the soybean PM29 gene based on the 5' coding sequences
in PM29 cDNA in DuPont EST database. The oligonucleotide sequences
of the GSP1 and GSP2 primers (SEQ ID NO:21 and SEQ ID NO:22,
respectively) contain recognition sites for the restriction enzyme
BAMH I.
[0211] The AP2 primer from the Universal GenomeWalker.TM. kit
contains a Sal I restriction site. The AP1 and the GSP1 primers
were used in the first round PCR using each of the adaptor ligated
genomic DNA populations (DraI, EcoRV, PvuII or StuI) under
conditions defined in the GenomeWalker.TM. protocol. Cycle
conditions were 94.degree. C. for 4 minutes; 94.degree. C. for 2
seconds and 72.degree. C. for 3 minutes, 7 cycles; 94.degree. C.
for 2 seconds and 67.degree. C. for 3 minutes, 32 cycles;
67.degree. C. for 4 minutes. The products from each of the first
run PCRs were diluted 50-fold. One microliter from each of the
diluted products was used as templates for the second PCR with the
AP2 and GSP2 as primers. Cycle conditions were 94.degree. C. for 4
minutes; 94.degree. C. for 2 seconds and 72.degree. C. for 3
minutes, 5 cycles; 94.degree. C. for 2 seconds and 67.degree. C.
for 3 minutes, 20 cycles; 67.degree. C. for 3 minutes. Agarose gels
were run to determine which PCR gave an optimal fragment length. A
679 by genomic fragment was detected and isolated from the
EcoRV-digested genomic DNA reaction. The genomic fragment was
digested with BamH I and Sal I and cloned into Bluescript KS.sup.+
vector for sequencing. Finally, sequencing data indicated that this
genomic fragment contained a 597 by soybean PM29 promoter sequence
as shown in SEQ ID NO:23.
Example 11
Construction of Galactinol Synthase Silencing Plasmids Driven by
PM29
[0212] Two oligonucleotides were designed to re-amplify the PM29
promoter with either BamH I or Nco I sites (SEQ ID NO:24 and SEQ ID
NO:25, respectively). The re-amplified PM29 promoter fragment was
digested with BamH I and Nco I, purified and cloned into the BamH I
and Nco I sites of plasmid pG4G (FIG. 6) to make the fusion between
the soybean PM29 promoter-GUS fusion (pSH43). The plasmid pG4G has
been described in U.S. Pat. No. 5,968,793, the contents of which
are hereby incorporated by reference.
Preparation of SH55 and SH49:
[0213] Plasmid pSH43 (described above) was digested with NcoI,
filled in with vent polymerase (obtained from New England Biolabs
Inc.) and subsequently digested with BamHI (5' end of the
promoter). The resulting promoter fragment was isolated and cloned
into pBluescript II SK (+) (Stratagene, Inc.) previously cut with
XbaI (and filled in by vent polymerase) and BamHI creating the
plasmids pBluescriptPM29. This construct contains a unique Not1
site at the 3' end of the promoter. Two copies of the
Eag1-ELVISLIVES sequence (SEQ ID NO:26) were added on the 5' site
of the Not1 site as described in EP1297163 A2 (PCT Publication No.
WO 2002/000904, which published Jan. 3, 2002).
[0214] The promoter fragment was isolated using a BamHI/Not1
digestion and ligated into pJMS10 plasmid previously cut with BamH1
(partial) and Not1. The pJMS10 plasmid also contains the
complementary strand of SEQ ID NO:26 (SEQ ID NO:27) 3' of the Not1
site. This ligation resulted in the following plasmid: SH55 (PM29
promoter-ELEL-Not1-ELEL-Phaseolin terminator) and SH49. SH49 is
identical to SH55 with the exception of a truncated ELEL sequence
(SEQ ID NO:28) at the 3' border of the Not1. The truncated sequence
is missing the "tgacca" of the ELEL sequence at the 3' border of
the Not1 and was identified after sequence verification of the
plasmid and probably originated during the PCR amplification of the
ELEL linker. This truncation has no effect on the ability of
silencing the GAS genes as is evident from Example 12.
Preparation of SH50:
[0215] A Not1 fragment containing the partial sequences of soybean
GAS1 (SEQ ID NO:14), GAS2 (SEQ ID NO:17) and GAS3 (SEQ ID NO:20)
was digested from pJMS10 (described above) and then ligated into
SH49 previously digested with Not1, creating the plasmid SH50 (SEQ
ID NO:29 and FIG. 7).
Example 12
Raffinose Family Oligosaccharide (RFO) Analysis of PM29 Driven
Transgenic Soybean Somatic Embryos and Mature Seeds
[0216] Soybean somatic embryos were transformed with a
seed-preferred expression vector SH50 (SEQ ID NO:29 and FIG. 7) by
the method described in Example 8.
[0217] Raffinose Family Oligosaccharides (galactinol, raffinose,
stachyose, etc.) of transgenic somatic embryos and seeds containing
the PM29 promoter driven recombinant expression construct described
in Example 11 was measured by thin layer chromatography. Somatic
embryos or seed chips were extracted with hexane then dried. The
dried material was resuspended in 80% methanol, incubated at room
temperature for 1-2 hours, centrifuged, and 2 microliters of the
supernatant is spotted onto a TLC plate (Kieselgel 60 CF, from EM
Scientific, Gibbstown, N.J.; Catalog No. 13749-6). The TLC was run
in ethylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5
hours. The air dried plates were sprayed with 2% sulfuric acid and
heated until the charred sugars were detected. As shown in FIG. 8
two lines show reduced levels of raffinose sugars (raffinose and
stachyose lowest bands) when compared to a to wild-type soybean.
The arrow indicates somatic embryos with reduced raffinose family
oligosaccharides. (WT=wild type control, S=sucrose, Rf=raffinose
and St=stachyose standard). FIG. 9 shows a TLC analysis of mature
seed chips from a soybean line transformed with SH50 and revealed
an almost complete reduction in raffinose and stachyose (RFO)
sugars in seeds when compared to wild-type soybean. The plate shows
that 13 out of 17 seeds from a single event show a dramatic
reduction in raffinose family oligosaccharides (RFO).
Carbohydrate Analysis of Transgenic Soybean Seeds
[0218] The carbohydrate composition of transgenic soybean seeds
containing the PM29 promoter driven recombinant expression
construct described in Example 11 (SH50) was measured by high
performance anion exchange chromatography/pulsed amperometric
detection (HPAE/PAD). Seed chips were extracted in ethanol (80%)
and heated to 70.degree. C. for 15 minutes. The samples were
centrifuged at 14,000 rpm for 5 minutes at 4.degree. C. and the
supernatant collected. The pellet was re-extracted two additional
times with 80% ethanol at 70.degree. C. The supernatants were
combined, dried down in a speedvac, and the pellet re-suspended in
water.
[0219] For HPAE analysis, the extracts were filtered through a 0.2
.mu.m Nylon-66 filter (Rainin, Emeryville, Calif.) and analyzed by
HPAE/PAD using a DX500 anion exchange analyzer (Dionex, Sunnyvale,
Calif.) equipped with a 250.times.4 mm CarboPac PA1 anion exchange
column and a 25.times.4 mm CarboPac PA guard column. Soluble
carbohydrates were separated with a 20 isocratic run in 150 mM NaOH
at a flow rate of 1.0 mL/min. Soluble sugars were identified by
comparison to standards (glucose, fructose, sucrose, raffinose,
stachyose, and verbascose) using HPAE/PAD.
[0220] A typical profile of a soybean mutant characterized by
highly reduced raffinose family oligosaccharides content (Hitz et
al. 2002. Plant Physiology Vol 128, pp 650-660) is shown in FIG. 10
(Mutant HE2, left). As a comparison, the carbohydrate profile of
soybeans transformed with SH50 as described in Example 12 is shown
on the right (Transgenic seed with HE2 phenotype). Furthermore, the
sucrose to RFO ratio of the transgenic and mutant was very similar
and more than 10 fold higher when compared to wild type. The %
decrease in RFO when compared to wild type of some transgenic seeds
are shown in Table 6 and indicate a % reduction ranging from 71% to
89%. In comparison, the percent reduction of the mutant was
85%.
TABLE-US-00006 TABLE 6 Sucrose to RFO ratio and % decrease in RFO
of transgenic soybean seeds (transformed with SH50) when compared
with Jack wild type. The % reduction in RFO of a known mutant is
included as a reference. A U V 1 Event S/RFO ratio % decrease in
RFO 2 AFS74042-5-1-1, seed 1 6.8 71 3 AFS4042-5-1-1, seed 5 15.9 87
4 AFS4042-5-1-1, seed 7 11.2 82 5 AFS4042-5-1-1, seed 12 12.7 85 6
AFS4042-5-1-1, seed 18 17.8 89 7 AFS4042-5-1-1, seed 20 12.5 82 8
AFS4042-5-1-3, seed 7 16.3 87 9 AFS4042-5-1-4, seed 2 14.6 85 10
AFS4042-5-1-4, seed 7 10.5 80 11 12 Jack-wild type 0 14 Low RFO
mutant 85
Example 13
Construction of Galactinol Synthase Silencing Plasmids Driven by
.beta.-Conglycinin and KTI3
[0221] Plasmid pJMS10 (FIG. 3) was prepared as described in Example
6.
[0222] Preparation of plasmid pDS3:
[0223] pKR57 (FIG. 11) (4479 bp; SEQ ID NO:30) was digested with
Eco RI and Not I, run on a 0.8%
Tris-Acetate-Ethylenediaminetetraacetic acid-agarose gel
(TAE-agarose gel) and a 3144 by fragment containing the
.beta.-conglycinin promoter, an origin of replication and a gene
encoding ampicillin resistance was purified using the Qiagen gel
extraction kit. pKR63 (FIG. 12) (5010 bp; SEQ ID NO:31) was
digested with Eco RI and Not I, run on a 0.8% TAE-agarose gel and a
2270 by fragment containing the KTi promoter was purified using the
Qiagen gel extraction kit. The isolated fragments were ligated
together and the ligation was transformed into E. coli and colonies
were selected on ampicillin. Bacterial colonies were selected and
grown overnight in LB media and appropriate antibiotic selection.
DNA was isolated from the resulting culture using a Qiagen miniprep
kit according to the manufacturer's protocol and then analyzed by
restriction digest. The resulting plasmid was named pDS1 (FIG. 13)
(5414 bp; SEQ ID NO:32).
[0224] pKR72 (FIG. 14) (7085 bp; SEQ ID NO:33) was digested with
Hind III, run on a 0.8% TAE-agarose gel and a 5303 by fragment
containing a gene that encodes resistance to hygromycin operably
linked to a prokaryotic promoter and a gene that encodes resistance
to hygromycin operably linked to a eukaryotic promoter were
purified using the Qiagen gel extraction kit. The fragment was
ligated to itself and the ligation was transformed into E. coli and
colonies were selected on hygromycin. Bacterial colonies were
selected and grown overnight in LB media and appropriate antibiotic
selection. DNA was isolated from the resulting culture using a
Qiagen miniprep kit according to the manufacturer's protocol and
then analyzed by restriction digest. The resulting plasmid was
named pDS2 (FIG. 15) (5303 bp; SEQ ID NO:34).
[0225] pDS2 was digested with Sal I and the ends were
dephosphorylated with calf intestinal alkaline phosphatase (CIAP)
according to the manufacture's instructions (Stratagene, San Diego,
Calif.). pDS1 was digested with Sal I and Fsp I, run on a 0.8%
TAE-agarose gel and a 2728 by fragment containing the KTi3 promoter
and the .beta.-conglycinin promoter in opposite orientations was
purified using the Qiagen gel extraction kit. The isolated
fragments were ligated together and the ligation was transformed
into E. coli and colonies were selected on hygromycin. Bacterial
colonies were selected and grown overnight in LB media and
appropriate antibiotic selection. DNA was isolated from the
resulting culture using a Qiagen miniprep kit according to the
manufacturer's protocol and then analyzed by restriction digest.
The resulting plasmids were named pDS3 [orientation 2 (FIG. 16, SEQ
ID NO: 35)].
[0226] Preparation of SH60:
[0227] pJMS10 (FIG. 3) was digested with Not1, run on a 0.8%
TAE-agarose gel and a 1585 by DNA fragment (SEQ ID NO:37)
comprising the partial sequences of GAS1 (SEQ ID NO:14), GAS2 (SEQ
ID NO:17) and GAS3 (SEQ ID NO:20) was purified using the Qiagen gel
extraction kit. pDS3 (orientation 2) (FIG. 16, SEQ ID NO: 35) was
digested with Not1, run on a 0.8% TAE-agarose gel and a 8031 by DNA
fragment was purified using the Qiagen gel extraction kit. The
isolated fragments were ligated together and the ligation was
transformed into E. coli and colonies were selected on hygromycin.
Bacterial colonies were selected and grown overnight in LB media
and appropriate antibiotic selection. DNA was isolated from the
resulting culture using a Qiagen miniprep kit according to the
manufacturer's protocol and then analyzed by restriction digest.
The resulting plasmid was named SH60 (FIG. 17, SEQ ID NO: 36).
Example 14
Reduction of Raffinose Family Oligosaccharide (RFO) in Transgenic
Soybean Somatic Embryos
[0228] SH60, as described in Example 13, was transformed into
soybean embryogenic suspension cultures using a protocol as
described in Example 8 above. Individual immature soybean embryos
were dried-down (by transferring them into an empty small petridish
that was seated on top of a 10 cm petridish containing some agar
gel to allow slow dry down) to mimic the last stages of soybean
seed development. Dried-down embryos are capable of producing
plants when transferred to soil or soil-less media. Storage
products produced by embryos at this stage are similar in
composition to storage products produced by zygotic embryos at a
similar stage of development and most importantly the storage
product profile is predictive of plants derived from a somatic
embryo line (PCT Publication No. WO 94/11516, which published on
May 26, 1994). Raffinose Family Oligosaccharides (raffinose,
stachyose) of transgenic somatic embryos containing the
B-conglycinin/KTI3 driven (SH60) recombinant expression construct
described in Example 13 was measured by thin layer chromatography.
Somatic embryos were extracted with hexane then dried. The dried
material was re-suspended in 80% methanol, incubated at room
temperature for 1-2 hours, centrifuged, and 2 .mu.l of the
supernatant is spotted onto a TLC plate (Kieselgel 60 CF, from EM
Scientific, Gibbstown, N.J.; Catalog No. 13749-6). The TLC was run
methylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5 hours.
The air dried plates were sprayed with 2% sulfuric acid and heated
until the charred sugars were detected. As shown in FIG. 18 the
embryos labeled "Low RFO embryos" show reduced levels of raffinose
sugars (raffinose and stachyose s) when compared to a to wild-type
soybean. Five out of eleven (45%) lines analyzed showed reduced
levels of RFOs, which is demonstrative of reduced galactinol
synthase expression (see Table 7).
