U.S. patent application number 16/589854 was filed with the patent office on 2020-05-21 for synthetic brassica-derived chloroplast transit peptides.
The applicant listed for this patent is Dow AgroSciences LLC. Invention is credited to Robert Cicchillo, Justin M. Lira, Andrew E. Robinson, Carla N. Yerkes.
Application Number | 20200157555 16/589854 |
Document ID | / |
Family ID | 48904120 |
Filed Date | 2020-05-21 |
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United States Patent
Application |
20200157555 |
Kind Code |
A1 |
Lira; Justin M. ; et
al. |
May 21, 2020 |
SYNTHETIC BRASSICA-DERIVED CHLOROPLAST TRANSIT PEPTIDES
Abstract
This disclosure concerns compositions and methods for targeting
peptides, polypeptides, and proteins to plastids of
plastid-containing cells. In some embodiments, the disclosure
concerns chloroplast transit peptides that may direct a polypeptide
to a plastid, and nucleic acid molecules encoding the same. In some
embodiments, the disclosure concerns methods for producing a
transgenic plant material (e.g., a transgenic plant) comprising a
chloroplast transit peptide, as well as plant materials produced by
such methods, and plant commodity products produced therefrom.
Inventors: |
Lira; Justin M.;
(Zionsville, IN) ; Cicchillo; Robert; (Zionsville,
IN) ; Yerkes; Carla N.; (Crawfordsville, IN) ;
Robinson; Andrew E.; (Brownsburg, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dow AgroSciences LLC |
Indianapolis |
IN |
US |
|
|
Family ID: |
48904120 |
Appl. No.: |
16/589854 |
Filed: |
October 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15352229 |
Nov 15, 2016 |
10428339 |
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16589854 |
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13757613 |
Feb 1, 2013 |
9493781 |
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15352229 |
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61625222 |
Apr 17, 2012 |
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61593555 |
Feb 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 114/11 20130101;
C12N 9/0069 20130101; C12N 15/8209 20130101; C12N 15/8245 20130101;
C12N 9/1092 20130101; Y02A 40/162 20180101; C12N 9/96 20130101;
C12N 15/8274 20130101; C12N 9/0071 20130101; C12N 9/1085 20130101;
C12N 15/8281 20130101; C12N 15/8286 20130101; C12Y 205/01019
20130101; A01N 57/20 20130101; C12N 15/8283 20130101; C12N 15/8205
20130101; C12N 15/8247 20130101; C12N 15/8221 20130101; C12N
15/8275 20130101; C07K 2319/08 20130101; C12N 15/62 20130101; C12Y
113/00 20130101; C07K 14/415 20130101; C12N 15/8241 20130101; C12N
15/8214 20130101; Y02A 40/146 20180101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; A01N 57/20 20060101
A01N057/20; C12N 9/02 20060101 C12N009/02; C12N 9/10 20060101
C12N009/10; C12N 9/96 20060101 C12N009/96; C12N 15/62 20060101
C12N015/62 |
Claims
1. A nucleic acid molecule comprising a polynucleotide that encodes
a chimeric protein comprising a synthetic chloroplast transit
peptide (CTP) and a linked 5-enolpyruvylshikimate 3-phosphate
synthase (EPSPS) polypeptide, wherein the amino acid sequence of
the synthetic CTP selected from the group consisting of: (A) a
first amino acid sequence between 25 and 41 amino acids in length,
wherein the first amino acid sequence is at least 90% identical to
a contiguous amino acid sequence of the same length in the first 41
amino acids of SEQ ID NO:1, linked to a second amino acid sequence
between 25 and 41 amino acids in length, wherein the second amino
acid sequence is at least 90% identical to a contiguous amino acid
sequence of the same length in the last 41 amino acids of SEQ ID
NO:2, and (B) a first amino acid sequence between 25 and 41 amino
acids in length, wherein the first amino acid sequence is at least
90% identical to a contiguous amino acid sequence of the same
length in the first 41 amino acids of SEQ ID NO:2, linked to a
second amino acid sequence between 25 and 41 amino acids in length,
wherein the second amino acid sequence is at least 90% identical to
a contiguous amino acid sequence of the same length in the last 41
amino acids of SEQ ID NO:1; and wherein the EPSPS polypeptide
comprises the amino acid sequences of [SEQ ID NOs:170-173 from
P11743.1US].
2. The nucleic acid molecule of claim 1, wherein the EPSPS
polypeptide comprises an amino acid sequence that is at least 90%
identical to [DGT-28, DGT-31, DGT-32, or DGT-33].
3. The nucleic acid molecule of claim 2, wherein the EPSPS
polypeptide comprises an amino acid sequence that is at least 95%
identical to [DGT-28, DGT-31, DGT-32, or DGT-33].
4. The nucleic acid molecule of claim 2, wherein the chimeric
protein comprises the amino acid sequence encoded by SEQ ID NO:34
or SEQ ID NO:35.
5. The nucleic acid molecule of claim 1, wherein the synthetic CTP
comprises SEQ ID NO:13.
6. The nucleic acid molecule of claim 1, wherein the synthetic CTP
is less than 70 amino acids in length.
7. The nucleic acid molecule of claim 1, wherein the synthetic CTP
is about 65 amino acids in length.
8. The nucleic acid molecule of claim 2, wherein the synthetic CTP
comprises less than 7 conservative amino acid substitutions to the
amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the
synthetic CTP is at least 90% identical to SEQ ID NO:3 or SEQ ID
NO:4.
9. The nucleic acid molecule of claim 2, wherein the synthetic CTP
comprises less than 4 conservative amino acid substitutions to the
amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the
synthetic CTP is at least 95% identical to SEQ ID NO:3 or SEQ ID
NO:4.
10. The nucleic acid molecule of claim 2, wherein the synthetic CTP
comprises 1 conservative amino acid substitution to the amino acid
sequence of SEQ ID NO:3 or SEQ ID NO:4, such that the synthetic CTP
is at least 98% identical to SEQ ID NO:3 or SEQ ID NO:4.
11. The nucleic acid molecule of claim 2, wherein the first amino
acid sequence of the CTP comprises amino acids 1-35 of SEQ ID NO:1
or amino acids 1-31 of SEQ ID NO:2.
12. The nucleic acid molecule of claim 2, wherein the second amino
acid sequence of the CTP comprises amino acids 32-61 of SEQ ID NO:2
or amino acids 36-69 of SEQ ID NO:1.
13. The nucleic acid molecule of claim 2, wherein the
polynucleotide is operably linked to a promoter operable in a plant
cell.
14. The nucleic acid molecule of claim 8, wherein the
polynucleotide is operably linked to a promoter operable in a plant
cell.
15. The nucleic acid molecule of claim 14, wherein the molecule is
a plant expression vector.
16. A transgenic plant material comprising the nucleic acid
molecule of claim 14 as a genomic nucleic acid molecule, wherein
the EPSPS polypeptide is targeted to chloroplasts of the plant
material.
17. The transgenic plant material of claim 16, wherein the plant
material is selected from the group consisting of a plant cell, a
plant tissue, a plant tissue culture, a callus culture, and a plant
part.
18. The transgenic plant material of claim 16, wherein the plant
material is a whole plant or seed.
19. The transgenic plant material of claim 18, wherein the plant
material is from a plant selected from the group consisting of
Arabidopsis, alfalfa, Brassica, beans, broccoli, cabbage, carrot,
cauliflower, celery, Chinese cabbage, cotton, cucumber, eggplant,
lettuce, melon, pea, pepper, peanut, potato, pumpkin, radish,
rapeseed, spinach, soybean, squash, sugarbeet, sunflower, tobacco,
tomato, watermelon, corn, onion, rice, sorghum, wheat, rye, millet,
sugarcane, oat, triticale, switchgrass, and turfgrass.
20. A method for producing a transgenic plant material, the method
comprising transforming a plant material with the nucleic acid
molecule of claim 15.
21. The method according to claim 20, wherein the plant material is
selected from the group consisting of a plant cell, a plant tissue,
a plant tissue culture, and a callus culture.
22. A transgenic plant commodity product produced from the plant
material of claim 16, wherein the commodity product comprises the
polynucleotide.
23. A method for growing a glyphosate-tolerant plant, the method
comprising: planting or cultivating the transgenic plant or seed of
claim 18 in a suitable growing environment.
24. The method according to claim 23, the method further
comprising: applying glyphosate to the growing environment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/352,229 filed Nov. 15, 2016, which is a
continuation of U.S. patent application Ser. No. 13/757,613 filed
Feb. 1, 2013, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/593,555 filed Feb. 1, 2012, and also to
U.S. Provisional Patent Application Ser. No. 61/625,222, filed Apr.
17, 2012, the disclosures of each of which is hereby incorporated
herein in its entirety by this reference.
[0002] STATEMENT ACCORDING TO 37 C.F.R. .sctn. 1.821(c) or
(e)--SEQUENCE LISTING SUBMITTED AS ASCII TEXT FILE
[0003] Pursuant to 37 C.F.R. .sctn. 1.821(c) or (e), a file
containing an ASCII text version of the Sequence Listing has been
submitted concomitant with this application, the contents of which
are hereby incorporated by reference.
TECHNICAL FIELD
[0004] This disclosure relates to compositions and methods for
genetically encoding and expressing polypeptides that are targeted
to plastids of plastid-containing cells. In certain embodiments,
the disclosure relates to amino acid sequences that target
polypeptides to chloroplasts (e.g., of higher plants), and/or
nucleic acid molecules encoding the same. In certain embodiments,
the disclosure relates to chimeric polypeptides comprising an amino
acid sequence that controls the transit of the chimeric
polypeptides to plastids, and/or nucleic acid molecules encoding
the same.
BACKGROUND
[0005] Plant cells contain distinct subcellular organelles,
referred to generally as "plastids," that are delimited by
characteristic membrane systems and perform specialized functions
within the cell. Particular plastids are responsible for
photosynthesis, as well as the synthesis and storage of certain
chemical compounds. All plastids are derived from proplastids that
are present in the meristematic regions of the plant. Proplastids
may develop into, for example: chloroplasts, etioplasts,
chromoplasts, gerontoplasts, leucoplasts, amyloplasts, elaioplasts,
and proteinoplasts. Plastids exist in a semi-autonomous fashion
within the cell, containing their own genetic system and protein
synthesis machinery, but relying upon a close cooperation with the
nucleo-cytoplasmic system in their development and biosynthetic
activities.
[0006] In photosynthetic leaf cells of higher plants, the most
conspicuous plastids are the chloroplasts. The most essential
function of chloroplasts is the performance of the light-driven
reactions of photosynthesis. But, chloroplasts also carry out many
other biosynthetic processes of importance to the plant cell. For
example, all of the cell's fatty acids are made by enzymes located
in the chloroplast stroma, using the ATP, NAOPH, and carbohydrates
readily available there. Moreover, the reducing power of
light-activated electrons drives the reduction of nitrite
(NO.sub.2.sup.-) to ammonia (NH.sub.3) in the chloroplast; this
ammonia provides the plant with nitrogen required for the synthesis
of amino acids and nucleotides.
[0007] The chloroplast also takes part in processes of particular
importance in the agrochemical industry. For example, it is known
that many herbicides act by blocking functions which are performed
within the chloroplast. Recent studies have identified the specific
target of several herbicides. For instance, triazine-derived
herbicides inhibit photosynthesis by displacing a plastoquinone
molecule from its binding site in the 32 kD polypeptide of the
photosystem II. This 32 kD polypeptide is encoded in the
chloroplast genome and synthesized by the organelle machinery.
Mutant plants have been obtained which are resistant to triazine
herbicides. These plants contain a mutant 32 kD polypeptide from
which the plastoquinone can no longer be displaced by triazine
herbicides. Sulfonylureas inhibit acetolactate synthase in the
chloroplast. Acetolactate synthase is involved in isoleucine and
valine synthesis. Glyphosate inhibits the function of 5-enol
pyruvyl-3-phosphoshikimate synthase (EPSPS), which is an enzyme
involved in the synthesis of aromatic amino acids. All these
enzymes are encoded by the nuclear genome, but they are
translocated into the chloroplast where the actual amino acid
synthesis takes place.
[0008] Most chloroplast proteins are encoded in the nucleus of the
plant cell, synthesized as larger precursor proteins in the
cytosol, and post-translationally imported into the chloroplast.
Import across the outer and inner envelope membranes into the
stroma is the major means for entry of proteins destined for the
stroma, the thylakoid membrane, and the thylakoid lumen.
Localization of imported precursor proteins to the thylakoid
membrane and thylakoid lumen is accomplished by four distinct
mechanisms, including two that are homologous to bacterial protein
transport systems. Thus, mechanisms for protein localization in the
chloroplast are, in part, derived from the prokaryotic
endosymbiont. Cline and Henry (1996), Annu. Rev. Cell. Dev. Biol.
12:1-26.
[0009] Precursor proteins destined for chloroplastic expression
contain N-terminal extensions known as chloroplast transit peptides
(CTPs). The transit peptide is instrumental for specific
recognition of the chloroplast surface and in mediating the
post-translational translocation of pre-proteins across the
chloroplastic envelope and, thence, to the various sub-compartments
within the chloroplast (e.g., stroma, thylakoid, and thylakoid
membrane). These N-terminal transit peptide sequences contain all
the information necessary for the import of the chloroplast protein
into plastids; the transit peptide sequences are necessary and
sufficient for plastid import.
[0010] Plant genes reported to have naturally-encoded transit
peptide sequences at their N-terminus include the chloroplast small
subunit of ribulose-1,5-bisphosphate caroxylase (RuBisCo) (de
Castro Silva-Filho et al. (1996), Plant Mol. Biol. 30:769-80;
Schnell et al. (1991), J. Biol. Chem. 266:3335-42); EPSPS (see,
e.g., Archer et al. (1990), J. Bioenerg. and Biomemb. 22:789-810,
and U.S. Pat. Nos. 6,867,293, 7,045,684, and Re. 36,449);
tryptophan synthase (Zhao et al. (1995), J. Biol. Chem.
270:6081-7); plastocyanin (Lawrence et al. (1997), J. Biol. Chem.
272:20357-63); chorismate synthase (Schmidt et al. (1993), J. Biol.
Chem. 268:27447-57); the light harvesting chlorophyll a/b binding
protein (LHBP) (Lamppa et al. (1988), J. Biol. Chem.
263:14996-14999); and chloroplast protein of Arabidopsis thaliana
(Lee et al. (2008), Plant Cell 20:1603-22). United States Patent
Publication No. US 2010/0071090 provides certain chloroplast
targeting peptides from Chlamydomonas sp.
[0011] However, the structural requirements for the information
encoded by chloroplast targeting peptides remains elusive, due to
their high level of sequence diversity and lack of common or
consensus sequence motifs, though it is possible that there are
distinct subgroups of chloroplast targeting peptides with
independent structural motifs. Lee et al. (2008), supra. Further,
not all of these sequences have been useful in the heterologous
expression of chloroplast-targeted proteins in higher plants.
BRIEF SUMMARY OF THE DISCLOSURE
[0012] Described herein are compositions and methods for plastid
targeting of polypeptides in a plant. In some embodiments, a
composition comprises a nucleic acid molecule comprising at least
one nucleotide sequence encoding a synthetic Brassica-derived
chloroplast transit peptide (e.g., a TraP8 peptide, and a TraP9
peptide) operably linked to a nucleotide sequence of interest. In
particular embodiments, such nucleic acid molecules may be useful
for expression and targeting of a polypeptide encoded by the
nucleotide sequence of interest in a monocot or dicot plant.
Further described are vectors comprising a nucleic acid molecule
comprising at least one nucleotide sequence encoding a synthetic
Brassica-derived chloroplast transit peptide operably linked to a
nucleotide sequence of interest.
[0013] In some embodiments, a nucleotide sequence encoding a
synthetic Brassica-derived CTP may be a nucleotide sequence that is
derived from a reference nucleotide sequence obtained from a
Brassica sp. gene (e.g., B. napus, B. rapa, B. juncea, and B.
carinata), or a functional variant thereof. In some embodiments, a
nucleotide sequence encoding a synthetic Brassica-derived CTP may
be a chimeric nucleotide sequence comprising a partial CTP-encoding
nucleotide sequence from a Brassica sp. gene, or a functional
variant thereof. In specific embodiments, a nucleotide sequence
encoding a synthetic Brassica-derived CTP may contain contiguous
nucleotide sequences obtained from each of a reference Brassica sp.
CTP, and a CTP from a different gene of the Brassica sp., a
different Brassica sp., or a different organism (e.g., a plant,
prokaryote, and lower photosynthetic eukaryote), or or functional
variants of any of the foregoing. In particular embodiments, a
contiguous nucleotide sequence may be obtained from an orthologous
nucleotide sequence of the reference Brassica CTP that is obtained
from a different organism's ortholog of the reference Brassica sp.
gene (e.g., a different Brassica sp. genome). In these and further
embodiments, a nucleotide sequence encoding a synthetic
Brassica-derived CTP may be a chimeric nucleotide sequence
comprising more than one CTP-encoding nucleotide sequence.
[0014] In some examples, a nucleotide sequence encoding a synthetic
Brassica-derived CTP may be a chimeric nucleotide sequence
comprising a partial CTP nucleotide sequence from either of B.
napus and B. rapa, or functional variants thereof. In specific
examples, a nucleotide sequence encoding a synthetic
Brassica-derived CTP may contain contiguous nucleotide sequences
obtained from each of B. napus and B. rapa, or functional variants
thereof.
[0015] In some embodiments, a composition comprises a nucleic acid
molecule comprising at least one Brassica-derived means for
targeting a polypeptide to a chloroplast. Further described are
nucleic acid molecules comprising a nucleic acid molecule
comprising at least one Brassica-derived means for targeting a
polypeptide to a chloroplast operably linked to a nucleotide
sequence of interest. In particular embodiments, such nucleic acid
molecules may be useful for expression and targeting of a
polypeptide encoded by the nucleotide sequence of interest in a
monocot or dicot plant. For the purposes of the present disclosure,
a Brassica-derived means for targeting a polypeptide to a
chloroplast refers to particular synthetic nucleotide sequences. In
particular embodiments, a Brassica-derived means for targeting a
polypeptide to a chloroplast is selected from the group consisting
of the nucleotide sequences encoding the polypeptides referred to
herein as TraP8 and TraP9.
[0016] Also described herein are plant materials (for example and
without limitation, plants, plant tissues, and plant cells)
comprising a nucleic acid molecule comprising at least one
nucleotide sequence encoding a synthetic Brassica-derived CTP
operably linked to a nucleotide sequence of interest. In some
embodiments, a plant material may have such a nucleic acid molecule
stably integrated in its genome. In some embodiments, a plant
material may transiently express a product of a nucleic acid
molecule comprising at least one nucleotide sequence encoding a
synthetic Brassica-derived CTP operably linked to a nucleotide
sequence of interest. In some embodiments, the plant material is a
plant cell from which a plant cannot be regenerated.
[0017] Methods are also described for expressing a nucleotide
sequence in a plastid-containing cell (e.g., a plant) in a plastid
(e.g., a chloroplast) of the plastid-containing cell. In particular
embodiments, a nucleic acid molecule comprising at least one
nucleotide sequence encoding a synthetic Brassica-derived CTP
operably linked to a nucleotide sequence of interest may be used to
transform a plant cell, such that a precursor fusion polypeptide
comprising the synthetic Brassica-derived CTP fused to an
expression product of the nucleotide sequence of interest is
produced in the cytoplasm of the plant cell, and the fusion
polypeptide is then transported in vivo into a chloroplast of the
plant cell. In some embodiments, the plant cell is not capable of
regeneration to a plant.
[0018] Further described are methods for the production of a
transgenic plant comprising a nucleic acid molecule comprising at
least one nucleotide sequence encoding a synthetic Brassica-derived
CTP operably linked to a nucleotide sequence of interest. Also
described are plant commodity products (e.g., seeds) produced from
such transgenic plants. In some embodiments, these transgenic
plants or plant commodity products contain transgenic cells from
which a plant cannot be regenerated.
[0019] The foregoing and other features will become more apparent
from the following detailed description of several embodiments,
which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 illustrates an mRNA molecule that is representative
of particular examples of synthetic Brassica-derived CTP-encoding
nucleotide sequences (for example, for TraP8 and TraP9) operably
linked to a nucleotide sequence of interest. In some embodiments,
an mRNA molecule (such as the one shown) may be transcribed from a
DNA molecule comprising an open reading frame including the
synthetic Brassica-derived CTP-encoding sequence operably linked to
the nucleotide sequence of interest. The nucleotide sequence of
interest may be, in some embodiments, a sequence encoding a peptide
of interest, for example and without limitation, a marker gene
product or peptide to be targeted to a plastid.
[0021] FIG. 2 illustrates a plasmid map of pDAB101977.
[0022] FIG. 3 illustrates a plasmid map of pDAB101978.
[0023] FIG. 4 illustrates a plasmid map of pDAB101908.
[0024] FIG. 5 includes a microscopy image showing that TraP8-YFP
infiltrated into tobacco leaf tissue was translocated into the
chloroplasts of the tobacco leaf tissue.
[0025] FIG. 6 includes a microscopy image showing that TraP9-YFP
infiltrated into tobacco leaf tissue was translocated into the
chloroplasts of the tobacco leaf tissue.
[0026] FIG. 7 includes a microscopy image showing that non-targeted
YFP controls that were infiltrated into tobacco leaf tissue were
not incorporated into the chloroplasts of the tobacco leaf
tissue.
[0027] FIG. 8 illustrates a plasmid map of pDAB106597.
[0028] FIG. 9 includes a microscopy image of the TraP8-YFP
construct transformed into maize protoplasts showing the
translocation into the chloroplasts of the maize protoplast.
[0029] FIG. 10 illustrates a plasmid map of pDAB105526.
[0030] FIG. 11 illustrates a plasmid map of pDAB105527.
[0031] FIG. 12 illustrates a plasmid map of pDAB109807.
[0032] FIG. 13 illustrates a plasmid map of pDAB107687.
[0033] FIG. 14 illustrates a plasmid map of pDAB111481.
[0034] FIG. 15 illustrates a plasmid map of pDAB111479.
[0035] FIG. 16 illustrates a plasmid map of pDAB111338.
[0036] FIG. 17 illustrates a plasmid map of pDAB112710.
[0037] FIG. 18 includes an alignment of the predicted chloroplast
transit peptides for the EPSPS protein from Brassica napus (SEQ ID
NO:1) and Brassica rapa (SEQ ID NO:2). The asterisk indicates where
the sequences were split and recombined to form TraP8 and
Trap9.
[0038] FIG. 19 illustrates a plasmid map of pDAB107527.
[0039] FIG. 20 illustrates a plasmid map of pDAB105530.
[0040] FIG. 21 illustrates a plasmid map of pDAB105531.
[0041] FIG. 22 illustrates a plasmid map of pDAB105532.
[0042] FIG. 23 illustrates a plasmid map of pDAB105533.
[0043] FIG. 24 illustrates a plasmid map of pDAB105534.
[0044] FIG. 25 illustrates a plasmid map of pDAB107532.
[0045] FIG. 26 illustrates a plasmid map of pDAB107534.
[0046] FIG. 27 illustrates a plasmid map of pDAB107533.
[0047] FIG. 28 illustrates a plasmid map of pDAB4104.
[0048] FIG. 29 illustrates a plasmid map of pDAB102715.
[0049] FIG. 30 illustrates a plasmid map of pDAB102716.
[0050] FIG. 31 illustrates a plasmid map of pDAB102717.
[0051] FIG. 32 illustrates a plasmid map of pDAB102785.
[0052] FIG. 33 illustrates a plasmid map of pDAB102719.
[0053] FIG. 34 illustrates a plasmid map of pDAB102718.
[0054] FIG. 35 illustrates a plasmid map of pDAB107663.
[0055] FIG. 36 illustrates a plasmid map of pDAB107664.
[0056] FIG. 37 illustrates a plasmid map of pDAB107665.
[0057] FIG. 38 illustrates a plasmid map of pDAB107666.
[0058] FIG. 39 illustrates a plasmid map of pDAB109812.
[0059] FIG. 40 illustrates a plasmid map of pDAB101556.
[0060] FIG. 41 illustrates a plasmid map of pDAB107698.
[0061] FIG. 42 illustrates a plasmid map of pDAB108384.
[0062] FIG. 43 illustrates a plasmid map of pDAB108385.
[0063] FIG. 44 illustrates a plasmid map of pDAB108386.
[0064] FIG. 45 illustrates a plasmid map of pDAB108387.
SEQUENCE LISTING
[0065] The nucleic acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. .sctn. 1.822. Only one
strand of each nucleic acid sequence is shown, but the
complementary strand is understood to be included by any reference
to the displayed strand. In the accompanying sequence listing:
[0066] SEQ ID NO:1 shows the amino acid of a Brassica napus EPSPS
chloroplast transit peptide.
[0067] SEQ ID NO:2 shows the amino acid of a Brassica rapa EPSPS
chloroplast transit peptide.
[0068] SEQ ID NO:3 shows the amino acid of a TraP8 chimeric
chloroplast transit peptide.
[0069] SEQ ID NO:4 shows the amino acid of a TraP9 chimeric
chloroplast transit peptide.
[0070] SEQ ID NO:5 shows a polynucleotide sequence encoding a TraP8
chimeric chloroplast transit peptide.
[0071] SEQ ID NO:6 shows a polynucleotide sequence encoding a TraP9
chimeric chloroplast transit peptide.
[0072] SEQ ID NO:7 shows a polynucleotide sequence encoding a
linker sequence.
[0073] SEQ ID NO:8 shows a polynucleotide sequence encoding a TraP8
v2 chimeric chloroplast transit peptide.
[0074] SEQ ID NO:9 shows a polynucleotide sequence encoding a TraP9
v2 chimeric chloroplast transit peptide.
[0075] SEQ ID NO:10 shows a polynucleotide sequence encoding a
cry2aa gene.
[0076] SEQ ID NO:11 shows a polynucleotide sequence encoding a
wp3ab1v6 gene.
[0077] SEQ ID NO:12 shows a polynucleotide sequence encoding a
wp3ab1v7 gene.
[0078] SEQ ID NO:13 shows a peptide having the amino acid sequence,
Ser-Val-Ser-Leu.
[0079] SEQ ID NO:14 shows a polynucleotide sequence encoding the
Brassica napus EPSPS chloroplast transit peptide of SEQ ID
NO:1.
[0080] SEQ ID NO:15 shows a polynucleotide sequence encoding the
Brassica rapa EPSPS chloroplast transit peptide of SEQ ID NO:2.
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0081] A chloroplast transit peptide (CTP) (or plastid transit
peptide) functions co-translationally or post-translationally to
direct a polypeptide comprising the CTP to a plastid (e.g., a
chloroplast). In some embodiments of the invention, either
endogenous chloroplast proteins or heterologous proteins may be
directed to a chloroplast by expression of such a protein as a
larger precursor polypeptide comprising a CTP. In particular
embodiments, the CTP may be derived from a nucleotide sequence
obtained from a Brassica sp. gene, for example and without
limitation, by incorporating at least one contiguous sequence from
a orthologous gene obtained from a different organism, or a
functional variant thereof.