TABLE-US-00007 TABLE 7 Positive Transformed Lines with Reduced
Galactinol Synthase Expression carbohydrate phenotype GAS1GAS2GAS3
lines with wild type 6 out of 11 RFO levels GAS1GAS2GAS3 lines with
reduced 5 out of 11 RFO levels Percent gene silencing 45%
Sequence CWU 1
1
3711151DNAGlycine maxCDS(71)..(1090) 1ctaagctctc ttttagtctt
actcacaaac acttttttca ctgcttccat tacgaacata 60tatttattat atg gct
cct gaa ctt gtc ccc acc gtt gtg aaa tcc agt 109 Met Ala Pro Glu Leu
Val Pro Thr Val Val Lys Ser Ser 1 5 10gct gcg ttc acg aaa ccc gcg
acc ctt cca agg cgt gcc tac gtg aca 157Ala Ala Phe Thr Lys Pro Ala
Thr Leu Pro Arg Arg Ala Tyr Val Thr 15 20 25ttc ctc gcc gga aac ggt
gac tac gtg aaa ggg gtg gtt ggc ctc gcc 205Phe Leu Ala Gly Asn Gly
Asp Tyr Val Lys Gly Val Val Gly Leu Ala30 35 40 45aaa ggg ttg cga
aag gtg aaa acc gcg tac ccg ttg gtg gtg gct gtc 253Lys Gly Leu Arg
Lys Val Lys Thr Ala Tyr Pro Leu Val Val Ala Val 50 55 60ctc ccc gat
gtg ccg gag gag cac cgt aag atc ctg gag tct cag ggc 301Leu Pro Asp
Val Pro Glu Glu His Arg Lys Ile Leu Glu Ser Gln Gly 65 70 75tgc atc
gtt cgc gag atc gaa ccc gtt tac cca ccc gaa aac caa acc 349Cys Ile
Val Arg Glu Ile Glu Pro Val Tyr Pro Pro Glu Asn Gln Thr 80 85 90cag
ttt gcc atg gct tat tac gtc atc aac tac tcc aag ctc cgt ata 397Gln
Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile 95 100
105tgg gag ttt gtg gag tac agc aag atg ata tac ttg gac gga gac att
445Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp
Ile110 115 120 125gag gta tat gag aac ata gac cac cta ttt gac cta
cct gat ggt aac 493Glu Val Tyr Glu Asn Ile Asp His Leu Phe Asp Leu
Pro Asp Gly Asn 130 135 140ttt tac gct gtg atg gat tgt ttc tgc gag
aag aca tgg agt cac acc 541Phe Tyr Ala Val Met Asp Cys Phe Cys Glu
Lys Thr Trp Ser His Thr 145 150 155cct cag tac aag gtg ggt tac tgc
cag caa tgc ccg gag aag gtg cgg 589Pro Gln Tyr Lys Val Gly Tyr Cys
Gln Gln Cys Pro Glu Lys Val Arg 160 165 170tgg ccc acc gaa ttg ggt
cag ccc cct tct ctt tac ttc aac gct ggc 637Trp Pro Thr Glu Leu Gly
Gln Pro Pro Ser Leu Tyr Phe Asn Ala Gly 175 180 185atg ttc gtg ttc
gaa ccc aac atc gcc acc tat cat gac cta ttg aaa 685Met Phe Val Phe
Glu Pro Asn Ile Ala Thr Tyr His Asp Leu Leu Lys190 195 200 205acg
gtg caa gtc acc act ccc acc tcg ttc gct gaa caa gat ttc ttg 733Thr
Val Gln Val Thr Thr Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu 210 215
220aac atg tac ttc aag gac att tac aag cca atc cct tta aat tac aat
781Asn Met Tyr Phe Lys Asp Ile Tyr Lys Pro Ile Pro Leu Asn Tyr Asn
225 230 235ctt gtc ctc gcc atg ctg tgg cgc cac ccg gaa aac gtt aaa
tta gac 829Leu Val Leu Ala Met Leu Trp Arg His Pro Glu Asn Val Lys
Leu Asp 240 245 250caa gtc aag gtt gtt cac tat tgc gca gcg ggg tcc
aag cca tgg aga 877Gln Val Lys Val Val His Tyr Cys Ala Ala Gly Ser
Lys Pro Trp Arg 255 260 265tat acg ggg aag gaa gag aat atg cag agg
gag gac ata aag atg ctg 925Tyr Thr Gly Lys Glu Glu Asn Met Gln Arg
Glu Asp Ile Lys Met Leu270 275 280 285gtg aag aaa tgg tgg gat atc
tac aat gat gct tcg ctt gac tac aag 973Val Lys Lys Trp Trp Asp Ile
Tyr Asn Asp Ala Ser Leu Asp Tyr Lys 290 295 300cca ttg atg aat gca
agt gaa gct cca gca gcg gat ggt gtt gac att 1021Pro Leu Met Asn Ala
Ser Glu Ala Pro Ala Ala Asp Gly Val Asp Ile 305 310 315gaa caa ttc
gtg cag gct cta tca gag gtt ggt cat gtt caa tat gtc 1069Glu Gln Phe
Val Gln Ala Leu Ser Glu Val Gly His Val Gln Tyr Val 320 325 330acc
gcg cct tca gca gct taa ttaagagggc acattcaaat cacgacaaaa 1120Thr
Ala Pro Ser Ala Ala 335aacaaccaag tgaaaaaaaa aaaaaaaaaa a
11512339PRTGlycine max 2Met Ala Pro Glu Leu Val Pro Thr Val Val Lys
Ser Ser Ala Ala Phe1 5 10 15Thr Lys Pro Ala Thr Leu Pro Arg Arg Ala
Tyr Val Thr Phe Leu Ala 20 25 30Gly Asn Gly Asp Tyr Val Lys Gly Val
Val Gly Leu Ala Lys Gly Leu 35 40 45Arg Lys Val Lys Thr Ala Tyr Pro
Leu Val Val Ala Val Leu Pro Asp 50 55 60Val Pro Glu Glu His Arg Lys
Ile Leu Glu Ser Gln Gly Cys Ile Val65 70 75 80Arg Glu Ile Glu Pro
Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala 85 90 95Met Ala Tyr Tyr
Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe 100 105 110Val Glu
Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp Ile Glu Val Tyr 115 120
125Glu Asn Ile Asp His Leu Phe Asp Leu Pro Asp Gly Asn Phe Tyr Ala
130 135 140Val Met Asp Cys Phe Cys Glu Lys Thr Trp Ser His Thr Pro
Gln Tyr145 150 155 160Lys Val Gly Tyr Cys Gln Gln Cys Pro Glu Lys
Val Arg Trp Pro Thr 165 170 175Glu Leu Gly Gln Pro Pro Ser Leu Tyr
Phe Asn Ala Gly Met Phe Val 180 185 190Phe Glu Pro Asn Ile Ala Thr
Tyr His Asp Leu Leu Lys Thr Val Gln 195 200 205Val Thr Thr Pro Thr
Ser Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr 210 215 220Phe Lys Asp
Ile Tyr Lys Pro Ile Pro Leu Asn Tyr Asn Leu Val Leu225 230 235
240Ala Met Leu Trp Arg His Pro Glu Asn Val Lys Leu Asp Gln Val Lys
245 250 255Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr
Thr Gly 260 265 270Lys Glu Glu Asn Met Gln Arg Glu Asp Ile Lys Met
Leu Val Lys Lys 275 280 285Trp Trp Asp Ile Tyr Asn Asp Ala Ser Leu
Asp Tyr Lys Pro Leu Met 290 295 300Asn Ala Ser Glu Ala Pro Ala Ala
Asp Gly Val Asp Ile Glu Gln Phe305 310 315 320Val Gln Ala Leu Ser
Glu Val Gly His Val Gln Tyr Val Thr Ala Pro 325 330 335Ser Ala
Ala31398DNAGlycine maxCDS(94)..(1089) 3gcacgaggtg atttttgctt
aattactaaa ccaaaccatt tcttattccc tcatcgaaac 60cttttctttc tatatatttc
ccttttcaat atc atg gca cct aac atc acc acc 114 Met Ala Pro Asn Ile
Thr Thr 1 5gtt gtt gcc aat gcc acc act gag caa tta ccc aaa gct cat
gga gga 162Val Val Ala Asn Ala Thr Thr Glu Gln Leu Pro Lys Ala His
Gly Gly 10 15 20agt agt ggg cgt gcc ttt gtg act ttt ctt gct gga aac
ggt gat tat 210Ser Ser Gly Arg Ala Phe Val Thr Phe Leu Ala Gly Asn
Gly Asp Tyr 25 30 35gta aag ggt gtt gtg ggt ttg gcc aaa gga ctg aga
aag gcc aaa agc 258Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu Arg
Lys Ala Lys Ser40 45 50 55atg tac cct ttg gtg gtt gct gtg tta cca
gat gtt cct gaa gaa cat 306Met Tyr Pro Leu Val Val Ala Val Leu Pro
Asp Val Pro Glu Glu His 60 65 70cgt gcg att ctc aaa tcc caa ggt tgc
att gtc agg gag att gaa cct 354Arg Ala Ile Leu Lys Ser Gln Gly Cys
Ile Val Arg Glu Ile Glu Pro 75 80 85gtg tac cct cct aag aac cag acc
cag ttc gcc atg gcc tat tat gtc 402Val Tyr Pro Pro Lys Asn Gln Thr
Gln Phe Ala Met Ala Tyr Tyr Val 90 95 100atc aat tac tcc aag cta
cgt att tgg gag ttc gtg gag tac cag aag 450Ile Asn Tyr Ser Lys Leu
Arg Ile Trp Glu Phe Val Glu Tyr Gln Lys 105 110 115atg ata tac cta
gac ggc gac atc caa gtt ttt gga aac att gac cac 498Met Ile Tyr Leu
Asp Gly Asp Ile Gln Val Phe Gly Asn Ile Asp His120 125 130 135ttg
ttt gat ctt cct aat aat tat ttc tat gcg gtg atg gat tgt ttc 546Leu
Phe Asp Leu Pro Asn Asn Tyr Phe Tyr Ala Val Met Asp Cys Phe 140 145
150tgc gag aag act tgg agc cac acc cct cag ttc cag att ggg tac tgc
594Cys Glu Lys Thr Trp Ser His Thr Pro Gln Phe Gln Ile Gly Tyr Cys
155 160 165caa cag tgc cct gat aag gtt caa tgg ccc tct cac ttt ggt
acc aaa 642Gln Gln Cys Pro Asp Lys Val Gln Trp Pro Ser His Phe Gly
Thr Lys 170 175 180cct cct cta tat ttc aat gct ggc atg ttt gtt tat
gag cct aat ctc 690Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val Tyr
Glu Pro Asn Leu 185 190 195aac acc tac cgt cat ctt ctc caa act gtc
caa gtc atc aag ccc acg 738Asn Thr Tyr Arg His Leu Leu Gln Thr Val
Gln Val Ile Lys Pro Thr200 205 210 215tcc ttt gct gag cag gac ttt
ctg aac atg tac ttc aag gac aag tac 786Ser Phe Ala Glu Gln Asp Phe
Leu Asn Met Tyr Phe Lys Asp Lys Tyr 220 225 230aag cca ata ccg aac
gtg tac aac ctt gtg ctg gcc atg ttg tgg cgt 834Lys Pro Ile Pro Asn
Val Tyr Asn Leu Val Leu Ala Met Leu Trp Arg 235 240 245cac cct gag
aat gtt gaa ctt gat caa gtt caa gtg gtt cat tac tgt 882His Pro Glu
Asn Val Glu Leu Asp Gln Val Gln Val Val His Tyr Cys 250 255 260gct
gct ggg tct aag cct tgg agg ttc act ggg aag gaa gag aac atg 930Ala
Ala Gly Ser Lys Pro Trp Arg Phe Thr Gly Lys Glu Glu Asn Met 265 270
275gat agg gaa gat atc aag atg ctt atg aag aag tgg tgg gac ata tat
978Asp Arg Glu Asp Ile Lys Met Leu Met Lys Lys Trp Trp Asp Ile
Tyr280 285 290 295gaa gat gag aca ctg gac tac aat aac aac tct gtc
aat gtg gaa cgt 1026Glu Asp Glu Thr Leu Asp Tyr Asn Asn Asn Ser Val
Asn Val Glu Arg 300 305 310ttc aca tca gta cta ttg gat gct ggg ggt
ttt cag ttt gtg cca gca 1074Phe Thr Ser Val Leu Leu Asp Ala Gly Gly
Phe Gln Phe Val Pro Ala 315 320 325cct tct gct gcc taa tatgattcac
agctacaaat taaagtctaa ttaacgacaa 1129Pro Ser Ala Ala 330agtatatatg
tattgttatt tgtttttgtt ttttttttcg tttttgggtc ttatgaacga
1189accacgtcta tagttttaat ttggatgacc tttttgtata caaagtcaca
tgtgacgtct 1249tacagctttt gattattatt aagatttaat tatatgagtc
ctttacttaa tttgttttca 1309ttgatcaaga gttgtggata tatatatata
tatatatata tctttaattt tattaaatga 1369aattttaagg caaaaaaaaa
aaaaaaaaa 13984331PRTGlycine max 4Met Ala Pro Asn Ile Thr Thr Val
Val Ala Asn Ala Thr Thr Glu Gln1 5 10 15Leu Pro Lys Ala His Gly Gly
Ser Ser Gly Arg Ala Phe Val Thr Phe 20 25 30Leu Ala Gly Asn Gly Asp
Tyr Val Lys Gly Val Val Gly Leu Ala Lys 35 40 45Gly Leu Arg Lys Ala
Lys Ser Met Tyr Pro Leu Val Val Ala Val Leu 50 55 60Pro Asp Val Pro
Glu Glu His Arg Ala Ile Leu Lys Ser Gln Gly Cys65 70 75 80Ile Val
Arg Glu Ile Glu Pro Val Tyr Pro Pro Lys Asn Gln Thr Gln 85 90 95Phe
Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp 100 105
110Glu Phe Val Glu Tyr Gln Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln
115 120 125Val Phe Gly Asn Ile Asp His Leu Phe Asp Leu Pro Asn Asn
Tyr Phe 130 135 140Tyr Ala Val Met Asp Cys Phe Cys Glu Lys Thr Trp
Ser His Thr Pro145 150 155 160Gln Phe Gln Ile Gly Tyr Cys Gln Gln
Cys Pro Asp Lys Val Gln Trp 165 170 175Pro Ser His Phe Gly Thr Lys
Pro Pro Leu Tyr Phe Asn Ala Gly Met 180 185 190Phe Val Tyr Glu Pro
Asn Leu Asn Thr Tyr Arg His Leu Leu Gln Thr 195 200 205Val Gln Val
Ile Lys Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn 210 215 220Met
Tyr Phe Lys Asp Lys Tyr Lys Pro Ile Pro Asn Val Tyr Asn Leu225 230
235 240Val Leu Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp
Gln 245 250 255Val Gln Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro
Trp Arg Phe 260 265 270Thr Gly Lys Glu Glu Asn Met Asp Arg Glu Asp
Ile Lys Met Leu Met 275 280 285Lys Lys Trp Trp Asp Ile Tyr Glu Asp
Glu Thr Leu Asp Tyr Asn Asn 290 295 300Asn Ser Val Asn Val Glu Arg
Phe Thr Ser Val Leu Leu Asp Ala Gly305 310 315 320Gly Phe Gln Phe
Val Pro Ala Pro Ser Ala Ala 325 33051417DNAGlycine
maxCDS(213)..(1184) 5ccaagatctt aaaatatctc ttccatacaa gtttgttttc
aaagtgtttt tgtctcccaa 60atcctactct tgtgaccaca agccttcact tcactctctc
tctctctctc tctctctctc 120tctctctctc tctctctttt ttgaaaccct
tttttctctt ctcaaaccaa accaagcaag 180caattatatt acactactca
ctcactgaga cc atg gct cct aat atc acc acc 233 Met Ala Pro Asn Ile
Thr Thr 1 5gtc acc gac gct caa gcc aag gcc gcc ggc ggg cgt ggc cgt
gcc tac 281Val Thr Asp Ala Gln Ala Lys Ala Ala Gly Gly Arg Gly Arg
Ala Tyr 10 15 20gtc acc ttc ctc gcc gga aac ggt gac tat gtg aaa ggt
gtc gtt ggc 329Val Thr Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly
Val Val Gly 25 30 35ttg gcc aaa ggt ctg agg aag gtg aaa agc atg tac
cct ctg gtg gtt 377Leu Ala Lys Gly Leu Arg Lys Val Lys Ser Met Tyr
Pro Leu Val Val40 45 50 55gca gtg tta ccc gat gtt cca gaa cat cac
cga aac att ctc acc tcc 425Ala Val Leu Pro Asp Val Pro Glu His His
Arg Asn Ile Leu Thr Ser 60 65 70caa ggt tgc att gtt aga gaa att gaa
ccc gtg tac cct cct gag aat 473Gln Gly Cys Ile Val Arg Glu Ile Glu
Pro Val Tyr Pro Pro Glu Asn 75 80 85cag acg cag ttc gcc atg gca tat
tac gtc atc aac tat tcc aag cta 521Gln Thr Gln Phe Ala Met Ala Tyr
Tyr Val Ile Asn Tyr Ser Lys Leu 90 95 100cgt att tgg gag ttt gtg
gag ttc agc aag atg ata tac cta gac ggt 569Arg Ile Trp Glu Phe Val
Glu Phe Ser Lys Met Ile Tyr Leu Asp Gly 105 110 115gat ata caa gtg
ttt gac aat att gac cac ttg ttt gac ttg cct gat 617Asp Ile Gln Val
Phe Asp Asn Ile Asp His Leu Phe Asp Leu Pro Asp120 125 130 135aac
tac ttt tat gcg gtg atg gac tgt ttt tgt gag ccc act tgg ggc 665Asn
Tyr Phe Tyr Ala Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly 140 145
150cac act ctg cag tat caa atc gga tac tgc cag cag tgc cct cat aag
713His Thr Leu Gln Tyr Gln Ile Gly Tyr Cys Gln Gln Cys Pro His Lys
155 160 165gtt cag tgg ccc act cac ttt ggg ccc aag cct cct ctc tat
ttc aat 761Val Gln Trp Pro Thr His Phe Gly Pro Lys Pro Pro Leu Tyr
Phe Asn 170 175 180gct ggc atg ttt gtt tat gag ccc aat ctg gat acc
tac cgt gac ctc 809Ala Gly Met Phe Val Tyr Glu Pro Asn Leu Asp Thr
Tyr Arg Asp Leu 185 190 195ctt caa act gtc caa gtc act aag ccc act
tcc ttt gct gaa cag gat 857Leu Gln Thr Val Gln Val Thr Lys Pro Thr
Ser Phe Ala Glu Gln Asp200 205 210 215ttt ttg aac atg tac ttc aag
gac aaa tat agg cca att cct aat gtc 905Phe Leu Asn Met Tyr Phe Lys
Asp Lys Tyr Arg Pro Ile Pro Asn Val 220 225 230tat aat ctt gtg ttg
gcc atg ctg tgg cgt cac cct gag aac gtt gag 953Tyr Asn Leu Val Leu
Ala Met Leu Trp Arg His Pro Glu Asn Val Glu 235 240 245ctt gaa aaa
gtt aaa gtg gtt cac tac tgt gct gct gga tct aag cct 1001Leu Glu Lys
Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro 250 255 260tgg
agg tac aca ggg aag gag gaa aat atg gag aga gaa gat atc aag 1049Trp
Arg Tyr Thr Gly Lys Glu Glu Asn Met Glu Arg Glu Asp Ile Lys 265 270
275atg ttg gtg aag aag tgg tgg gat ata tat gag gat gag act ttg gac
1097Met Leu Val Lys Lys Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu
Asp280 285 290 295tac aac aat cca ttc aac gtg gat agg ttc act gcg
gca ctt ttg gag 1145Tyr Asn Asn Pro Phe Asn Val Asp Arg Phe Thr Ala
Ala Leu Leu Glu 300 305 310gtt ggt gaa gtc aag ttc gtc cgt gcc cca
tct gct gct tagagtgtct 1194Val Gly Glu Val Lys Phe Val Arg Ala Pro
Ser Ala Ala 315 320ttggaaatca agtgtgatcc aagtacatag gataagatat
acagacccat acatcattaa 1254gttttatgtg tttttaaagt gtttagagga
cctttttatg tgtccctttt ttcttttttc 1314tttttcaatt ctgccattgt
aaagcagtga aataccatgt ccttaatttt attattggat 1374atgaatttta
ttttgtacgt
tctctaaaaa aaaaaaaaaa aaa 14176324PRTGlycine max 6Met Ala Pro Asn
Ile Thr Thr Val Thr Asp Ala Gln Ala Lys Ala Ala1 5 10 15Gly Gly Arg
Gly Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 20 25 30Tyr Val
Lys Gly Val Val Gly Leu Ala Lys Gly Leu Arg Lys Val Lys 35 40 45Ser
Met Tyr Pro Leu Val Val Ala Val Leu Pro Asp Val Pro Glu His 50 55
60His Arg Asn Ile Leu Thr Ser Gln Gly Cys Ile Val Arg Glu Ile Glu65
70 75 80Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala Met Ala Tyr
Tyr 85 90 95Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu
Phe Ser 100 105 110Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe
Asp Asn Ile Asp 115 120 125His Leu Phe Asp Leu Pro Asp Asn Tyr Phe
Tyr Ala Val Met Asp Cys 130 135 140Phe Cys Glu Pro Thr Trp Gly His
Thr Leu Gln Tyr Gln Ile Gly Tyr145 150 155 160Cys Gln Gln Cys Pro
His Lys Val Gln Trp Pro Thr His Phe Gly Pro 165 170 175Lys Pro Pro
Leu Tyr Phe Asn Ala Gly Met Phe Val Tyr Glu Pro Asn 180 185 190Leu
Asp Thr Tyr Arg Asp Leu Leu Gln Thr Val Gln Val Thr Lys Pro 195 200
205Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Lys
210 215 220Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu Ala Met
Leu Trp225 230 235 240Arg His Pro Glu Asn Val Glu Leu Glu Lys Val
Lys Val Val His Tyr 245 250 255Cys Ala Ala Gly Ser Lys Pro Trp Arg
Tyr Thr Gly Lys Glu Glu Asn 260 265 270Met Glu Arg Glu Asp Ile Lys
Met Leu Val Lys Lys Trp Trp Asp Ile 275 280 285Tyr Glu Asp Glu Thr
Leu Asp Tyr Asn Asn Pro Phe Asn Val Asp Arg 290 295 300Phe Thr Ala
Ala Leu Leu Glu Val Gly Glu Val Lys Phe Val Arg Ala305 310 315
320Pro Ser Ala Ala7334PRTPisum sativum 7Met Ala Pro Glu Ile Val Gln
Thr Ser Thr Lys Pro Val Thr Gly Phe1 5 10 15Thr Lys Leu Lys Arg Ala
Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 20 25 30Tyr Val Lys Gly Val
Ile Gly Leu Ala Lys Gly Leu Arg Lys Val Lys 35 40 45Thr Ala Tyr Pro
Leu Val Val Ala Val Leu Pro Asp Val Pro Glu Glu 50 55 60His Arg Glu
Met Leu Glu Ser Gln Gly Cys Ile Val Arg Glu Ile Gln65 70 75 80Pro
Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala Met Ala Tyr Tyr 85 90
95Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Tyr Ser
100 105 110Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Tyr Glu Asn
Ile Asp 115 120 125His Leu Phe Asp Leu Pro Asp Gly Tyr Phe Tyr Ala
Val Met Asp Cys 130 135 140Phe Cys Glu Lys Thr Trp Ser His Thr Pro
Gln Tyr Lys Ile Gly Tyr145 150 155 160Cys Gln Gln Cys Pro Glu Lys
Val Gln Trp Pro Lys Glu Met Gly Glu 165 170 175Pro Pro Ser Leu Tyr
Phe Asn Ala Gly Met Phe Leu Phe Glu Pro Ser 180 185 190Val Glu Thr
Tyr Asp Asp Leu Leu Lys Thr Cys Gln Val Thr Ala Pro 195 200 205Thr
Pro Phe Ala Asp Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Ile 210 215
220Tyr Arg Pro Ile Pro Leu Val Tyr Asn Leu Val Leu Ala Met Leu
Trp225 230 235 240Arg His Pro Glu Asn Val Glu Leu Arg Lys Val Lys
Val Val His Tyr 245 250 255Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr
Thr Gly Lys Glu Glu Asn 260 265 270Met Gln Arg Glu Asp Ile Lys Met
Leu Val Gln Lys Trp Leu Asp Ile 275 280 285Tyr Ser Asp Ser Ser Leu
Asp Tyr Lys Lys Asn Leu Ser Gly Asn Cys 290 295 300Glu Thr Gln Arg
Asn Asp Val Glu Glu Pro Phe Val Gln Ala Leu Ser305 310 315 320Glu
Val Gly Arg Val Arg Tyr Val Thr Ala Pro Ser Ala Ala 325
3308335PRTArabidopsis thaliana 8Met Ala Pro Glu Ile Asn Thr Lys Leu
Thr Val Pro Val His Ser Ala1 5 10 15Thr Gly Gly Glu Lys Arg Ala Tyr
Val Thr Phe Leu Ala Gly Thr Gly 20 25 30Asp Tyr Val Lys Gly Val Val
Gly Leu Ala Lys Gly Leu Arg Lys Ala 35 40 45Lys Ser Lys Tyr Pro Leu
Val Val Ala Val Leu Pro Asp Val Pro Glu 50 55 60Asp His Arg Lys Gln
Leu Val Asp Gln Gly Cys Val Val Lys Glu Ile65 70 75 80Glu Pro Val
Tyr Pro Pro Glu Asn Gln Thr Glu Phe Ala Met Ala Tyr 85 90 95Tyr Val
Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Tyr 100 105
110Asn Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe Asp Asn Ile
115 120 125Asp His Leu Phe Asp Leu Pro Asn Gly Gln Phe Tyr Ala Val
Met Asp 130 135 140Cys Phe Cys Glu Lys Thr Trp Ser His Ser Pro Gln
Tyr Lys Ile Gly145 150 155 160Tyr Cys Gln Gln Cys Pro Asp Lys Val
Thr Trp Pro Glu Ala Lys Leu 165 170 175Gly Pro Lys Pro Pro Leu Tyr
Phe Asn Ala Gly Met Phe Val Tyr Glu 180 185 190Pro Asn Leu Ser Thr
Tyr His Asn Leu Leu Glu Thr Val Lys Ile Val 195 200 205Pro Pro Thr
Leu Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys 210 215 220Asp
Ile Tyr Lys Pro Ile Pro Pro Val Tyr Asn Leu Val Leu Ala Met225 230
235 240Leu Trp Arg His Pro Glu Asn Ile Glu Leu Asp Gln Val Lys Val
Val 245 250 255His Tyr Cys Ala Ala Gly Ala Lys Pro Trp Arg Phe Thr
Gly Glu Glu 260 265 270Glu Asn Met Asp Arg Glu Asp Ile Lys Met Leu
Val Lys Lys Trp Trp 275 280 285Asp Ile Tyr Asn Asp Glu Ser Leu Asp
Tyr Lys Asn Val Val Ile Gly 290 295 300Asp Ser His Lys Lys Gln Gln
Thr Leu Gln Gln Phe Ile Glu Ala Leu305 310 315 320Ser Glu Ala Gly
Ala Leu Gln Tyr Val Lys Ala Pro Ser Ala Ala 325 330
3359328PRTGlycine max 9Met Ala Pro Asn Ile Thr Thr Val Lys Thr Thr
Ile Thr Asp Ala Gln1 5 10 15Ala Lys Val Ala Thr Asp His Gly Arg Ala
Tyr Val Thr Phe Leu Ala 20 25 30Gly Asn Gly Asp Tyr Val Lys Gly Val
Val Gly Leu Ala Lys Gly Leu 35 40 45Arg Lys Val Lys Ser Met Tyr Pro
Leu Val Val Ala Val Leu Pro Asp 50 55 60Val Pro Gln Asp His Arg Asn
Ile Leu Thr Ser Gln Gly Cys Ile Val65 70 75 80Arg Glu Ile Glu Pro
Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala 85 90 95Met Ala Tyr Tyr
Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe 100 105 110Val Glu
Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe 115 120
125Asp Asn Ile Asp His Leu Phe Asp Leu Pro Asp Asn Tyr Phe Tyr Ala
130 135 140Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly His Thr Lys
Gln Tyr145 150 155 160Gln Ile Gly Tyr Cys Gln Gln Cys Pro His Lys
Val Gln Trp Pro Thr 165 170 175His Phe Gly Pro Lys Pro Pro Leu Tyr
Phe Asn Ala Gly Met Phe Val 180 185 190Tyr Glu Pro Asn Leu Ala Thr
Tyr Arg Asp Leu Leu Gln Thr Val Gln 195 200 205Val Thr Gln Pro Thr
Ser Phe Ala Glu Gln Asp Phe Leu Asn Ile Tyr 210 215 220Phe Lys Asp
Lys Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu225 230 235
240Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Lys Val Lys
245 250 255Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr
Thr Gly 260 265 270Lys Glu Glu Asn Met Glu Arg Glu Asp Ile Lys Met
Leu Val Lys Lys 275 280 285Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu
Asp Tyr Asn Asn Pro Leu 290 295 300Asn Val Asp Lys Phe Thr Ala Ala
Leu Met Glu Val Gly Glu Val Lys305 310 315 320Phe Val Arg Ala Pro
Ser Ala Ala 325101406DNAGlycine max 10gtttgttttc aaagtgtgtt
ttgtttccca aatcctactc ttgtgaccac aacccttcct 60cctctttctt ttgaaacctc
tttttttcta ttccccaacc aaacaagcaa acgctactca 120ctcatcatca
ctgagatcat ggctcctaat atcaccactg tcaaaaccac catcaccgac
180gctcaagcca aggtcgccac cgatcatggt cgtgcctacg tcaccttcct
cgccggaaac 240ggtgactatg tgaaaggtgt cgttggcttg gcaaaaggtc
tgagaaaagt gaagagcatg 300taccctctgg tggttgcagt gctacccgat
gttccccaag atcaccgcaa cattctcacc 360tcccaaggtt gcattgttag
agagattgag cccgtgtacc ccccagagaa tcaaacccag 420tttgccatgg
catattacgt catcaactat tccaagctac gtatttggga gtttgtggag
480tacagcaaga tgatatacct agacggtgat atccaagttt ttgacaacat
tgaccacttg 540tttgacttgc ctgataacta cttctatgcg gtgatggact
gtttctgtga gccaacttgg 600ggccacacta aacaatatca gatcggttac
tgccagcagt gcccccataa ggttcagtgg 660cccactcact ttgggcccaa
acctcctctc tatttcaatg ctggcatgtt tgtgtatgag 720cccaatttgg
ctacttaccg tgacctcctt caaacagtcc aagtcaccca gcccacttcc
780tttgctgaac aggatttttt gaacatgtac ttcaaggaca aatataggcc
aattcctaat 840gtctacaatc ttgtgctggc catgctgtgg cgtcaccctg
agaacgttga gcttgacaaa 900gttaaagtgg ttcactactg tgctgctggg
tctaagcctt ggaggtacac tgggaaggag 960gagaatatgg agagagaaga
tatcaagatg ttagtgaaaa agtggtggga tatatatgag 1020gatgagactt
tggactacaa caatccactc aatgtggata agttcactgc ggcacttatg
1080gaggttggtg aagtcaagtt cgtccgtgcc ccatctgctg cttaagagtg
tctttggaaa 1140tcaagtgtga tccaagtaca tgtacaaagt catacatcat
tacattaact tttatgtatt 1200tctaaaagtc atacatcatt acattaagtt
ttatgtattt ctaaagtctt aagacttaag 1260aggacctttt ttatkkkkcc
cgcttttctt tttttctttt tccaattctg tcattgtaaa 1320gsrgagaata
ccgtatcctt aattttataa atggatatga attttatttg tactaaaggg
1380ggggccggta ccaattcgcc tatagt 1406111350DNAGlycine max
11gcacgagaaa caaccaacct cttcagtgat ctttgattag tactaagcta aaccatttct
60tattccctca aaatcaaaac ctttttcttt ctagctattt cccttttcaa atcatgccac
120ctaacatcac caccgttgtt gccaatgtca ccaccgagca attacccaag
gctcgtggag 180gaagtgggcg tgccttcgtg acctttcttg ctgggaacgg
tgattacgta aagggtgtcg 240tgggtttggc caaaggactg agaaaggcca
aaagcatgta ccctttggtg gttgctgtgt 300taccagatgt tcctgaagaa
catcgtgaga ttctcaaatc ccaaggttgc attgtcaggg 360agattgaacc
tgtgtaccct cctgagaacc agacccagtt cgccatggcc tattatgtca
420tcaattactc caagctacgt atttgggagt tcgtggagta caagaagacg
atatacctag 480acggtgacat ccaagtattt ggaaacatag accacttgtt
tgatctgcct gataattatt 540tctatgcggt gatggattgt ttctgcgaga
agacttggag ccacacccct cagttccaga 600ttgggtactg ccaacagtgc
cctgataagg ttcaatggcc ctctcacttt ggttccaaac 660ctcctctata
tttcaatgct ggcatgtttg tttatgagcc taatctcgac acctaccgtg
720atcttctcca aactgtccaa ctcaccaagc ccacttcttt tgctgagcag
gactttctca 780acatgtactt caaggacaag tacaagccaa taccgaacat
gtacaacctt gtgctggcca 840tgttgtggcg tcaccctgaa aatgttgaac
ttgataaagt tcaagtggtt cattactgtg 900ctgctgggtc taagccttgg
aggttcactg ggaaggaaga gaacatggat agggaagata 960tcaagatgct
tgtgaagaag tggtgggaca tatatgaaga tgagacactg gactacaata
1020acaactctgt caacgtggaa cgtttcacat cggcactatt ggatgctggg
ggctttcagt 1080ttgtgccagc accttctgct gcctaatatg cttattattt
acagctacaa attaatgtta 1140attaacgaca aagtatatgt attgttattt
gctttttttc gtttttgggt cttatatatg 1200aaggaacaac gtctatggtt
ttaatttgga tgaccttctt gtatacaaag ccacatgtga 1260tctcatacag
cttttgatta ttattaagaa attagaggac cttttattat gagtccttta
1320cttaaaaaaa aaaaaaaaaa aaaaaaaaaa 13501236DNAArtificialPrimer
12aagcttgcgg ccgcgtcatc aactattcca agctac 361336DNAArtificialPrimer
13aagcttctcg agtcacttcc cagtgtacct ccaagg 3614519DNAGlycine Max
14gtcatcaact attccaagct acgtatttgg gagtttgtgg agtacagcaa gatgatatac
60ctagacggtg atatccaagt ttttgacaac attgaccact tgtttgactt gcctgataac
120tacttctatg cggtgatgga ctgtttctgt gagccaactt ggggccacac
taaacaatat 180cagatcggtt actgccagca gtgcccccat aaggttcagt
ggcccactca ctttgggccc 240aaacctcctc tctatttcaa tgctggcatg
tttgtgtatg agcccaattt ggctacttac 300cgtgacctcc ttcaaacagt
ccaagtcacc cagcccactt cctttgctga acaggatttt 360ttgaacatgt
acttcaagga caaatatagg ccaattccta atgtctacaa tcttgtgctg
420gccatgctgt ggcgtcaccc tgagaacgtt gagcttgaca aagttaaagt
ggttcactac 480tgtgctgctg ggtctaagcc ttggaggtac actgggaag
5191534DNAArtificialPrimer 15aagcttctcg aggtcatcaa ttactccaag ctac
341643DNAArtificialPrimer 16agcttgcggc cgcctgcagt tacttcccag
tgaacctcca agg 4317519DNAGlycine max 17gtcatcaatt actccaagct
acgtatttgg gagttcgtgg agtacaagaa gacgatatac 60ctagacggtg acatccaagt
atttggaaac atagaccact tgtttgatct gcctgataat 120tatttctatg
cggtgatgga ttgtttctgc gagaagactt ggagccacac ccctcagttc
180cagattgggt actgccaaca gtgccctgat aaggttcaat ggccctctca
ctttggttcc 240aaacctcctc tatatttcaa tgctggcatg tttgtttatg
agcctaatct cgacacctac 300cgtgatcttc tccaaactgt ccaactcacc
aagcccactt cttttgctga gcaggacttt 360ctcaacatgt acttcaagga
caagtacaag ccaataccga acatgtacaa ccttgtgctg 420gccatgttgt
ggcgtcaccc tgaaaatgtt gaacttgata aagttcaagt ggttcattac
480tgtgctgctg ggtctaagcc ttggaggttc actgggaag
5191842DNAArtificialPrimer 18aagcttgcgg ccgcctgcag gtcatcaact
actccaagct cc 421937DNAArtificialPrimer 19aagcttgcgg ccgctacttc
cccgtatatc tccatgg 3720519DNAGlycine max 20gtcatcaact actccaagct
ccgtatatgg gagtttgtgg agtacagcaa gatgatatac 60ttggacggag acattgaggt
atatgagaac atagaccacc tatttgacct acctgatggt 120aacttttacg
ctgtgatgga ttgtttctgc gagaagacat ggagtcacac ccctcagtac
180aaggtgggtt actgccagca atgcccggag aaggtgcggt ggcccaccga
attgggtcag 240cccccttctc tttacttcaa cgctggcatg ttcgtgttcg
aacccaacat cgccacctat 300catgacctat tgaaaacggt gcaagtcacc
actcccacct cgttcgctga acaagatttc 360ttgaacatgt acttcaagga
catttacaag ccaatccctt taaattacaa tcttgtcctc 420gccatgctgt
ggcgccaccc ggaaaacgtt aaattagacc aagtcaaggt tgttcactat
480tgcgcagcgg ggtccaagcc atggagatat acggggaag
5192126DNAArtificialPrimer 21tcttctgttc ttgccgttgc tttctc
262233DNAArtificialPrimer 22cgcggatccg acttgctcct tggcagcact ggt
3323597DNAGlycine max 23agagttttta taagttattt tatacatgaa ttaattttaa
cttgtgaaaa aaattatttt 60cttcttataa gtatttatga caaagcttat ataaacatag
tcttaatttc actcagaaaa 120acagaggagg aaaacttgtt gtatgaagcc
cggctatttc atccattatc catatttgga 180tcgaaaagag aaggaaagtg
tcattttata tgtgtataaa aagtatttca tccataagta 240atgataagat
aattgtgtat gtaacattat taatgtattt aaattaaaat cataaattat
300tttaaacaat tcttattcgt tagtgacacg ataacggata agctaataat
atatctatgg 360ttttctgtga acgtggcagc atattgatgg gaatagctct