[0082] In an exemplary embodiment, nucleic acid sequences, each
encoding a CTP, were isolated from EPSPS gene sequences obtained
from Brassica napus (NCBI Database Accession No. P17688) and
Brassica rapa (NCBI Database Accession No. AAS80163). The
CTP-encoding nucleic acid sequences were isolated by analyzing the
EPSPS gene sequence with the ChloroP prediction server. Emanuelsson
et al. (1999), Protein Science 8:978-84 (available at
cbs.dtu.dk/services/ChloroP). The predicted protein products of the
isolated CTP-encoding sequences are approximately 60-70 amino
acid-long transit peptides. In this example, the native B. napus
CTP was used as a reference sequence to design exemplary synthetic
Brassica-derived CTPs by fusing contiguous sequences from the other
CTPs at a particular position in the B. napus CTP. This design
process illustrates the development of a novel synthetic CTP,
according to some aspects, from a Brassica sp. nucleic acid
sequence. These exemplary synthetic Brassica-derived CTPs are
referred to throughout this disclosure as TraP8 and TraP9. These
exemplary synthetic TraPs were tested for plastid-targeting
function and were found to exhibit plastid targeting that was at
least as favorable as that observed for the native Brassica
sequences individually.
[0083] In a further exemplary embodiment, nucleic acid sequences,
each encoding a synthetic TraP peptide of the invention, were
synthesized independently and operably linked to a nucleic acid
sequence encoding a yellow fluorescent protein (YFP) to produce
synthetic nucleic acid molecules, each encoding a chimeric TraP:YFP
fusion polypeptide. Such nucleic acid molecules, each encoding a
chimeric TraP:YFP polypeptide, were each introduced into a binary
vector, such that each TraP:YFP-encoding nucleic acid sequence was
operably linked to an AtUbi 10 promoter.
[0084] In yet a further exemplary embodiment, binary vectors
comprising a TraP:YFP-encoding nucleic acid sequence operably
linked to an AtUbi 10 promoter each were independently, transiently
transformed into tobacco (Nicotiana benthamiana) via
Agrobacterium-mediated transformation. Confocal microscopy and
Western blot analysis confirmed that each TraP successfully
targeted YFP to tobacco chloroplasts.
[0085] In a further exemplary embodiment, nucleic acid sequences,
each encoding a synthetic TraP peptide of the invention, were
synthesized independently and operably linked to a nucleic acid
sequence encoding an agronomically important gene sequence. The
TraP sequences were fused to herbicide tolerant traits (e.g. dgt-28
and dgt-14) to produce synthetic nucleic acid molecules, each
encoding a chimeric TraP:DGT-28 or TraP:DGT-14 fusion polypeptide.
Such nucleic acid molecules, each encoding a chimeric TraP:DGT-28
or TraP:DGT-14 polypeptide, were each introduced into a binary
vector, such that each TraP:dgt-28 or TraP:dgt-14-encoding nucleic
acid sequence was operably linked to a promoter and other gene
regulatory elements. The binary containing the TraP:dgt-28 or
TraP:dgt-14-encoding nucleic acid sequence was used to transform
varopis plant species. The transgenic plants were assayed for
herbicide tolerance as a result of the expression and translocation
of the DGT-28 or DGT-14 enzymes to the chloroplast.
[0086] In a further exemplary embodiment, nucleic acid sequences,
each encoding a synthetic TraP peptide of the invention, were
synthesized independently and operably linked to a nucleic acid
sequence encoding an agronomically important gene sequence. The
TraP sequences were fused to genes conferring insect tolerance
traits (e.g. cry2Aa and vip3ab1) to produce synthetic nucleic acid
molecules, each encoding a chimeric TraP:Cry2Aa or TraP:Vip3Ab1
fusion polypeptide. Such nucleic acid molecules, each encoding a
chimeric TraP:Cry2Aa or TraP: Vip3Ab1 polypeptide, were each
introduced into a binary vector, such that each TraP: Cry2Aa or
TraP: Vip3Ab1--encoding nucleic acid sequence was operably linked
to a promoter and other gene regulatory elements. The binary
containing the TraP: Cry2Aa or TraP: Vip3Ab1-encoding nucleic
acid-sequence was used to transform various plant species. The
transgenic plants were bioassayed for insect resistance as a result
of the expression and translocation of the Cry2Aa or Vip3Ab1
enzymes to the chloroplast.
[0087] In view of the aforementioned detailed working examples,
synthetic Brassica-derived CTP sequences of the invention, and
nucleic acids encoding the same, may be used to direct any
polypeptide to a plastid in a broad range of plastid-containing
cells. For example, by methods made available to those of skill in
the art by the present disclosure, a chimeric polypeptide
comprising a synthetic Brassica-derived CTP sequence fused to the
N-terminus of any second peptide sequence may be introduced into
(or expressed in) a plastid-containing host cell for plastid
targeting of the second peptide sequence. Thus, in particular
embodiments, a TraP peptide of the invention may provide increased
efficiency of import and processing of a peptide for which plastid
expression is desired, when compared to a native CTP.
II. Abbreviations
[0088] CTP chloroplast transit peptide [0089] Bt Bacillus
thuringiensis [0090] EPSPS 3-enolpyruvylshikimate-5-phosphate
synthetase [0091] YFP yellow fluorescent protein [0092] T.sub.i
tumor-inducing (plasmids derived from A. tumefaciens) [0093] T-DNA
transfer DNA
III. Terms
[0094] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0095] Chloroplast transit peptide: As used herein, the term
"chloroplast transit peptide" (CTP) (or "plastid transit peptide")
may refer to an amino acid sequence that, when present at the
N-terminus of a polypeptide, directs the import of the polypeptide
into a plastid of a plastid-containing cell (e.g., a plant cell,
such as in a whole plant or plant cell culture). A CTP is generally
necessary and sufficient to direct the import of a protein into a
plastid (e.g., a primary, secondary, or tertiary plastid, such as a
chloroplast) of a host cell. A putative chloroplast transit peptide
may be identified by one of several available algorithms (e.g.,
PSORT, and ChloroP (available at cbs.dtu.dk/services/ChloroP)).
ChloroP may provide particularly good prediction of CTPs.
Emanuelsson et al. (1999), Protein Science 8:978-84. However,
prediction of functional CTPs is not achieved at 100% efficiency by
any existing algorithm. Therefore, it is important to verify that
an identified putative CTP does indeed function as intended in,
e.g., an in vitro, or in vivo methodology.
[0096] Chloroplast transit peptides may be located at the
N-terminus of a polypeptide that is imported into a plastid. The
CTP may facilitate co- or post-translational transport of a
polypeptide comprising the CTP into the plastid. Chloroplast
transit peptides typically comprise between about 40 and about 100
amino acids, and such CTPs have been observed to contain certain
common characteristics. For example: CTPs contain very few, if any,
negatively charged amino acids (such as aspartic acid, glutamic
acid, asparagines, or glutamine); the N-terminal regions of CTPs
lack charged amino acids, glycine, and proline; the central region
of a CTP also is likely to contain a very high proportion of basic
or hydroxylated amino acids (such as serine and threonine); and the
C-terminal region of a CTP is likely to be rich in arginine, and
have the ability to comprise an amphipathic beta-sheet structure.
Plastid proteases may cleave the CTP from the remainder of a
polypeptide comprising the CTP after importation of the polypeptide
into the plastid.
[0097] Contact: As used herein, the term "contact with" or "uptake
by" a cell, tissue, or organism (e.g., a plant cell; plant tissue;
and plant), with regard to a nucleic acid molecule, includes
internalization of the nucleic acid molecule into the organism, for
example and without limitation: contacting the organism with a
composition comprising the nucleic acid molecule; and soaking of
organisms with a solution comprising the nucleic acid molecule.
[0098] Endogenous: As used herein, the term "endogenous" refers to
substances (e.g., nucleic acid molecules and polypeptides) that
originate from within a particular organism, tissue, or cell. For
example, an "endogenous" polypeptide expressed in a plant cell may
refer to a polypeptide that is normally expressed in cells of the
same type from non-genetically engineered plants of the same
species. In some examples, an endogenous gene (e.g., an EPSPS gene)
from a Brassica sp. may be used to obtain a reference Brassica CTP
sequence.
[0099] Expression: As used herein, "expression" of a coding
sequence (for example, a gene or a transgene) refers to the process
by which the coded information of a nucleic acid transcriptional
unit (including, e.g., genomic DNA or cDNA) is converted into an
operational, non-operational, or structural part of a cell, often
including the synthesis of a protein. Gene expression can be
influenced by external signals; for example, exposure of a cell,
tissue, or organism to an agent that increases or decreases gene
expression. Expression of a gene can also be regulated anywhere in
the pathway from DNA to RNA to protein. Regulation of gene
expression occurs, for example, through controls acting on
transcription, translation, RNA transport and processing,
degradation of intermediary molecules such as mRNA, or through
activation, inactivation, compartmentalization, or degradation of
specific protein molecules after they have been made, or by
combinations thereof. Gene expression can be measured at the RNA
level or the protein level by any method known in the art, for
example and without limitation: Northern blot; RT-PCR; Western
blot; or in vitro; in situ; and in vivo protein activity
assay(s).
[0100] Genetic material: As used herein, the term "genetic
material" includes all genes, and nucleic acid molecules, such as
DNA and RNA.
[0101] Heterologous: As used herein, the term "heterologous" refers
to substances (e.g., nucleic acid molecules and polypeptides) that
do not originate from within a particular organism, tissue, or
cell. For example, a "heterologous" polypeptide expressed in a
plant cell may refer to a polypeptide that is not normally
expressed in cells of the same type from non-genetically engineered
plants of the same species (e.g., a polypeptide that is expressed
in different cells of the same organism or cells of a different
organism).
[0102] Isolated: As used herein, the term "isolated" refers to
molecules (e.g., nucleic acid molecules and polypeptides) that are
substantially separated or purified away from other molecules of
the same type (e.g., other nucleic acid molecules and other
polypeptides) with which the molecule is normally associated in the
cell of the organism in which the molecule naturally occurs. For
example, an isolated nucleic acid molecule may be substantially
separated or purified away from chromosomal DNA or extrachromosomal
DNA in the cell of the organism in which the nucleic acid molecule
naturally occurs. Thus, the term includes recombinant nucleic acid
molecules and polypeptides that are biochemically purified such
that other nucleic acid molecules, polypeptides, and cellular
components are removed. The term also includes recombinant nucleic
acid molecules, chemically-synthesized nucleic acid molecules, and
recombinantly produced polypeptides.
[0103] The term "substantially purified," as used herein, refers to
a molecule that is separated from other molecules normally
associated with it in its native state. A substantially purified
molecule may be the predominant species present in a composition. A
substantially purified molecule may be, for example, at least 60%
free, at least 75% free, or at least 90% free from other molecules
besides a solvent present in a natural mixture. The term
"substantially purified" does not refer to molecules present in
their native state.
[0104] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" refers to a polymeric form of nucleotides, which may
include both sense and anti-sense strands of RNA, cDNA, genomic
DNA, and synthetic forms and mixed polymers of the above. A
nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a
modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." A nucleic acid molecule is usually at least 10
bases in length, unless otherwise specified. The term includes
single- and double-stranded forms of DNA. Nucleic acid molecules
include dimeric (so-called in tandem) forms, and the transcription
products of nucleic acid molecules. A nucleic acid molecule can
include either or both naturally occurring and modified nucleotides
linked together by naturally occurring and/or non-naturally
occurring nucleotide linkages.
[0105] Nucleic acid molecules may be modified chemically or
biochemically, or may contain non-natural or derivatized nucleotide
bases, as will be readily appreciated by those of skill in the art.
Such modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications (e.g., uncharged
linkages: for example, methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.; charged linkages: for example,
phosphorothioates, phosphorodithioates, etc.; pendent moieties: for
example, peptides; intercalators: for example, acridine, psoralen,
etc.; chelators; alkylators; and modified linkages: for example,
alpha anomeric nucleic acids, etc.). The term "nucleic acid
molecule" also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hairpinned, circular, and padlocked conformations.
[0106] As used herein with respect to DNA, the term "coding
sequence," "structural nucleotide sequence," or "structural nucleic
acid molecule" refers to a nucleotide sequence that is ultimately
translated into a polypeptide, via transcription and mRNA, when
placed under the control of appropriate regulatory sequences. With
respect to RNA, the term "coding sequence" refers to a nucleotide
sequence that is translated into a peptide, polypeptide, or
protein. The boundaries of a coding sequence are determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. Coding sequences include, but are not
limited to: genomic DNA; cDNA; ESTs; and recombinant nucleotide
sequences.
[0107] In some embodiments, the invention includes nucleotide
sequences that may be isolated, purified, or partially purified,
for example, using separation methods such as, for example,
ion-exchange chromatography; by exclusion based on molecular size
or by affinity; by fractionation techniques based on solubility in
different solvents; and methods of genetic engineering such as
amplification, cloning, and subcloning.
[0108] Sequence identity: The term "sequence identity" or
"identity," as used herein in the context of two nucleic acid or
polypeptide sequences, may refer to the residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window.
[0109] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences, and amino acid
sequences) over a comparison window, wherein the portion of the
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 nucleotide 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 comparison window, and multiplying the
result by 100 to yield the percentage of sequence identity.
[0110] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981), Adv. Appl. Math. 2:482;
Needleman and Wunsch (1970), J. Mol. Biol. 48:443; Pearson and
Lipman (1988), Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and
Sharp (1988), Gene 73:237-44; Higgins and Sharp (1989), CABIOS
5:151-3; Corpet et al. (1988), Nucleic Acids Res. 16:10881-90;
Huang et al. (1992), Comp. Appl. Biosci. 8:155-65; Pearson et al.
(1994), Methods Mol. Biol. 24:307-31; Tatiana et al. (1999), FEMS
Microbiol. Lett. 174:247-50. A detailed consideration of sequence
alignment methods and homology calculations can be found in, e.g.,
Altschul et al. (1990), J. Mol. Biol. 215:403-10.
[0111] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default BLOSUM62 matrix set to default
parameters. Nucleic acid sequences with even greater similarity to
the reference sequences will show increasing percentage identity
when assessed by this method.
[0112] Specifically hybridizable/Specifically complementary: As
used herein, the terms "Specifically hybridizable" and
"specifically complementary" are terms that indicate a sufficient
degree of complementarity, such that stable and specific binding
occurs between the nucleic acid molecule and a target nucleic acid
molecule. Hybridization between two nucleic acid molecules involves
the formation of an anti-parallel alignment between the nucleic
acid sequences of the two nucleic acid molecules. The two molecules
are then able to form hydrogen bonds with corresponding bases on
the opposite strand to form a duplex molecule that, if it is
sufficiently stable, is detectable using methods well known in the
art. A nucleic acid molecule need not be 100% complementary to its
target sequence to be specifically hybridizable. However, the
amount of sequence complementarity that must exist for
hybridization to be specific is a function of the hybridization
conditions used.
[0113] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Calculations regarding hybridization conditions
required for attaining particular degrees of stringency are known
to those of ordinary skill in the art, and are discussed, for
example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, chapters 9 and 11; and Hames and
Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985.
Further detailed instruction and guidance with regard to the
hybridization of nucleic acids may be found, for example, in
Tijssen, "Overview of principles of hybridization and the strategy
of nucleic acid probe assays," in Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2, Elsevier, N Y, 1993; and Ausubel et al.,
Eds., Current Protocols in Molecular Biology, Chapter 2, Greene
Publishing and Wiley-Interscience, N Y, 1995.
[0114] As used herein, "stringent conditions" encompass conditions
under which hybridization will only occur if there is less than 20%
mismatch between the hybridization molecule and a homologous
sequence within the target nucleic acid molecule. "Stringent
conditions" include further particular levels of stringency. Thus,
as used herein, "moderate stringency" conditions are those under
which molecules with more than 20% sequence mismatch will not
hybridize; conditions of "high stringency" are those under which
sequences with more than 10% mismatch will not hybridize; and
conditions of "very high stringency" are those under which
sequences with more than 5% mismatch will not hybridize.
[0115] The following are representative, non-limiting hybridization
conditions. [0116] High Stringency condition (detects sequences
that share at least 90% sequence identity): Hybridization in
5.times.SSC buffer at 65.degree. C. for 16 hours; wash twice in
2.times.SSC buffer at room temperature for 15 minutes each; and
wash twice in 0.5.times.SSC buffer at 65.degree. C. for 20 minutes
each. [0117] Moderate Stringency condition (detects sequences that
share at least 80% sequence identity): Hybridization in
5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours;
wash twice in 2.times.SSC buffer at room temperature for 5-20
minutes each; and wash twice in 1.times.SSC buffer at 55-70.degree.
C. for 30 minutes each. [0118] Non-stringent control condition
(sequences that share at least 50% sequence identity will
hybridize): Hybridization in 6.times.SSC buffer at room temperature
to 55.degree. C. for 16-20 hours; wash at least twice in
2.times.-3.times.SSC buffer at room temperature to 55.degree. C.
for 20-30 minutes each.
[0119] As used herein, the term "substantially homologous" or
"substantial homology," with regard to a contiguous nucleic acid
sequence, refers to contiguous nucleotide sequences that hybridize
under stringent conditions to the reference nucleic acid sequence.
For example, nucleic acid sequences that are substantially
homologous to a reference nucleic acid sequence are those nucleic
acid sequences that hybridize under stringent conditions (e.g., the
Moderate Stringency conditions set forth, supra) to the reference
nucleic acid sequence. Substantially homologous sequences may have
at least 80% sequence identity. For example, substantially
homologous sequences may have from about 80% to 100% sequence
identity, such as about 81%; about 82%; about 83%; about 84%; about
85%; about 86%; about 87%; about 88%; about 89%; about 90%; about
91%; about 92%; about 93%; about 94% about 95%; about 96%; about
97%; about 98%; about 98.5%; about 99%; about 99.5%; and about
100%. The property of substantial homology is closely related to
specific hybridization. For example, a nucleic acid molecule is
specifically hybridizable when there is a sufficient degree of
complementarity to avoid non-specific binding of the nucleic acid
to non-target sequences under conditions where specific binding is
desired, for example, under stringent hybridization conditions.
[0120] As used herein, the term "ortholog" (or "orthologous")
refers to a gene in two or more species that has evolved from a
common ancestral nucleotide sequence, and may retain the same
function in the two or more species.
[0121] As used herein, two nucleic acid sequence molecules are said
to exhibit "complete complementarity" when every nucleotide of a
sequence read in the 5' to 3' direction is complementary to every
nucleotide of the other sequence when read in the 3' to 5'
direction. A nucleotide sequence that is complementary to a
reference nucleotide sequence will exhibit a sequence identical to
the reverse complement sequence of the reference nucleotide
sequence. These terms and descriptions are well defined in the art
and are easily understood by those of ordinary skill in the
art.
[0122] When determining the percentage of sequence identity between
amino acid sequences, it is well-known by those of skill in the art
that the identity of the amino acid in a given position provided by
an alignment may differ without affecting desired properties of the
polypeptides comprising the aligned sequences. In these instances,
the percent sequence identity may be adjusted to account for
similarity between conservatively substituted amino acids. These
adjustments are well-known and commonly used by those of skill in
the art. See, e.g., Myers and Miller (1988), Computer Applications
in Biosciences 4:11-7.
[0123] Embodiments of the invention include functional variants of
exemplary plastid transit peptide amino acid sequences, and nucleic
acid sequences encoding the same. A functional variant of an
exemplary transit peptide sequence may be, for example, a fragment
of an exemplary transit peptide amino acid sequence (such as an
N-terminal or C-terminal fragment), or a modified sequence of a
full-length exemplary transit peptide amino acid sequence or
fragment of an exemplary transit peptide amino acid sequence. An
exemplary transit peptide amino acid sequence may be modified in
some embodiments be introducing one or more conservative amino acid
substitutions. A "conservative" amino acid substitution is one in
which the amino acid residue is replaced by an amino acid residue
having a similar functional side chain, similar size, and/or
similar hydrophobicity. Families of amino acids that may be used to
replace another amino acid of the same family in order to introduce
a conservative substitution are known in the art. For example,
these amino acid families include: Basic amino acids (e.g., lysine,
arginine, and histidine); acidic amino acids (e.g., aspartic acid
and glutamic acid); uncharged (at physiological pH) polar amino
acids (e.g., glycine, asparagines, glutamine, serine, threonine,
tyrosine, and cytosine); non-polar amino acids (e.g., alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
and tryptophan); beta-branched amino acids (e.g., threonine,
valine, and isoleucine); and aromatic amino acids (e.g., tyrosine,
phenylalanine, tryptophan, and histidine). See, e.g., Sambrook et
al. (Eds.), supra; and Innis et al., PCR Protocols: A Guide to
Methods and Applications, 1990, Academic Press, NY, USA.
[0124] Operably linked: A first nucleotide sequence is "operably
linked" with a second nucleotide sequence when the first nucleotide
sequence is in a functional relationship with the second nucleotide
sequence. For instance, a promoter is operably linked to a coding
sequence if the promoter affects the transcription or expression of
the coding sequence. When recombinantly produced, operably linked
nucleotide sequences are generally contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
However, nucleotide sequences need not be contiguous to be operably
linked.
[0125] The term, "operably linked," when used in reference to a
regulatory sequence and a coding sequence, means that the
regulatory sequence affects the expression of the linked coding
sequence. "Regulatory sequences," or "control elements," refer to
nucleotide sequences that influence the timing and level/amount of
transcription, RNA processing or stability, or translation of the
associated coding sequence. Regulatory sequences may include
promoters; translation leader sequences; introns; enhancers;
stem-loop structures; repressor binding sequences; termination
sequences; polyadenylation recognition sequences; etc. Particular
regulatory sequences may be located upstream and/or downstream of a
coding sequence operably linked thereto. Also, particular
regulatory sequences operably linked to a coding sequence may be
located on the associated complementary strand of a double-stranded
nucleic acid molecule.
[0126] When used in reference to two or more amino acid sequences,
the term "operably linked" means that the first amino acid sequence
is in a functional relationship with at least one of the additional
amino acid sequences. For instance, a transit peptide (e.g., a CTP)
is operably linked to a second amino acid sequence within a
polypeptide comprising both sequences if the transit peptide
affects expression or trafficking of the polypeptide or second
amino acid sequence.
[0127] Promoter: As used herein, the term "promoter" refers to a
region of DNA that may be upstream from the start of transcription,
and that may be involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A promoter
may be operably linked to a coding sequence for expression in a
cell, or a promoter may be operably linked to a nucleotide sequence
encoding a signal sequence which may be operably linked to a coding
sequence for expression in a cell. A "plant promoter" may be a
promoter capable of initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred."
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific." A "cell type-specific" promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter may be a promoter which may be under
environmental control. Examples of environmental conditions that
may initiate transcription by inducible promoters include anaerobic
conditions and the presence of light. Tissue-specific,
tissue-preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which may be active under
most environmental conditions.
[0128] Any inducible promoter can be used in some embodiments of
the invention. See Ward et al. (1993), Plant Mol. Biol. 22:361-366.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent. Exemplary inducible promoters
include, but are not limited to: Promoters from the ACEI system
that responds to copper; Int gene from maize that responds to
benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and
the inducible promoter from a steroid hormone gene, the
transcriptional activity of which may be induced by a
glucocorticosteroid hormone (Schena et al. (1991), Proc. Natl.
Acad. Sci. USA 88:0421).
[0129] Exemplary constitutive promoters include, but are not
limited to: Promoters from plant viruses, such as the 35S promoter
from CaMV; promoters from rice actin genes; ubiquitin promoters;
pEMU; MAS; maize H3 histone promoter; and the ALS promoter,
Xba1/NcoI fragment 5' to the Brassica napus ALS3 structural gene
(or a nucleotide sequence similarity to said Xba1/NcoI fragment)
(International PCT Publication No. WO 96/30530).
[0130] Additionally, any tissue-specific or tissue-preferred
promoter may be utilized in some embodiments of the invention.
Plants transformed with a nucleic acid molecule comprising a coding
sequence operably linked to a tissue-specific promoter may produce
the product of the coding sequence exclusively, or preferentially,
in a specific tissue. Exemplary tissue-specific or tissue-preferred
promoters include, but are not limited to: A root-preferred
promoter, such as that from the phaseolin gene; a leaf-specific and
light-induced promoter such as that from cab or rubisco; an
anther-specific promoter such as that from LAT52; a pollen-specific
promoter such as that from Zm13; and a microspore-preferred
promoter such as that from apg.
[0131] Transformation: As used herein, the term "transformation" or
"transduction" refers to the transfer of one or more nucleic acid
molecule(s) into a cell. A cell is "transformed" by a nucleic acid
molecule transduced into the cell when the nucleic acid molecule
becomes stably replicated by the cell, either by incorporation of
the nucleic acid molecule into the cellular genome, or by episomal
replication. As used herein, the term "transformation" encompasses
all techniques by which a nucleic acid molecule can be introduced
into such a cell. Examples include, but are not limited to:
transfection with viral vectors; transformation with plasmid
vectors; electroporation (Fromm et al. (1986), Nature 319:791-3);
lipofection (Feigner et al. (1987), Proc. Natl. Acad. Sci. USA
84:7413-7); microinjection (Mueller et al. (1978), Cell 15:579-85);
Agrobacterium-mediated transfer (Fraley et al. (1983), Proc. Natl.
Acad. Sci. USA 80:4803-7); direct DNA uptake; and microprojectile
bombardment (Klein et al. (1987), Nature 327:70).
[0132] Transgene: An exogenous nucleic acid sequence. In some
examples, a transgene may be a sequence that encodes a polypeptide
comprising at least one synthetic Brassica-derived CTP. In
particular examples, a transgene may encode a polypeptide
comprising at least one synthetic Brassica-derived CTP and at least
an additional peptide sequence (e.g., a peptide sequence that
confers herbicide-resistance), for which plastid expression is
desirable. In these and other examples, a transgene may contain
regulatory sequences operably linked to a coding sequence of the
transgene (e.g., a promoter). For the purposes of this disclosure,
the term "transgenic" when used to refer to an organism (e.g., a
plant), refers to an organism that comprises the exogenous nucleic
acid sequence. In some examples, the organism comprising the
exogenous nucleic acid sequence may be an organism into which the
nucleic acid sequence was introduced via molecular transformation
techniques. In other examples, the organism comprising the
exogenous nucleic acid sequence may be an organism into which the
nucleic acid sequence was introduced by, for example, introgression
or cross-pollination in a plant.
[0133] Transport: As used herein, the terms "transport(s),"
"target(s)," and "transfer(s)" refers to the property of certain
amino acid sequences of the invention that facilitates the movement
of a polypeptide comprising the amino acid sequence from the
nucleus of a host cell into a plastid of the host cell. In
particular embodiments, such an amino acid sequence (i.e., a
synthetic Brassica-derived CTP sequence) may be capable of
transporting about 100%, at least about 95%, at least about 90%, at
least about 85%, at least about 80%, at least about 70%, at least
about 60%, and/or at least about 50% of a polypeptide comprising
the amino acid sequence into plastids of a host cell.