gcatgttgaa caagtggcac 420ggtacctagc gtgccttgct cttcttttgt
ctaggcttgg tttggttcgc atcttccttc 480tcatataaat cctccaccac
gtcgagtttt ctgttcaaat taaatcgttc aacactggaa 540ctctttgata
taatatagaa agagacagag agagagagac agacaagaag aacaagg
5972442DNAArtificialPrimer 24cgcggatcca gagtttttat aagttatttt
atacatgaat ta 422539DNAArtificialPrimer 25ccttgaccat ggttgttctt
cttgtctgtc tctctctct 392681DNAArtificialtwo copies of
Eag1-ELVISLIVES 26cggccggagc tggtcatctc gctcatcgtc gagtcggcgg
ccggagctgg tcatctcgct 60catcgtcgag tcggcggccg c
812781DNAArtificialComplementary strand of two copies of
Eag1-ELVISLIVES 27gcggccgccg actcgacgat gagcgagatg accagctccg
gccgccgact cgacgatgag 60cgagatgacc agctccggcc g
812869DNAArtificialtruncated version of the two copies of
ELVISLIVES linker 28gcggccgccg actcgacgat gagcgagatg accagctccg
gccgccgact cgacgatgag 60cgagagctc 69298810DNAArtificialPlasmid SH50
29ggccgccgac tcgacgatga gcgagatgac cagctccggc cgccgactcg acgatgagcg
60agagctccgg ccgcaagtat gaactaaaat gcacgtaggt gtaagagctc atggagagca
120tggaatattg tatccgacca tgtaacagta taataactga gctccatctc
acttcttcta 180tgaataaaca aaggatgtta tgatatatta acactctatc
tatgcacctt attgttctat 240gataaatttc ctcttattat tataaatcat
ctgaatcgtg acggcttatg gaatgcttca 300aatagtacaa aaacaaatgt
gtactataag actttctaaa caattctaac tttagcattg 360tgaacgagac
ataagtgtta agaagacata acaattataa tggaagaagt ttgtctccat
420ttatatatta
tatattaccc acttatgtat tatattagga tgttaaggag acataacaat
480tataaagaga gaagtttgta tccatttata tattatatac tacccattta
tatattatac 540ttatccactt atttaatgtc tttataaggt ttgatccatg
atatttctaa tattttagtt 600gatatgtata tgaaagggta ctatttgaac
tctcttactc tgtataaagg ttggatcatc 660cttaaagtgg gtctatttaa
ttttattgct tcttacagat aaaaaaaaaa ttatgagttg 720gtttgataaa
atattgaagg atttaaaata ataataaata acatataata tatgtatata
780aatttattat aatataacat ttatctataa aaaagtaaat attgtcataa
atctatacaa 840tcgtttagcc ttgctggacg aatctcaatt atttaaacga
gagtaaacat atttgacttt 900ttggttattt aacaaattat tatttaacac
tatatgaaat tttttttttt atcagcaaag 960aataaaatta aattaaggag
gacaatggtg tcccaatcct tatacaacca acttccacaa 1020gaaagtcaag
tcagagacaa caaaaaaaca agcaaaggaa attttttaat ttgagttgtc
1080ttgtttgctg cataatttat gcagtaaaac actacacata acccttttag
cagtaaagca 1140atggttgacc gtgtgcttag cttcttttat tttatttttt
tatcagcaaa gaataaataa 1200aataaaatga gacacttcag ggatgtttca
acggatccaa gcttggcgcg ccgttctata 1260gtgtcaccta aatcgtatgt
gtatgataca taaggttatg tattaattgt agccgcgttc 1320taacgacaat
atgtccatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
1380aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
gtctgctccc 1440ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
tgcatgtgtc agaggttttc 1500accgtcatca ccgaaacgcg cgagacgaaa
gggcctcgtg atacgcctat ttttataggt 1560taatgtcatg accaaaatcc
cttaacgtga gttttcgttc cactgagcgt cagaccccgt 1620agaaaagatc
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
1680aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
taccaactct 1740ttttccgaag gtaactggct tcagcagagc gcagatacca
aatactgtcc ttctagtgta 1800gccgtagtta ggccaccact tcaagaactc
tgtagcaccg cctacatacc tcgctctgct 1860aatcctgtta ccagtggctg
ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 1920aagacgatag
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
1980gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
agcattgaga 2040aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
ccggtaagcg gcagggtcgg 2100aacaggagag cgcacgaggg agcttccagg
gggaaacgcc tggtatcttt atagtcctgt 2160cgggtttcgc cacctctgac
ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2220cctatggaaa
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
2280tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
ttaccgcctt 2340tgagtgagct gataccgctc gccgcagccg aacgaccgag
cgcagcgagt cagtgagcga 2400ggaagcggaa gagcgcccaa tacgcaaacc
gcctctcccc gcgcgttggc cgattcatta 2460atgcaggttg atcagattcg
acatcgatct agtaacatag atgacaccgc gcgcgataat 2520ttatcctagt
ttgcgcgcta tattttgttt tctatcgcgt attaaatgta taattgcggg
2580actctaatca taaaaaccca tctcataaat aacgtcatgc attacatgtt
aattattaca 2640tgcttaacgt aattcaacag aaattatatg ataatcatcg
caagaccggc aacaggattc 2700aatcttaaga aactttattg ccaaatgttt
gaacgatctg cttcgacgca ctccttcttt 2760aggtacctca ctattccttt
gccctcggac gagtgctggg gcgtcggttt ccactatcgg 2820cgagtacttc
tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac
2880gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc
gcccaagctg 2940catcatcgaa attgccgtca accaagctct gatagagttg
gtcaagacca atgcggagca 3000tatacgcccg gagccgcggc gatcctgcaa
gctccggatg cctccgctcg aagtagcgcg 3060tctgctgctc catacaagcc
aaccacggcc tccagaagaa gatgttggcg acctcgtatt 3120gggaatcccc
gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg
3180tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg
gggcagtcct 3240cggcccaaag catcagctca tcgagagcct gcgcgacgga
cgcactgacg gtgtcgtcca 3300tcacagtttg ccagtgatac acatggggat
cagcaatcgc gcatatgaaa tcacgccatg 3360tagtgtattg accgattcct
tgcggtccga atgggccgaa cccgctcgtc tggctaagat 3420cggccgcagc
gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg
3480tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa
taggtcaggc 3540tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg
gagcgcggcc gatgcaaagt 3600gccgataaac ataacgatct ttgtagaaac
catcggcgca gctatttacc cgcaggacat 3660atccacgccc tcctacatcg
aagctgaaag cacgagattc ttcgccctcc gagagctgca 3720tcaggtcgga
gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga
3780gttcaggctt tttcatggtt taataagaag agaaaagagt tcttttgtta
tggctgaagt 3840aatagagaaa tgagctcgag cgtgtcctct ccaaatgaaa
tgaacttcct tatatagagg 3900aagggtcttg cgaaggatag tgggattgtg
cgtcatccct tacgtcagtg gagatgtcac 3960atcaatccac ttgctttgaa
gacgtggttg gaacgtcttc tttttccacg atgctcctcg 4020tgggtggggg
tccatctttg ggaccactgt cggcagaggc atcttgaatg atagcctttc
4080ctttatcgca atgatggcat ttgtaggagc caccttcctt ttctactgtc
ctttcgatga 4140agtgacagat agctgggcaa tggaatccga ggaggtttcc
cgaaattatc ctttgttgaa 4200aagtctcaat agccctttgg tcttctgaga
ctgtatcttt gacatttttg gagtagacca 4260gagtgtcgtg ctccaccatg
ttgacgaaga ttttcttctt gtcattgagt cgtaaaagac 4320tctgtatgaa
ctgttcgcca gtcttcacgg cgagttctgt tagatcctcg atttgaatct
4380tagactccat gcatggcctt agattcagta ggaactacct ttttagagac
tccaatctct 4440attacttgcc ttggtttatg aagcaagcct tgaatcgtcc
atactggaat agtacttctg 4500atcttgagaa atatgtcttt ctctgtgttc
ttgatgcaat tagtcctgaa tcttttgact 4560gcatctttaa ccttcttggg
aaggtatttg atctcctgga gattgttact cgggtagatc 4620gtcttgatga
gacctgctgc gtaggcctct ctaaccatct gtgggtcagc attctttctg
4680aaattgaaga ggctaacctt ctcattatca gtggtgaaca tagtgtcgtc
accttcacct 4740tcgaacttcc ttcctagatc gtaaagatag aggaaatcgt
ccattgtaat ctccggggca 4800aaggagatct cttttggggc tggatcactg
ctgggccttt tggttcctag cgtgagccag 4860tgggcttttt gctttggtgg
gcttgttagg gccttagcaa agctcttggg cttgagttga 4920gcttctcctt
tggggatgaa gttcaacctg tctgtttgct gacttgttgt gtacgcgtca
4980gctgctgctc ttgcctctgt aatagtggca aatttcttgt gtgcaactcc
gggaacgccg 5040tttgttgccg cctttgtaca accccagtca tcgtatatac
cggcatgtgg accgttatac 5100acaacgtagt agttgatatg agggtgttga
atacccgatt ctgctctgag aggagcaact 5160gtgctgttaa gctcagattt
ttgtgggatt ggaattggat cgatctcgat cccgcgaaat 5220taatacgact
cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
5280tttaagaagg agatataccc atggaaaagc ctgaactcac cgcgacgtct
gtcgagaagt 5340ttctgatcga aaagttcgac agcgtctccg acctgatgca
gctctcggag ggcgaagaat 5400ctcgtgcttt cagcttcgat gtaggagggc
gtggatatgt cctgcgggta aatagctgcg 5460ccgatggttt ctacaaagat
cgttatgttt atcggcactt tgcatcggcc gcgctcccga 5520ttccggaagt
gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
5580gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
gttctgcagc 5640cggtcgcgga ggctatggat gcgatcgctg cggccgatct
tagccagacg agcgggttcg 5700gcccattcgg accgcaagga atcggtcaat
acactacatg gcgtgatttc atatgcgcga 5760ttgctgatcc ccatgtgtat
cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg 5820tcgcgcaggc
tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
5880tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
ataacagcgg 5940tcattgactg gagcgaggcg atgttcgggg attcccaata
cgaggtcgcc aacatcttct 6000tctggaggcc gtggttggct tgtatggagc
agcagacgcg ctacttcgag cggaggcatc 6060cggagcttgc aggatcgccg
cggctccggg cgtatatgct ccgcattggt cttgaccaac 6120tctatcagag
cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
6180acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
cgcagaagcg 6240cggccgtctg gaccgatggc tgtgtagaag tactcgccga
tagtggaaac cgacgcccca 6300gcactcgtcc gagggcaaag gaatagtgag
gtacagcttg gatcgatccg gctgctaaca 6360aagcccgaaa ggaagctgag
ttggctgctg ccaccgctga gcaataacta gcataacccc 6420ttggggcctc
taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
6480gatcgggcgc gccgtcgacg gtatcgataa gcttgatatc gaattcctgc
agcccggggg 6540atccagagtt tttataagtt attttataca tgaattaatt
ttaacttgtg aaaaaaatta 6600ttttcttctt ataagtattt atgacaaagc
ttatataaac atagtcttaa tttcactcag 6660aaaaacagag gaggaaaact
tgttgtatga agcccggcta tttcatccat tatccatatt 6720tggatcgaaa
agagaaggaa agtgtcattt tatatgtgta taaaaagtat ttcatccata
6780agtaatgata agataattgt gtatgtaaca ttattaatgt atttaaatta
aaatcataaa 6840ttattttaaa caattcttat tcgttagtga cacgataacg
gataagctaa taatatatct 6900atggttttct gtgaacgtgg cagcatattg
atgggaatag ctctgcatgt tgaacaagtg 6960gcacggtacc tagcgtgcct
tgctcttctt ttgtctaggc ttggtttggt tcgcatcttc 7020cttctcatat
aaatcctcca ccacgtcgag ttttctgttc aaattaaatc gttcaacact
7080ggaactcttt gatataatat agaaagagac agagagagag agacagacaa
gaagaacaac 7140catgctagag cggccggagc tggtcatctc gctcatcgtc
gagtcggcgg ccggagctgg 7200tcatctcgct catcgtcgag tcggcggccg
cgtcatcaac tattccaagc tacgtatttg 7260ggagtttgtg gagtacagca
agatgatata cctagacggt gatatccaag tttttgacaa 7320cattgaccac
ttgtttgact tgcctgataa ctacttctat gcggtgatgg actgtttctg
7380tgagccaact tggggccaca ctaaacaata tcagatcggt tactgccagc
agtgccccca 7440taaggttcag tggcccactc actttgggcc caaacctcct
ctctatttca atgctggcat 7500gtttgtgtat gagcccaatt tggctactta
ccgtgacctc cttcaaacag tccaagtcac 7560ccagcccact tcctttgctg
aacaggattt tttgaacatg tacttcaagg acaaatatag 7620gccaattcct
aatgtctaca atcttgtgct ggccatgctg tggcgtcacc ctgagaacgt
7680tgagcttgac aaagttaaag tggttcacta ctgtgctgct gggtctaagc
cttggaggta 7740cactgggaag tgactcgagg tcatcaatta ctccaagcta
cgtatttggg agttcgtgga 7800gtacaagaag acgatatacc tagacggtga
catccaagta tttggaaaca tagaccactt 7860gtttgatctg cctgataatt
atttctatgc ggtgatggat tgtttctgcg agaagacttg 7920gagccacacc
cctcagttcc agattgggta ctgccaacag tgccctgata aggttcaatg
7980gccctctcac tttggttcca aacctcctct atatttcaat gctggcatgt
ttgtttatga 8040gcctaatctc gacacctacc gtgatcttct ccaaactgtc
caactcacca agcccacttc 8100ttttgctgag caggactttc tcaacatgta
cttcaaggac aagtacaagc caataccgaa 8160catgtacaac cttgtgctgg
ccatgttgtg gcgtcaccct gaaaatgttg aacttgataa 8220agttcaagtg
gttcattact gtgctgctgg gtctaagcct tggaggttca ctgggaagta
8280actgcaggtc atcaactact ccaagctccg tatatgggag tttgtggagt
acagcaagat 8340gatatacttg gacggagaca ttgaggtata tgagaacata
gaccacctat ttgacctacc 8400tgatggtaac ttttacgctg tgatggattg
tttctgcgag aagacatgga gtcacacccc 8460tcagtacaag gtgggttact
gccagcaatg cccggagaag gtgcggtggc ccaccgaatt 8520gggtcagccc
ccttctcttt acttcaacgc tggcatgttc gtgttcgaac ccaacatcgc
8580cacctatcat gacctattga aaacggtgca agtcaccact cccacctcgt
tcgctgaaca 8640agatttcttg aacatgtact tcaaggacat ttacaagcca
atccctttaa attacaatct 8700tgtcctcgcc atgctgtggc gccacccgga
aaacgttaaa ttagaccaag tcaaggttgt 8760tcactattgc gcagcggggt
ccaagccatg gagatatacg gggaagtagc 8810304479DNAArtificialPlasmid
pKR57 30ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
gtttcctgtg 60tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
aaagtgtaaa 120gcctggggtg cctaatgagt gagctaactc acattaattg
cgttgcgctc actgcccgct 180ttccagtcgg gaaacctgtc gtgccagctg
cattaatgaa tcggccaacg cgcggggaga 240ggcggtttgc gtattgggcg
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 300gttcggctgc
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
360tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
caggaaccgt 420aaaaaggccg cgttgctggc gtttttccat aggctccgcc
cccctgacga gcatcacaaa 480aatcgacgct caagtcagag gtggcgaaac
ccgacaggac tataaagata ccaggcgttt 540ccccctggaa gctccctcgt
gcgctctcct gttccgaccc tgccgcttac cggatacctg 600tccgcctttc
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
660agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
cgttcagccc 720gaccgctgcg ccttatccgg taactatcgt cttgagtcca
acccggtaag acacgactta 780tcgccactgg cagcagccac tggtaacagg
attagcagag cgaggtatgt aggcggtgct 840acagagttct tgaagtggtg
gcctaactac ggctacacta gaaggacagt atttggtatc 900tgcgctctgc
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
960caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
gcgcagaaaa 1020aaaggatctc aagaagatcc tttgatcttt tctacggggt
ctgacgctca gtggaacgaa 1080aactcacgtt aagggatttt ggtcatgaga
ttatcaaaaa ggatcttcac ctagatcctt 1140ttaaattaaa aatgaagttt
taaatcaatc taaagtatat atgagtaaac ttggtctgac 1200agttaccaat
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
1260atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
accatctggc 1320cccagtgctg caatgatacc gcgagaccca cgctcaccgg
ctccagattt atcagcaata 1380aaccagccag ccggaagggc cgagcgcaga
agtggtcctg caactttatc cgcctccatc 1440cagtctatta attgttgccg
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 1500aacgttgttg
ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
1560ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
gtgcaaaaaa 1620gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
agttggccgc agtgttatca 1680ctcatggtta tggcagcact gcataattct
cttactgtca tgccatccgt aagatgcttt 1740tctgtgactg gtgagtactc
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 1800tgctcttgcc
cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
1860ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
gctgttgaga 1920tccagttcga tgtaacccac tcgtgcaccc aactgatctt
cagcatcttt tactttcacc 1980agcgtttctg ggtgagcaaa aacaggaagg
caaaatgccg caaaaaaggg aataagggcg 2040acacggaaat gttgaatact
catactcttc ctttttcaat attattgaag catttatcag 2100ggttattgtc
tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
2160gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
tattatcatg 2220acattaacct ataaaaatag gcgtatcacg aggccctttc
gtctcgcgcg tttcggtgat 2280gacggtgaaa acctctgaca catgcagctc
ccggagacgg tcacagcttg tctgtaagcg 2340gatgccggga gcagacaagc
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 2400tggcttaact
atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
2460ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
attcaggctg 2520cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
tattacgcca gctggcgaaa 2580gggggatgtg ctgcaaggcg attaagttgg
gtaacgccag ggttttccca gtcacgacgt 2640tgtaaaacga cggccagtga
attcgagctc ggtacccggg gatcctctag acgtacgttg 2700aaacatccct
gaagtgtctc attttatttt atttattctt tgctgataaa aaaataaaat
2760aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta
tgtgtagtgt 2820tttactgcat aaattatgca gcaaacaaga caactcaaat
taaaaaattt cctttgcttg 2880tttttttgtt gtctctgact tgactttctt
gtggaagttg gttgtataag gattgggaca 2940ccattgtcct tcttaattta
attttattct ttgctgataa aaaaaaaaat ttcatatagt 3000gttaaataat
aatttgttaa ataaccaaaa agtcaaatat gtttactctc gtttaaataa
3060ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat
ttactttttt 3120atagataaat gttatattat aataaattta tatacatata
ttatatgtta tttattatta 3180ttttaaatcc ttcaatattt tatcaaacca
actcataatt ttttttttat ctgtaagaag 3240caataaaatt aaatagaccc
actttaagga tgatccaacc tttatacaga gtaagagagt 3300tcaaatagta
ccctttcata tacatatcaa ctaaaatatt agaaatatca tggatcaaac
3360cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt
atataatata 3420taaatggata caaacttctc tctttataat tgttatgtct
ccttaacatc ctaatataat 3480acataagtgg gtaatatata atatataaat
ggagacaaac ttcttccatt ataattgtta 3540tgtcttctta acacttatgt
ctcgttcaca atgctaaggt tagaattgtt tagaaagtct 3600tatagtacac
atttgttttt gtactatttg aagcattcca taagccgtca cgattcagat
3660gatttataat aataagagga aatttatcat agaacaataa ggtgcataga
tagagtgtta 3720atatatcata acatcctttg tttattcata gaagaagtga
gatggagctc agttattata 3780ctgttacatg gtcggataca atattccatg
ctctccatga gctcttacac ctacatgcat 3840tttagttcat acttgcggcc
gcagtatatc ttaaattctt taatacggtg tactaggata 3900ttgaactggt
tcttgatgat gaaaacctgg gccgagattg cagctattta tagtcatagg
3960tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac
gggtgttgaa 4020ataaacaaaa atgaggtggc ggaagagaat acgagtttga
ggttgggtta gaaacaacaa 4080atgtgagggc tcatgatggg ttgagttggt
gaatgttttg ggctgctcga ttgacacctt 4140tgtgagtacg tgttgttgtg
catggctttt ggggtccagt ttttttttct tgacgcggcg 4200atcctgatca
gctagtggat aagtgatgtc cactgtgtgt gattgcgttt ttgtttgaat
4260tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt
cttcttttgt 4320tccttggctt tttcttatga tccaagagac tagtcagtgt
tgtggcattc gagactacca 4380agattaatta tgatggggga aggataagta
actgattagt acggactgtt accaaattaa 4440ttaataagcg gcaaatgaag
ggcatggatc ggccggcct 4479315010DNAArtificialPlasmid pKR63
31ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
60tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
120gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
tcgggaaacc 180tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
gagaggcggt ttgcgtattg 240ggcgctcttc cgcttcctcg ctcactgact
cgctgcgctc ggtcgttcgg ctgcggcgag 300cggtatcagc tcactcaaag
gcggtaatac ggttatccac agaatcaggg gataacgcag 360gaaagaacat
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
420tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
cgctcaagtc 480agaggtggcg aaacccgaca ggactataaa gataccaggc
gtttccccct ggaagctccc 540tcgtgcgctc tcctgttccg accctgccgc
ttaccggata cctgtccgcc tttctccctt 600cgggaagcgt ggcgctttct
catagctcac gctgtaggta tctcagttcg gtgtaggtcg 660ttcgctccaa
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
720ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
ctggcagcag 780ccactggtaa caggattagc agagcgaggt atgtaggcgg
tgctacagag ttcttgaagt 840ggtggcctaa ctacggctac actagaagga
cagtatttgg tatctgcgct ctgctgaagc 900cagttacctt cggaaaaaga
gttggtagct cttgatccgg caaacaaacc accgctggta 960gcggtggttt
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
1020atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
cgttaaggga 1080ttttggtcat gagattatca aaaaggatct tcacctagat
ccttttaaat taaaaatgaa 1140gttttaaatc aatctaaagt atatatgagt
aaacttggtc tgacagttac caatgcttaa 1200tcagtgaggc acctatctca
gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1260ccgtcgtgta
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
1320taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
ccagccggaa 1380gggccgagcg cagaagtggt cctgcaactt tatccgcctc
catccagtct attaattgtt 1440gccgggaagc tagagtaagt agttcgccag
ttaatagttt gcgcaacgtt gttgccattg 1500ctacaggcat cgtggtgtca
cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1560aacgatcaag
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
1620gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
gttatggcag 1680cactgcataa ttctcttact gtcatgccat ccgtaagatg
cttttctgtg actggtgagt 1740actcaaccaa gtcattctga gaatagtgta
tgcggcgacc gagttgctct tgcccggcgt 1800caatacggga taataccgcg
ccacatagca gaactttaaa agtgctcatc attggaaaac 1860gttcttcggg
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
1920ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
tctgggtgag 1980caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
ggcgacacgg aaatgttgaa 2040tactcatact cttccttttt caatattatt
gaagcattta tcagggttat tgtctcatga
2100gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
cgcacatttc 2160cccgaaaagt gccacctgac gtctaagaaa ccattattat
catgacatta acctataaaa 2220ataggcgtat cacgaggccc tttcgtctcg
cgcgtttcgg tgatgacggt gaaaacctct 2280gacacatgca gctcccggag
acggtcacag cttgtctgta agcggatgcc gggagcagac 2340aagcccgtca
gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
2400catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
cacagatgcg 2460taaggagaaa ataccgcatc aggcgccatt cgccattcag
gctgcgcaac tgttgggaag 2520ggcgatcggt gcgggcctct tcgctattac
gccagctggc gaaaggggga tgtgctgcaa 2580ggcgattaag ttgggtaacg
ccagggtttt cccagtcacg acgttgtaaa acgacggcca 2640gtgaattcga
gctcggtacc cggggatcct ctagagtcga cctgcaggtc ctcgaagaga
2700agggttaata acacattttt taacattttt aacacaaatt ttagttattt
aaaaatttat 2760taaaaaattt aaaataagaa gaggaactct ttaaataaat
ctaacttaca aaatttatga 2820tttttaataa gttttcacca ataaaaaatg
tcataaaaat atgttaaaaa gtatattatc 2880aatattctct ttatgataaa
taaaaagaaa aaaaaaataa aagttaagtg aaaatgagat 2940tgaagtgact
ttaggtgtgt ataaatatat caaccccgcc aacaatttat ttaatccaaa
3000tatattgaag tatattattc catagccttt atttatttat atatttatta
tataaaagct 3060ttatttgttc taggttgttc atgaaatatt tttttggttt
tatctccgtt gtaagaaaat 3120catgtgcttt gtgtcgccac tcactattgc
agctttttca tgcattggtc agattgacgg 3180ttgattgtat ttttgttttt
tatggttttg tgttatgact taagtcttca tctctttatc 3240tcttcatcag
gtttgatggt tacctaatat ggtccatggg tacatgcatg gttaaattag
3300gtggccaact ttgttgtgaa cgatagaatt ttttttatat taagtaaact
atttttatat 3360tatgaaataa taataaaaaa aatattttat cattattaac
aaaatcatat tagttaattt 3420gttaactcta taataaaaga aatactgtaa
cattcacatt acatggtaac atctttccac 3480cctttcattt gttttttgtt
tgatgacttt ttttcttgtt taaatttatt tcccttcttt 3540taaatttgga
atacattatc atcatatata aactaaaata ctaaaaacag gattacacaa
3600atgataaata ataacacaaa tatttataaa tctagctgca atatatttaa
actagctata 3660tcgatattgt aaaataaaac tagctgcatt gatactgata
aaaaaatatc atgtgctttc 3720tggactgatg atgcagtata cttttgacat
tgcctttatt ttatttttca gaaaagcttt 3780cttagttctg ggttcttcat
tatttgtttc ccatctccat tgtgaattga atcatttgct 3840tcgtgtcaca
aatacaattt agntaggtac atgcattggt cagattcacg gtttattatg
3900tcatgactta agttcatggt agtacattac ctgccacgca tgcattatat
tggttagatt 3960tgataggcaa atttggttgt caacaatata aatataaata
atgtttttat attacgaaat 4020aacagtgatc aaaacaaaca gttttatctt
tattaacaag attttgtttt tgtttgatga 4080cgttttttaa tgtttacgct
ttcccccttc ttttgaattt agaacacttt atcatcataa 4140aatcaaatac
taaaaaaatt acatatttca taaataataa cacaaatatt tttaaaaaat
4200ctgaaataat aatgaacaat attacatatt atcacgaaaa ttcattaata
aaaatattat 4260ataaataaaa tgtaatagta gttatatgta ggaaaaaagt
actgcacgca taatatatac 4320aaaaagatta aaatgaacta ttataaataa
taacactaaa ttaatggtga atcatatcaa 4380aataatgaaa aagtaaataa
aatttgtaat taacttctat atgtattaca cacacaaata 4440ataaataata
gtaaaaaaaa ttatgataaa tatttaccat ctcataagat atttaaaata
4500atgataaaaa tatagattat tttttatgca actagctagc caaaaagaga
acacgggtat 4560atataaaaag agtaccttta aattctactg tacttccttt
attcctgacg tttttatatc 4620aagtggacat acgtgaagat tttaattatc
agtctaaata tttcattagc acttaatact 4680tttctgtttt attcctatcc
tataagtagt cccgattctc ccaacattgc ttattcacac 4740aactaactaa
gaaagtcttc catagccccc caagcggccg cgacacaagt gtgagagtac
4800taaataaatg ctttggttgt acgaaatcat tacactaaat aaaataatca
aagcttatat 4860atgccttccg ctaaggccga atgcaaagaa attggttctt
tctcgttatc ttttgccact 4920tttactagta cgtattaatt actacttaat
catctttgtt tacggctcat tatatccggc 4980cggcctaaag ggcggatccc
ccgggctgca 5010325414DNAArtificialPlasmid pDS1 32aattcgagct
cggtacccgg ggatcctcta gagtcgacct gcaggtcctc gaagagaagg 60gttaataaca
cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa
120aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa
tttatgattt 180ttaataagtt ttcaccaata aaaaatgtca taaaaatatg
ttaaaaagta tattatcaat 240attctcttta tgataaataa aaagaaaaaa
aaaataaaag ttaagtgaaa atgagattga 300agtgacttta ggtgtgtata
aatatatcaa ccccgccaac aatttattta atccaaatat 360attgaagtat
attattccat agcctttatt tatttatata tttattatat aaaagcttta
420tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta
agaaaatcat 480gtgctttgtg tcgccactca ctattgcagc tttttcatgc
attggtcaga ttgacggttg 540attgtatttt tgttttttat ggttttgtgt
tatgacttaa gtcttcatct ctttatctct 600tcatcaggtt tgatggttac
ctaatatggt ccatgggtac atgcatggtt aaattaggtg 660gccaactttg
ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat
720gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag
ttaatttgtt 780aactctataa taaaagaaat actgtaacat tcacattaca
tggtaacatc tttccaccct 840ttcatttgtt ttttgtttga tgactttttt
tcttgtttaa atttatttcc cttcttttaa 900atttggaata cattatcatc
atatataaac taaaatacta aaaacaggat tacacaaatg 960ataaataata
acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg
1020atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg
tgctttctgg 1080actgatgatg cagtatactt ttgacattgc ctttatttta
tttttcagaa aagctttctt 1140agttctgggt tcttcattat ttgtttccca
tctccattgt gaattgaatc atttgcttcg 1200tgtcacaaat acaatttagn
taggtacatg cattggtcag attcacggtt tattatgtca 1260tgacttaagt
tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga
1320taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt
acgaaataac 1380agtgatcaaa acaaacagtt ttatctttat taacaagatt
ttgtttttgt ttgatgacgt 1440tttttaatgt ttacgctttc ccccttcttt
tgaatttaga acactttatc atcataaaat 1500caaatactaa aaaaattaca
tatttcataa ataataacac aaatattttt aaaaaatctg 1560aaataataat
gaacaatatt acatattatc acgaaaattc attaataaaa atattatata
1620aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa
tatatacaaa 1680aagattaaaa tgaactatta taaataataa cactaaatta
atggtgaatc atatcaaaat 1740aatgaaaaag taaataaaat ttgtaattaa
cttctatatg tattacacac acaaataata 1800aataatagta aaaaaaatta
tgataaatat ttaccatctc ataagatatt taaaataatg 1860ataaaaatat
agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata
1920taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt
ttatatcaag 1980tggacatacg tgaagatttt aattatcagt ctaaatattt
cattagcact taatactttt 2040ctgttttatt cctatcctat aagtagtccc
gattctccca acattgctta ttcacacaac 2100taactaagaa agtcttccat
agccccccaa gcggccgcag tatatcttaa attctttaat 2160acggtgtact
aggatattga actggttctt gatgatgaaa acctgggccg agattgcagc
2220tatttatagt cataggtctt gttaacatgc atggacattt ggccacgggg
tggcatgcag 2280tttgacgggt gttgaaataa acaaaaatga ggtggcggaa
gagaatacga gtttgaggtt 2340gggttagaaa caacaaatgt gagggctcat
gatgggttga gttggtgaat gttttgggct 2400gctcgattga cacctttgtg
agtacgtgtt gttgtgcatg gcttttgggg tccagttttt 2460ttttcttgac
gcggcgatcc tgatcagcta gtggataagt gatgtccact gtgtgtgatt
2520gcgtttttgt ttgaatttta tgaacttaga cattgctatg caaaggatac
tctcattgtg 2580ttttgtcttc ttttgttcct tggctttttc ttatgatcca
agagactagt cagtgttgtg 2640gcattcgaga ctaccaagat taattatgat
gggggaagga taagtaactg attagtacgg 2700actgttacca aattaattaa
taagcggcaa atgaagggca tggatcggcc ggcctctaga 2760gtcgacctgc
aggcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
2820ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
gtaaagcctg 2880gggtgcctaa tgagtgagct aactcacatt aattgcgttg
cgctcactgc ccgctttcca 2940gtcgggaaac ctgtcgtgcc agctgcatta
atgaatcggc caacgcgcgg ggagaggcgg 3000tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3060gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