[0134] Vector: A nucleic acid molecule as introduced into a cell,
for example, to produce a transformed cell. A vector may include
nucleic acid sequences that permit it to replicate in the host
cell, such as an origin of replication. Examples of vectors
include, but are not limited to: a plasmid; cosmid; bacteriophage;
or virus that carries exogenous DNA into a cell. A vector may also
include one or more genes, antisense molecules, and/or selectable
marker genes and other genetic elements known in the art. A vector
may transduce, transform, or infect a cell, thereby causing the
cell to express the nucleic acid molecules and/or proteins encoded
by the vector. A vector optionally includes materials to aid in
achieving entry of the nucleic acid molecule into the cell (e.g., a
liposome, protein coating, etc.).
[0135] Unless specifically indicated or implied, the terms "a,"
"an," and "the" signify "at least one," as used herein.
[0136] Unless otherwise specifically explained, all technical and
scientific terms used herein have the same meaning as commonly
understood by those of ordinary skill in the art to which this
disclosure belongs. Definitions of common terms in molecular
biology can be found in, for example, Lewin B., Genes V, Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994
(ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular Biology and
Biotechnology: A Comprehensive Desk Reference, VCH Publishers,
Inc., 1995 (ISBN 1-56081-569-8). All percentages are by weight and
all solvent mixture proportions are by volume unless otherwise
noted. All temperatures are in degrees Celsius.
IV. Nucleic Acid Molecules Comprising a Synthetic Brassica-Derived
CTP-Encoding Sequence
[0137] In some embodiments, this disclosure provides a nucleic acid
molecule comprising at least one nucleotide sequence encoding a
synthetic Brassica-derived CTP operably linked to a nucleotide
sequence of interest. In particular embodiments, the nucleotide
sequence of interest may be a nucleotide sequence that encodes a
polypeptide of interest. In particular examples, a single nucleic
acid molecule is provided that encodes a polypeptide wherein a
TraP8 or TraP9 sequence is fused to the N-terminus of a polypeptide
of interest.
[0138] A synthetic Brassica-derived CTP may be derived from a
Brassica EPSPS gene. In particular examples of such embodiments,
the Brassica EPSPS gene may be one that comprises the nucleic acid
sequence set forth as SEQ ID NO:14, or a homologous nucleic acid
sequence from a different EPSPS gene, or may be an ortholog of the
Brassica EPSPS gene comprising the nucleic acid sequence set forth
as SEQ ID NO:14 (for example, the Brassica EPSPS gene comprising
the nucleic acid sequence set forth as SEQ ID NO:15).
[0139] In some embodiments, a synthetic Brassica-derived
chloroplast transit peptide may be a chimeric Brassica-derived CTP.
A synthetic chimeric Brassica-derived CTP may be derived from a
reference Brassica CTP sequence by joining a first contiguous amino
acid sequence comprised within the reference Brassica CTP sequence
to a second contiguous amino acid sequence comprised within a
different CTP sequence (e.g., a second Brassica CTP sequence). In
particular embodiments, the different CTP sequence comprising the
second contiguous amino acid sequence may be encoded by a
homologous gene sequence from a genome other than that of the
Brassica sp. from which the reference sequence was obtained (e.g.,
a different Brassica sp., a plant other than a Brassica sp.; a
lower photosynthetic eukaryote, for example, a Chlorophyte; and a
prokaryote, for example, a Cyanobacterium or Agrobacterium). Thus,
a nucleotide sequence encoding a synthetic Brassica-derived CTP may
be derived from a reference Brassica CTP-encoding gene sequence by
fusing a nucleotide sequence that encodes a contiguous amino acid
sequence of the reference Brassica CTP sequence with a nucleotide
sequence that encodes the contiguous amino acid sequence from a
different CTP sequence that is homologous to the remainder of the
reference Brassica CTP sequence. In these and other examples, the
contiguous amino acid sequence of the reference Brassica CTP
sequence may be located at the 5' end or the 3' end of the
synthetic Brassica-derived CTP.
[0140] In some embodiments, a synthetic chimeric Brassica-derived
CTP may be derived from a plurality of Brassica CTP sequences
(including a reference Brassica CTP sequence) by joining a
contiguous amino acid sequence comprised within one Brassica CTP
sequence to a contiguous amino acid sequence comprised within a
different Brassica CTP sequence. In particular embodiments, the
plurality of Brassica CTP sequences may be encoded by orthologous
gene sequences in different Brassica species. In some examples, the
plurality of Brassica CTP sequences may be exactly two Brassica CTP
sequences. Thus, a nucleotide sequence encoding a synthetic
chimeric Brassica-derived CTP may be derived from two homologous
(e.g., substantially homologous) Brassica CTP-encoding gene
sequences (e.g., orthologous gene sequences) by fusing the
nucleotide sequence that encodes a contiguous amino acid sequence
of one of the Brassica CTP sequences with the nucleotide sequence
that encodes the contiguous amino acid sequence from the other of
the Brassica CTP sequences that is homologous to the remainder of
the first Brassica CTP sequence. TraP8 and TraP9 are illustrative
examples of such a synthetic chimeric Brassica-derived CTP.
[0141] One of ordinary skill in the art will understand that,
following the selection of a first contiguous amino acid sequence
within a reference Brassica CTP sequence, the identification and
selection of the contiguous amino acid sequence from the remainder
of a homologous CTP sequence according to the foregoing derivation
process is unambiguous and automatic. In some examples, the first
contiguous amino acid sequence may be between about 25 and about 41
amino acids in length (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 amino acids in length).
In some embodiments, the first contiguous amino acid sequence
within the reference Brassica CTP sequence is defined by the
position at the 3' end of a "SVSL" (SEQ ID NO:13) motif that is
conserved within some Brassica EPSPS genes.
[0142] Examples of synthetic chimeric Brassica-derived CTP
sequences according to the foregoing process are represented by SEQ
ID NO:3 and SEQ ID NO:4. In view of the degeneracy of the genetic
code, the genus of nucleotide sequences encoding these peptides
will be immediately envisioned by one of ordinary skill in the art.
Examples of such polynucleotide sequences include SEQ ID NOs: 5, 6,
8, and 9. These particular examples illustrate the structural
features of synthetic chimeric Brassica-derived CTPs by
incorporating contiguous sequences from a homologous CTP from one
of several ESPSP orthologs of a B. napus ESPSP gene.
[0143] Some embodiments include functional variants of a synthetic
Brassica-derived chloroplast transit peptide, and/or nucleic acids
encoding the same. Such functional variants include, for example
and without limitation: a synthetic Brassica-derived CTP-encoding
sequence that is derived from a homolog and/or ortholog of one or
both of the Brassica CTP-encoding sequences set forth as SEQ ID
NOs:14 and/or SEQ ID NO:15, and/or a CTP encoded thereby; a nucleic
acid that encodes a synthetic Brassica-derived CTP that comprises a
contiguous amino acid sequence within SEQ ID NO:1 and/or SEQ ID
NO:2, and/or a CTP encoded thereby; a truncated synthetic
Brassica-derived CTP-encoding sequence that comprises a contiguous
nucleic acid sequence within one of SEQ ID NOs:5, 6, 8, and 9; a
truncated synthetic Brassica-derived CTP-encoding sequence that
comprises a contiguous nucleic acid sequence that is substantially
homologous to one of SEQ ID NOs: 5, 6, 8, and 9; a truncated
synthetic Brassica-derived CTP that comprises a contiguous amino
acid sequence within one of SEQ ID NOs: 3 and 4; a nucleic acid
that encodes a synthetic Brassica-derived CTP comprising a
contiguous amino acid sequence within one of SEQ ID NOs: 5, 6, 8,
and 9, and/or a CTP encoded thereby; a nucleic acid that encodes a
synthetic Brassica-derived CTP comprising a contiguous amino acid
sequence within one of SEQ ID NOs: 3 and 4 that has one or more
conservative amino acid substitutions, and/or a CTP encoded
thereby; and a nucleic acid that encodes a synthetic
Brassica-derived CTP comprising a contiguous amino acid sequence
within one of SEQ ID NOs: 3 and 4 that has one or more
non-conservative amino acid substitutions that are demonstrated to
direct an operably linked peptide to a plastid in a
plastid-containing cell, and/or a CTP encoded thereby.
[0144] Thus, some embodiments of the invention include a nucleic
acid molecule comprising a nucleotide sequence encoding a synthetic
chimeric Brassica-derived CTP comprising one or more conservative
amino acid substitutions. Such a nucleic acid molecule may be
useful, for example, in facilitating manipulation of a CTP-encoding
sequence of the invention in molecular biology techniques. For
example, in some embodiments, a CTP-encoding sequence of the
invention may be introduced into a suitable vector for sub-cloning
of the sequence into an expression vector, or a CTP-encoding
sequence of the invention may be introduced into a nucleic acid
molecule that facilitates the production of a further nucleic acid
molecule comprising the CTP-encoding sequence operably linked to a
nucleotide sequence of interest. In these and further embodiments,
one or more amino acid positions in the sequence of a synthetic
chimeric Brassica-derived CTP may be deleted. For example, the
sequence of a synthetic chimeric Brassica-derived CTP may be
modified such that the amino acid(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions in the
sequence are deleted. An alignment of homologous CTP sequences may
be used to provide guidance as to which amino acids may be deleted
without affecting the function of the synthetic CTP.
[0145] In particular examples, a synthetic Brassica-derived
chloroplast transit peptide is less than 80 amino acids in length.
For example, a synthetic Brassica-derived CTP may be 79, 78, 77,
76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60,
or fewer amino acids in length. In certain examples, a synthetic
Brassica-derived CTP may be about 65, about 68, about 72, or about
74 amino acids in length. In these and further examples, a
synthetic Brassica-derived CTP may comprise an amino acid sequence
set forth in one of SEQ ID NOs: 3 and 4, or a functional variant of
any of the foregoing. Thus, a synthetic Brassica-derived CTP may
comprise an amino acid sequence comprising one of SEQ ID NOs: 3 and
4 or a functional variant thereof, wherein the length of the
synthetic Brassica-derived CTP is less than 80 amino acids in
length. In certain examples, a synthetic Brassica-derived CTP may
comprise an amino acid sequence that is, e.g., at least 80%, at
least 85%, at least 90%, at least 92%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical to one of SEQ ID NOs: 3 and 4.
[0146] All of the nucleotide sequences that encode a particular
synthetic Brassica-derived CTP, for example, the TraP8 peptide of
SEQ ID NO:3 and the TraP9 peptide of SEQ ID NO:4, or functional
variants of any of the foregoing including any specific deletions
and/or conservative amino acid substitutions, will be recognizable
by those of skill in the art in view of the present disclosure. The
degeneracy of the genetic code provides a finite number of coding
sequences for a particular amino acid sequence. The selection of a
particular sequence to encode a synthetic Brassica-derived CTP is
within the discretion of the practitioner. Different coding
sequences may be desirable in different applications. For example,
to increase expression of the synthetic Brassica-derived CTP in a
particular host, a coding sequence may be selected that reflects
the codon usage bias of the host. By way of example, a synthetic
Brassica-derived CTP may be encoded by a nucleotide sequence set
forth as one of SEQ ID NOs: 5, 6, 8, and 9.
[0147] In nucleic acid molecules provided in some embodiments of
the invention, the last codon of a nucleotide sequence encoding a
synthetic Brassica-derived CTP and the first codon of a nucleotide
sequence of interest may be separated by any number of nucleotide
triplets, e.g., without coding for an intron or a "STOP." In some
examples, a sequence encoding the first amino acids of a mature
protein normally associated with a chloroplast transit peptide in a
natural precursor polypeptide may be present between the last codon
of a nucleotide sequence encoding a synthetic Brassica-derived CTP
and the first codon of a nucleotide sequence of interest. A
sequence separating a nucleotide sequence encoding a synthetic
Brassica-derived CTP and the first codon of a nucleotide sequence
of interest may, for example, consist of any sequence, such that
the amino acid sequence encoded is not likely to significantly
alter the translation of the chimeric polypeptide and its
translocation to a plastid. In these and further embodiments, the
last codon of a nucleotide sequence encoding a synthetic
Brassica-derived chloroplast transit peptide may be fused in
phase-register with the first codon of the nucleotide sequence of
interest directly contiguous thereto, or separated therefrom by no
more than a short peptide sequence, such as that encoded by a
synthetic nucleotide linker (e.g., a nucleotide linker that may
have been used to achieve the fusion).
[0148] In some embodiments, it may be desirable to modify the
nucleotides of a nucleotide sequence of interest and/or a synthetic
Brassica-derived CTP-encoding sequence fused thereto in a single
coding sequence, for example, to enhance expression of the coding
sequence in a particular host. The genetic code is redundant with
64 possible codons, but most organisms preferentially use a subset
of these codons. The codons that are utilized most often in a
species are called optimal codons, and those not utilized very
often are classified as rare or low-usage codons. Zhang et al.
(1991), Gene 105:61-72. Codons may be substituted to reflect the
preferred codon usage of a particular host in a process sometimes
referred to as "codon optimization." Optimized coding sequences
containing codons preferred by a particular prokaryotic or
eukaryotic host may be prepared, for example, to increase the rate
of translation or to produce recombinant RNA transcripts having
desirable properties (e.g., a longer half-life, as compared with
transcripts produced from a non-optimized sequence).
[0149] Any polypeptide may be targeted to a plastid of a
plastid-containing cell by incorporation of a synthetic
Brassica-derived CTP sequence. For example, a polypeptide may be
linked to a synthetic Brassica-derived CTP sequence in some
embodiments, so as to direct the polypeptide to a plastid in a cell
wherein the linked polypeptide-CTP molecule is expressed. In
particular embodiments, a polypeptide targeted to a plastid by
incorporation of a synthetic Brassica-derived CTP sequence may be,
for example, a polypeptide that is normally expressed in a plastid
of a cell wherein the polypeptide is natively expressed. For
example and without limitation, a polypeptide targeted to a plastid
by incorporation of a synthetic Brassica-derived CTP sequence may
be a polypeptide involved in herbicide resistance, virus
resistance, bacterial pathogen resistance, insect resistance,
nematode resistance, or fungal resistance. See, e.g., U.S. Pat.
Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. A
polypeptide targeted to a plastid by incorporation of a synthetic
Brassica-derived CTP sequence may alternatively be, for example and
without limitation, a polypeptide involved in plant vigor or yield
(including polypeptides involved in tolerance for extreme
temperatures, soil conditions, light levels, water levels, and
chemical environment), or a polypeptide that may be used as a
marker to identify a plant comprising a trait of interest (e.g., a
selectable marker gene product, a polypeptide involved in seed
color, etc.).
[0150] Non-limiting examples of polypeptides involved in herbicide
resistance that may be linked to a synthetic Brassica-derived CTP
sequence in some embodiments of the invention include: acetolactase
synthase (ALS), mutated ALS, and precursors of ALS (see, e.g., U.S.
Pat. No. 5,013,659); EPSPS (see, e.g., U.S. Pat. Nos. 4,971,908 and
6,225,114), such as a CP4 EPSPS, a class III EPSPS, or a class IV
EPSPS; enzymes that modify a physiological process that occurs in a
plastid, including photosynthesis, and synthesis of fatty acids,
amino acids, oils, arotenoids, terpenoids, starch, etc. Other
non-limiting examples of polypeptides that may be linked to a
synthetic Brassica-derived chloroplast transit peptide in
particular embodiments include: zeaxanthin epoxidase, choline
monooxygenase, ferrochelatase, omega-3 fatty acid desaturase,
glutamine synthetase, starch modifying enzymes, polypeptides
involved in synthesis of essential amino acids, provitamin A,
hormones, Bt toxin proteins, etc. Nucleotide sequences encoding the
aforementioned peptides are known in the art, and such nucleotide
sequences may be operably linked to a nucleotide sequence encoding
a synthetic Brassica-derived CTP to be expressed into a polypeptide
comprising the polypeptide of interest linked to the synthetic
Brassica-derived CTP. Furthermore, additional nucleotide sequences
encoding any of the aforementioned polypeptides may be identified
by those of skill in the art (for example, by cloning of genes with
high homology to other genes encoding the particular polypeptide).
Once such a nucleotide sequence has been identified, it is a
straightforward process to design a nucleotide sequence comprising
a synthetic Brassica-derived CTP-encoding sequence operably linked
to the identified nucleotide sequence, or a sequence encoding an
equivalent polypeptide.
V. Expression of Polypeptides Comprising a Synthetic
Brassica-Derived Chloroplast Transit Peptide
[0151] In some embodiments, at least one nucleic acid molecule(s)
comprising a nucleotide sequence encoding a polypeptide comprising
at least one synthetic Brassica-derived CTP, or functional
equivalent thereof, may be introduced into a cell, tissue, or
organism for expression of the polypeptide therein. In particular
embodiments, a nucleic acid molecule may comprise a nucleotide
sequence of interest operably linked to a nucleotide sequence
encoding a synthetic Brassica-derived CTP. For example, a nucleic
acid molecule may comprise a coding sequence encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP and at least
an additional peptide sequence encoded by a nucleotide sequence of
interest. In some embodiments, a nucleic acid molecule of the
invention may be introduced into a plastid-containing host cell,
tissue, or organism (e.g., a plant cell, plant tissue, and plant),
such that a polypeptide may be expressed from the nucleic acid
molecule in the plastid-containing host cell, tissue, or organism,
wherein the expressed polypeptide comprises at least one synthetic
Brassica-derived CTP and at least an additional peptide sequence
encoded by a nucleotide sequence of interest. In certain examples,
the synthetic Brassica-derived CTP of such an expressed polypeptide
may facilitate targeting of a portion of the polypeptide comprising
at least the additional peptide sequence to a plastid of the host
cell, tissue, or organism.
[0152] In some embodiments, a nucleic acid molecule of the
invention may be introduced into a plastid-containing cell by one
of any of the methodologies known to those of skill in the art. In
particular embodiments, a host cell, tissue, or organism may be
contacted with a nucleic acid molecule of the invention in order to
introduce the nucleic acid molecule into the cell, tissue, or
organism. In particular embodiments, a cell may be transformed with
a nucleic acid molecule of the invention such that the nucleic acid
molecule is introduced into the cell, and the nucleic acid molecule
is stably integrated into the genome of the cell. In some
embodiments, a nucleic acid molecule comprising at least one
nucleotide sequence encoding a synthetic Brassica-derived CTP
operably linked to a nucleotide sequence of interest may be used
for transformation of a cell, for example, a plastid-containing
cell (e.g., a plant cell). In order to initiate or enhance
expression, a nucleic acid molecule may comprise one or more
regulatory sequences, which regulatory sequences may be operably
linked to the nucleotide sequence encoding a polypeptide comprising
at least one synthetic Brassica-derived CTP.
[0153] A nucleic acid molecule may, for example, be a vector system
including, for example, a linear or a closed circular plasmid. In
particular embodiments, the vector may be an expression vector.
Nucleic acid sequences of the invention may, for example, be
inserted into a vector, such that the nucleic acid sequence is
operably linked to one or more regulatory sequences. Many vectors
are available for this purpose, and selection of the particular
vector may depend, for example, on the size of the nucleic acid to
be inserted into the vector and the particular host cell to be
transformed with the vector. A vector typically contains various
components, the identity of which depend on a function of the
vector (e.g., amplification of DNA and expression of DNA), and the
particular host cell(s) with which the vector is compatible.
[0154] Some embodiments may include a plant transformation vector
that comprises a nucleotide sequence comprising at least one of the
above-described regulatory sequences operatively linked to one or
more nucleotide sequence(s) encoding a polypeptide comprising at
least one synthetic Brassica-derived CTP. The one or more
nucleotide sequences may be expressed, under the control of the
regulatory sequence(s), in a plant cell, tissue, or organism to
produce a polypeptide comprising a synthetic Brassica-derived CTP
that targets at least a portion of the polypeptide to a plastid of
the plant cell, tissue, or organism.
[0155] In some embodiments, a regulatory sequence operably linked
to a nucleotide sequence encoding a polypeptide comprising at least
one synthetic Brassica-derived CTP, may be a promoter sequence that
functions in a host cell, such as a bacterial cell wherein the
nucleic acid molecule is to be amplified, or a plant cell wherein
the nucleic acid molecule is to be expressed. Promoters suitable
for use in nucleic acid molecules of the invention include those
that are inducible, viral, synthetic, or constitutive, all of which
are well known in the art. Non-limiting examples of promoters that
may be useful in embodiments of the invention are provided by: U.S.
Pat. No. 6,437,217 (maize RS81 promoter); U.S. Pat. No. 5,641,876
(rice actin promoter); U.S. Pat. No. 6,426,446 (maize RS324
promoter); U.S. Pat. No. 6,429,362 (maize PR-1 promoter); U.S. Pat.
No. 6,232,526 (maize A3 promoter); U.S. Pat. No. 6,177,611
(constitutive maize promoters); U.S. Pat. Nos. 5,322,938,
5,352,605, 5,359,142, and 5,530,196 (35S promoter); U.S. Pat. No.
6,433,252 (maize L3 oleosin promoter); U.S. Pat. No. 6,429,357
(rice actin 2 promoter, and rice actin 2 intron); U.S. Pat. No.
6,294,714 (light-inducible promoters); U.S. Pat. No. 6,140,078
(salt-inducible promoters); U.S. Pat. No. 6,252,138
(pathogen-inducible promoters); U.S. Pat. No. 6,175,060
(phosphorous deficiency-inducible promoters); U.S. Pat. No.
6,388,170 (bidirectional promoters); U.S. Pat. No. 6,635,806
(gamma-coixin promoter); and U.S. patent application Ser. No.
09/757,089 (maize chloroplast aldolase promoter).
[0156] Additional exemplary promoters include the nopaline synthase
(NOS) promoter (Ebert et al. (1987), Proc. Natl. Acad. Sci. USA
84(16):5745-9); the octopine synthase (OCS) promoter (which is
carried on tumor-inducing plasmids of Agrobacterium tumefaciens);
the caulimovirus promoters such as the cauliflower mosaic virus
(CaMV) 19S promoter (Lawton et al. (1987), Plant Mol. Biol.
9:315-24); the CaMV 35S promoter (Odell et al. (1985), Nature
313:810-2; the figwort mosaic virus 35S-promoter (Walker et al.
(1987), Proc. Natl. Acad. Sci. USA 84(19):6624-8); the sucrose
synthase promoter (Yang and Russell (1990), Proc. Natl. Acad. Sci.
USA 87:4144-8); the R gene complex promoter (Chandler et al.
(1989), Plant Cell 1:1175-83); the chlorophyll a/b binding protein
gene promoter; CaMV35S (U.S. Pat. Nos. 5,322,938, 5,352,605,
5,359,142, and 5,530,196); FMV35S (U.S. Pat. Nos. 6,051,753, and
5,378,619); a PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1
promoter (U.S. Pat. No. 6,677,503); and AGRtu.nos promoters
(GenBank Accession No. V00087; Depicker et al. (1982), J. Mol.
Appl. Genet. 1:561-73; Bevan et al. (1983), Nature 304:184-7).
[0157] In particular embodiments, nucleic acid molecules of the
invention may comprise a tissue-specific promoter. A
tissue-specific promoter is a nucleotide sequence that directs a
higher level of transcription of an operably linked nucleotide
sequence in the tissue for which the promoter is specific, relative
to the other tissues of the organism. Examples of tissue-specific
promoters include, without limitation: tapetum-specific promoters;
anther-specific promoters; pollen-specific promoters (See, e.g.,
U.S. Pat. No. 7,141,424, and International PCT Publication No. WO
99/042587); ovule-specific promoters; (See, e.g., U.S. Patent
Application No. 2001/047525 A1); fruit-specific promoters (See,
e.g., U.S. Pat. Nos. 4,943,674, and 5,753,475); and seed-specific
promoters (See, e.g., U.S. Pat. Nos. 5,420,034, and 5,608,152). In
some embodiments, a developmental stage-specific promoter (e.g., a
promoter active at a later stage in development) may be used in a
composition or method of the invention.
[0158] Additional regulatory sequences that may in some embodiments
be operably linked to a nucleic acid molecule include 5' UTRs
located between a promoter sequence and a coding sequence that
function as a translation leader sequence. The translation leader
sequence is present in the fully-processed mRNA, and it may affect
processing of the primary transcript, and/or RNA stability.
Examples of translation leader sequences include maize and petunia
heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus
coat protein leaders, plant rubisco leaders, and others. See, e.g.,
Turner and Foster (1995), Molecular Biotech. 3(3):225-36.
Non-limiting examples of 5' UTRs are provided by: GmHsp (U.S. Pat.
No. 5,659,122); PhDnaK (U.S. Pat. No. 5,362,865); AtAnt1; TEV
(Carrington and Freed (1990), J. Virol. 64:1590-7); and AGRtunos
(GenBank Accession No. V00087; and Bevan et al. (1983), Nature
304:184-7).
[0159] Additional regulatory sequences that may in some embodiments
be operably linked to a nucleic acid molecule also include 3'
non-translated sequences, 3' transcription termination regions, or
poly-adenylation regions. These are genetic elements located
downstream of a nucleotide sequence, and include polynucleotides
that provide polyadenylation signal, and/or other regulatory
signals capable of affecting transcription or mRNA processing. The
polyadenylation signal functions in plants to cause the addition of
polyadenylate nucleotides to the 3' end of the mRNA precursor. The
polyadenylation sequence can be derived from a variety of plant
genes, or from T-DNA genes. A non-limiting example of a 3'
transcription termination region is the nopaline synthase 3' region
(nos 3; Fraley et al. (1983), Proc. Natl. Acad. Sci. USA
80:4803-7). An example of the use of different 3' nontranslated
regions is provided in Ingelbrecht et al., (1989), Plant Cell
1:671-80. Non-limiting examples of polyadenylation signals include
one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al.
(1984), EMBO J. 3:1671-9) and AGRtu.nos (GenBank Accession No.
E01312).
[0160] A recombinant nucleic acid molecule or vector of the present
invention may comprise a selectable marker that confers a
selectable phenotype on a transformed cell, such as a plant cell.
Selectable markers may also be used to select for plants or plant
cells that comprise recombinant nucleic acid molecule of the
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g., kanamycin, Geneticin (G418), bleomycin,
hygromycin, etc.), or herbicide resistance (e.g., glyphosate,
etc.). Examples of selectable markers include, but are not limited
to: a neo gene which codes for kanamycin resistance and can be
selected for using kanamycin, G418, etc.; a pat or bar gene which
codes for bialaphos resistance; a mutant EPSP synthase gene which
encodes glyphosate resistance; a nitrilase gene which confers
resistance to bromoxynil; a mutant acetolactate synthase gene (ALS)
which confers imidazolinone or sulfonylurea resistance; and a
methotrexate resistant DHFR gene. Multiple selectable markers are
available that confer resistance to ampicillin, bleomycin,
chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin,
methotrexate, phosphinothricin, puromycin, spectinomycin,
rifampicin, streptomycin and tetracycline, and the like. Examples
of such selectable markers are illustrated in, e.g., U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047.
[0161] A recombinant nucleic acid molecule or vector of the present
invention may also or alternatively include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson et al. (1987), Plant Mol. Biol.
Rep. 5:387-405); an R-locus gene, which encodes a product that
regulates the production of anthocyanin pigments (red color) in
plant tissues (Dellaporta et al. (1988), "Molecular cloning of the
maize R-nj allele by transposon tagging with Ac." In 18th Stadler
Genetics Symposium, P. Gustafson and R. Appels, eds. (New York:
Plenum), pp. 263-82); a .beta.-lactamase gene (Sutcliffe et al.