3120ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 3180ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc acaaaaatcg 3240acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg cgtttccccc 3300tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3360ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
3420ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 3480ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg acttatcgcc 3540actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg gtgctacaga 3600gttcttgaag tggtggccta
actacggcta cactagaagg acagtatttg gtatctgcgc 3660tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
3720caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 3780atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga acgaaaactc 3840acgttaaggg attttggtca tgagattatc
aaaaaggatc ttcacctaga tccttttaaa 3900ttaaaaatga agttttaaat
caatctaaag tatatatgag taaacttggt ctgacagtta 3960ccaatgctta
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
4020tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
ctggccccag 4080tgctgcaatg ataccgcgag acccacgctc accggctcca
gatttatcag caataaacca 4140gccagccgga agggccgagc gcagaagtgg
tcctgcaact ttatccgcct ccatccagtc 4200tattaattgt tgccgggaag
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4260tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
4320ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
aaaaagcggt 4380tagctccttc ggtcctccga tcgttgtcag aagtaagttg
gccgcagtgt tatcactcat 4440ggttatggca gcactgcata attctcttac
tgtcatgcca tccgtaagat gcttttctgt 4500gactggtgag tactcaacca
agtcattctg agaatagtgt atgcggcgac cgagttgctc 4560ttgcccggcg
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
4620cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
tgagatccag 4680ttcgatgtaa cccactcgtg cacccaactg atcttcagca
tcttttactt tcaccagcgt 4740ttctgggtga gcaaaaacag gaaggcaaaa
tgccgcaaaa aagggaataa gggcgacacg 4800gaaatgttga atactcatac
tcttcctttt tcaatattat tgaagcattt atcagggtta 4860ttgtctcatg
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
4920gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
tcatgacatt 4980aacctataaa aataggcgta tcacgaggcc ctttcgtctc
gcgcgtttcg gtgatgacgg 5040tgaaaacctc tgacacatgc agctcccgga
gacggtcaca gcttgtctgt aagcggatgc 5100cgggagcaga caagcccgtc
agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 5160taactatgcg
gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
5220gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
ggctgcgcaa 5280ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
cgccagctgg cgaaaggggg 5340atgtgctgca aggcgattaa gttgggtaac
gccagggttt tcccagtcac gacgttgtaa 5400aacgacggcc agtg
5414337085DNAArtificialPlasmid pKR72 33gtacggatcc gtcgacggcg
cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag
aggccccaag gggttatgct agttattgct cagcggtggc 120agcagccaac
tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc
180tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat
cggcgagtac 240ttctacacag ccatcggtcc agacggccgc gcttctgcgg
gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga cgattgcgtc
gcatcgaccc tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc
tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420ccggagccgc
ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg
480ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt
attgggaatc 540cccgaacatc gcctcgctcc agtcaatgac cgctgttatg
cggccattgt ccgtcaggac 600attgttggag ccgaaatccg cgtgcacgag
gtgccggact tcggggcagt cctcggccca 660aagcatcagc tcatcgagag
cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720ttgccagtga
tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta
780ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa
gatcggccgc 840agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca
gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac cctgtgcacg
gcgggagatg caataggtca ggctctcgct 960gaattcccca atgtcaagca
cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020aacataacga
tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg
1080ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct
gcatcaggtc 1140ggagacgctg tcgaactttt cgatcagaaa cttctcgaca
gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc cttcttaaag
ttaaacaaaa ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga
gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320atcccacaaa
aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt
1380caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg
tatatacgat 1440gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg
gagttgcaca caagaaattt 1500gccactatta cagaggcaag agcagcagct
gacgcgtaca caacaagtca gcaaacagac 1560aggttgaact tcatccccaa
aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620ctaacaagcc
caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc
1680agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga
cgatttcctc 1740tatctttacg atctaggaag gaagttcgaa ggtgaaggtg
acgacactat gttcaccact 1800gataatgaga aggttagcct cttcaatttc
agaaagaatg ctgacccaca gatggttaga 1860gaggcctacg cagcaggtct
catcaagacg atctacccga gtaacaatct ccaggagatc 1920aaataccttc
ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag
1980aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg
gacgattcaa 2040ggcttgcttc ataaaccaag gcaagtaata gagattggag
tctctaaaaa ggtagttcct 2100actgaatcta aggccatgca tggagtctaa
gattcaaatc gaggatctaa cagaactcgc 2160cgtgaagact ggcgaacagt
tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220cttcgtcaac
atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt
2280ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg
gaaacctcct 2340cggattccat tgcccagcta tctgtcactt catcgaaagg
acagtagaaa aggaaggtgg 2400ctcctacaaa tgccatcatt gcgataaagg
aaaggctatc attcaagatg cctctgccga 2460cagtggtccc aaagatggac
ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520aaccacgtct
tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc
2580acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat
ttcatttgga 2640gaggacacgc tcgagctcat ttctctatta cttcagccat
aacaaaagaa ctcttttctc 2700ttcttattaa accatgaaaa agcctgaact
caccgcgacg tctgtcgaga agtttctgat 2760cgaaaagttc gacagcgtct
ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820tttcagcttc
gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
2880tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
cgattccgga 2940agtgcttgac attggggaat tcagcgagag cctgacctat
tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc tgcctgaaac
cgaactgccc gctgttctgc agccggtcgc 3060ggaggccatg gatgcgatcg
ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120cggaccgcaa
ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
3180tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
ccgtcgcgca 3240ggctctcgat gagctgatgc tttgggccga ggactgcccc
gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca atgtcctgac
ggacaatggc cgcataacag cggtcattga 3360ctggagcgag gcgatgttcg
gggattccca atacgaggtc gccaacatct tcttctggag 3420gccgtggttg
gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
3480tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
aactctatca 3540gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga ctgtcgggcg
tacacaaatc gcccgcagaa gcgcggccgt 3660ctggaccgat ggctgtgtag
aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720tccgagggca
aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca
3780aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
gatgattatc 3840atataatttc tgttgaatta cgttaagcat gtaataatta
acatgtaatg catgacgtta 3900tttatgagat gggtttttat gattagagtc
ccgcaattat acatttaata cgcgatagaa 3960aacaaaatat agcgcgcaaa
ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020gatcgatgtc
gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg
4080tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcaatgctca
cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc
tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg
caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg
attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt
4980tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
tcccggagac 5040ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg gctggcttaa
ctatgcggca tcagagcaga ttgtactgag 5160agtgcaccat atggacatat
tgtcgttaga acgcggctac aattaataca taaccttatg 5220tatcatacac
atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat
5280ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata
aaataaaaga 5340agctaagcac acggtcaacc attgctctac tgctaaaagg
gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac aagacaactc
aaattaaaaa atttcctttg cttgtttttt 5460tgttgtctct gacttgactt
tcttgtggaa gttggttgta taaggattgg gacaccattg 5520tccttcttaa
tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa
5580taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa
ataattgaga 5640ttcgtccagc aaggctaaac gattgtatag atttatgaca
atatttactt ttttatagat 5700aaatgttata ttataataaa tttatataca
tatattatat gttatttatt attattttaa 5760atccttcaat attttatcaa
accaactcat aatttttttt ttatctgtaa gaagcaataa 5820aattaaatag
acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat
5880agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc
aaaccttata 5940aagacattaa ataagtggat aagtataata tataaatggg
tagtatataa tatataaatg 6000gatacaaact tctctcttta taattgttat
gtctccttaa catcctaata taatacataa 6060gtgggtaata tataatatat
aaatggagac aaacttcttc cattataatt gttatgtctt 6120cttaacactt
atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt
6180acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc
agatgattta 6240taataataag aggaaattta tcatagaaca ataaggtgca
tagatagagt gttaatatat 6300cataacatcc tttgtttatt catagaagaa
gtgagatgga gctcagttat tatactgtta 6360catggtcgga tacaatattc
catgctctcc atgagctctt acacctacat gcattttagt 6420tcatacttgc
ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac
6480tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca
taggtcttgt 6540taacatgcat ggacatttgg ccacggggtg gcatgcagtt
tgacgggtgt tgaaataaac
6600aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca
acaaatgtga 6660gggctcatga tgggttgagt tggtgaatgt tttgggctgc
tcgattgaca cctttgtgag 6720tacgtgttgt tgtgcatggc ttttggggtc
cagttttttt ttcttgacgc ggcgatcctg 6780atcagctagt ggataagtga
tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840aacttagaca
ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg
6900gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact
accaagatta 6960attatgatgg gggaaggata agtaactgat tagtacggac
tgttaccaaa ttaattaata 7020agcggcaaat gaagggcatg gatcaaaagc
ttggatctcc tgcaggatct ggccggccgg 7080atctc
7085345303DNAArtificialPlasmid pDS2 34agcttggatc tcctgcagga
tctggccggc cggatctcgt acggatccgt cgacggcgcg 60cccgatcatc cggatatagt
tcctcctttc agcaaaaaac ccctcaagac ccgtttagag 120gccccaaggg
gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
180cgggctttgt tagcagccgg atcgatccaa gctgtacctc actattcctt
tgccctcgga 240cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt
ctacacagcc atcggtccag 300acggccgcgc ttctgcgggc gatttgtgta
cgcccgacag tcccggctcc ggatcggacg 360attgcgtcgc atcgaccctg
cgcccaagct gcatcatcga aattgccgtc aaccaagctc 420tgatagagtt
ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca
480agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
caaccacggc 540ctccagaaga agatgttggc gacctcgtat tgggaatccc
cgaacatcgc ctcgctccag 600tcaatgaccg ctgttatgcg gccattgtcc
gtcaggacat tgttggagcc gaaatccgcg 660tgcacgaggt gccggacttc
ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 720tgcgcgacgg
acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga
780tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
ttgcggtccg 840aatgggccga acccgctcgt ctggctaaga tcggccgcag
cgatcgcatc catagcctcc 900gcgaccggct gcagaacagc gggcagttcg
gtttcaggca ggtcttgcaa cgtgacaccc 960tgtgcacggc gggagatgca
ataggtcagg ctctcgctga attccccaat gtcaagcact 1020tccggaatcg
ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa
1080ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
gaagctgaaa 1140gcacgagatt cttcgccctc cgagagctgc atcaggtcgg
agacgctgtc gaacttttcg 1200atcagaaact tctcgacaga cgtcgcggtg
agttcaggct tttccatggg tatatctcct 1260tcttaaagtt aaacaaaatt
atttctagag ggaaaccgtt gtggtctccc tatagtgagt 1320cgtattaatt
tcgcgggatc gagatcgatc caattccaat cccacaaaaa tctgagctta
1380acagcacagt tgctcctctc agagcagaat cgggtattca acaccctcat
atcaactact 1440acgttgtgta taacggtcca catgccggta tatacgatga
ctggggttgt acaaaggcgg 1500caacaaacgg cgttcccgga gttgcacaca
agaaatttgc cactattaca gaggcaagag 1560cagcagctga cgcgtacaca
acaagtcagc aaacagacag gttgaacttc atccccaaag 1620gagaagctca
actcaagccc aagagctttg ctaaggccct aacaagccca ccaaagcaaa
1680aagcccactg gctcacgcta ggaaccaaaa ggcccagcag tgatccagcc
ccaaaagaga 1740tctcctttgc cccggagatt acaatggacg atttcctcta
tctttacgat ctaggaagga 1800agttcgaagg tgaaggtgac gacactatgt
tcaccactga taatgagaag gttagcctct 1860tcaatttcag aaagaatgct
gacccacaga tggttagaga ggcctacgca gcaggtctca 1920tcaagacgat
ctacccgagt aacaatctcc aggagatcaa ataccttccc aagaaggtta
1980aagatgcagt caaaagattc aggactaatt gcatcaagaa cacagagaaa
gacatatttc 2040tcaagatcag aagtactatt ccagtatgga cgattcaagg
cttgcttcat aaaccaaggc 2100aagtaataga gattggagtc tctaaaaagg
tagttcctac tgaatctaag gccatgcatg 2160gagtctaaga ttcaaatcga
ggatctaaca gaactcgccg tgaagactgg cgaacagttc 2220atacagagtc
ttttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac
2280gacactctgg tctactccaa aaatgtcaaa gatacagtct cagaagacca
aagggctatt 2340gagacttttc aacaaaggat aatttcggga aacctcctcg
gattccattg cccagctatc 2400tgtcacttca tcgaaaggac agtagaaaag