(1978), Proc. Natl. Acad. Sci. USA 75:3737-41); a gene which
encodes an enzyme for which various chromogenic substrates are
known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene
(Ow et al. (1986), Science 234:856-9); a xy/E gene that encodes a
catechol dioxygenase that can convert chromogenic catechols
(Zukowski et al. (1983), Gene 46(2-3):247-55); an amylase gene
(Ikatu et al. (1990) Bio/Technol. 8:241-2); a tyrosinase gene which
encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin (Katz et al. (1983),
1 Gen. Microbiol. 129:2703-14); and an .alpha.-galactosidase.
[0162] Suitable methods for transformation of host cells include
any method by which DNA can be introduced into a cell, for example
and without limitation: by transformation of protoplasts (see,
e.g., U.S. Pat. No. 5,508,184); by desiccation/inhibition-mediated
DNA uptake (see, e.g., Potrykus et al. (1985), Mol. Gen. Genet.
199:183-8); by electroporation (see, e.g., U.S. Pat. No.
5,384,253); by agitation with silicon carbide fibers (see, e.g.,
U.S. Pat. Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated
transformation (see, e.g., U.S. Pat. Nos. 5,563,055, 5,591,616,
5,693,512, 5,824,877, 5,981,840, and 6,384,301); and by
acceleration of DNA-coated particles (see, e.g., U.S. Pat. Nos.
5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and
6,403,865); etc. Through the application of techniques such as
these, the cells of virtually any species may be stably
transformed. In some embodiments, transforming DNA is integrated
into the genome of the host cell. In the case of multicellular
species, transgenic cells may be regenerated into a transgenic
organism. Alternatively, the transgenic cells may not be capable of
regeneration to a plant. Any of these techniques may be used to
produce a transgenic plant, for example, comprising one or more
nucleic acid sequences of the invention in the genome of the
transgenic plant.
[0163] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria which genetically
transform plant cells. The T.sub.i and R.sub.i plasmids of A.
tumefaciens and A. rhizogenes, respectively, carry genes
responsible for genetic transformation of the plant. The T.sub.i
(tumor-inducing)-plasmids contain a large segment, known as T-DNA,
which is transferred to transformed plants. Another segment of the
T.sub.i plasmid, the vir region, is responsible for T-DNA transfer.
The T-DNA region is bordered by terminal repeats. In some modified
binary vectors, the tumor-inducing genes have been deleted, and the
functions of the vir region are utilized to transfer foreign DNA
bordered by the T-DNA border sequences. The T-region may also
contain, for example, a selectable marker for efficient recovery of
transgenic plants and cells, and a multiple cloning site for
inserting sequences for transfer such as a synthetic
Brassica-derived CTP-encoding nucleic acid.
[0164] Thus, in some embodiments, a plant transformation vector may
be derived from a T.sub.i plasmid of A. tumefaciens (See, e.g.,
U.S. Pat. Nos. 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and
European Patent EP 0 122 791) or a Ri plasmid of A. rhizogenes.
Additional plant transformation vectors include, for example and
without limitation, those described by Herrera-Estrella et al.
(1983), Nature 303:209-13; Bevan et al. (1983), Nature 304:184-7;
Klee et al. (1985), Bio/Technol. 3:637-42; and in European Patent
EP 0 120 516, and those derived from any of the foregoing. Other
bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium that
interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants. These plant-associated
symbiotic bacteria can be made competent for gene transfer by
acquisition of both a disarmed T.sub.i plasmid and a suitable
binary vector.
[0165] After providing exogenous DNA to recipient cells,
transformed cells are generally identified for further culturing
and plant regeneration. In order to improve the ability to identify
transformed cells, one may desire to employ a selectable or
screenable marker gene, as previously set forth, with the vector
used to generate the transformant. In the case where a selectable
marker is used, transformed cells are identified within the
potentially transformed cell population by exposing the cells to a
selective agent or agents. In the case where a screenable marker is
used, cells may be screened for the desired marker gene trait.
[0166] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In some
embodiments, any suitable plant tissue culture media (e.g., MS and
N6 media) may be modified by including further substances, such as
growth regulators. Tissue may be maintained on a basic media with
growth regulators until sufficient tissue is available to begin
plant regeneration efforts, or following repeated rounds of manual
selection, until the morphology of the tissue is suitable for
regeneration (e.g., at least 2 weeks), then transferred to media
conducive to shoot formation. Cultures are transferred periodically
until sufficient shoot formation has occurred. Once shoots are
formed, they are transferred to media conducive to root formation.
Once sufficient roots are formed, plants can be transferred to soil
for further growth and maturity.
[0167] To confirm the presence of a nucleic acid molecule of
interest (for example, a nucleotide sequence encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP) in a
regenerating plant, a variety of assays may be performed. Such
assays include, for example: molecular biological assays, such as
Southern and Northern blotting, PCR, and nucleic acid sequencing;
biochemical assays, such as detecting the presence of a protein
product, e.g., by immunological means (ELISA and/or Western blots)
or by enzymatic function; plant part assays, such as leaf or root
assays; and analysis of the phenotype of the whole regenerated
plant.
[0168] By way of example, integration events may be analyzed by PCR
amplification using, e.g., oligonucleotide primers specific for a
nucleotide sequence of interest. PCR genotyping is understood to
include, but not be limited to, polymerase-chain reaction (PCR)
amplification of genomic DNA derived from isolated host plant
tissue predicted to contain a nucleic acid molecule of interest
integrated into the genome, followed by standard cloning and
sequence analysis of PCR amplification products. Methods of PCR
genotyping have been well described (see, e.g., Rios, G. et al.
(2002), Plant J. 32:243-53), and may be applied to genomic DNA
derived from any plant species (e.g., Z. mays or G. max) or tissue
type, including cell cultures.
[0169] A transgenic plant formed using Agrobacterium-dependent
transformation methods typically contains a single recombinant DNA
sequence inserted into one chromosome. The single recombinant DNA
sequence is referred to as a "transgenic event" or "integration
event." Such transgenic plants are heterozygous for the inserted
DNA sequence. In some embodiments, a transgenic plant homozygous
with respect to a transgene may be obtained by sexually mating
(selfing) an independent segregant transgenic plant that contains a
single exogenous gene sequence to itself, for example, an F.sub.0
plant, to produce F.sub.1 seed. One fourth of the F.sub.1 seed
produced will be homozygous with respect to the transgene.
Germinating F.sub.1 seed results in plants that can be tested for
heterozygosity, typically using a SNP assay or a thermal
amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0170] In particular embodiments, copies of at least one
polypeptide comprising at least one synthetic Brassica-derived CTP
are produced in a plastid-containing cell, into which has been
introduced at least one nucleic acid molecule(s) comprising a
nucleotide sequence encoding the at least one polypeptide
comprising at least one synthetic Brassica-derived CTP. Each
polypeptide comprising at least one synthetic Brassica-derived CTP
may be expressed from multiple nucleic acid sequences introduced in
different transformation events, or from a single nucleic acid
sequence introduced in a single transformation event. In some
embodiments, a plurality of such polypeptides is expressed under
the control of a single promoter. In other embodiments, a plurality
of such polypeptides is expressed under the control of multiple
promoters. Single polypeptides may be expressed that comprise
multiple peptide sequences, each of which peptide sequences is to
be targeted to a plastid.
[0171] In addition to direct transformation of a plant with a
recombinant nucleic acid molecule, transgenic plants can be
prepared by crossing a first plant having at least one transgenic
event with a second plant lacking such an event. For example, a
recombinant nucleic acid molecule comprising a nucleotide sequence
encoding a polypeptide comprising at least one synthetic
Brassica-derived CTP may be introduced into a first plant line that
is amenable to transformation, to produce a transgenic plant, which
transgenic plant may be crossed with a second plant line to
introgress the nucleotide sequence that encodes the polypeptide
into the second plant line.
VI. Plant Materials Comprising a Synthetic Brassica-Derived
Chloroplast Transit Peptide-Directed Polypeptide
[0172] In some embodiments, a plant cell is provided, wherein the
plant cell comprises a nucleotide sequence encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP. In
particular embodiments, such a plant cell may be produced by
transformation of a plant cell that is not capable of regeneration
to produce a plant. In some embodiments, a plant is provided,
wherein the plant comprises a plant cell comprising a nucleotide
sequence encoding a polypeptide comprising at least one synthetic
Brassica-derived CTP. In particular embodiments, such a plant may
be produced by transformation of a plant tissue or plant cell, and
regeneration of a whole plant. In further embodiments, such a plant
may be obtained from a commercial source, or through introgression
of a nucleic acid comprising a nucleotide sequence encoding a
polypeptide comprising at least one synthetic Brassica-derived CTP
into a germplasm. In particular embodiments, such a plant comprises
plant cells comprising a nucleotide sequence encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP that are not
capable of regeneration to produce a plant. Plant materials
comprising a plant cell comprising a nucleotide sequence encoding a
polypeptide comprising at least one synthetic Brassica-derived CTP
are also provided. Such a plant material may be obtained from a
plant comprising the plant cell.
[0173] A transgenic plant, nonregenerable plant cell, or plant
material comprising a nucleotide sequence encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP may in some
embodiments exhibit one or more of the following characteristics:
expression of the polypeptide in a cell of the plant; expression of
a portion of the polypeptide in a plastid of a cell of the plant;
import of the polypeptide from the cytosol of a cell of the plant
into a plastid of the cell; plastid-specific expression of the
polypeptide in a cell of the plant; and/or localization of the
polypeptide in a cell of the plant. Such a plant may additionally
have one or more desirable traits other than expression of the
encoded polypeptide. Such traits may include, for example:
resistance to insects, other pests, and disease-causing agents;
tolerances to herbicides; enhanced stability, yield, or shelf-life;
environmental tolerances; pharmaceutical production; industrial
product production; and nutritional enhancements.
[0174] A transgenic plant according to the invention may be any
plant capable of being transformed with a nucleic acid molecule of
the invention. Accordingly, the plant may be a dicot or monocot.
Non-limiting examples of dicotyledonous plants usable in the
present methods include Arabidopsis, alfalfa, beans, broccoli,
cabbage, carrot, cauliflower, celery, Chinese cabbage, cotton,
cucumber, eggplant, lettuce, melon, pea, pepper, peanut, potato,
pumpkin, radish, rapeseed, spinach, soybean, squash, sugarbeet,
sunflower, tobacco, tomato, and watermelon. Non-limiting examples
of monocotyledonous plants usable in the present methods include
corn, Brassica, onion, rice, sorghum, wheat, rye, millet,
sugarcane, oat, triticale, switchgrass, and turfgrass. Transgenic
plants according to the invention may be used or cultivated in any
manner.
[0175] Some embodiments also provide commodity products containing
one or more nucleotide sequences encoding a polypeptide comprising
at least one synthetic Brassica-derived CTP, for example, a
commodity product produced from a recombinant plant or seed
containing one or more of such nucleotide sequences. Commodity
products containing one or more nucleotide sequences encoding a
polypeptide comprising at least one synthetic Brassica-derived CTP
include, for example and without limitation: food products, meals,
oils, or crushed or whole grains or seeds of a plant comprising one
or more nucleotide sequences encoding a polypeptide comprising at
least one synthetic Brassica-derived CTP. The detection of one or
more nucleotide sequences encoding a polypeptide comprising at
least one synthetic Brassica-derived CTP in one or more commodity
or commodity products is de facto evidence that the commodity or
commodity product was at least in part produced from a plant
comprising one or more nucleotide sequences encoding a polypeptide
comprising at least one synthetic Brassica-derived CTP. In
particular embodiments, a commodity product of the invention
comprise a detectable amount of a nucleic acid sequence encoding a
polypeptide comprising at least one synthetic Brassica-derived CTP.
In some embodiments, such commodity products may be produced, for
example, by obtaining transgenic plants and preparing food or feed
from them.
[0176] In some embodiments, a transgenic plant, nonregenerable
plant cell, or seed comprising a transgene comprising a nucleotide
sequence encoding a polypeptide comprising at least one synthetic
Brassica-derived CTP also may comprise at least one other
transgenic event in its genome, including without limitation: a
transgenic event from which is transcribed an iRNA molecule; a gene
encoding an insecticidal protein (e.g., an Bacillus thuringiensis
insecticidal protein); an herbicide tolerance gene (e.g., a gene
providing tolerance to glyphosate); and a gene contributing to a
desirable phenotype in the transgenic plant (e.g., increased yield,
altered fatty acid metabolism, or restoration of cytoplasmic male
sterility).
VII. Synthetic Brassica-Derived Chloroplast Transit
Peptide-Mediated Localization of Gene Products to Plastids
[0177] Some embodiments of the present invention provide a method
for expression and/or localization of a gene product to a plastid
(e.g., a chloroplast). In particular embodiments, the gene product
may be a marker gene product, for example, a fluorescent molecule.
Expression of the gene product as part of a polypeptide also
comprising a synthetic Brassica-derived CTP may provide a system to
evaluate the plastid-localizing capabilities of a particular
synthetic Brassica-derived CTP sequence. In some embodiments,
expression of a marker gene product as part of a synthetic
Brassica-derived CTP-containing polypeptide is utilized to target
expression of the marker gene product to a plastid of a cell
wherein the polypeptide is expressed. In certain embodiments, such
a marker gene product is localized in plastid(s) of the host cell.
For example, the marker gene product may be expressed at higher
levels in the plastid(s) than in the cytosol or other organelles of
the host cell; the marker gene product may be expressed at much
higher levels in the plastid(s); the marker gene product may be
expressed essentially only in the plastid(s); or the marker gene
product may be expressed entirely in the plastid(s), such that
expression in the cytosol or non-plastid organelles cannot be
detected.
[0178] In some embodiments, a polypeptide comprising a functional
variant of a synthetic Brassica-derived CTP, wherein the
polypeptide is operably linked to a marker gene product is used to
evaluate the characteristics of the functional variant peptide. For
example, the sequence of a synthetic Brassica-derived CTP may be
varied, e.g., by introducing at least one conservative mutation(s)
into the synthetic Brassica-derived CTP, and the resulting variant
peptide may be linked to a marker gene product. After expression in
a suitable host cell (for example, a cell wherein one or more
regulatory elements in the expression construct are operable),
expression of the marker gene product may be determined. By
comparing the sub-cellular localization of the marker gene product
between the reference synthetic Brassica-derived CTP-marker
construct and the variant peptide-marker construct, it may be
determined whether the variant peptide provides, for example,
greater plastid localization, or substantially identical plastid
localization. Such a variant may be considered a functional
variant. By identifying functional variants of synthetic
Brassica-derived CTP that provide greater plastic localization, the
mutations in such variants may be incorporated into further
variants of synthetic Brassica-derived CTPs. Performing multiple
rounds of this evaluation process, and subsequently incorporating
identified favorable mutations in a synthetic Brassica-derived CTP
sequence, may yield an iterative process for optimization of a
synthetic Brassica-derived CTP sequence. Such optimized synthetic
Brassica-derived CTP sequences, and nucleotide sequences encoding
the same, are considered part of the present invention, whether or
not such optimized synthetic Brassica-derived CTP sequences may be
further optimized by additional mutation.
[0179] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the extent they are not inconsistent with the explicit details of
this disclosure, and are so incorporated to the same extent as if
each reference were individually and specifically indicated to be
incorporated by reference and were set forth in its entirety
herein. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0180] The following Examples are provided to illustrate certain
particular features and/or aspects. These Examples should not be
construed to limit the disclosure to the particular features or
aspects described.
EXAMPLES
Example 1: Design and Production of Chimeric Chloroplast Transit
Peptide (TraP) Sequences
[0181] Plastids are cytoplasmic organelles found in higher plant
species and are present in all plant tissues. Choloroplasts are a
specific type of plastid found in green photosynthetic tissues
which are responsible for essential physiological functions. For
example, one such primary physiological function is the synthesis
of aromatic amino acids required by the plant. Nuclear encoded
enzymes are required in this biosynthetic pathway and are
transported from the cytoplasm to the interior of the chloroplast.
These nuclear encoded enzymes usually possess an N-terminal transit
peptide that interacts with the chloroplast membrane to facilitate
transport of the peptide to the stroma of the chloroplast. Bruce B.
(2000) Chloroplast transit peptides: structure, function, and
evolution. Trends Cell Bio. 10:440-447. Upon import, stromal
peptidases cleave the transit peptide, leaving the mature
functional protein imported within the chloroplast. Richter S,
Lamppa G K. (1999) Stromal processing peptidase binds transit
peptides and initiates their ATP-dependent turnover in
chloroplasts. Journ. Cell Bio. 147:33-43. The chloroplast transit
peptides are variable sequences which are highly divergent in
length, composition and organization. Bruce B. (2000) Chloroplast
transit peptides: structure, function, and evolution. Trends Cell
Bio. 10:440-447. The sequence similarities of chloroplast transit
peptides diverge significantly amongst homologous proteins from
different plant species. The amount of divergence between
chloroplast transit peptides is unexpected given that the
homologous proteins obtained from different plant species typically
share relatively high levels of sequence similarity when comparing
the processed mature functional protein.
[0182] Novel chimeric chloroplast transit peptide sequences were
designed, produced and tested in planta. The novel chimeric
chloroplast transit peptides were shown to possess efficacious
translocation and processing properties for the import of agronomic
important proteins within the chloroplast. Initially, native
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein
sequences from different plant species were analyzed via the
ChloroP.TM. computer program to identify putative chloroplast
transit peptide sequences (Emanuelsson O, Nielsen H, von Heijne G,
(1999) ChloroP, a neural network-based method for predicting
chloroplast transit peptides and their cleavage sites, Protein
Science 8; 978-984), available at
http://www.cbs.dtu.dk/services/ChloroP/. After the native
chloroplast transit peptides were identified, a first chloroplast
transit peptide sequence was aligned with a second chloroplast
transit peptide sequences from a second organism. FIG. 18
illustrates the alignment of the EPSPS chloroplast transit peptide
sequences of Brassica napus (NCBI Accession No: P17688) and
Brassica rapa (NCBI Accession No: AAS80163). Utilizing the
chloroplast transit peptide sequence alignment, novel chimeric
chloroplast transit peptides were designed by combining the first
half of the chloroplast transit peptide sequence from the first
organism with the second half of the chloroplast transit peptide
sequence from the second organism in an approximate ratio of 1:1.
Exemplary sequences of the newly designed chimeric chloroplast
transit peptides are TraP8 (SEQ ID NO:3) and TraP9 (SEQ ID NO:4).
These novel chimeric chloroplast transit peptide sequences are
derived from the EPSPS proteins of Brassica napus [ATCC Accession
No: P17688] and Brassica rapa [ATCC Accession No: AAS80163]. The
TraP8 (SEQ ID NO:3) chimeric chloroplast transit peptide sequence
comprises an N-terminus which is derived from Brassica napus, and
the C-terminus of the chloroplast transit peptide is derived from
Brassica rapa. The TraP9 (SEQ ID NO:4) chloroplast transit peptide
sequence comprises an N-terminus which is derived from Brassica
rapa, and the C-terminus of the chloroplast transit peptide is
derived from Brassica napus. The chimeric chloroplast transit
peptides were tested via multiple assays which included a transient
in planta expression system and transgenically as a stable
transformation event comprising a gene expression element fused to
an agronomic important transgene sequence.
Example 2: Transient in Planta Testing of Chimeric Chloroplast
Transit Peptide (TraP) Sequences
Tobacco Transient Assay:
[0183] The Trap8 and TraP9 chimeric chloroplast transit peptide
sequences were initially tested via a transient in planta assay.
Polynucleotide sequences which encode the Trap8 (SEQ ID NO:5) and
TraP9 (SEQ ID NO:6) chimeric chloroplast transit peptide sequences
were synthesized. A linker sequence (SEQ ID NO:7) was incorporated
between the TraP sequence and the yfp coding sequence. The
resulting constructs contained two plant transcription units (PTU).
The first PTU was comprised of the Arabidopsis thaliana Ubiquitin
10 promoter (AtUbi10 promoter; Callis, et al., (1990) J. Biol.
Chem., 265: 12486-12493), Trap yellow fluorescent protein fusion
gene (TraP-YFP; US Patent App. 2007/0298412), and Agrobacterium
tumefaciens ORF 23 3' untranslated region (AtuORF23 3'UTR; U.S.
Pat. No. 5,428,147). The second PTU was comprised of the Cassava
Vein Mosaic Virus promoter (CsVMV promoter; Verdaguer et al.,
(1996) Plant Molecular Biology, 31:1129-1139), phosphinothricin
acetyl transferase (PAT; Wohlleben et al., (1988) Gene, 70: 25-37),
and Agrobacterium tumefaciens ORF 1 3' untranslated region (AtuORF1
3'UTR; Huang et al., (1990) J Bacteriol., 172:1814-1822). Construct
pDAB101977 contains the TraP8 chimeric chloroplast transit peptide
(FIG. 2). Construct pDAB101978 contains the TraP9 chimeric
chloroplast transit peptide (FIG. 3). A control plasmid, 101908,
which did not contain a chloroplast transit peptide sequence
upstream of the yfp gene was built and included in the studies
(FIG. 4). The constructs were confirmed via restriction enzyme
digestion and sequencing. Finally, the constructs were transformed
into Agrobacterium tumefaciens and stored as glycerol stocks.
[0184] From an Agrobacterium glycerol stock, a loop full of frozen
culture was inoculated into 2 ml of YPD (100 .mu.g/ml
spectinomycin) in a 14 ml sterile tube. The inoculated media was
incubated at 28.degree. C. overnight with shaking at 200 rpm. The
following day about 100 .mu.l of the culture was used to inoculate
25 ml of YPD (100 .mu.g/ml spectinomycin) in a 125 ml sterile
tri-baffled flask, and incubated overnight at 28.degree. C.
overnight with shaking at 200 rpm. The following day the cultures
were diluted to an OD.sub.600 of 0.5 in sterile ddH.sub.20 (pH
8.0). The diluted Agrobacterium strain was mixed with a second
Agrobacterium strain containing the P19 helper protein at a ratio
of 1:1. The culture were used for tobacco leaf infiltration via the
method of Voinnet O, Rivas S, Mestre P, and Baulcombe D., (2003) An
enhanced transient expression system in plants based on suppression
of gene silencing by the p19 protein of tomato bushy stunt virus,
The Plant Journal, 33:949-956. Infiltrated tobacco plants were
placed in a Conviron.TM. set at 16 hr of light at 24.degree. C. for
at least three days until being assayed.
Microscopy Results:
[0185] Agrobacterium-infiltrated tobacco leaves were severed from
the plant, and placed into a petri-dish with water to prevent
dehydration. The infiltrated tobacco leaves were observed under
blue light excitation with long-pass filter glasses held in place
using a Dark Reader Hand Lamp.TM. (Clare Chemical Research Co.;
Dolores, Colo.) to identify undamaged areas of the leaf that were
successfully expressing the YFP reporter proteins. Specifically
identified leaf areas were dissected from the leaf and mounted in
water for imaging by confocal microscopy (Leica TCS-SP5 AOBS.TM.;
Buffalo Grove, Ill.). The YFP reporter protein was excited by a 514
nm laser line, using a multi-line argon-ion laser. The width of the
detection slits was adjusted using a non-expressing (dark) control
leaf sample to exclude background leaf autofluoresence. Chlorophyll
autofluorescence was simultaneously collected in a second channel
for direct comparison to the fluorescent reporter protein signal
for determination of chloroplastic localization.
[0186] The microscopy imaging results indicated that the YFP
fluorescent protein comprising a TraP8 or TraP9 chloroplast transit
peptide accumulated within the chloroplasts located in the
cytoplasm of the tobacco cells as compared to the control YFP
fluorescent proteins which did not translocate into the
chloroplasts of the cytoplasm of the tobacco cells (FIG. 5 and FIG.
6). These microscopy imaging results suggest that the translocation
of the YFP protein into the chloroplast was a result of the TraP8
or TraP9 chloroplast transit peptide. As shown in FIG. 5 and FIG. 6
the YFP fluorescence signal is localized in the chloroplasts which
also fluoresce red due to auto-fluorescence under the microscopy
imaging conditions. Comparatively, FIG. 7 provides a microscopy
image of tobacco leaf tissue infiltrated with the control construct
pDAB101908 that does not contain a chloroplast transit peptide. The
chloroplasts in this image only fluoresce red due to
auto-fluorescence under the microscopy imaging conditions, and are
devoid of any YFP fluorescence signal that is exhibited in the TraP
infiltrated tobacco cells. Rather, the YFP fluorescence signal in
the control tobacco plant cells is expressed diffusely throughout
the cytoplasm of the tobacco plant cells.
Western Blot Results:
[0187] Samples of the infiltrated tobacco plants were assayed via
Western blotting. Leaf punches were collected and subjected to
bead-milling. About 100-200 mg of leaf material was mixed with 2
BBs (steel balls) (Daisy; Rogers, Ark.) and 500 ml of PBST for 3
minutes in a Kleco.TM. bead mill. The samples were then spun down
in a centrifuge at 14,000.times.g at 4.degree. C. The supernatant
was removed and either analyzed directly via Western blot or
immunoprecipitated. The immunoprecipitations were performed using
the Pierce Direct IP Kit.TM. (Thermo Scientific; Rockford, Ill.)
following the manufacturer's protocol. Approximately, 50 .mu.g of
anti-YFP was bound to the resin. The samples were incubated with
the resin overnight at 4.degree. C. Next, the samples were washed
and eluted the following morning and prepped for analysis by
combining equal volumes of 2.times.8M Urea sample buffer and then
boiling the samples for 5 minutes. The boiled samples were run on a
4-12% SDS-Bis Tris gel in MOPS buffer for 40 minutes. The gel was
then blotted using the Invitrogen iBlot.TM. (Life Technologies;
Carlsbad, Calif.) following the manufacturer's protocol. The
blotted membrane was blocked for 10 minutes using 5% non-fat dry
milk in PBS-Tween solution. The membrane was probed with the
primary antibody (monoclonal anti-GFP in rabbit) used at a 1:1000
dilution in the 5% non-fat dry milk in PBS-Tween solution for 1
hour. Next, the membrane was rinsed three times for five minutes
with PBS-Tween to remove all unbound primary antibody. The membrane
was probed with a secondary monoclonal anti-rabbit in goat antibody
(Life Technologies) used at a 1:1000 dilution, for 60 minutes. The
membrane was washed as previously described and developed by adding
Themo BCIP/NBT substrate. The colormetric substrate was allowed to
develop for 5-10 minutes and then the blots were rinsed with water
before being dried.
[0188] The Western blot results indicated that the YFP protein was
expressed in the infiltrated tobacco cells. Both, the pDAB101977
and pDAB101978 infiltrated tobacco plant leaf tissues expressed the
YFP protein as indicated by the presence of a protein band which
reacted to the YFP antibodies and was equivalent in size to the YFP
protein band obtained from tobacco plant leaf tissue infiltrated
with the YFP control construct. Moreover, these results indicated
that the TraP chimeric chloroplast transit peptides were processed
and cleaved from the YFP protein. The TraP8-YFP and TraP9-YFP
constructs express a pre-processed protein band that is larger in
molecular weight than the control YFP protein. The presence of
bands on the Western blot which are equivalent in size to the
control YFP indicate that the TraP8 and TraP9 chloroplast transit
peptide sequences were processed, thereby reducing the size of the
YFP to a molecular weight size which is equivalent to the YFP
control.
Maize Protoplast Transient Assay:
[0189] The Trap8 chimeric chloroplast transit peptide-encoding
polynucleotide sequence (SEQ ID NO:5) and the linker-encoding
polynucleotide sequence (SEQ ID NO:7) were cloned upstream of the
yellow fluorescent protein gene and incorporated into construct
pDAB106597 (FIG. 8) for testing via the maize protoplast transient
in planta assay. The resulting constructs contained a single plant
transcription unit (PTU). The PTU was comprised of the Zea mays
Ubiquitin 1 promoter (ZmUbi1 promoter; Christensen, A., Sharrock
R., and Quail P., (1992) Maize polyubiquitin genes: structure,
thermal perturbation of expression and transcript splicing, and
promoter activity following transfer to protoplasts by
electroporation, Plant Molecular Biology, 18:675-689), Trap yellow
fluorescent protein fusion gene (TraP8-YFP; US Patent App.
2007/0298412), and Zea mays Peroxidase 5 3' untranslated region
(ZmPer5 3'UTR; U.S. Pat. No. 6,384,207). The constructs were
confirmed via restriction enzyme digestion and sequencing.
[0190] Seed of Zea mays var. B104 were surface sterilized by
shaking vigorously in 50% Clorox (3% sodium hypochlorite),
containing 2-3 drops of Tween 20, for about 20 minutes. The seeds
were rinsed thoroughly with sterile distilled water. The sterile
seed were plated onto 1/2 MS medium in Phytatrays or similar type
boxes, and allowed to grow in the dark (28.degree. C.) for 12 to 20
days. A maize protoplast transient assay was used to obtain and
transfect maize protoplasts from leaves of B104-maize. This maize
protoplast assay is a modification of the system described by Yoo,
S.-D., Cho, Y.-H., and Sheen, J., (2007), Arabidopsis Mesophyll
Protoplasts: A Versitile Cell System for Transient Gene Expression
Analysis, Nature Protocols, 2:1565-1572. The solutions were
prepared as described by Yoo et. al., (2007), with the exception
that the mannitol concentration used for the following experiments
was change to 0.6 M.
[0191] Transfection of 100 to 500 .mu.l of protoplasts
(1-5.times.10.sup.5) was completed by adding the protoplasts to a 2
ml microfuge tube containing about 40 .mu.g of plasmid DNA
(pDAB106597), at room temperature. The volume of DNA was preferably
kept to about 10% of the protoplast volume. The protoplasts and DNA
were occasionally mixed during a 5 minute incubation period. An
equal volume of PEG solution was slowly added to the protoplasts
and DNA, 2 drops at a time with mixing inbetween the addition of
the drops of PEG solution. The tubes were allowed to incubate for
about 10 minutes with occasional gentle mixing. Next, 1 ml of W5+
solution was added and mixed by inverting the tube several times.
The tube(s) were centrifuged for 5 minutes at 75.times.g at a
temperature of 4.degree. C. Finally, the supernatant was removed
and the pellet was resuspended in 1 ml of WI solution and the
protoplasts were placed into a small Petri plate (35.times.10 mm)
or into 6-well multiwell plates and incubated overnight in the dark
at room temperature. Fluorescence of YFP was viewed by microscopy
after 12 hours of incubation. The microscopy conditions previously
described were used for the imaging.
[0192] The microscopy imaging results indicated that the YFP
fluorescent protein comprising a TraP8 chimeric chloroplast transit
peptide accumulated within the chloroplasts located in the
cytoplasm of the maize cells as compared to the control YFP
fluorescent proteins which did not translocate into the
chloroplasts of the cytoplasm of the maize cells (FIG. 9). These
microscopy imaging results suggest that the translocation of the
YFP protein into the chloroplast was a result of the TraP8 chimeric
chloroplast transit peptide.
Example 3: Chimeric Chloroplast Transit Peptide (TraP) Sequences
for Expression of Agronomically Important Transgenes in
Arabidopsis
[0193] A single amino acid mutation (G96A) in the Escherichia coli
5-enolpyruvylshikimate 3-phosphate synthase enzyme (EPSP synthase)
can result in glyphosate insensitivity (Padgette et al., (1991);
Eschenburg et al., (2002); Priestman et al., (2005); Haghani et
al., (2008)). While this mutation confers tolerance to glyphosate,
it is also known to adversely affect binding of EPSP synthase with
its natural substrate, phosphoenolpyruvate (PEP). The resulting
change in substrate binding efficiency can render a mutated enzyme
unsuitable for providing in planta tolerance to glyphosate.
[0194] The NCBI Genbank database was screened in silico for EPSP
synthase protein and polynucleotide sequences that naturally
contain an alanine at an analogous position within the EPSP
synthase enzyme as that of the G96A mutation which was introduced
into the E. coli version of the enzyme (Padgette et al., (1991);
Eschenburg et al., (2002); Priestman et al., (2005); Haghani et
al., (2008)).
[0195] One enzyme that was identified to contain a natural alanine
at this position was DGT-28 (GENBANK ACC NO: ZP_06917240.1) from
Streptomyces sviceus ATCC29083. Further in silico data mining
revealed three other unique Streptomyces enzymes with greater
homology to DGT-28; DGT-31 (GENBANK ACC NO: YP_004922608.1); DGT-32
(GENBANK ACC NO: ZP_04696613); and DGT-33 (GENBANK ACC NO: NC
010572). Each of these enzymes contains a natural alanine at an
analogous position within the EPSP synthase enzyme as that of the
G96A mutation that was introduced into the E. coli version of the
enzyme. FIG. 1.
[0196] Because EPSP synthase proteins from different organisms are
of different lengths, the numbering of the mutation for the E. coli
version of the EPSP synthase enzyme does not necessarily correspond
with the numbering of the mutation for the EPSP synthase enzymes
from the other organisms. These identified EPSP synthase enzymes
were not previously characterized in regard to glyphosate tolerance
or PEP substrate affinity. Furthermore, these EPSP synthase enzymes
represent a new class of EPSP synthase enzymes and do not contain
any sequence motifs that have been used to characterize previously
described Class I (plant derived sequences further described in
U.S. Pat. No. RE39247), II (bacterially derived sequences further
described in U.S. Pat. No. RE39247), and III (bacterially derived
sequences further described in International Patent Application WO
2006/110586) EPSP synthase enzymes.
[0197] The novel DGT-14, DGT-28, DGT-31, DGT-32, and DGT-33 enzymes
were characterized for glyphosate tolerance and PEP substrate
affinity by comparison to Class I EPSP synthase enzymes. The
following Class I enzymes; DGT-1 from Glycine max, DGT-3 from
Brassica napus (GENBANK ACC NO: P17688), and DGT-7 from Triticum
aestivum (GENBANK ACC NO: EU977181) were for comparison. The Class
I EPSP synthase enzymes and mutant variants thereof were
synthesized and evaluated. A mutation introduced into the plant
EPSP synthase enzymes consisted of the Glycine to Alanine mutation
made within the EPSP synthase enzyme at a similar location as that
of the G96A mutation from the E. coli version of the enzyme. In
addition, Threonine to Isoleucine and Proline to Serine mutations
were introduced within these Class I EPSP synthase enzymes at
analogous positions as that of amino acid 97 (T to I) and amino
acid 101 (P to S) in the EPSP synthase of E. coli as described in
Funke et al., (2009).
DGT14:
[0198] Transgenic T.sub.1 Arabidopsis plants containing the TraP8
and TraP9 chimeric chloroplast transit peptides fused to the dgt-14
transgene were produced using the floral dip method from Clough and
Bent (1998), Plant J. 16:735-743. Transgenic Arabidopsis plants
were obtained and confirmed to contain the transgene via molecular
confirmation. The transgenic plants were sprayed with differing
rates of glyphosate. A distribution of varying concentrations of
glyphosate rates, including elevated rates, were applied in this
study to determine the relative levels of resistance (105, 420,
1,680 or 3,360 g ae/ha). The typical 1.times. field usage rate of
glyphosate is 1,120 g ae/ha. The T.sub.1 Arabidopsis plants that
were used in this study were variable in copy number for the dgt-14
transgene. The low copy dgt-14 T.sub.1 Arabidopsis plants were
identified using molecular confirmation assays, and self-pollinated
and used to produce T.sub.2 plants. Table 1 shows the resistance
for dgt-14 transgenic plants, as compared to control plants
comprising a glyphosate herbicide resistance gene, dgt-1 (as
described in U.S. Patent Filing No. 12558351, incorporated herein
by reference in its entirety), and wildtype controls.
[0199] The Arabidopsis T.sub.1 transformants were first selected
from the background of untransformed seed using a glufosinate
selection scheme. Three flats, or 30,000 seed, were analyzed for
each T.sub.1 construct. The selected T.sub.1 plants were
molecularly characterized and the plants were subsequently
transplanted to individual pots and sprayed with various rates of
commercial glyphosate as previously described. The dose response of
these plants is presented in terms of % visual injury 2 weeks after
treatment (WAT). Data are presented in the tables below which show
individual plants exhibiting little or no injury (<20%),
moderate injury (20-40%), or severe injury (>40%). An arithmetic
mean and standard deviation is presented for each construct used
for Arabidopsis transformation. The range in individual response is
also indicated in the last column for each rate and transformation.
Wildtype, non-transformed Arabidopsis (c.v. Columbia) served as a
glyphosate sensitive control.
[0200] The level of plant response varied in the T.sub.1
Arabidopsis plants. This variance can be attributed to the fact
each plant represents an independent transformation event and thus
the copy number of the gene of interest varies from plant to plant.
An overall population injury average by rate is presented in Table
1 to demonstrate the tolerance provided by each of the dgt-14
constructs linked with either the TraP8 v2 or TraP9 v2 chloroplast
transit peptide versus the dgt-1 and non-transformed wildtype
controls for varying rates of glyphosate. The events contained
dgt-14 linked with TraP8 v2 (SEQ ID NO:8) which is contained in
construct pDAB105526 (FIG. 10) and TraP9 v2 (SEQ ID NO:9) which is
contained in construct pDAB105527 (FIG. 11). Data from the
glyphosate selection of T.sub.1 plants demonstrated that when
dgt-14 was linked with these chloroplast transit peptides, robust
tolerance to high levels of glyphosate was provided. Comparatively,
the non-transformed (or wild-type) controls did not provide
tolerance to the treatment of high concentrations of glyphosate
when treated with similar rates of glyphosate. In addition, there
were instances when events that were shown to contain three or more
copies of dgt-14 were more susceptible to elevated rates of
glyphosate. These instances are demonstrated within the percent
visual injury range shown in Table 1. It is likely that the
presence of high copy numbers of the transgenes within the
Arabidopsis plants result in transgene silencing or other
epigenetic effects which resulted in sensitivity to glyphosate,
despite the presence of the dgt-14 transgene.
TABLE-US-00001 TABLE 1 dgt-14 transformed T.sub.1 Arabidopsis
response to a range of glyphosate rates applied postemergence,
compared to a dgt-1 (T.sub.2) segregating population, and a
non-transformed control. Visual % injury 2 weeks after application.
% Injury Range (No. Replicates) % Injury Analysis <20% 20-40%
>40% Ave Std dev Range (%) 0 20-50 0-70 25-85 0-90 0 0-50 0-70
15-85 5-85 dgt-1 (pDAB3759) 0 30-60 30 40-100 45-65 Non-transformed
control 0 100 100 100 100
[0201] Selected T.sub.1 Arabidopsis plants which were identified to
contain low-copy numbers of transgene insertions (1-3 copies) were
self-fertilized to produce a second generation for additional
assessment of glyphosate tolerance. The second generation
Arabidopsis plants (T.sub.2) which contained 1-3 copies of the
dgt-14 transgene fused to the TraP8 and TraP9 chimeric chloroplast
transit peptides were further characterized for glyphosate
tolerance and glufosinate tolerance (glufosinate resistance
indicated that the PAT expression cassette was intact and did not
undergo rearrangements during the selfing of the T.sub.1 plants).
In the T.sub.2 generation hemizygous and homozygous plants were
available for testing for each event and therefore were included
for each rate of glyphosate tested. Hemizygous plants contain two
different alleles at a locus as compared to homozygous plants which
contain the same two alleles at a locus. The copy number and ploidy
levels of the T.sub.2 plants were confirmed using molecular
analysis protocols. Likewise, glyphosate was applied using the
methods and rates as previously described. The dose response of the
plants is presented in terms of % visual injury 2 weeks after
treatment (WAT). Data are presented as a histogram of individuals
exhibiting little or no injury (<20%), moderate injury (20-40%),
or severe injury (>40%). An arithmetic mean and standard
deviation are presented for each construct used for Arabidopsis
transformation. The range in individual response is also indicated
in the last column for each rate and transformation. Wildtype,
non-transformed Arabidopsis (cv. Columbia) served as a glyphosate
sensitive control. In addition, plants comprising a glyphosate
herbicide resistance gene, dgt-1 (as described in U.S. Patent
Filing No. 12558351, incorporated herein by reference in its
entirety) were included as a positive control.
[0202] In the T.sub.2 generation both single copy and low-copy (two
or three copy) dgt-14 events were characterized for glyphosate
tolerance. An overall population injury average by rate is
presented in Table 2 to demonstrate the tolerance provided by each
of the dgt-14 constructs linked with a chloroplast transit peptide
versus the dgt-1 and non-transformed wildtype controls for varying
rates of glyphosate. The T.sub.2 generation events contained dgt-14
linked with TraP8 v2 (pDAB105526) and TraP9 v2 (pDAB105527). Both
of these events are highly resistant to glyphosate. The results
indicated that the injury range for the T.sub.2 Arabidopsis plants
was less than 20% for all concentrations of glyphosate that were
tested. Comparatively, the non-transformed (or wild-type) controls
did not provide tolerance to the treatment of high concentrations
of glyphosate when treated with similar rates of glyphosate.
Overall, the results showed that plants containing and expressing
DGT-14 fused to the TraP8 and TraP9 chimeric transit peptide
proteins yielded commercial level resistance to glyphosate at
levels of up to 3 times the field rate (1120 g ae/ha).
TABLE-US-00002 TABLE 2 dgt-14 transformed T.sub.2 Arabidopsis
response to a range of glyphosate rates applied postemergence,
compared to a dgt-1 (T.sub.2) segregating population, and a
non-transformed control. Visual % injury 2 weeks after application.
Data represents a selected single copy line from each construct
that segregated as a single locus in the heritability screen. %
Injury Range (No. Replicates) % Injury Analysis <20% 20-40%
>40% Ave Std dev Range (%) dgt-1 (pDAB3759) Non-transformed
control
[0203] Randomly selected T.sub.2 Arabidopsis plants which were
identified to contain low-copy numbers of transgene insertions (1-3
copies) were self-fertilized to produce a third generation for
additional assessment of glyphosate tolerance. Arabidopsis seed
from the third generation (T.sub.3) were planted and evaluated for
glyphosate tolerance using the same protocols as previously
described. The events tested in the T.sub.3 generation contained
replicates from each line that were homozygous (as determined by
using a glufosinate resistance screen to identify if any of the
advanced plants showed segregation of the transgenes). These Events
were assayed via LC-MS-MS to confirm that the plants expressed the
DGT-14 protein. The results of the T.sub.3 generation for overall
population injury average by rate of glyphosate is presented in
Table 3 which shows the tolerance to glyphosate provided by each of
the dgt-14 constructs for varying rates of glyphosate. Exemplary
resistant T.sub.3 Events comprised dgt-14 linked with TraP8 v2
(pDAB105526) and TraP9 v2 (pDAB105527). Both of these Events are
highly resistant to glyphosate. The results indicated that the
injury range for the T.sub.3Arabidopsis plants was less than 20%
for all concentrations of glyphosate that were tested.
Comparatively, the non-transformed (or wild-type) controls did not
provide tolerance to the treatment of high concentrations of
glyphosate when treated with similar rates of glyphosate. Overall,
the results showed that plants containing and expressing DGT-14
yielded commercial level resistance to glyphosate at levels of up
to 3 times the field rate (1120 g ae/ha).
TABLE-US-00003 TABLE 3 dgt-14 transformed T.sub.3 Arabidopsis
response to a range of glyphosate rates applied postemergence,
compared to a dgt-1 (T.sub.2) segregating population, and a
non-transformed control. Visual % injury 2 weeks after application.
Data represents a selected single copy population from each
construct that segregated as a single locus in the T.sub.2
heritability screen. % Injury Range (No. Replicates) % Injury
Analysis <20% 20-40% >40% Ave Std dev Range (%) dgt-1
(pDAB3759) Non-transformed control
[0204] The data show that expression of a glyphosate-resistant
enzyme (e.g., DGT-28), when targeted to the chloroplast of a plant
cell by a TraP transit peptide in a fusion protein, is capable of
conferring glyphosate resistance to the plant cell and plants
comprised of these cells.
DGT-28, DGT-31, DGT-32, and DGT-33:
[0205] The newly-designed, dicotyledonous plant optimized dgt-28 v5
polynucleotide sequence is listed in SEQ ID NO:16. The
newly-designed, monocotyledonous plant optimized dgt-28 v6
polynucleotide sequence is listed in SEQ ID NO:17; this sequence
was slightly modified by including an alanine at the second amino
acid position to introduce a restriction enzyme site. The resulting
DNA sequences have a higher degree of codon diversity, a desirable
base composition, contains strategically placed restriction enzyme
recognition sites, and lacks sequences that might interfere with
transcription of the gene, or translation of the product mRNA.
[0206] Synthesis of DNA fragments comprising SEQ ID NO:16 and SEQ
ID NO:17 containing additional sequences, such as 6-frame stops
(stop codons located in all six reading frames that are added to
the 3' end of the coding sequence), and a 5' restriction site for
cloning were performed by commercial suppliers (DNA2.0, Menlo Park,
Calif.). The synthetic nucleic acid molecule was then cloned into
expression vectors and transformed into plants or bacteria as
described in the Examples below.
[0207] Similar codon optimization strategies were used to design
dgt-1, dgt-3 v2 (G173A), dgt-3 v3 (G173A; P178S), dgt-3 v4 (T174I;
P178S), dgt-7 v4 (T168I; P172S), dgt-32 v3, dgt-33 v3, and dgt-31
v3. The codon optimized version of these genes are listed as SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:24, and SEQ ID NO:25, respectively.
[0208] Plant Binary Vector Construction.
[0209] Standard cloning methods were used in the construction of
entry vectors containing a chloroplast transit peptide
polynucleotide sequence joined to dgt-28 as an in-frame fusion. The
entry vectors containing a transit peptide (TraP) fused to dgt-28
were assembled using the IN-FUSION.TM. Advantage Technology
(Clontech, Mountain View, Calif.). As a result of the fusion, the
first amino acid, methionine, was removed from dgt-28. Transit
peptides TraP4 v2 (SEQ ID NO:26), TraP5 v2 (SEQ ID NO:27), TraP8 v2
(SEQ ID NO:28), TraP9 v2 (SEQ ID NO:29), TraP12 v2 (SEQ ID NO:30),
and TraP13 v2 (SEQ ID NO:31) were each synthesized by DNA2.0 (Menlo
Park, Calif.) and fused to the 5' end fragment of dgt-28, up to and
including a unique AccI restriction endonuclease recognition
site.
[0210] Binary plasmids which contained the various TraP and dgt-28
expression cassettes were driven by the Arabidopsis thaliana
Ubiquitin 10 promoter (AtUbi10 v2; Callis, et al., (1990) 1 Biol.
Chem., 265: 12486-12493) and flanked by the Agrobacterium
tumefaciens open reading frame twenty-three 3' untranslated region
(AtuORF23 3' UTR v1; U.S. Pat. No. 5,428,147).
[0211] The assembled TraP and dgt-28 expression cassettes were
engineered using GATEWAY.RTM. Technology (Invitrogen, Carlsbad,
Calif.) and transformed into plants via Agrobacterium-mediated
plant transformation. Restriction endonucleases were obtained from
New England BioLabs (NEB; Ipswich, Mass.) and T4 DNA Ligase
(Invitrogen) was used for DNA ligation. Gateway reactions were
performed using GATEWAY.RTM. LR CLONASE.RTM. enzyme mix
(Invitrogen) for assembling one entry vector into a single
destination vector which contained the selectable marker cassette
Cassava Vein Mosaic Virus promoter (CsVMV v2; Verdaguer et al.,
(1996) Plant Mol. Biol., 31: 1129-1139)--DSM-2 (U.S. Pat. App. No.
2007/086813)--Agrobacterium tumefaciens open reading frame one 3'
untranslated region (AtuORF1 3' UTR v6; Huang et al., (1990) J.
Bacteriol. 172:1814-1822). Plasmid preparations were performed
using NUCLEOSPIN.RTM. Plasmid Kit (Macherey-Nagel Inc., Bethlehem,
Pa.) or the Plasmid Midi Kit (Qiagen) following the instructions of
the suppliers. DNA fragments were isolated using QIAquick.TM. Gel
Extraction Kit (Qiagen) after agarose Tris-acetate gel
electrophoresis.
[0212] Colonies of all assembled plasmids were initially screened
by restriction digestion of miniprep DNA. Plasmid DNA of selected
clones was sequenced by a commercial sequencing vendor
(Eurofins.TM. MWG Operon, Huntsville, Ala.). Sequence data were
assembled and analyzed using the SEQUENCHER.TM. software (Gene
Codes Corp., Ann Arbor, Mich.).
[0213] The following binary constructs express the various
TraP:dgt-28 fusion gene sequences: pDAB107527 (FIG. 19) contains
TraP4 v2:dgt-28 v5 (SEQ ID NO:32); pDAB105530 (FIG. 20) contains
TraP5 v2: dgt-28 v5 (SEQ ID NO:33); pDAB105531 (FIG. 21) contains
TraP8 v2: dgt-28 v5 (SEQ ID NO:34); PDAB105532 (FIG. 22) contains
TraP9 v2: dgt-28 v5 (SEQ ID NO:35); pDAB105533 (FIG. 23) contains
TraP12 v2: dgt-28 v5 (SEQ ID NO:36); and pDAB105534 (FIG. 24)
contains TraP13 v2:dgt-28 v5 (SEQ ID NO:37). The dgt-28 v5 sequence
of pDAB105534 was modified wherein the first codon (GCA) was
changed to (GCT).
[0214] Additional Plant Binary Vector Construction.
[0215] Cloning strategies similar to those described above were
used to construct binary plasmids which contain dgt-31, dgt-32,
dgt-33, dgt-1, dgt-3, and dgt-7.
[0216] The microbially derived genes; dgt-31, dgt-32, and dgt-33,
were fused with different chloroplast transit peptides than
previously described. The following chloroplast transit peptides
were used; TraP14 v2 (SEQ ID NO:38), TraP23 v2 (SEQ ID NO:39),
TraP24 v2 (SEQ ID NO:40). pDAB107532 (FIG. 25) contains dgt-32 v3
fused to TraP14 v2 (SEQ ID NO:41), pDAB107534 (FIG. 26) contains
dgt-33 v3 fused to TraP24 v2 (SEQ ID NO:42), and pDAB107533 (FIG.
27) contains dgt-31 v3 fused to TraP23 v2 (SEQ ID NO:43). The dgt
expression cassettes were driven by the Arabidopsis thaliana
Ubiquitin 10 promoter (AtUbi10 promoter v2) and flanked by the
Agrobacterium tumefaciens open reading frame twenty-three 3'
untranslated region (AtuORF23 3' UTR v1). A DSM-2 selectable marker
cassette containing Cassava Vein Mosaic Virus promoter (CsVMV
v2)--DSM-2--Agrobacterium tumefaciens open reading frame one 3'
untranslated region (AtuORF1 3' UTR v6) was also present in the
binary vector.
[0217] Additional binaries are constructed wherein dgt-31 v3,
dgt-32 v3, and dgt-33 v3 are fused to the previously described
chloroplast transit peptide sequences. For example, the TraP8 v2
sequence is fused to dgt-31 v3, dgt-32 v3, and dgt-33 v3, and
cloned into binary vectors as described above.
[0218] Binary vectors containing the Class I genes (dgt-1, dgt-3,
and dgt-7) were constructed. The following binary vectors were
constructed and transformed into plants: pDAB4104 (FIG. 28), which
contains the dgt-1 v4 sequence as described in U.S. Patent
Application Publication No. 2011/0124503, which is flanked by the
Nicotiana tabacum Osmotin sequences as described in U.S. Patent
Application Publication No. 2009/0064376; pDAB102715 (FIG. 29);
pDAB102716 (FIG. 30); pDAB102717 (FIG. 31); and pDAB102785 (FIG.
32). The various TraP chloroplast transit peptides that were fused
to dgt-28, dgt-31, dgt-32, and dgt-33 were not added to the Class I
genes, as these plant derived sequences possess native plant
chloroplast transit peptides. These vectors are described in
further detail in Table 4.
TABLE-US-00004 TABLE 4 Description of the binary vectors which
contain a Class I EPSP synthase gene (i.e., dgt-1, dgt-3, or
dgt-7). EPSPS Name Description mutation pDAB4104 RB7 MAR v2 ::
CsVMV promoter TI PS v2/NtOsm 5' UTR v2/dgt-1 v4/NtOsm 3' UTR
v2/AtuORF24 3' UTR v2 :: AtUbi10 promoter v4/pat v3/AtuORF1 3'UTR
v3 binary vector pDAB102715 AtUbi10 promoter v2/dgt-3 GA
v2/AtuORF23 3'UTR v1 :: CsVMV promoter v2/pat v9/AtuORF1 3'UTR v6
binary vector pDAB102716 AtUbi10 promoter v2/dgt-3 GA PS
v3/AtuORF23 3'UTR v1 :: CsVMV promoter v2/pat v9/AtuORF1 3'UTR v6
binary vector pDAB102717 AtUbi10 promoter v2/dgt-3 TI PS
v4/AtuORF23 3'UTR v1 :: CsVMV promoter v2/pat v9/AtuORF1 3'UTR v6
binary vector pDAB102785 AtUbi10 promoter v2/dgt-7 TI PS
v4/AtuORF23 3'UTR :: CsVMV promoter v2/DSM-2 v2/AtuORF1 3'UTR v6
binary vector
[0219] Arabidopsis thaliana Transformation.
[0220] Arabidopsis was transformed using the floral dip method from
Clough and Bent (1998). A selected Agrobacterium colony containing
one of the binary plasmids described above was used to inoculate
one or more 100 mL pre-cultures of YEP broth containing
spectinomycin (100 mg/L) and kanamycin (50 mg/L). The culture was
incubated overnight at 28.degree. C. with constant agitation at 225
rpm. The cells were pelleted at approximately 5000.times.g for 10
minutes at room temperature, and the resulting supernatant
discarded. The cell pellet was gently resuspended in 400 mL dunking
media containing: 5% (w/v) sucrose, 10 .mu.g/L 6-benzylaminopurine,
and 0.04% Silwet.TM. L-77. Plants approximately 1 month old were
dipped into the media for 5-10 minutes with gentle agitation. The
plants were laid down on their sides and covered with transparent
or opaque plastic bags for 2-3 hours, and then placed upright. The
plants were grown at 22.degree. C., with a 16-hour light/8-hour
dark photoperiod. Approximately 4 weeks after dipping, the seeds
were harvested.