gaaggtggct cctacaaatg ccatcattgc 2460gataaaggaa aggctatcat
tcaagatgcc tctgccgaca gtggtcccaa agatggaccc 2520ccacccacga
ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg
2580gattgatgtg acatctccac tgacgtaagg gatgacgcac aatcccacta
tccttcgcaa 2640gacccttcct ctatataagg aagttcattt catttggaga
ggacacgctc gagctcattt 2700ctctattact tcagccataa caaaagaact
cttttctctt cttattaaac catgaaaaag 2760cctgaactca ccgcgacgtc
tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc 2820gacctgatgc
agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg
2880cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
tcgttatgtt 2940tatcggcact ttgcatcggc cgcgctcccg attccggaag
tgcttgacat tggggaattc 3000agcgagagcc tgacctattg catctcccgc
cgtgcacagg gtgtcacgtt gcaagacctg 3060cctgaaaccg aactgcccgc
tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct 3120gcggccgatc
ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa
3180tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
tcactggcaa 3240actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
ctctcgatga gctgatgctt 3300tgggccgagg actgccccga agtccggcac
ctcgtgcacg cggatttcgg ctccaacaat 3360gtcctgacgg acaatggccg
cataacagcg gtcattgact ggagcgaggc gatgttcggg 3420gattcccaat
acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag
3480cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
gcggctccgg 3540gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
gcttggttga cggcaatttc 3600gatgatgcag cttgggcgca gggtcgatgc
gacgcaatcg tccgatccgg agccgggact 3660gtcgggcgta cacaaatcgc
ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa 3720gtactcgccg
atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagtga
3780ggtacctaaa gaaggagtgc gtcgaagcag atcgttcaaa catttggcaa
taaagtttct 3840taagattgaa tcctgttgcc ggtcttgcga tgattatcat
ataatttctg ttgaattacg 3900ttaagcatgt aataattaac atgtaatgca
tgacgttatt tatgagatgg gtttttatga 3960ttagagtccc gcaattatac
atttaatacg cgatagaaaa caaaatatag cgcgcaaact 4020aggataaatt
atcgcgcgcg gtgtcatcta tgttactaga tcgatgtcga atcgatcaac
4080ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
gcgctcttcc 4140gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
tgcggcgagc ggtatcagct 4200cactcaaagg cggtaatacg gttatccaca
gaatcagggg ataacgcagg aaagaacatg 4260tgagcaaaag gccagcaaaa
ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 4320cataggctcc
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
4380aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
cgtgcgctct 4440cctgttccga ccctgccgct taccggatac ctgtccgcct
ttctcccttc gggaagcgtg 4500gcgctttctc aatgctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag 4560ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat 4620cgtcttgagt
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
4680aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac 4740tacggctaca ctagaaggac agtatttggt atctgcgctc
tgctgaagcc agttaccttc 4800ggaaaaagag ttggtagctc ttgatccggc
aaacaaacca ccgctggtag cggtggtttt 4860tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4920ttttctacgg
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
4980acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
tttcggtgat 5040gacggtgaaa acctctgaca catgcagctc ccggagacgg
tcacagcttg tctgtaagcg 5100gatgccggga gcagacaagc ccgtcagggc
gcgtcagcgg gtgttggcgg gtgtcggggc 5160tggcttaact atgcggcatc
agagcagatt gtactgagag tgcaccatat ggacatattg 5220tcgttagaac
gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg
5280tgacactata gaacggcgcg cca 5303358031DNAArtificialPlasmid pDS3
(orientation 2) 35tcgactctag aggccggccg atccatgccc ttcatttgcc
gcttattaat taatttggta 60acagtccgta ctaatcagtt acttatcctt cccccatcat
aattaatctt ggtagtctcg 120aatgccacaa cactgactag tctcttggat
cataagaaaa agccaaggaa caaaagaaga 180caaaacacaa tgagagtatc
ctttgcatag caatgtctaa gttcataaaa ttcaaacaaa 240aacgcaatca
cacacagtgg acatcactta tccactagct gatcaggatc gccgcgtcaa
300gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa
aggtgtcaat 360cgagcagccc aaaacattca ccaactcaac ccatcatgag
ccctcacatt tgttgtttct 420aacccaacct caaactcgta ttctcttccg
ccacctcatt tttgtttatt tcaacacccg 480tcaaactgca tgccaccccg
tggccaaatg tccatgcatg ttaacaagac ctatgactat 540aaatagctgc
aatctcggcc caggttttca tcatcaagaa ccagttcaat atcctagtac
600accgtattaa agaatttaag atatactgcg gccgcttggg gggctatgga
agactttctt 660agttagttgt gtgaataagc aatgttggga gaatcgggac
tacttatagg ataggaataa 720aacagaaaag tattaagtgc taatgaaata
tttagactga taattaaaat cttcacgtat 780gtccacttga tataaaaacg
tcaggaataa aggaagtaca gtagaattta aaggtactct 840ttttatatat
acccgtgttc tctttttggc tagctagttg cataaaaaat aatctatatt
900tttatcatta ttttaaatat cttatgagat ggtaaatatt tatcataatt
ttttttacta 960ttatttatta tttgtgtgtg taatacatat agaagttaat
tacaaatttt atttactttt 1020tcattatttt gatatgattc accattaatt
tagtgttatt atttataata gttcatttta 1080atctttttgt atatattatg
cgtgcagtac ttttttccta catataacta ctattacatt 1140ttatttatat
aatattttta ttaatgaatt ttcgtgataa tatgtaatat tgttcattat
1200tatttcagat tttttaaaaa tatttgtgtt attatttatg aaatatgtaa
tttttttagt 1260atttgatttt atgatgataa agtgttctaa attcaaaaga
agggggaaag cgtaaacatt 1320aaaaaacgtc atcaaacaaa aacaaaatct
tgttaataaa gataaaactg tttgttttga 1380tcactgttat ttcgtaatat
aaaaacatta tttatattta tattgttgac aaccaaattt 1440gcctatcaaa
tctaaccaat ataatgcatg cgtggcaggt aatgtactac catgaactta
1500agtcatgaca taataaaccg tgaatctgac caatgcatgt acctanctaa
attgtatttg 1560tgacacgaag caaatgattc aattcacaat ggagatggga
aacaaataat gaagaaccca 1620gaactaagaa agcttttctg aaaaataaaa
taaaggcaat gtcaaaagta tactgcatca 1680tcagtccaga aagcacatga
tattttttta tcagtatcaa tgcagctagt tttattttac 1740aatatcgata
tagctagttt aaatatattg cagctagatt tataaatatt tgtgttatta
1800tttatcattt gtgtaatcct gtttttagta ttttagttta tatatgatga
taatgtattc 1860caaatttaaa agaagggaaa taaatttaaa caagaaaaaa
agtcatcaaa caaaaaacaa 1920atgaaagggt ggaaagatgt taccatgtaa
tgtgaatgtt acagtatttc ttttattata 1980gagttaacaa attaactaat
atgattttgt taataatgat aaaatatttt ttttattatt 2040atttcataat
ataaaaatag tttacttaat ataaaaaaaa ttctatcgtt cacaacaaag
2100ttggccacct aatttaacca tgcatgtacc catggaccat attaggtaac
catcaaacct 2160gatgaagaga taaagagatg aagacttaag tcataacaca
aaaccataaa aaacaaaaat 2220acaatcaacc gtcaatctga ccaatgcatg
aaaaagctgc aatagtgagt ggcgacacaa 2280agcacatgat tttcttacaa
cggagataaa accaaaaaaa tatttcatga acaacctaga 2340acaaataaag
cttttatata ataaatatat aaataaataa aggctatgga ataatatact
2400tcaatatatt tggattaaat aaattgttgg cggggttgat atatttatac
acacctaaag 2460tcacttcaat ctcattttca cttaactttt attttttttt
tctttttatt tatcataaag 2520agaatattga taatatactt tttaacatat
ttttatgaca ttttttattg gtgaaaactt 2580attaaaaatc ataaattttg
taagttagat ttatttaaag agttcctctt cttattttaa 2640attttttaat
aaatttttaa ataactaaaa tttgtgttaa aaatgttaaa aaatgtgtta
2700ttaacccttc tcttcgagga cctgcaggtc gacggcgcgc ccgatcatcc
ggatatagtt 2760cctcctttca gcaaaaaacc cctcaagacc cgtttagagg
ccccaagggg ttatgctagt 2820tattgctcag cggtggcagc agccaactca
gcttcctttc gggctttgtt agcagccgga 2880tcgatccaag ctgtacctca
ctattccttt gccctcggac gagtgctggg gcgtcggttt 2940ccactatcgg
cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg
3000atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca
tcgaccctgc 3060gcccaagctg catcatcgaa attgccgtca accaagctct
gatagagttg gtcaagacca 3120atgcggagca tatacgcccg gagccgcggc
gatcctgcaa gctccggatg cctccgctcg 3180aagtagcgcg tctgctgctc
catacaagcc aaccacggcc tccagaagaa gatgttggcg 3240acctcgtatt
gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg
3300ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg
ccggacttcg 3360gggcagtcct cggcccaaag catcagctca tcgagagcct
gcgcgacgga cgcactgacg 3420gtgtcgtcca tcacagtttg ccagtgatac
acatggggat cagcaatcgc gcatatgaaa 3480tcacgccatg tagtgtattg
accgattcct tgcggtccga atgggccgaa cccgctcgtc 3540tggctaagat
cggccgcagc gatcgcatcc atagcctccg cgaccggctg cagaacagcg
3600ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg
ggagatgcaa 3660taggtcaggc tctcgctgaa ttccccaatg tcaagcactt
ccggaatcgg gagcgcggcc 3720gatgcaaagt gccgataaac ataacgatct
ttgtagaaac catcggcgca gctatttacc 3780cgcaggacat atccacgccc
tcctacatcg aagctgaaag cacgagattc ttcgccctcc 3840gagagctgca
tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac
3900gtcgcggtga gttcaggctt ttccatgggt atatctcctt cttaaagtta
aacaaaatta 3960tttctagagg gaaaccgttg tggtctccct atagtgagtc
gtattaattt cgcgggatcg 4020agatcgatcc aattccaatc ccacaaaaat
ctgagcttaa cagcacagtt gctcctctca 4080gagcagaatc gggtattcaa
caccctcata tcaactacta cgttgtgtat aacggtccac 4140atgccggtat
atacgatgac tggggttgta caaaggcggc aacaaacggc gttcccggag
4200ttgcacacaa gaaatttgcc actattacag aggcaagagc agcagctgac
gcgtacacaa 4260caagtcagca aacagacagg ttgaacttca tccccaaagg
agaagctcaa ctcaagccca 4320agagctttgc taaggcccta acaagcccac
caaagcaaaa agcccactgg ctcacgctag 4380gaaccaaaag gcccagcagt
gatccagccc caaaagagat ctcctttgcc ccggagatta 4440caatggacga
tttcctctat ctttacgatc taggaaggaa gttcgaaggt gaaggtgacg
4500acactatgtt caccactgat aatgagaagg ttagcctctt caatttcaga
aagaatgctg 4560acccacagat ggttagagag gcctacgcag caggtctcat
caagacgatc tacccgagta 4620acaatctcca ggagatcaaa taccttccca
agaaggttaa agatgcagtc aaaagattca 4680ggactaattg catcaagaac
acagagaaag acatatttct caagatcaga agtactattc 4740cagtatggac
gattcaaggc ttgcttcata aaccaaggca agtaatagag attggagtct
4800ctaaaaaggt agttcctact gaatctaagg ccatgcatgg agtctaagat
tcaaatcgag 4860gatctaacag aactcgccgt gaagactggc gaacagttca
tacagagtct tttacgactc 4920aatgacaaga agaaaatctt cgtcaacatg
gtggagcacg acactctggt ctactccaaa 4980aatgtcaaag atacagtctc
agaagaccaa agggctattg agacttttca acaaaggata 5040atttcgggaa
acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca
5100gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa
ggctatcatt 5160caagatgcct ctgccgacag tggtcccaaa gatggacccc
cacccacgag gagcatcgtg 5220gaaaaagaag acgttccaac cacgtcttca
aagcaagtgg attgatgtga catctccact 5280gacgtaaggg atgacgcaca
atcccactat ccttcgcaag acccttcctc tatataagga 5340agttcatttc
atttggagag gacacgctcg agctcatttc tctattactt cagccataac
5400aaaagaactc ttttctcttc ttattaaacc atgaaaaagc ctgaactcac
cgcgacgtct 5460gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
acctgatgca gctctcggag 5520ggcgaagaat ctcgtgcttt cagcttcgat
gtaggagggc gtggatatgt cctgcgggta 5580aatagctgcg ccgatggttt
ctacaaagat cgttatgttt atcggcactt tgcatcggcc 5640gcgctcccga
ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
5700atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
actgcccgct 5760gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
cggccgatct tagccagacg 5820agcgggttcg gcccattcgg accgcaagga
atcggtcaat acactacatg gcgtgatttc 5880atatgcgcga ttgctgatcc
ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc 5940agtgcgtccg
tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
6000gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
caatggccgc 6060ataacagcgg tcattgactg gagcgaggcg atgttcgggg
attcccaata cgaggtcgcc 6120aacatcttct tctggaggcc gtggttggct
tgtatggagc agcagacgcg ctacttcgag 6180cggaggcatc cggagcttgc
aggatcgccg cggctccggg cgtatatgct ccgcattggt 6240cttgaccaac
tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
6300ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
acaaatcgcc 6360cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
tactcgccga tagtggaaac 6420cgacgcccca gcactcgtcc gagggcaaag
gaatagtgag gtacctaaag aaggagtgcg 6480tcgaagcaga tcgttcaaac
atttggcaat aaagtttctt aagattgaat cctgttgccg 6540gtcttgcgat
gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca
6600tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg
caattataca 6660tttaatacgc gatagaaaac aaaatatagc gcgcaaacta
ggataaatta tcgcgcgcgg 6720tgtcatctat gttactagat cgatgtcgaa
tcgatcaacc tgcattaatg aatcggccaa 6780cgcgcgggga gaggcggttt
gcgtattggg cgctcttccg cttcctcgct cactgactcg 6840ctgcgctcgg
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
6900ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
ccagcaaaag 6960gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
ataggctccg cccccctgac 7020gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa acccgacagg actataaaga 7080taccaggcgt ttccccctgg
aagctccctc gtgcgctctc ctgttccgac cctgccgctt 7140accggatacc
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc
7200tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
gcacgaaccc 7260cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
gtcttgagtc caacccggta 7320agacacgact tatcgccact ggcagcagcc
actggtaaca ggattagcag agcgaggtat 7380gtaggcggtg ctacagagtt
cttgaagtgg tggcctaact acggctacac tagaaggaca 7440gtatttggta
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
7500tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
gcagcagatt 7560acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
tttctacggg gtctgacgct 7620cagtggaacg aaaactcacg ttaagggatt
ttggtcatga cattaaccta taaaaatagg 7680cgtatcacga ggccctttcg
tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 7740atgcagctcc
cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
7800cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
tgcggcatca 7860gagcagattg tactgagagt gcaccatatg gacatattgt
cgttagaacg cggctacaat 7920taatacataa ccttatgtat catacacata
cgatttaggt gacactatag aacggcgcgc 7980caagcttgga tctcctgcag
gatctggccg gccggatctc gtacggatcc g 8031369616DNAArtificialPLasmid
SH60 36ggccgcgtca tcaactattc caagctacgt atttgggagt ttgtggagta
cagcaagatg 60atatacctag acggtgatat ccaagttttt gacaacattg accacttgtt