[0221] Selection of Transformed Plants.
[0222] Freshly harvested T.sub.1 seed [containing the dgt and DSM-2
expression cassettes] was allowed to dry for 7 days at room
temperature. T.sub.1 seed was sown in 26.5.times.51-cm germination
trays, each receiving a 200 mg aliquot of stratified T.sub.1 seed
(.about.10,000 seed) that had previously been suspended in 40 mL of
0.1% agarose solution and stored at 4.degree. C. for 2 days to
complete dormancy requirements and ensure synchronous seed
germination.
[0223] Sunshine Mix LP5 was covered with fine vermiculite and
subirrigated with Hoagland's solution until wet, then allowed to
gravity drain. Each 40 mL aliquot of stratified seed was sown
evenly onto the vermiculite with a pipette and covered with
humidity domes for 4-5 days. Domes were removed 1 day prior to
initial transformant selection using glufosinate postemergence
spray (selecting for the co-transformed DSM-2 gene).
[0224] Seven days after planting (DAP) and again 11 DAP, T.sub.1
plants (cotyledon and 2-4-1f stage, respectively) were sprayed with
a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate, Bayer
Crop Sciences, Kansas City, Mo.) at a spray volume of 10 mL/tray
(703 L/ha) using a DeVilbiss compressed air spray tip to deliver an
effective rate of 280 g ai/ha glufosinate per application.
Survivors (plants actively growing) were identified 4-7 days after
the final spraying and transplanted individually into 3-inch pots
prepared with potting media (Metro Mix 360). Transplanted plants
were covered with humidity domes for 3-4 days and placed in a
22.degree. C. growth chamber as before or moved to directly to the
greenhouse. Domes were subsequently removed and plants reared in
the greenhouse (22.+-.5.degree. C., 50.+-.30% RH, 14 h light:10
dark, minimum 500 .mu.E/m.sup.2s.sup.1 natural+supplemental light).
Molecular confirmation analysis was completed on the surviving
T.sub.1 plants to confirm that the glyphosate tolerance gene had
stably integrated into the genome of the plants.
[0225] Molecular Confirmation.
[0226] The presence of the dgt-28 and DSM-2 transgenes within the
genome of Arabidopsis plants that were transformed with pDAB107527,
pDAB105530, pDAB105531, pDAB105532, pDAB105533, or pDAB105534 was
confirmed. The presence of these polynucleotide sequences was
confirmed via hydrolysis probe assays, gene expression cassette PCR
(also described as plant transcription unit PCR PTU PCR), Southern
blot analysis, and Quantitative Reverse Transcription PCR
analyses.
[0227] The T.sub.1 Arabidopsis plants were initially screened via a
hydrolysis probe assay, analogous to TAQMAN.TM., to confirm the
presence of the DSM-2 and dgt-28 transgenes. Events were screened
via gene expression cassette PCR to determine whether the dgt
expression cassette completely integrated into the plant genomes
without rearrangement. The data generated from these studies were
used to determine the transgene copy number and identify select
Arabidopsis events for self fertilization and advancement to the
T.sub.2 generation. The advanced T.sub.2 Arabidopsis plants were
also screened via hydrolysis probe assays to confirm the presence
and to estimate the copy number of the DSM-2 and dgt genes within
the plant chromosome. Finally, a Southern blot assay was used to
confirm the estimated copy number on a subset of the T.sub.1
Arabidopsis plants.
[0228] Similar assays were used to confirm the presence of the
dgt-1 transgene from plants transformed with pDAB4101, the presence
of the dgt-32 transgene from plants transformed with pDAB107532,
the presence of the dgt-33 transgene from plants transformed with
pDAB107534, the presence of the dgt-3 transgene from plants
transformed with pDAB102715, the presence of the dgt-3 transgene
from plants transformed with pDAB102716, the presence of the dgt-3
transgene from plants transformed with pDAB102717, and the presence
of the dgt-7 transgene from plants transformed with pDAB102785.
[0229] Hydrolysis Probe Assay.
[0230] Copy number was determined in the T.sub.1 and T.sub.2
Arabidopsis plants using the hydrolysis probe assay described
below. Plants with varying numbers of transgenes were identified
and advanced for subsequent glyphosate tolerance studies.
[0231] Tissue samples were collected in 96-well plates and
lyophilized for 2 days. Tissue maceration was performed with a
KLECO.TM. tissue pulverizer and tungsten beads (Environ Metal INC.,
Sweet Home, Oreg.). Following tissue maceration, the genomic DNA
was isolated in high-throughput format using the Biosprint.TM. 96
Plant kit (Qiagen.TM. Germantown, Md.) according to the
manufacturer's suggested protocol. Genomic DNA was quantified by
QUANT-IT.TM. PICO GREEN DNA ASSAY KIT (Molecular Probes,
Invitrogen, Carlsbad, Calif.). Quantified genomic DNA was adjusted
to around 2 ng/.mu.L for the hydrolysis probe assay using a
BIOROBOT3000.TM. automated liquid handler (Qiagen, Germantown,
Md.). Transgene copy number determination by hydrolysis probe assay
was performed by real-time PCR using the LIGHTCYCLER.RTM. 480
system (Roche Applied Science, Indianapolis, Ind.). Assays were
designed for DSM-2, dgt-28 and the internal reference gene, TAFIII5
(Genbank ID: NC 003075; Duarte et al., (201) BMC Evol. Biol.,
10:61).\
[0232] For amplification, LIGHTCYCLER.RTM. 480 Probes Master mix
(Roche Applied Science, Indianapolis, Ind.) was prepared at a
1.times. final concentration in a 10 .mu.L volume multiplex
reaction containing 0.1 .mu.M of each primer for DSM-2 and dgt-28,
0.4 .mu.M of each primer for TAFIII5 and 0.2 .mu.M of each
probe.
[0233] Table 5. A two-step amplification reaction was performed
with an extension at 60.degree. C. for 40 seconds with fluorescence
acquisition. All samples were run and the averaged Cycle threshold
(Ct) values were used for analysis of each sample. Analysis of real
time PCR data was performed using LightCycler.TM. software release
1.5 using the relative quant module and is based on the
.DELTA..DELTA.Ct method. For this, a sample of genomic DNA from a
single copy calibrator and known 2 copy check were included in each
run. The copy number results of the hydrolysis probe screen were
determined for the T.sub.1 and T.sub.2 transgenic Arabidopsis
plants.
TABLE-US-00005 TABLE 5 Primer and probe Information for hydrolysis
probe assay of DSM-2, dgt-28 and internal reference gene (TAFII15).
Primer Name Sequence DSM2A 5' AGCCACATCCCAGTAACGA 3' (SEQ ID NO:
44) DSM2S 5' CCTCCCTCTTTGACGCC 3' (SEQ ID NO: 45) DSM2 Cy5 probe 5'
CAGCCCAATGAGGCATCAGC 3' (SEQ ID NO: 46) DGT28F 5'
CTTCAAGGAGATTTGGGATTTGT 3' (SEQ ID NO: 47) DGT28R 5'
GAGGGTCGGCATCGTAT 3' (SEQ ID NO: 48) UPL154 probe Cat# 04694406001
(Roche, Indianapolis, IN) TAFFY-HEX probe 5'
AGAGAAGTTTCGACGGATTTCGGGC 3' (SEQ ID NO: 49) TAFII15-F 5'
GAGGATTAGGGTTTCAACGGAG 3' (SEQ ID NO: 50) TAFII15-R 5'
GAGAATTGAGCTGAGACGAGG 3' (SEQ ID NO: 51)
[0234] Dgt-28 Integration Confirmation Via Southern Blot
Analysis.
[0235] Southern blot analysis was used to establish the integration
pattern of the inserted T-strand DNA fragment and identify events
which contained dgt-28. Data were generated to demonstrate the
integration and integrity of the transgene inserts within the
Arabidopsis genome. Southern blot data were used to identify simple
integration of an intact copy of the T-strand DNA. Detailed
Southern blot analysis was conducted using a PCR amplified probe
specific to the dgt-28 gene expression cassette. The hybridization
of the probe with genomic DNA that had been digested with specific
restriction enzymes identified genomic DNA fragments of specific
molecular weights, the patterns of which were used to identify full
length, simple insertion T.sub.1 transgenic events for advancement
to the next generation.
[0236] Tissue samples were collected in 2 mL conical tubes
(Eppendorf.TM.) and lyophilized for 2 days. Tissue maceration was
performed with a KLECKO.TM. tissue pulverizer and tungsten beads.
Following tissue maceration, the genomic DNA was isolated using a
CTAB isolation procedure. The genomic DNA was further purified
using the Qiagen.TM. Genomic Tips kit. Genomic DNA was quantified
by Quant-IT.TM. Pico Green DNA assay kit (Molecular Probes,
Invitrogen, Carlsbad, Calif.). Quantified genomic DNA was adjusted
to 4 .mu.g for a consistent concentration.
[0237] For each sample, 4 .mu.g of genomic DNA was thoroughly
digested with the restriction enzyme SwaI (New England Biolabs,
Beverley, Mass.) and incubated at 25.degree. C. overnight, then
NsiI was added to the reaction and incubated at 37.degree. C. for 6
hours. The digested DNA was concentrated by precipitation with
Quick Precipitation Solution.TM. (Edge Biosystems, Gaithersburg,
Md.) according to the manufacturer's suggested protocol. The
genomic DNA was then resuspended in 25 .mu.L of water at 65.degree.
C. for 1 hour. Resuspended samples were loaded onto a 0.8% agarose
gel prepared in 1.times.TAE and electrophoresed overnight at 1.1
V/cm in 1.times.TAE buffer. The gel was sequentially subjected to
denaturation (0.2 M NaOH/0.6 M NaCl) for 30 minutes, and
neutralization (0.5 M Tris-HCl (pH 7.5)/1.5 M NaCl) for 30
minutes.
[0238] Transfer of DNA fragments to nylon membranes was performed
by passively wicking 20.times.SSC solution overnight through the
gel onto treated IMMOBILON.TM. NY+ transfer membrane (Millipore,
Billerica, Mass.) by using a chromatography paper wick and paper
towels. Following transfer, the membrane was briefly washed with
2.times.SSC, cross-linked with the STRATALINKER.TM. 1800
(Stratagene, LaJolla, Calif.), and vacuum baked at 80.degree. C.
for 3 hours.
[0239] Blots were incubated with pre-hybridization solution
(Perfect Hyb plus, Sigma, St. Louis, Mo.) for 1 hour at 65.degree.
C. in glass roller bottles using a model 400 hybridization
incubator (Robbins Scientific, Sunnyvale, Calif.). Probes were
prepared from a PCR fragment containing the entire coding sequence.
The PCR amplicon was purified using QIAEX.TM. II gel extraction kit
and labeled with .alpha..sup.32P-dCTP via the Random RT Prime
IT.TM. labeling kit (Stratagene, La Jolla, Calif.). Blots were
hybridized overnight at 65.degree. C. with denatured probe added
directly to hybridization buffer to approximately 2 million counts
per blot per mL. Following hybridization, blots were sequentially
washed at 65.degree. C. with 0.1.times.SSC/0.1% SDS for 40 minutes.
Finally, the blots were exposed to storage phosphor imaging screens
and imaged using a Molecular Dynamics Storm 860.TM. imaging
system.
[0240] The Southern blot analyses completed in this study were used
to determine the copy number and confirm that selected events
contained the dgt-28 transgene within the genome of
Arabidopsis.
[0241] dgt-28 Gene Expression Cassette Confirmation Via PCR
Analysis.
[0242] The presence of the dgt-28 gene expression cassette
contained in the T.sub.1 plant events was detected by an end point
PCR reaction. Primers (Table 6) specific to the AtUbi10 promoter v2
and AtuORF23 3'UTR v1 regions of the dgt-28 gene expression
cassette were used for detection.
TABLE-US-00006 TABLE 6 Oligonucleotide primers used for dgt-28 gene
expression cassette confirmation. Primer Name Sequence Forward
oligo 5' CTGCAGGTCAACGGATCAGGATAT 3' (SEQ ID NO: 52) Reverse oligo
5' TGGGCTGAATTGAAGACATGCTCC 3' (SEQ ID NO: 53)
[0243] The PCR reactions required a standard three step PCR cycling
protocol to amplify the gene expression cassette. All of the PCR
reactions were completed using the following PCR conditions:
94.degree. C. for three minutes followed by 35 cycles of 94.degree.
C. for thirty seconds, 60.degree. C. for thirty seconds, and
72.degree. C. for three minutes. The reactions were completed using
the EX-TAQ.TM. PCR kit (TaKaRa Biotechnology Inc. Otsu, Shiga,
Japan) per manufacturer's instructions. Following the final cycle,
the reaction was incubated at 72.degree. C. for 10 minutes. TAE
agarose gel electrophoresis was used to determine the PCR amplicon
size. PCR amplicons of an expected size indicated the presence of a
full length gene expression cassette was present in the genome of
the transgenic Arabidopsis events.
[0244] dgt-28 Relative Transcription Confirmation Via Quantitative
Reverse Transcription PCR Analysis.
[0245] Tissue samples of dgt-28 transgenic plants were collected in
96-well plates and frozen at 80.degree. C. Tissue maceration was
performed with a KLECO.TM. tissue pulverizer and tungsten beads
(Environ Metal INC., Sweet Home, Oreg.). Following tissue
maceration, the Total RNA was isolated in high-throughput format
using the Qiagen.TM. Rneasy 96 kit (Qiagen.TM., Germantown, Md.)
according to the manufacturer's suggested protocol which included
the optional DnaseI treatment on the column. This step was
subsequently followed by an additional DnaseI (Ambion.TM., Austin,
Tex.) treatment of the eluted total RNA. cDNA synthesis was carried
out using the total RNA as template with the High Capacity cDNA
Reverse Transcription.TM. kit (Applied Biosystems, Austin, Tex.)
following the manufacturer's suggested procedure with the addition
of the oligonucleotide, TVN. Quantification of expression was
completed by hydrolysis probe assay and was performed by real-time
PCR using the LIGHTCYCLER.RTM.480 system (Roche Applied Science,
Indianapolis, Ind.). Assays were designed for dgt-28 and the
internal reference gene "unknown protein" (Genbank Accession
Number: AT4G24610) using the LIGHTCYCLER.RTM. Probe Design Software
2.0. For amplification, LIGHTCYCLER.RTM.480 Probes Master mix
(Roche Applied Science, Indianapolis, Ind.) was prepared at
1.times. final concentration in a 10 .mu.L volume singleplex
reaction containing 0.4 .mu.M of each primer, and 0.2 .mu.M of each
probe. Table 7.
TABLE-US-00007 TABLE 7 PCR primers used for quantitative reverse
transcription PCR analysis of dgt-28. Primer Name Sequence
AT26410LP 5' CGTCCACAAAGCTGAATGTG 3' (SEQ ID NO: 54) AT26410RP 5'
CGAAGTCATGGAAGCCACTT3' (SEQ ID NO: 55) UPL146 Cat# 04694325001
(Roche, Indianapolis, IN) DGT28F 5' CTTCAAGGAGATTTGGGATTTGT3' (SEQ
ID NO: 56) DGT28R 5' GAGGGTCGGCATCGTAT 3' (SEQ ID NO: 57) UPL154
probe Cat# 04694406001 (Roche, Indianapolis, IN)
[0246] A two-step amplification reaction was performed with an
extension at 60.degree. C. for 40 seconds with fluorescence
acquisition. All samples were run in triplicate and the averaged
Cycle threshold (Ct) values were used for analysis of each sample.
A minus reverse transcription reaction was run for each sample to
ensure that no gDNA contamination was present. Analysis of real
time PCR data was performed based on the .DELTA..DELTA.Ct method.
This assay was used to determine the relative expression of dgt-28
in transgenic Arabidopsis events which were determined to be
hemizygous and homozygous. The relative transcription levels of the
dgt-28 mRNA ranged from 2.5 fold to 207.5 fold higher than the
internal control. These data indicate that dgt-28 transgenic plants
contained a functional dgt-28 gene expression cassette, and the
plants were capable of transcribing the dgt-28 transgene.
[0247] Western Blotting Analysis.
[0248] DGT-28 was detected in leaf samples obtained from transgenic
Arabidopsis thaliana plants. Plant extracts from dgt-28 transgenic
plants and DGT-28 protein standards were incubated with NUPAGE.RTM.
LDS sample buffer (Invitrogen, Carlsbad, Calif.) containing DTT at
90.degree. C. for 10 minutes and electrophoretically separated in
an acrylamide precast gel. Proteins were then electro-transferred
onto nitrocellulose membrane using the manufacturer's protocol.
After blocking with the WESTERNBREEZE.RTM. Blocking Mix
(Invitrogen) the DGT-28 protein was detected by anti-DGT-28
antiserum followed by goat anti-rabbit phosphatase. The detected
protein was visualized by chemiluminescence substrate BCIP/NBT
Western Analysis Reagent (KPL, Gaithersburg, Md.). Production of an
intact DGT-28 protein via Western blot indicated that the dgt-28
transgenic plants which were assayed expressed the DGT-28
protein.
[0249] Transgenic T.sub.1 Arabidopsis plants containing the dgt-28
transgene were sprayed with differing rates of glyphosate. Elevated
rates were applied in this study to determine the relative levels
of resistance (105, 420, 1,680 or 3,360 g ae/ha). A typical
1.times. usage rate of glyphosate that will control non-transformed
Arabidopsis is 420 g ae/ha. Glyphosate formulations with the
addition of ammonium sulfate were applied to the T.sub.1 plants
with a track sprayer calibrated at 187 L/ha. The T.sub.1
Arabidopsis plants that were used in this study were variable copy
number for the dgt-28 transgene. The low copy dgt-28 T.sub.1
Arabidopsis plants were self-pollinated and used to produce T.sub.2
plants. Table 8 shows the comparison of dgt-28 transgenic plants,
drawn to a glyphosate herbicide resistance gene, dgt-1, and
wildtype controls. Table 9 shows the comparison of dgt-32, and
dgt-33 drawn to a glyphosate herbicide resistance gene, dgt-1, and
wildtype controls. Table 10 shows the comparison of the novel
bacterial EPSP synthase enzymes to the Class I EPSP synthase
enzymes and the controls at a glyphosate rate of 1,680 g ae/ha.
[0250] Results of Glyphosate Selection of Transformed Dgt-28
Arabidopsis Plants.
[0251] The Arabidopsis T.sub.1 transformants were first selected
from the background of untransformed seed using a glufosinate
selection scheme. Three flats or 30,000 seed were analyzed for each
T.sub.1 construct. The T.sub.1 plants selected above were
molecularly characterized and representative plants with variable
copy number were subsequently transplanted to individual pots and
sprayed with various rates of commercial glyphosate as previously
described. The response of these plants is presented in terms of %
visual injury 2 weeks after treatment (WAT). Data are presented in
a table which shows individual plants exhibiting little or no
injury (<20%), moderate injury (20-40%), or severe injury
(>40%). An arithmetic mean and standard deviation is presented
for each construct used for Arabidopsis transformation. The range
in individual response is also indicated in the last column for
each rate and transformation. Wild-type, non-transformed
Arabidopsis (c.v. Columbia) served as a glyphosate sensitive
control.
[0252] The level of plant response varied. This variance can be
attributed to the fact each plant represents an independent
transformation event and thus the copy number of the gene of
interest varies from plant to plant. It was noted that some plants
which contained the transgene were not tolerant to glyphosate; a
thorough analysis to determine whether these plants expressed the
transgene was not completed. It is likely that the presence of high
copy numbers of the transgene within the T.sub.1 Arabidopsis plants
resulted in transgene silencing or other epigenetic effects which
resulted in sensitivity to glyphosate, despite the presence of the
dgt-28 transgene.
[0253] An overall population injury average by rate is presented in
Table 10 for rates of glyphosate at 1,680 g ae/ha to demonstrate
the significant difference between the plants transformed with
dgt-3, dgt-7, dgt-28, dgt-32, and dgt-33 versus the dgt-1 and
wild-type controls.
[0254] The tolerance provided by the novel bacterial EPSP synthases
varied depending upon the specific enzyme. DGT-28, DGT-32, and
DGT-33 unexpectedly provided significant tolerance to glyphosate.
The dgt genes imparted herbicide resistance to individual T.sub.1
Arabidopsis plants across all transit peptides tested. As such, the
use of additional chloroplast transit peptides (i.e., TraP8-dgt-32
or TraP8-dgt-33) would provide protection to glyphosate with
similar injury levels as reported within a given treatment.
TABLE-US-00008 TABLE 8 dgt-28 transformed T.sub.1 Arabidopsis
response to a range of glyphosate rates applied postemergence,
compared to a dgt-1 (T.sub.4) homozygous resistant population, and
a non-transformed control. Visual % injury 14 days after
application. % Injury % Injury Averages <20% 20-40% >40% Ave
Std dev Range (%) pDAB107527: TraP4 v2 - dgt-28 v5 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 0 0 3.8 7.5
0-15 420 g ae/ha glyphosate 2 1 1 28.8 28.1 0-65 1680 g ae/ha
glyphosate 0 2 2 55.0 26.8 35-85 3360 g ae/ha glyphosate 0 2 2 43.8
18.0 30-70 pDAB105530: TraP5 v2 - dgt-28 v5 0 g ae/ha glyphosate 6
0 0 0.0 0.0 0 105 g ae/ha glyphosate 2 2 2 39.3 37.4 8-100 420 g
ae/ha glyphosate 1 4 1 33.0 26.6 8-85 1680 g ae/ha glyphosate 0 4 2
47.5 27.5 25-85 3360 g ae/ha glyphosate 0 0 6 76.7 13.7 50-85
pDAB105531: TraP8 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0
0 105 g ae/ha glyphosate 3 1 0 10.8 10.4 0-25 420 g ae/ha
glyphosate 3 0 1 22.8 18.6 8-50 1680 g ae/ha glyphosate 4 0 0 5.3
3.8 0-8 3360 g ae/ha glyphosate 0 4 0 29.3 6.8 22-35 pDAB105532:
TraP9 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g
ae/ha glyphosate 3 0 1 17.5 28.7 0-60 420 g ae/ha glyphosate 1 1 2
39.5 25.1 18-70 1680 g ae/ha glyphosate 3 0 1 26.3 36.1 5-80 3360 g
ae/ha glyphosate 3 0 1 25.8 32.9 8-75 pDAB105533: TraP12 v2 -
dgt-28 v5 0 g ae/ha glyphosate 5 0 0 0.0 0.0 0 105 g ae/ha
glyphosate 4 1 0 10.0 10.0 0-25 420 g ae/ha glyphosate 1 1 3 53.6
34.6 8-85 1680 g ae/ha glyphosate 4 1 0 11.0 8.2 0-20 3360 g ae/ha
glyphosate 0 2 3 55.0 25.5 25-80 pDAB105534: TraP13 v2 - dgt-28 v5
0 g ae/ha glyphosate 5 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 0 1
14.0 20.6 0-50 420 g ae/ha glyphosate 3 1 1 17.6 19.5 0-50 1680 g
ae/ha glyphosate 3 0 2 39.0 47.1 5-100 3360 g ae/ha glyphosate 2 2
1 31.2 22.3 18-70 pDAB4104: dgt-1 (transformed control) 0 g ae/ha
glyphosate 5 0 0 0.0 0.0 0 105 g ae/ha glyphosate 0 0 4 80.0 0.0 80
420 g ae/ha glyphosate 0 0 4 80.0 0.0 80 1680 g ae/ha glyphosate 0
0 4 80.0 0.0 80 3360 g ae/ha glyphosate 0 0 4 81.3 2.5 80-85 WT
(non-transformed control) 0 g ae/ha glyphosate 5 0 0 0.0 0.0 0 105
g ae/ha glyphosate 0 0 4 100.0 0.0 100 420 g ae/ha glyphosate 0 0 4
100.0 0.0 100 1680 g ae/ha glyphosate 0 0 4 100.0 0.0 100 3360 g
ae/ha glyphosate 0 0 4 100.0 0.0 100
TABLE-US-00009 TABLE 9 dgt-32, and dgt-33 transformed T.sub.1
Arabidopsis response to a range of glyphosate rates applied
postemergence, compared to a dgt-1 (T.sub.4) homozygous resistant
population, and a non-transformed control. Visual % injury 14 days
after application. % Injury % Injury Averages <20% 20-40%
>40% Ave Std dev Range (%) pDAB107532: TraP14 v2 - dgt-32 v3 0 g
ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha glyphosate 4 0 0 0.0
0.0 0 420 g ae/ha glyphosate 2 0 2 30.0 29.4 0-60 1680 g ae/ha
glyphosate 3 0 1 17.5 21.8 5-50 3360 g ae/ha glyphosate 0 3 1 35.0
30.0 20-80 pDAB107534: TraP24 v2 - dgt-33 v3 0 g ae/ha glyphosate 4
0 0 0.0 0.0 0 105 g ae/ha glyphosate 2 2 0 21.3 14.9 5-40 420 g
ae/ha glyphosate 1 1 2 46.3 30.9 5-70 1680 g ae/ha glyphosate 1 0 3
62.5 38.8 5-90 3360 g ae/ha glyphosate 1 0 3 62.0 36.0 8-80
pDAB4104: dgt-1 (transformed control) 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 105 g ae/ha glyphosate 0 2 3 42.5 15.0 20-50 420 g ae/ha
glyphosate 0 1 2 38.8 11.1 25-50 1680 g ae/ha glyphosate 0 0 4 79.0
19.4 50-90 3360 g ae/ha glyphosate 0 0 4 50.0 0.0 50 WT
(non-transformed control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105
g ae/ha glyphosate 0 0 4 85.0 0.0 85 420 g ae/ha glyphosate 0 0 4
100.0 0.0 100 1680 g ae/ha glyphosate 0 0 4 100.0 0.0 100 3360 g
ae/ha glyphosate 0 0 4 100.0 0.0 100
TABLE-US-00010 TABLE 10 dgt-28, dgt-32, dgt-33, dgt-3, and dgt-7
transformed T.sub.1 Arabidopsis response to glyphosate applied
postemergence at 1,680 g ae/ha, compared to a dgt-1 (T.sub.4)
homozygous resistant population, and a non-transformed control.
Visual % injury 14 days after application. % Injury % Injury
<20% 20-40% >40% Ave Std dev Range (%) Bacterial pDAB07527
TraP4 v2 - dgt-28 v5 0 2 2 55.0 26.8 35-85 Enzymes pDAB105530 TraP5
v2 - dgt -28 v5 0 4 2 47.5 27.5 25-85 pDAB105531 TraP8 v2 - dgt -28
v5 4 0 0 5.3 3.8 0-8 pDAB105532 TraP9 v2 - dgt -28 v5 3 0 1 26.3
36.1 5-80 pDAB105533 Trap12 v2 - dgt -28 v5 4 1 0 11.0 8.2 0-20
pDAB105534 TraP13 v2 - dgt -28 v5 3 0 2 39.0 47.1 5-100 pDAB107532
TraP14 v2 - dgt-32 v3 3 0 1 17.5 21.8 5-50 pDAB107534 TraP24 v2 -
dgt-33 v3 1 0 3 62.5 38.8 5-90 Class I pDAB102715 dgt-3 v2 4 0 3 42
48 0-100 Enzymes pDAB102716 dgt-3 v3 2 0 1 14 23 0-40 pDAB102717
dgt-3 v4 3 2 1 28 35 10-100 pDAB102785 dgt-7 v4 0 1 1 45 21 30-60
pDAB4104 dgt-1 (transformed 0 0 4 80.0 0.0 80 control) -- WT
(non-transfor 0 0 4 100.0 0.0 100 med control)
[0255] dgt-28 as a Selectable Marker.
[0256] The use of dgt-28 as a selectable marker for glyphosate
selection agent is tested with the Arabidopsis transformed plants
described above. Approximately 50 T.sub.4 generation Arabidopsis
seed (homozygous for dgt-28) are spiked into approximately 5,000
wildtype (sensitive to glyphosate) seed. The seeds are germinated
and plantlets are sprayed with a selecting dose of glyphosate.
Several treatments of glyphosate are compared; each tray of plants
receives either one or two application timings of glyphosate in one
of the following treatment schemes: 7 DAP (days after planting), 11
DAP, or 7 followed by 11 DAP. Since all plants also contain a
glufosinate resistance gene in the same transformation vector,
dgt-28 containing plants selected with glyphosate can be directly
compared to DSM-2 or pat containing plants selected with
glufosinate.
[0257] Glyphosate treatments are applied with a DeVilbiss.TM. spray
tip as previously described. Transgenic plants containing dgt-28
are identified as "resistant" or "sensitive" 17 DAP. Treatments of
26.25-1680 g ae/ha glyphosate applied 7 and 11 days after planting
(DAP), show effective selection for transgenic Arabidopsis plants
that contain dgt-28. Sensitive and resistant plants are counted and
the number of glyphosate tolerant plants is found to correlate with
the original number of transgenic seed containing the dgt-28
transgene which are planted. These results indicate that dgt-28 can
be effectively used as an alternative selectable marker for a
population of transformed Arabidopsis.
[0258] Heritability.
[0259] Confirmed transgenic T.sub.1 Arabidopsis events were
self-pollinated to produce T.sub.2 seed. These seed were progeny
tested by applying Ignite.TM. herbicide containing glufosinate (200
g ae/ha) to 100 random T.sub.2 siblings. Each individual T.sub.2
plant was transplanted to 7.5-cm square pots prior to spray
application (track sprayer at 187 L/ha applications rate). The
T.sub.1 families (T.sub.2 plants) segregated in the anticipated 3
Resistant: 1 Sensitive model for a dominantly inherited single
locus with Mendelian inheritance as determined by Chi square
analysis (P>0.05). The percentage of T.sub.1 families that
segregated with the expected Mendelian inheritance are illustrated
in Table 11, and demonstrate that the dgt-28 trait is passed via
Mendelian inheritance to the T.sub.2 generation. Seed were
collected from 5 to 15 T.sub.2 individuals (T.sub.3 seed).
Twenty-five T.sub.3 siblings from each of 3-4 randomly-selected
T.sub.2 families were progeny tested as previously described. Data
showed no segregation and thus demonstrated that dgt-28 and dgt-3
are stably integrated within the chromosome and inherited in a
Mendelian fashion to at least three generations.
TABLE-US-00011 TABLE 11 Percentage of T.sub.1 families (T.sub.2
plants) segregating as single Mendelian inheritance for a progeny
test of 100 plants. T1 Families Tested Gene of Interest Segregating
at 1 Locus (%) dgt-3 v2 64% dgt-3 v3 60% dgt-3 v4 80% dgt-7 v4 63%
TraP5 v2 - dgt-28 v5 100% TraP8 v2 - dgt-28 v5 100% TraP9 v2 -
dgt-28 v5 100% TraP12 v2 - dgt-28 v5 50% TraP13 v2 - dgt-28 v5 75%
yfp Transgenic Control 100% Plants
[0260] T.sub.2 Arabidopsis Data.
[0261] The second generation plants (T.sub.2) of selected T.sub.1
Arabidopsis events which contained low copy numbers of the dgt-28
transgene were further characterized for glyphosate tolerance.
Glyphosate was applied as described previously. The response of the
plants is presented in terms of % visual injury 2 weeks after
treatment (WAT). Data are presented as a histogram of individuals
exhibiting little or no injury (<20%), moderate injury (20-40%),
or severe injury (>40%). An arithmetic mean and standard
deviation are presented for each construct used for Arabidopsis
transformation. The range in individual response is also indicated
in the last column for each rate and transformation. Wild-type,
non-transformed Arabidopsis (cv. Columbia) served as a glyphosate
sensitive control. In the T.sub.2 generation hemizygous and
homozygous plants were available for testing for each event and
therefore were included for each rate of glyphosate tested.
Hemizygous plants contain two different alleles at a locus as
compared to homozygous plants which contain the same two alleles at
a locus. Variability of response to glyphosate is expected in the
T.sub.2 generation as a result of the difference in gene dosage for
hemizygous as compared to homozygous plants. The variability in
response to glyphosate is reflected in the standard deviation and
range of response.
[0262] In the T.sub.2 generation both single copy and multi-copy
dgt-28 events were characterized for glyphosate tolerance. Within
an event, single copy plants showed similar levels of tolerance to
glyphosate. Characteristic data for a single copy T.sub.2 event are
presented in Table 12. Events containing dgt-28 linked with TraP5
v2 did not provide robust tolerance to glyphosate as compared with
the dgt-28 constructs which contained other TraP transit peptides.
However, the dgt-28 TraP5 constructs did provide a low level of
glyphosate tolerance as compared to the non-transformed Columbia
control. There were instances when events that were shown to
contain two or more copies of dgt-28 were more susceptible to
elevated rates of glyphosate (data not shown). This increase in
sensitivity to glyphosate is similar to the data previously
described for the T.sub.1 plants which also contained high copy
numbers of the dgt-28 transgene. It is likely that the presence of
high copy numbers of the transgene within the Arabidopsis plants
result in transgene silencing or other epigenetic effects which
resulted in sensitivity to glyphosate, despite the presence of the
dgt-28 transgene.
[0263] These events contained dgt-28 linked with TraP5 v2
(pDAB105530), TraP12 v2 (pDAB105533) and TraP13 v2
(pDAB105534).
[0264] In addition to dgt-28, T.sub.2 Arabidopsis events
transformed with dgt-3 are presented in Table 13. As described for
the dgt-28 events in Table 12, the data table contains a
representative event that is characteristic of the response to
glyphosate for each construct. For the dgt-3 characterization,
constructs containing a single PTU (plant transformation unit) with
the dgt-3 gene being driven by the AtUbi10 promoter (pDAB102716,
FIG. 30 and pDAB102715, FIG. 29) were compared to constructs with
the same gene containing 2 PTUs of the gene (pDAB102719, FIG. 33;
pDAB102718, FIG. 34). The constructs which contained 2 PTU used the
AtUbi10 promoter to drive one copy of the gene and the CsVMV
promoter to drive the other copy. The use of the double PTU was
incorporated to compare the dgt-3 transgenic plants with dgt-28
transgenic plants which contained two copies of the transgene. Data
demonstrated that single copy T.sub.2 dgt-3 events with only a
single PTU were more susceptible to glyphosate than single copy
dgt-28 events tested, but were more tolerant than the
non-transformed control. T.sub.1 families containing 2 PTUs of the
dgt-3 gene provided a higher level of visual tolerance to
glyphosate compared to the 1 PTU constructs. In both instances the
T.sub.1 families were compared to the dgt-1 and wildtype controls.
T.sub.2 data demonstrate that dgt-28 provides robust tolerance as
single copy events.
TABLE-US-00012 TABLE 12 Response of selected individual T.sub.2
Arabidopsis events containing dgt-28 to glyphosate applied
postemergence at varying rates, compared to a dgt-1 (T.sub.4)
homozygous resistant population, and a non-transformed control.
Visual % injury 14 days after application. % Injury % Injury 1 copy
<20% 20-40% >40% Ave Std dev Range (%) pDAB105530: TraP5 v2 -
dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha
glyphosate 0 0 4 75.0 17.8 50-90 840 g ae/ha glyphosate 0 0 4 80.0
20.0 50-90 1680 g ae/ha glyphosate 0 0 4 75.0 10.8 60-85 3360 g
ae/ha glyphosate 0 0 4 76.3 4.8 70-80 pDAB105531: TraP8 v2 - dgt-28
v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 4 0
0 0.5 1.0 0-2 840 g ae/ha glyphosate 4 0 0 1.3 2.5 0-5 1680 g ae/ha
glyphosate 4 0 0 7.5 5.0 5-15 3360 g ae/ha glyphosate 4 0 0 7.5 6.5
0-15 pDAB105532: TraP9 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 420 g ae/ha glyphosate 4 0 0 2.0 4.0 0-8 840 g ae/ha
glyphosate 4 0 0 9.0 2.0 8-12 1680 g ae/ha glyphosate 4 0 0 7.3 4.6
2-12 3360 g ae/ha glyphosate 4 0 0 11.0 1.2 10-12 pDAB105533:
TraP12 v2 - dgt-28 v5 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g
ae/ha glyphosate 4 0 0 0.0 0.0 0 840 g ae/ha glyphosate 4 0 0 0.0
0.0 0 1680 g ae/ha glyphosate 4 0 0 0.0 0.0 0 3360 g ae/ha
glyphosate 3 1 0 13.3 7.9 8-25 pDAB105534: TraP13 v2 - dgt-28 v5 0
g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha glyphosate 3 1 0 5.0
10.0 0-20 840 g ae/ha glyphosate 3 1 0 5.0 10.0 0-20 1680 g ae/ha
glyphosate 2 2 0 10.0 11.5 0-20 3360 g ae/ha glyphosate 2 2 0 15.0
12.2 5-30 WT (non-transformed control) 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 420 g ae/ha glyphosate 0 0 4 100.0 0.0 100 840 g ae/ha
glyphosate 0 0 4 100.0 0.0 100 1680 g ae/ha glyphosate 0 0 4 100.0
0.0 100 3360 g ae/ha glyphosate 0 0 4 100.0 0.0 100 pDAB4104: dgt-1
(transformed control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g
ae/ha glyphosate 0 4 0 37.5 2.9 35-40 840 g ae/ha glyphosate 0 0 4
45.0 0.0 45 1680 g ae/ha glyphosate 0 0 4 47.5 2.9 45-50 3360 g
ae/ha glyphosate 0 0 4 50.0 0.0 50
TABLE-US-00013 TABLE 13 Response of selected T.sub.2 Arabidopsis
events transformed with dgt-3 to glyphosate applied postemergence
at varying rates. Visual % injury 14 days after application. %
Injury % Injury 1 copy seg <20% 20-40% >40% Ave Std dev Range
(%) pDAB102716: dgt-3 v3 (1 PTU) 0 g ae/ha glyphosate 4 0 0 0 0 0
420 g ae/ha glyphosate 1 1 2 39 25 15-65 840 g ae/ha glyphosate 0 2
2 50 23 30-70 1680 g ae/ha glyphosate 0 1 3 69 19 40-80 3360 g
ae/ha glyphosate 0 0 4 79 6 70-85 pDAB102719: dgt-3 v3 (2 PTU) 0 g
ae/ha glyphosate 4 0 0 0 0 0 420 g ae/ha glyphosate 0 4 0 20 0 20
840 g ae/ha glyphosate 0 3 1 38 5 35-45 1680 g ae/ha glyphosate 3 1
0 15 7 10-25 3360 g ae/ha glyphosate 2 2 0 21 8 15-30 pDAB102715:
dgt-3 v2 (1 PTU) 0 g ae/ha glyphosate 4 0 0 0 0 0 420 g ae/ha
glyphosate 2 2 0 26 16 10-40 840 g ae/ha glyphosate 0 2 2 55 17
40-70 1680 g ae/ha glyphosate 0 2 2 56 22 35-75 3360 g ae/ha
glyphosate 0 0 4 65 17 50-80 pDAB102718: dgt-3 v2 (2 PTU) 0 g ae/ha
glyphosate 4 0 0 0 0 0 420 g ae/ha glyphosate 4 0 0 5 7 0-15 840 g
ae/ha glyphosate 2 2 0 23 10 15-35 1680 g ae/ha glyphosate 3 0 1 20
20 5-50 3360 g ae/ha glyphosate 1 1 2 36 22 15-60
[0265] T.sub.3 Arabidopsis Data.
[0266] The third generation plants (T.sub.3) of selected T.sub.2
Arabidopsis events which contained low copy numbers of the dgt-28
transgene were further characterized for glyphosate tolerance.
Twenty-five plants per line were selected with glufosinate as
previously described and lines from every construct tested did not
segregate for the selectable marker gene. Glyphosate was applied as
described previously. The response of the plants is presented in
terms of % visual injury 2 weeks after treatment (WAT). Data are
presented as a histogram of individuals exhibiting little or no
injury (<20%), moderate injury (20-40%), or severe injury
(>40%). An arithmetic mean and standard deviation are presented
for each construct used for Arabidopsis transformation. The range
in individual response is also indicated in the last column for
each rate and transformation. Wild-type, non-transformed
Arabidopsis (cv. Columbia) served as a glyphosate-sensitive
control.
TABLE-US-00014 TABLE 14 Response of selected individual T.sub.3
Arabidopsis events containing dgt-28 to glyphosate applied
postemergence at varying rates, compared to a dgt-1 (T.sub.4)
homozygous resistant population, and a non-transformed control.
Visual % injury 14 days after application. % Injury Range (No.
Replicates) % Injury Analysis <20% 20-40% >40% Ave Std dev
Range (%)
[0267] Selection of Transformed Plants.
[0268] Freshly harvested T.sub.1 seed [dgt-31, dgt-32, and dgt-33
v1 gene] were allowed to dry at room temperature and shipped to
Indianapolis for testing. T.sub.1 seed was sown in 26.5.times.51-cm
germination trays (T.O. Plastics Inc., Clearwater, Minn.), each
receiving a 200 mg aliquots of stratified T.sub.1 seed
(.about.10,000 seed) that had previously been suspended in 40 mL of
0.1% agarose solution and stored at 4.degree. C. for 2 days to
complete dormancy requirements and ensure synchronous seed
germination.
[0269] Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue,
Wash.) was covered with fine vermiculite and subirrigated with
Hoagland's solution until wet, then allowed to gravity drain. Each
40 mL aliquot of stratified seed was sown evenly onto the
vermiculite with a pipette and covered with humidity domes
(KORD.TM. Products, Bramalea, Ontario, Canada) for 4-5 days. Domes
were removed once plants had germinated prior to initial
transformant selection using glufosinate postemergence spray
(selecting for the co-transformed dsm-2 gene).
[0270] Six days after planting (DAP) and again 10 DAP, T.sub.1
plants (cotyledon and 2-4-1f stage, respectively) were sprayed with
a 0.1% solution of IGNITE.TM. herbicide (280 g ai/L glufosinate,
Bayer Crop Sciences, Kansas City, Mo.) at a spray volume of 10
mL/tray (703 L/ha) using a DeVilbiss.TM. compressed air spray tip
to deliver an effective rate of 200 g ae/ha glufosinate per
application. Survivors (plants actively growing) were identified
4-7 days after the final spraying. Surviving plants were
transplanted individually into 3-inch pots prepared with potting
media (Metro Mix 360.TM.). Plants reared in the greenhouse at least
1 day prior to tissue sampling for copy number analyses.
[0271] T.sub.1 plants were sampled and copy number analysis for the
dgt-31, dgt-32, and dgt-33 v1 gene were completed. T.sub.1 plants
were then assigned to various rates of glyphosate so that a range
of copies were among each rate. For Arabidopsis, 26.25 g ae/ha
glyphosate is an effective dose to distinguish sensitive plants
from ones with meaningful levels of resistance. Elevated rates were
applied to determine relative levels of resistance (105, 420, 1680,
or 3360 g ae/ha). Table 15 shows the comparisons drawn to
dgt-1.
[0272] All glyphosate herbicide applications were made by track
sprayer in a 187 L/ha spray volume. Glyphosate used was of the
commercial Durango dimethylamine salt formulation (480 g ae/L, Dow
AgroSciences, LLC). Low copy T.sub.1 plants that exhibited
tolerance to either glufosinate or glyphosate were further accessed
in the T.sub.2 generation.
[0273] The first Arabidopsis transformations were conducted using
dgt-31, dgt-32, and dgt-33 v1. T.sub.1 transformants were first
selected from the background of untransformed seed using a
glufosinate selection scheme. Three flats or 30,000 seed were
analyzed for each T.sub.1 construct. Transformation frequency was
calculated and results of T1 dgt-31, dgt-32, and dgt-33 constructs
are listed in Table 15.
TABLE-US-00015 TABLE 15 Transformation frequency of T1 dgt-31,
dgt-32, and dgt-33 Arabidopsis constructs selected with glufosinate
for selection of the selectable marker gene DSM-2. Transformation
Construct Cassette Frequency (%) pDAB107532 AtUbi10/TraP14 dgt-32
v1 0.47 pDAB107533 AtUbi10/TraP23 dgt-31 v1 0.36 pDAB107534
AtUbi10/TraP24 dgt-33 v1 0.68
[0274] T.sub.1 plants selected above were subsequently transplanted
to individual pots and sprayed with various rates of commercial
glyphosate. Table 16 compares the response of dgt-31, dgt-32, and
dgt-33 v1 and control genes to impart glyphosate resistance to
Arabidopsis T.sub.1 transformants. Response is presented in terms
of % visual injury 2 WAT. Data are presented as a histogram of
individuals exhibiting little or no injury (<20%), moderate
injury (20-40%), or severe injury (>40%). An arithmetic mean and
standard deviation is presented for each treatment. The range in
individual response is also indicated in the last column for each
rate and transformation. Wild-type non-transformed Arabidopsis (cv.
Columbia) served as a glyphosate sensitive control. The DGT-31 (v1)
gene with transit peptide TraP23 imparted slight herbicide
tolerance to individual T.sub.1 Arabidopsis plants compared to the
negative control, but the gene exhibited improved tolerance with
transit peptide TraP8. Both DGT-32 and DGT-33 demonstrated robust
tolerance to glyphosate at the rates tested with TraP8 and with
their respective differing chloroplast transit peptide (TraP14 and
TraP24 respectively). Within a given treatment, the level of plant
response varied greatly, which can be attributed to the fact each
plant represents an independent transformation event and thus the
copy number of the gene of interest varies from plant to plant. Of
important note, at each glyphosate rate tested, there were
individuals that were more tolerant than others. An overall
population injury average by rate is presented in Table 16 to
demonstrate the significant difference between the plants
transformed with dgt-31, dgt-32, and dgt-33 v1 versus the dgt-1 v1
or Wild-type controls.
TABLE-US-00016 TABLE 16 dgt-31, dgt-32, and dgt-33 v1 transformed
T.sub.1 Arabidopsis response to a range of glyphosate rates applied
postemergence, compared to a dgt-1 (T4) homozygous resistant
population, or a non-transformed control. Visual % injury 2 weeks
after treatment. % Injury % Injury 20- Std. Range Averages <20%
40% >40% Ave Dev. (%) TraP23 dgt-31 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 105 g ae/ha 0 0 4 81.3 2.5 80-85 420 g ae/ha 0 0 4 97.3
4.9 90-100 1680 g ae/ha 0 0 4 90.0 7.1 85-100 3360 g ae/ha 0 0 4
91.3 6.3 85-100 TraP14 dgt-32 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
105 g ae/ha 4 0 0 0.0 0.0 0 420 g ae/ha 2 0 2 30.0 29.4 0-60 1680 g
ae/ha 3 0 1 17.5 21.8 5-50 3360 g ae/ha 0 3 1 35.0 30.0 20-80
TraP24 dgt-33 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 2 2
0 21.3 14.9 5-40 420 g ae/ha 1 1 2 46.3 30.9 5-70 1680 g ae/ha 1 0
3 62.5 38.8 5-90 3360 g ae/ha 1 0 3 62.0 36.0 8-80 TraP8 dgt-31 0 g
ae/ha glyphosate 4 0 0 0.0 0.0 0.0 105 g ae/ha glyphosate 0 1 3 0.0
43.8 17.0 420 g ae/ha glyphosate 1 2 1 0.0 43.8 32.5 1680 g ae/ha
glyphosate 0 1 3 0.0 71.3 27.8 3360 g ae/ha glyphosate 0 0 4 0.0
81.3 8.5 TraP8 dgt-32 0 g ae/ha glyphosate 4 0 4 0.0 0.0 0.0 105 g
ae/ha glyphosate 4 0 0 0.0 0.0 0.0 420 g ae/ha glyphosate 4 0 0 0.0
7.5 5.0 1680 g ae/ha glyphosate 3 1 0 0.0 10.8 9.6 3360 g ae/ha
glyphosate 4 0 0 0.0 12.8 3.2 TraP8 dgt-33 0 g ae/ha glyphosate 4 0
0 0.0 0.0 0.0 105 g ae/ha glyphosate 4 0 0 0.0 0.0 0.0 420 g ae/ha
glyphosate 4 0 0 0.0 2.5 3.8 1680 g ae/ha glyphosate 4 0 0 0.0 6.3
2.5 3360 g ae/ha glyphosate 3 1 0 0.0 20.0 13.5 dgt-1 (transformed
control 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 0 1 3 42.5
15.0 20-50 420 g ae/ha 0 2 2 38.8 11.1 25-50 1680 g ae/ha 0 0 4
79.0 19.4 50-90 3360 g ae/ha 0 0 4 50.0 0.0 50 WT (non-transformed
control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 105 g ae/ha 0 0 4
85.0 0.0 85 420 g ae/ha 0 0 4 100.0 0.0 100 1680 g ae/ha 0 0 4
100.0 0.0 100 3360 g ae/ha 0 0 4 100.0 0.0 100
[0275] Maize Transformation.
[0276] Standard cloning methods, as described above, were used in
the construction of binary vectors for use in Agrobacterium
tumefaciens-mediated transformation of maize. Table 17 lists the
vectors which were constructed for maize transformation. The
following gene elements were used in the vectors which contained
dgt-28; the Zea mays Ubiquitin 1 promoter (ZmUbi1; U.S. Pat. No.
5,510,474) was used to drive the dgt-28 coding sequence which is
flanked by a Zea mays Lipase 3' untranslated region (ZmLip 3'UTR;
U.S. Pat. No. 7,179,902), the selectable marker cassette consists
of the Zea mays Ubiquitin 1 promoter which was used to drive the
aad-1 coding sequence (U.S. Pat. No. 7,838,733) which is flanked by
a Zea mays Lipase 3' untranslated region. The aad-1 coding sequence
confers tolerance to the phenoxy auxin herbicides, such as,
2,4-dichlorophenoxyacetic acid (2,4-D) and to
aryloxyphenoxypropionate (AOPP) herbicides.
[0277] The dgt-28 constructs were built as standard binary vectors
and Agrobacterium superbinary system vectors (Japan Tobacco, Tokyo,
JP). The standard binary vectors include; pDAB107663, pDAB107664,
pDAB107665, and pDAB107665. The Agrobacterium superbinary system
vectors include pDAB108384, pDAB108385, pDAB108386, and
pDAB108387.
[0278] Additional constructs were completed which contain a yellow
fluorescent protein (yfp); US Patent Application 2007/0298412)
reporter gene. pDAB109812 contains a yfp reporter gene cassette
which is driven by the Zea mays Ubiquitin 1 promoter and flanked by
the Zea mays per 5 3' untranslated region (Zm per5 3'UTR; U.S. Pat.
No. 7,179,902), the selectable marker cassette consists of the
sugar cane bacilliform virus promoter (SCBV; U.S. Pat. No.
5,994,123) which is used to drive the expression of aad-1 and is
flanked by the Zea mays Lipase 3' untranslated region. pDAB101556
contains a yfp cassette which is driven by the Zea mays Ubiquitin 1
promoter and flanked by the Zea mays per 5 3' untranslated region,
the selectable marker cassette consists of the Zea mays Ubiquitin 1
promoter which is used to drive the expression of aad-1 and is
flanked by the Zea mays Lipase 3' untranslated region. pDAB107698
contains a dgt-28 cassette which is driven by the Zea mays
Ubiquitin 1 promoter and is flanked by a Zea mays Lipase 3'
untranslated region, an yfp cassette which is driven by the Zea
mays Ubiquitin 1 promoter and flanked by the Zea mays per 5 3'
untranslated region, the selectable marker cassette consists of the
sugar cane bacilliform virus promoter which is used to drive the
expression of aad-1 and is flanked by the Zea mays Lipase 3'
untranslated region. All three of these constructs are standard
binary vectors.
TABLE-US-00017 TABLE 17 Maize Transformation Vectors Plasmid FIG.
No. No: Description of Gene Elements pDAB107663 35 ZmUbi1/TraP4
dgt-28/ZmLip 3'UTR :: ZmUbi1/aad-1/ZmLip 3'UTR binary vector
pDAB107664 36 ZmUbi1/TraP8 dgt-28/ZmLip 3'UTR :: ZmUbi1/aad-1/ZmLip
3'UTR binary vector pDAB107665 37 ZmUbi1/TraP23 dgt-28/ZmLip 3'UTR
:: ZmUbi1/aad-1/ZmLip 3'UTR binary vector pDAB107666 38
ZmUbi1/TraP5 dgt-28/ZmLip 3'UTR :: ZmUbi1/aad-1/ZmLip 3'UTR binary
vector pDAB109812 39 ZmUbi1/yfp/ZmPer5 3'UTR :: SCBV/aad-1/ZmLip
3'UTR binary vector pDAB101556 40 ZmUbi1/yfp/ZmPer5 3'UTR ::
ZmUbi1/aad-1/ZmLip 3'UTR binary vector pDAB107698 41 ZmUbi1/TraP8
dgt-28/ZmLip 3'UTR :: ZmUbi1/yfp/ZmLip 3'UTR::SCBV/aad-1/ZmLip
3'UTR pDAB108384 42 ZmUbi1/TraP4 dgt-28/ZmLip 3'UTR::
ZmUbi1/aad-1/ZmLip 3'UTR superbinary vector pDAB108385 43
ZmUbi1/TraP8 dgt-28/ZmLip 3'UTR :: ZmUbi1/aad-1/ZmLip 3'UTR
superbinary precursor pDAB108386 44 ZmUbi1/TraP23 dgt-28/ZmLip
3'UTR :: ZmUbi1/aad-1/ZmLip 3'UTR superbinary precursor pDAB108387
45 ZmUbi1/TraP5 dgt-28/ZmLip 3'UTR::ZmUbi1/aad-1/ZmLip 3'UTR
superbinary precursor
[0279] Ear Sterilization and Embryo Isolation.
[0280] To obtain maize immature embryos, plants of the Zea mays
inbred line B104 were grown in the greenhouse and were self or
sib-pollinated to produce ears. The ears were harvested
approximately 9-12 days post-pollination. On the experimental day,
ears were surface-sterilized by immersion in a 20% solution of
sodium hypochlorite (5%) and shaken for 20-30 minutes, followed by
three rinses in sterile water. After sterilization, immature
zygotic embryos (1.5-2.4 mm) were aseptically dissected from each
ear and randomly distributed into micro-centrifuge tubes containing
liquid infection media (LS Basal Medium, 4.43 gm/L; N6 Vitamin
Solution [1000.times.], 1.00 mL/L; L-proline, 700.0 mg/L; Sucrose,
68.5 gm/L; D(+) Glucose, 36.0 gm/L; 10 mg/ml of 2,4-D, 150
.mu.L/L). For a given set of experiments, pooled embryos from three
ears were used for each transformation.