tgacttgcct 120gataactact tctatgcggt gatggactgt ttctgtgagc
caacttgggg ccacactaaa 180caatatcaga tcggttactg ccagcagtgc
ccccataagg ttcagtggcc cactcacttt 240gggcccaaac ctcctctcta
tttcaatgct ggcatgtttg tgtatgagcc caatttggct 300acttaccgtg
acctccttca aacagtccaa gtcacccagc ccacttcctt tgctgaacag
360gattttttga acatgtactt caaggacaaa tataggccaa ttcctaatgt
ctacaatctt 420gtgctggcca tgctgtggcg tcaccctgag aacgttgagc
ttgacaaagt taaagtggtt 480cactactgtg ctgctgggtc taagccttgg
aggtacactg ggaagtgact cgaggtcatc 540aattactcca agctacgtat
ttgggagttc gtggagtaca agaagacgat atacctagac 600ggtgacatcc
aagtatttgg aaacatagac cacttgtttg atctgcctga taattatttc
660tatgcggtga tggattgttt ctgcgagaag acttggagcc acacccctca
gttccagatt 720gggtactgcc aacagtgccc tgataaggtt caatggccct
ctcactttgg ttccaaacct 780cctctatatt tcaatgctgg catgtttgtt
tatgagccta atctcgacac ctaccgtgat 840cttctccaaa ctgtccaact
caccaagccc acttcttttg ctgagcagga ctttctcaac 900atgtacttca
aggacaagta caagccaata ccgaacatgt acaaccttgt gctggccatg
960ttgtggcgtc accctgaaaa tgttgaactt gataaagttc aagtggttca
ttactgtgct 1020gctgggtcta agccttggag
gttcactggg aagtaactgc aggtcatcaa ctactccaag 1080ctccgtatat
gggagtttgt ggagtacagc aagatgatat acttggacgg agacattgag
1140gtatatgaga acatagacca cctatttgac ctacctgatg gtaactttta
cgctgtgatg 1200gattgtttct gcgagaagac atggagtcac acccctcagt
acaaggtggg ttactgccag 1260caatgcccgg agaaggtgcg gtggcccacc
gaattgggtc agcccccttc tctttacttc 1320aacgctggca tgttcgtgtt
cgaacccaac atcgccacct atcatgacct attgaaaacg 1380gtgcaagtca
ccactcccac ctcgttcgct gaacaagatt tcttgaacat gtacttcaag
1440gacatttaca agccaatccc tttaaattac aatcttgtcc tcgccatgct
gtggcgccac 1500ccggaaaacg ttaaattaga ccaagtcaag gttgttcact
attgcgcagc ggggtccaag 1560ccatggagat atacggggaa gtagcggccg
cttggggggc tatggaagac tttcttagtt 1620agttgtgtga ataagcaatg
ttgggagaat cgggactact tataggatag gaataaaaca 1680gaaaagtatt
aagtgctaat gaaatattta gactgataat taaaatcttc acgtatgtcc
1740acttgatata aaaacgtcag gaataaagga agtacagtag aatttaaagg
tactcttttt 1800atatataccc gtgttctctt tttggctagc tagttgcata
aaaaataatc tatattttta 1860tcattatttt aaatatctta tgagatggta
aatatttatc ataatttttt ttactattat 1920ttattatttg tgtgtgtaat
acatatagaa gttaattaca aattttattt actttttcat 1980tattttgata
tgattcacca ttaatttagt gttattattt ataatagttc attttaatct
2040ttttgtatat attatgcgtg cagtactttt ttcctacata taactactat
tacattttat 2100ttatataata tttttattaa tgaattttcg tgataatatg
taatattgtt cattattatt 2160tcagattttt taaaaatatt tgtgttatta
tttatgaaat atgtaatttt tttagtattt 2220gattttatga tgataaagtg
ttctaaattc aaaagaaggg ggaaagcgta aacattaaaa 2280aacgtcatca
aacaaaaaca aaatcttgtt aataaagata aaactgtttg ttttgatcac
2340tgttatttcg taatataaaa acattattta tatttatatt gttgacaacc
aaatttgcct 2400atcaaatcta accaatataa tgcatgcgtg gcaggtaatg
tactaccatg aacttaagtc 2460atgacataat aaaccgtgaa tctgaccaat
gcatgtacct anctaaattg tatttgtgac 2520acgaagcaaa tgattcaatt
cacaatggag atgggaaaca aataatgaag aacccagaac 2580taagaaagct
tttctgaaaa ataaaataaa ggcaatgtca aaagtatact gcatcatcag
2640tccagaaagc acatgatatt tttttatcag tatcaatgca gctagtttta
ttttacaata 2700tcgatatagc tagtttaaat atattgcagc tagatttata
aatatttgtg ttattattta 2760tcatttgtgt aatcctgttt ttagtatttt
agtttatata tgatgataat gtattccaaa 2820tttaaaagaa gggaaataaa
tttaaacaag aaaaaaagtc atcaaacaaa aaacaaatga 2880aagggtggaa
agatgttacc atgtaatgtg aatgttacag tatttctttt attatagagt
2940taacaaatta actaatatga ttttgttaat aatgataaaa tatttttttt
attattattt 3000cataatataa aaatagttta cttaatataa aaaaaattct
atcgttcaca acaaagttgg 3060ccacctaatt taaccatgca tgtacccatg
gaccatatta ggtaaccatc aaacctgatg 3120aagagataaa gagatgaaga
cttaagtcat aacacaaaac cataaaaaac aaaaatacaa 3180tcaaccgtca
atctgaccaa tgcatgaaaa agctgcaata gtgagtggcg acacaaagca
3240catgattttc ttacaacgga gataaaacca aaaaaatatt tcatgaacaa
cctagaacaa 3300ataaagcttt tatataataa atatataaat aaataaaggc
tatggaataa tatacttcaa 3360tatatttgga ttaaataaat tgttggcggg
gttgatatat ttatacacac ctaaagtcac 3420ttcaatctca ttttcactta
acttttattt tttttttctt tttatttatc ataaagagaa 3480tattgataat
atacttttta acatattttt atgacatttt ttattggtga aaacttatta
3540aaaatcataa attttgtaag ttagatttat ttaaagagtt cctcttctta
ttttaaattt 3600tttaataaat ttttaaataa ctaaaatttg tgttaaaaat
gttaaaaaat gtgttattaa 3660cccttctctt cgaggacctg caggtcgacg
gcgcgcccga tcatccggat atagttcctc 3720ctttcagcaa aaaacccctc
aagacccgtt tagaggcccc aaggggttat gctagttatt 3780gctcagcggt
ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatcga
3840tccaagctgt acctcactat tcctttgccc tcggacgagt gctggggcgt
cggtttccac 3900tatcggcgag tacttctaca cagccatcgg tccagacggc
cgcgcttctg cgggcgattt 3960gtgtacgccc gacagtcccg gctccggatc
ggacgattgc gtcgcatcga ccctgcgccc 4020aagctgcatc atcgaaattg
ccgtcaacca agctctgata gagttggtca agaccaatgc 4080ggagcatata
cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt
4140agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg
ttggcgacct 4200cgtattggga atccccgaac atcgcctcgc tccagtcaat
gaccgctgtt atgcggccat 4260tgtccgtcag gacattgttg gagccgaaat
ccgcgtgcac gaggtgccgg acttcggggc 4320agtcctcggc ccaaagcatc
agctcatcga gagcctgcgc gacggacgca ctgacggtgt 4380cgtccatcac
agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac
4440gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg
ctcgtctggc 4500taagatcggc cgcagcgatc gcatccatag cctccgcgac
cggctgcaga acagcgggca 4560gttcggtttc aggcaggtct tgcaacgtga
caccctgtgc acggcgggag atgcaatagg 4620tcaggctctc gctgaattcc
ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg 4680caaagtgccg
ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca
4740ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
ccctccgaga 4800gctgcatcag gtcggagacg ctgtcgaact tttcgatcag
aaacttctcg acagacgtcg 4860cggtgagttc aggcttttcc atgggtatat
ctccttctta aagttaaaca aaattatttc 4920tagagggaaa ccgttgtggt
ctccctatag tgagtcgtat taatttcgcg ggatcgagat 4980cgatccaatt
ccaatcccac aaaaatctga gcttaacagc acagttgctc ctctcagagc
5040agaatcgggt attcaacacc ctcatatcaa ctactacgtt gtgtataacg
gtccacatgc 5100cggtatatac gatgactggg gttgtacaaa ggcggcaaca
aacggcgttc ccggagttgc 5160acacaagaaa tttgccacta ttacagaggc
aagagcagca gctgacgcgt acacaacaag 5220tcagcaaaca gacaggttga
acttcatccc caaaggagaa gctcaactca agcccaagag 5280ctttgctaag
gccctaacaa gcccaccaaa gcaaaaagcc cactggctca cgctaggaac
5340caaaaggccc agcagtgatc cagccccaaa agagatctcc tttgccccgg
agattacaat 5400ggacgatttc ctctatcttt acgatctagg aaggaagttc
gaaggtgaag gtgacgacac 5460tatgttcacc actgataatg agaaggttag
cctcttcaat ttcagaaaga atgctgaccc 5520acagatggtt agagaggcct
acgcagcagg tctcatcaag acgatctacc cgagtaacaa 5580tctccaggag
atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac
5640taattgcatc aagaacacag agaaagacat atttctcaag atcagaagta
ctattccagt 5700atggacgatt caaggcttgc ttcataaacc aaggcaagta
atagagattg gagtctctaa 5760aaaggtagtt cctactgaat ctaaggccat
gcatggagtc taagattcaa atcgaggatc 5820taacagaact cgccgtgaag
actggcgaac agttcataca gagtctttta cgactcaatg 5880acaagaagaa
aatcttcgtc aacatggtgg agcacgacac tctggtctac tccaaaaatg
5940tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa
aggataattt 6000cgggaaacct cctcggattc cattgcccag ctatctgtca
cttcatcgaa aggacagtag 6060aaaaggaagg tggctcctac aaatgccatc
attgcgataa aggaaaggct atcattcaag 6120atgcctctgc cgacagtggt
cccaaagatg gacccccacc cacgaggagc atcgtggaaa 6180aagaagacgt
tccaaccacg tcttcaaagc aagtggattg atgtgacatc tccactgacg
6240taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata
taaggaagtt 6300catttcattt ggagaggaca cgctcgagct catttctcta
ttacttcagc cataacaaaa 6360gaactctttt ctcttcttat taaaccatga
aaaagcctga actcaccgcg acgtctgtcg 6420agaagtttct gatcgaaaag
ttcgacagcg tctccgacct gatgcagctc tcggagggcg 6480aagaatctcg
tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
6540gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca
tcggccgcgc 6600tcccgattcc ggaagtgctt gacattgggg aattcagcga
gagcctgacc tattgcatct 6660cccgccgtgc acagggtgtc acgttgcaag
acctgcctga aaccgaactg cccgctgttc 6720tgcagccggt cgcggaggcc
atggatgcga tcgctgcggc cgatcttagc cagacgagcg 6780ggttcggccc
attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
6840gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac
accgtcagtg 6900cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
cgaggactgc cccgaagtcc 6960ggcacctcgt gcacgcggat ttcggctcca
acaatgtcct gacggacaat ggccgcataa 7020cagcggtcat tgactggagc
gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 7080tcttcttctg
gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
7140ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc
attggtcttg 7200accaactcta tcagagcttg gttgacggca atttcgatga
tgcagcttgg gcgcagggtc 7260gatgcgacgc aatcgtccga tccggagccg
ggactgtcgg gcgtacacaa atcgcccgca 7320gaagcgcggc cgtctggacc
gatggctgtg tagaagtact cgccgatagt ggaaaccgac 7380gccccagcac
tcgtccgagg gcaaaggaat agtgaggtac ctaaagaagg agtgcgtcga
7440agcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg
ttgccggtct 7500tgcgatgatt atcatataat ttctgttgaa ttacgttaag
catgtaataa ttaacatgta 7560atgcatgacg ttatttatga gatgggtttt
tatgattaga gtcccgcaat tatacattta 7620atacgcgata gaaaacaaaa
tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 7680atctatgtta
ctagatcgat gtcgaatcga tcaacctgca ttaatgaatc ggccaacgcg
7740cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
gactcgctgc 7800gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
aaaggcggta atacggttat 7860ccacagaatc aggggataac gcaggaaaga
acatgtgagc aaaaggccag caaaaggcca 7920ggaaccgtaa aaaggccgcg
ttgctggcgt ttttccatag gctccgcccc cctgacgagc 7980atcacaaaaa
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
8040aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
ccgcttaccg 8100gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
ttctcaatgc tcacgctgta 8160ggtatctcag ttcggtgtag gtcgttcgct
ccaagctggg ctgtgtgcac gaaccccccg 8220ttcagcccga ccgctgcgcc
ttatccggta actatcgtct tgagtccaac ccggtaagac 8280acgacttatc
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
8340gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
aggacagtat 8400ttggtatctg cgctctgctg aagccagtta ccttcggaaa
aagagttggt agctcttgat 8460ccggcaaaca aaccaccgct ggtagcggtg
gtttttttgt ttgcaagcag cagattacgc 8520gcagaaaaaa aggatctcaa
gaagatcctt tgatcttttc tacggggtct gacgctcagt 8580ggaacgaaaa
ctcacgttaa gggattttgg tcatgacatt aacctataaa aataggcgta
8640tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
tgacacatgc 8700agctcccgga gacggtcaca gcttgtctgt aagcggatgc
cgggagcaga caagcccgtc 8760agggcgcgtc agcgggtgtt ggcgggtgtc
ggggctggct taactatgcg gcatcagagc 8820agattgtact gagagtgcac
catatggaca tattgtcgtt agaacgcggc tacaattaat 8880acataacctt
atgtatcata cacatacgat ttaggtgaca ctatagaacg gcgcgccaag
8940cttggatctc ctgcaggatc tggccggccg gatctcgtac ggatccgtcg
actctagagg 9000ccggccgatc catgcccttc atttgccgct tattaattaa
tttggtaaca gtccgtacta 9060atcagttact tatccttccc ccatcataat
taatcttggt agtctcgaat gccacaacac 9120tgactagtct cttggatcat
aagaaaaagc caaggaacaa aagaagacaa aacacaatga 9180gagtatcctt
tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac gcaatcacac
9240acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa
aaaaaaactg 9300gaccccaaaa gccatgcaca acaacacgta ctcacaaagg
tgtcaatcga gcagcccaaa 9360acattcacca actcaaccca tcatgagccc
tcacatttgt tgtttctaac ccaacctcaa 9420actcgtattc tcttccgcca
cctcattttt gtttatttca acacccgtca aactgcatgc 9480caccccgtgg
ccaaatgtcc atgcatgtta acaagaccta tgactataaa tagctgcaat
9540ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc
gtattaaaga 9600atttaagata tactgc 9616371585DNAArtificialNot1
fragment 37ggccgcgtca tcaactattc caagctacgt atttgggagt ttgtggagta
cagcaagatg 60atatacctag acggtgatat ccaagttttt gacaacattg accacttgtt
tgacttgcct 120gataactact tctatgcggt gatggactgt ttctgtgagc
caacttgggg ccacactaaa 180caatatcaga tcggttactg ccagcagtgc
ccccataagg ttcagtggcc cactcacttt 240gggcccaaac ctcctctcta
tttcaatgct ggcatgtttg tgtatgagcc caatttggct 300acttaccgtg
acctccttca aacagtccaa gtcacccagc ccacttcctt tgctgaacag
360gattttttga acatgtactt caaggacaaa tataggccaa ttcctaatgt
ctacaatctt 420gtgctggcca tgctgtggcg tcaccctgag aacgttgagc
ttgacaaagt taaagtggtt 480cactactgtg ctgctgggtc taagccttgg
aggtacactg ggaagtgact cgaggtcatc 540aattactcca agctacgtat
ttgggagttc gtggagtaca agaagacgat atacctagac 600ggtgacatcc
aagtatttgg aaacatagac cacttgtttg atctgcctga taattatttc
660tatgcggtga tggattgttt ctgcgagaag acttggagcc acacccctca
gttccagatt 720gggtactgcc aacagtgccc tgataaggtt caatggccct
ctcactttgg ttccaaacct 780cctctatatt tcaatgctgg catgtttgtt
tatgagccta atctcgacac ctaccgtgat 840cttctccaaa ctgtccaact
caccaagccc acttcttttg ctgagcagga ctttctcaac 900atgtacttca
aggacaagta caagccaata ccgaacatgt acaaccttgt gctggccatg
960ttgtggcgtc accctgaaaa tgttgaactt gataaagttc aagtggttca
ttactgtgct 1020gctgggtcta agccttggag gttcactggg aagtaactgc
aggtcatcaa ctactccaag 1080ctccgtatat gggagtttgt ggagtacagc
aagatgatat acttggacgg agacattgag 1140gtatatgaga acatagacca
cctatttgac ctacctgatg gtaactttta cgctgtgatg 1200gattgtttct
gcgagaagac atggagtcac acccctcagt acaaggtggg ttactgccag
1260caatgcccgg agaaggtgcg gtggcccacc gaattgggtc agcccccttc
tctttacttc 1320aacgctggca tgttcgtgtt cgaacccaac atcgccacct
atcatgacct attgaaaacg 1380gtgcaagtca ccactcccac ctcgttcgct
gaacaagatt tcttgaacat gtacttcaag 1440gacatttaca agccaatccc
tttaaattac aatcttgtcc tcgccatgct gtggcgccac 1500ccggaaaacg
ttaaattaga ccaagtcaag gttgttcact attgcgcagc ggggtccaag
1560ccatggagat atacggggaa gtagc 1585
* * * * *