Agrobacterium Culture Initiation:
[0281] Glycerol stocks of Agrobacterium containing the binary
transformation vectors described above were streaked on AB minimal
medium plates containing appropriate antibiotics and were grown at
20.degree. C. for 3-4 days. A single colony was picked and streaked
onto YEP plates containing the same antibiotics and was incubated
at 28.degree. C. for 1-2 days.
[0282] Agrobacterium Culture and Co-Cultivation.
[0283] Agrobacterium colonies were taken from the YEP plate,
suspended in 10 mL of infection medium in a 50 mL disposable tube,
and the cell density was adjusted to OD.sub.600 nm of 0.2-0.4 using
a spectrophotometer. The Agrobacterium cultures were placed on a
rotary shaker at 125 rpm, room temperature, while embryo dissection
was performed. Immature zygotic embryos between 1.5-2.4 mm in size
were isolated from the sterilized maize kernels and placed in 1 mL
of the infection medium) and washed once in the same medium. The
Agrobacterium suspension (2 mL) was added to each tube and the
tubes were placed on a shaker platform for 10-15 minutes. The
embryos were transferred onto co-cultivation media (MS Salts, 4.33
gm/L; L-proline, 700.0 mg/L; Myo-inositol, 100.0 mg/L; Casein
enzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3 mg/L;
Sucrose, 30.0 gm/L; Gelzan.TM., 3.00 gm/L; Modified MS-Vitamin
[1000.times.], 1.00 ml/L; 8.5 mg/ml AgNo.sub.3, 15.0 mg/L; DMSO,
100 .mu.M), oriented with the scutellum facing up and incubated at
25.degree. C., under 24-hour light at 50 .mu.mole m.sup.-2
sec.sup.-1 light intensity for 3 days.
[0284] Callus Selection and Regeneration of Putative Events.
[0285] Following the co-cultivation period, embryos were
transferred to resting media (MS Salts, 4.33 gm/L; L-proline, 700.0
mg/L; 1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; MES
[(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.500 gm/L;
Casein enzymatic hydrolysate 100.0 mg/L; 30 mM Dicamba-KOH, 3.3
mg/L; Sucrose, 30.0 gm/L; Gelzan 2.30 gm/L; Modified MS-Vitamin
[1000.times.], 1.00 ml/L; 8.5 mg/ml AgNo3, 15.0 mg/L;
Carbenicillin, 250.0 mg/L) without selective agent and incubated
under 24-hour light at 50 .mu.mole m.sup.-2 sec.sup.-1 light
intensity and at 25.degree. C. for 3 days.
[0286] Growth inhibition dosage response experiments suggested that
glyphosate concentrations of 0.25 mM and higher were sufficient to
inhibit cell growth in the untransformed B104 maize line. Embryos
were transferred onto Selection 1 media containing 0.5 mM
glyphosate (MS Salts, 4.33 gm/L; L-proline, 700.0 mg/L;
Myo-inositol, 100.0 mg/L; MES [(2-(n-morpholino)-ethanesulfonic
acid), free acid] 0.500 gm/L; Casein enzymatic hydrolysate 100.0
mg/L; 30 mM Dicamba-KOH, 3.3 mg/L; Sucrose, 30.0 gm/L; Gelzan.TM.
2.30 gm/L; Modified MS-Vitamin [1000.times.], 1.00 ml/L; 8.5 mg/ml
AgNo3, 15.0 mg/L; Carbenicillin, 250.0 mg/L) and incubated in
either dark and/or under 24-hour light at 50 .mu.mole m.sup.-2
sec.sup.-1 light intensity for 7-14 days at 28.degree. C.
[0287] Proliferating embryogenic calli were transferred onto
Selection 2 media containing 1.0 mM glyphosate (MS Salts, 4.33
gm/L; 1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; L-proline,
700.0 mg/L; MES [(2-(n-morpholino)-ethanesulfonic acid), free acid]
0.500 gm/L; Casein enzymatic hydrolysate 100.0 mg/L; 30 mM
Dicamba-KOH, 3.3 mg/L; Sucrose, 30.0 gm/L; Gelzan.TM. 2.30 gm/L;
Modified MS-Vitamin [1000.times.], 1.00 ml/L; 8.5 mg/mL AgNo3, 15.0
mg/L; Carbenicillin, 250.0 mg/L; R-Haloxyfop acid 0.1810 mg/L), and
were incubated in either dark and/or under 24-hour light at 50
.mu.mole m.sup.-2 sec.sup.-1 light intensity for 14 days at
28.degree. C. This selection step allowed transgenic callus to
further proliferate and differentiate. The callus selection period
lasted for three to four weeks.
[0288] Proliferating, embryogenic calli were transferred onto
PreReg media containing 0.5 mM glyphosate (MS Salts, 4.33 gm/L;
1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; L-proline, 350.0
mg/L; MES [(2-(n-morpholino)-ethanesulfonic acid), free acid] 0.250
gm/L; Casein enzymatic hydrolysate 50.0 mg/L; NAA-NaOH 0.500 mg/L;
ABA-EtOH 2.50 mg/L; BA 1.00 mg/L; Sucrose, 45.0 gm/L; Gelzan.TM.
2.50 gm/L; Modified MS-Vitamin [1000.times.], 1.00 ml/L; 8.5 mg/ml
AgNo3, 1.00 mg/L; Carbenicillin, 250.0 mg/L) and cultured under
24-hour light at 50 .mu.mole m.sup.-2 sec.sup.-1 light intensity
for 7 days at 28.degree. C.
[0289] Embryogenic calli with shoot-like buds were transferred onto
Regeneration media containing 0.5 mM glyphosate (MS Salts, 4.33
gm/L; 1,2,3,5/4,6-Hexahydroxycyclohexane, 100.0 mg/L; Sucrose, 60.0
gm/L; Gellan Gum G434.TM. 3.00 gm/L; Modified MS-Vitamin
[1000.times.], 1.00 ml/L; Carbenicillin, 125.0 mg/L) and cultured
under 24-hour light at 50 .mu.mole m.sup.-2 sec.sup.-1 light
intensity for 7 days.
[0290] Small shoots with primary roots were transferred to rooting
media (MS Salts, 4.33 gm/L; Modified MS-Vitamin [1000.times.], 1.00
ml/L; 1,2,3,5/4,6-Hexahydroxycyclohexane, 100 mg/L; Sucrose, 60.0
gm/L; Gellan Gum G434.TM. 3.00 gm/L; Carbenicillin, 250.0 mg/L) in
phytotrays and were incubated under 16/8 hr. light/dark at 140-190
.mu.mole m.sup.-2 sec.sup.-1 light intensity for 7 days at
27.degree. C. Putative transgenic plantlets were analyzed for
transgene copy number using the protocols described above and
transferred to soil.
[0291] Molecular Confirmation of the Presence of the Dgt-28 and
Aad-1 Transgenes within Maize Plants.
[0292] The presence of the dgt-28 and aad-1 polynucleotide
sequences were confirmed via hydrolysis probe assays. Isolated
T.sub.0 Maize plants were initially screened via a hydrolysis probe
assay, analogous to TAQMAN.TM., to confirm the presence of a aad-1
and dgt-28 transgenes. The data generated from these studies were
used to determine the transgene copy number and used to select
transgenic maize events for back crossing and advancement to the
T.sub.1 generation.
[0293] Tissue samples were collected in 96-well plates, tissue
maceration was performed with a KLECO.TM. tissue pulverizer and
stainless steel beads (Hoover Precision Products, Cumming, Ga.), in
Qiagen.TM. RLT buffer. Following tissue maceration, the genomic DNA
was isolated in high-throughput format using the Biosprint 96.TM.
Plant kit (Qiagen, Germantown, Md.) according to the manufacturer's
suggested protocol. Genomic DNA was quantified by Quant-IT.TM. Pico
Green DNA assay kit (Molecular Probes, Invitrogen, Carlsbad,
Calif.). Quantified genomic DNA was adjusted to around 2 ng/.mu.L
for the hydrolysis probe assay using a BIOROBOT3000.TM. automated
liquid handler (Qiagen, Germantown, Md.). Transgene copy number
determination by hydrolysis probe assay, analogous to TAQMAN.RTM.
assay, was performed by real-time PCR using the LIGHTCYCLER.RTM.
480 system (Roche Applied Science, Indianapolis, Ind.). Assays were
designed for aad-1, dgt-28 and an internal reference gene Invertase
(Genbank Accession No: U16123.1) using the LIGHTCYCLER.RTM. Probe
Design Software 2.0. For amplification, LIGHTCYCLER.RTM.480 Probes
Master mix (Roche Applied Science, Indianapolis, Ind.) was prepared
at 1.times. final concentration in a 10 .mu.L volume multiplex
reaction containing 0.4 .mu.M of each primer for aad-1 and dgt-28
and 0.2 .mu.M of each probe (Table 18).
[0294] A two-step amplification reaction was performed with an
extension at 60.degree. C. for 40 seconds with fluorescence
acquisition. All samples were run and the averaged Cycle threshold
(Ct) values were used for analysis of each sample. Analysis of real
time PCR data was performed using LightCycler.RTM. software release
1.5 using the relative quant module and is based on the
.DELTA..DELTA.Ct method. Controls included a sample of genomic DNA
from a single copy calibrator and known two copy check that were
included in each run. Table 19 lists the results of the hydrolysis
probe assays.
TABLE-US-00018 TABLE 18 Primer and probe sequences used for
hydrolysis probe assay of aad-1, dgt-28 and internal reference
(Invertase). Oligonu- SEQ cleotide Gene ID Name Detected NO: Oligo
Sequence GAAD1F aad-1 58 TGTTCGGTTCCCTCTACCAA forward primer GAAD1P
aad-1 59 CACAGAACCGTCGCTTCAGCAACA probe GAAD1R aad-1 60
CAACATCCATCACCTTGACTGA reverse primer IV-Probe Invertase 61
CGAGCAGACCGCCGTGTACTTCTACC probe IVF-Taq Invertase 62
TGGCGGACGACGACTTGT forward primer IVR-Taq Invertase 63
AAAGTTTGGAGGCTGCCGT reverse primer zmDGT28 dgt-28 64
TTCAGCACCCGTCAGAAT F forward primer zmDGT28 dgt-28 65
TGCCGAGAACTTGAGGAGGT FAM probe zmDGT28 dgt-28 66 TGGTCGCCATAGCTTGT
R reverse primer
TABLE-US-00019 TABLE 19 T.sub.0 copy amount results for dgt-28
events. Low copy events consisted of 1-2 transgene copies, single
copy numbers are listed in parenthesis. High copy events contained
3 or more transgene copies. Plasmid used for # of Low Copy # of
High Copy Transformation Events (single copy) Events pDAB107663 43
(31) 10 pDAB107664 30 (24) 5 pDAB107665 40 (27) 10 pDAB107666 24
(12) 12 pDAB109812 2 (1) 0 pDAB101556 25 (15) 10 pDAB107698 3 (1)
2
[0295] Herbicide Tolerance in Dgt-28 Transformed Corn.
[0296] Zea mays dgt-28 transformation events (T.sub.0) were allowed
to acclimate in the greenhouse and were grown until plants had
transitioned from tissue culture to greenhouse growing conditions
(i.e., 2-4 new, normal looking leaves had emerged from the whorl).
Plants were grown at 27.degree. C. under 16 hour light:8 hour dark
conditions in the greenhouse. The plants were then treated with
commercial formulations of DURANGO DMA.TM. (containing the
herbicide glyphosate) with the addition of 2% w/v ammonium-sulfate.
Herbicide applications were made with a track sprayer at a spray
volume of 187 L/ha, 50-cm spray height. T.sub.0 plants were sprayed
with a range of glyphosate from 280-4480 g ae/ha glyphosate, which
is capable of significant injury to untransformed corn lines. A
lethal dose is defined as the rate that causes >95% injury to
the B104 inbred.
[0297] The results of the T.sub.0 dgt-28 corn plants demonstrated
that tolerance to glyphosate was achieved at rates up to 4480 g
ae/ha. A specific media type was used in the T.sub.0 generation.
Minimal stunting and overall plant growth of transformed plants
compared to the non-transformed controls demonstrated that dgt-28
provides robust tolerance to glyphosate when linked to the TraP5,
TraP8, and TraP23 chloroplast transit peptides.
[0298] Selected T.sub.0 plants are selfed or backcrossed for
further characterization in the next generation. 100 chosen dgt-28
lines containing the T.sub.1 plants are sprayed with 140-1120 g
ae/ha glufosinate or 105-1680 g ae/ha glyphosate. Both the
selectable marker and glyphosate resistant gene are constructed on
the same plasmid. Therefore, if one herbicide tolerant gene is
selected for by spraying with an herbicide, both genes are believed
to be present. At 14 DAT, resistant and sensitive plants are
counted to determine the percentage of lines that segregated as a
single locus, dominant Mendelian trait (3R:1S) as determined by Chi
square analysis. These data demonstrate that dgt-28 is inheritable
as a robust glyphosate resistance gene in a monocot species.
Increased rates of glyphosate are applied to the T.sub.1 or F.sub.1
survivors to further characterize the tolerance and protection that
is provided by the dgt-28 gene.
[0299] Post-Emergence Herbicide Tolerance in Dgt-28 Transformed
T.sub.0 Corn.
[0300] To events of dgt-28 linked with TraP4, TraP5, TraP8 and
TraP23 were generated by Agrobacterium transformation and were
allowed to acclimate under controlled growth chamber conditions
until 2-4 new, normal looking leaves had emerged from the whorl.
Plants were assigned individual identification numbers and sampled
for copy number analyses of both dgt-28 and aad-1. Based on copy
number analyses, plants were selected for protein expression
analyses. Plants were transplanted into larger pots with new
growing media and grown at 27.degree. C. under 16 hour light:8 hour
dark conditions in the greenhouse. Remaining plants that were not
sampled for protein expression were then treated with commercial
formulations of DURANGO DMA.TM. (glyphosate) with the addition of
2% w/v ammonium-sulfate. Treatments were distributed so that each
grouping of plants contained T.sub.0 events of varying copy number.
Herbicide applications were made with a track sprayer at a spray
volume of 187 L/ha, 50-cm spray height. T.sub.0 plants were sprayed
with a range of glyphosate from 280-4480 g ae/ha glyphosate capable
of significant injury to untransformed corn lines. A lethal dose is
defined as the rate that causes >95% injury to the B104 inbred.
B104 was the genetic background of the transformants.
[0301] Results of T.sub.0 dgt-28 corn plants demonstrate that
tolerance to glyphosate was achieved up to 4480 g ae/ha. Table 20.
Minimal stunting and overall plant growth of transformed plants
compared to the non-transformed controls demonstrated that dgt-28
provides robust protection to glyphosate when linked to TraP5,
TraP8, and TraP23.
TABLE-US-00020 TABLE 20 Response of T.sub.0 dgt-28 events of
varying copy numbers to rates of glyphosate ranging from 280-4480 g
ae/ha + 2.0% w/v ammonium sulfate 14 days after treatment. % Injury
% Injury 20- Std. Range Application Rate <20% 40% >40% Ave
Dev. (%) TraP4 dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 280 g
ae/ha 5 0 0 1.0 2.2 0-5 560 g ae/ha 6 0 0 2.0 4.0 0-10 1120 g ae/ha
12 0 0 1.3 3.1 0-10 2240 g ae/ha 7 0 0 1.7 4.5 0-12 4480 g ae/ha 7
0 0 1.1 3.0 0-8 TraP8 dgt-28 0 g ae/ha glyphosate 6 0 0 0.0 0.0 0
280 g ae/ha 5 1 0 6.7 8.8 0-20 560 g ae/ha 0 2 0 20.0 0.0 20 1120 g
ae/ha 7 0 0 1.4 2.4 0-5 2240 g ae/ha 3 1 0 7.5 15.0 0-30 4480 g
ae/ha 6 0 0 1.7 4.1 0-10 TraP23 dgt-28 0 g ae/ha glyphosate 6 0 0
0.8 2.0 0-5 280 g ae/ha 7 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 1.3 2.5
0-5 1120 g ae/ha 10 2 0 3.3 7.8 0-20 2240 g ae/ha 6 0 0 1.3 3.3 0-8
4480 g ae/ha 6 1 0 4.3 7.9 0-20 TraP5 dgt-28 0 g ae/ha glyphosate 4
0 0 0.0 0.0 0 280 g ae/ha 7 1 0 5.0 14.1 0-40 560 g ae/ha 8 0 0 0.6
1.8 0-5 1120 g ae/ha 7 1 0 5.0 14.1 0-40 2240 g ae/ha 8 0 0 0.0 0.0
0 4480 g ae/ha 8 0 0 0.0 0.0 0
[0302] Protein expression analyses by standard ELISA demonstrated a
mean range of DGT-28 protein from 12.6-22.5 ng/cm.sup.2 across the
constructs tested.
[0303] Confirmation of Glyphosate Tolerance in the F.sub.1
Generation Under Greenhouse Conditions.
[0304] Single copy T.sub.0 plants that were not sprayed were
backcrossed to the non-transformed background B104 for further
characterization in the next generation. In the T.sub.1 generation,
glyphosate tolerance was assessed to confirm the inheritance of the
dgt-28 gene. For T.sub.1 plants, the herbicide ASSURE II.TM. (35 g
ae/ha quizalofop-methyl) was applied at the V1 growth stage to
select for the AAD-1 protein. Both the selectable marker and
glyphosate resistant gene are constructed on the same plasmid.
Therefore if one gene is selected, both genes are believed to be
present. After 7 DAT, resistant and sensitive plants were counted
and null plants were removed from the population. These data
demonstrate that dgt-28 (v1) is heritable as a robust glyphosate
resistance gene in a monocot species. Plants were sampled for
characterization of DGT-28 protein by standard ELISA and RNA
transcript level. Resistant plants were sprayed with 560-4480 g
ae/ha glyphosate as previously described. The data demonstrate
robust tolerance of dgt-28 linked with the chloroplast transit
peptides TraP4, TraP5, TraP8 and TraP23 up to 4480 g ae/ha
glyphosate. Table 21.
TABLE-US-00021 TABLE 21 Response of F.sub.1 single copy dgt-28
events to rates of glyphosate ranging from 560-4480 g ae/ha + 2.0%
w/v ammonium sulfate 14 days after treatment. % Injury % Injury 20-
Std. Range Application Rate <20% 40% >40% Ave Dev. (%)
B104/TraP4::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha
4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 9.0 1.2 8-10 2240 g ae/ha 4 0 0
2.5 2.9 0-5 4480 g ae/ha 4 0 0 0.0 0.0 0 B104/TraP8::dgt-28 0 g
ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 1.3 2.5 0-5 1120
g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 5.0 4.1 0-10 4480 g
ae/ha 4 0 0 6.3 2.5 5-10 B104/TraP23::dgt-28 0 g ae/ha glyphosate 4
0 0 0.0 0.0 0 560 g ae/ha 3 1 0 10.0 10.0 5-25 1120 g ae/ha 2 2 0
18.8 11.8 10-35 2240 g ae/ha 4 0 0 12.5 2.9 10-15 4480 g ae/ha 3 1
0 10.0 7.1 5-20 B104/TraP5::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0
0.0 0 560 g ae/ha 4 0 0 8.0 0.0 8 1120 g ae/ha 4 0 0 11.3 3.0 8-15
2240 g ae/ha 4 0 0 12.5 2.9 10-15 4480 g ae/ha 4 0 0 10.0 2.5 10-15
Non-transformed B104 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g
ae/ha 0 0 4 100.0 0.0 100 1120 g ae/ha 0 0 4 100.0 0.0 100 2240 g
ae/ha 0 0 4 100.0 0.0 100 4480 g ae/ha 0 0 4 100.0 0.0 100
[0305] Protein expression data demonstrate a range of mean DGT-28
protein from 42.2-88.2 ng/cm.sup.2 across T.sub.1 events and
constructs tested, establishing protein expression in the T.sub.1
generation.
[0306] Characterization of dgt-28 corn under field conditions.
Single copy T.sub.1 events were sent to a field location to create
both hybrid hemizygous and inbred homozygous seed for additional
characterization. Hybrid seeds were created by crossing T.sub.1
events in the maize transformation line B104 to the inbred line
4XP811 generating hybrid populations segregating 1:1
(hemizygous:null) for the event. The resulting seeds were shipped
to 2 separate locations. A total of five single copy events per
construct were planted at each location in a randomized complete
block design in triplicate. The fields were designed for glyphosate
applications to occur at the V4 growth stage and a separate
grouping of plants to be applied at the V8 growth stage. The
4XP811/B104 conventional hybrid was used as a negative control.
[0307] Experimental rows were treated with 184 g ae/ha ASSURE
II.TM. (106 g ai/L quizalofop-methyl) to eliminate null segregants.
All experimental entries segregated 1:1 (sensitive:resistant)
(p=0.05) with respect to the ASSURE II.TM. application. Selected
resistant plants were sampled from each event for quantification of
the DGT-28 protein by standard ELISA.
[0308] Quizalofop-methyl resistant plants were treated with the
commercial herbicide DURANGO DMA.TM. (480 g ae/L glyphosate) with
the addition of 2.5% w/v ammonium-sulfate at either the V4 or V8
growth stages. Herbicide applications were made with a boom sprayer
calibrated to deliver a volume of 187 L/ha, 50-cm spray height.
Plants were sprayed with a range of glyphosate from 1120-4480 g
ae/ha glyphosate, capable of significant injury to untransformed
corn lines. A lethal dose is defined as the rate that causes
>95% injury to the 4XP811 inbred. Visual injury assessments were
taken for the percentage of visual chlorosis, percentage of
necrosis, percentage of growth inhibition and total visual injury
at 7, 14 and 21 DAT (days after treatment). Assessments were
compared to the untreated checks for each line and the negative
controls.
[0309] Visual injury data for all assessment timings demonstrated
robust tolerance up to 4480 g ae/ha DURANGO DMA.TM. at both
locations and application timings. Representative events for the V4
application are presented from one location and are consistent with
other events, application timings and locations. Table 22. One
event from the construct containing dgt-28 linked with TraP23
(pDAB107665) was tolerant to the ASSURE II.TM. selection for the
AAD-1 protein, but was sensitive to all rates of glyphosate
applied.
TABLE-US-00022 TABLE 22 Response of dgt-28 events applied with a
range of glyphosate from 1120-4480 g ae/ha + 2.5% w/v ammonium
sulfate at the V4 growth stage. % Injury % Injury 20- Std. Range
Application Rate <20% 40% >40% Ave Dev. (%) 4XPB11//B104/
TraP4::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0
0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 4480 g ae/ha 4 0 0 0.0 0.0
0 4XPB11//B104/ TraP8::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 4480 g
ae/ha 4 0 0 0.0 0.0 0 4XPB11//B104/ TraP23::dgt-28 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g
ae/ha 4 0 0 0.0 0.0 0 4480 g ae/ha 4 0 0 0.0 0.0 0 4XPB11//B104/
TraP5::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0
0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0 0.0 0 4480 g ae/ha 4 0 0 0.0 0.0
0 Non-transformed 4XPB11//B104 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
1120 g ae/ha 0 0 4 100.0 0.0 100 2240 g ae/ha 0 0 4 100.0 0.0 100
4480 g ae/ha 0 0 4 100.0 0.0 100
[0310] Additional assessments were made during the reproductive
growth stage for the 4480 g ae/ha glyphosate rate. Visual
assessments of tassels, pollination timing and ear fill were
similar to the untreated checks of each line for all constructs,
application timings and locations. Quantification results for the
DGT-28 protein demonstrated a range of mean protein expression from
186.4-303.0 ng/cm.sup.2. Data demonstrates robust tolerance of
dgt-28 transformed corn under field conditions through the
reproductive growth stages up to 4480 g ae/ha glyphosate. Data also
demonstrated DGT-28 protein detection and function based on spray
tolerance results.
[0311] Confirmation of Heritability and Tolerance of Dgt-28 Corn in
the Homozygous State.
[0312] Seed from the T.sub.1S2 were planted under greenhouse
conditions as previously described. The same five single copy lines
that were characterized under field conditions were characterized
in the homogeneous state. Plants were grown until the V3 growth
stage and separated into three rates of glyphosate ranging from
1120-4480 g ae/ha glyphosate (DURANGO DMA.TM.) and four replicates
per treatment. Applications were made in a track sprayer as
previously described and were formulated in 2.0% w/v ammonium
sulfate. An application of ammonium sulfate served as an untreated
check for each line. Visual assessments were taken 7 and 14 days
after treatment as previously described. Data demonstrated robust
tolerance up to 4480 g ae/ha glyphosate for all events tested.
Table 23.
TABLE-US-00023 TABLE 23 Response of homozygous dgt-28 events
applied with a range of glyphosate from 1120-4480 g ae/ha + 2.0%
w/v ammonium sulfate. % Injury % Injury 20- Std. Range Application
Rate <20% 40% >40% Ave Dev. (%) TraP4::dgt-28 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g
ae/ha 4 0 0 3.8 2.5 0-5 4480 g ae/ha 4 0 0 14.3 1.5 12-15
TraP8::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0
0 0.0 0.0 0 2240 g ae/ha 4 0 0 9.0 1.2 8-10 4480 g ae/ha 4 0 0 11.3
2.5 10-15 TraP23::dgt-28 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 1120
g ae/ha 4 0 0 4.5 3.3 0-8 2240 g ae/ha 4 0 0 7.5 2.9 5-10 4480 g
ae/ha 4 0 0 15.0 0.0 15 TraP5::dgt-28 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 1120 g ae/ha 4 0 0 1.3 2.5 0-5 2240 g ae/ha 4 0 0 9.0 2.0
8-12 4480 g ae/ha 4 0 0 15.0 2.4 12-18 Non-transformed B104 0 g
ae/ha glyphosate 4 0 0 0.0 0.0 0 1120 g ae/ha 0 0 4 100.0 0.0 100
2240 g ae/ha 0 0 4 100.0 0.0 100 4480 g ae/ha 0 0 4 100.0 0.0
100
[0313] The line from pDAB107665 that was not tolerant under field
conditions demonstrated no tolerance to glyphosate and therefore
consistent with field observations (data not shown). With the
exception of the one line previously mentioned, all replicates that
were treated with glyphosate from the lines were not sensitive to
glyphosate. Therefore data demonstrates heritability to a
homogeneous population of dgt-28 corn in a Mendelian fashion.
Expression of the DGT-28 protein by standard ELISA demonstrated a
range of mean protein expression from 27.5-65.8 ng/cm.sup.2 across
single copy events that were tolerant to glyphosate. Data
demonstrates functional protein and stability of the DGT-28 protein
across generations.
[0314] Postemergence Herbicide Tolerance Use of Glyphosate as a
Selectable Marker.
[0315] As previously described, To transformed plants were moved
from tissue culture and acclimated in the greenhouse. The events
tested contained dgt-28 linked to TraP5, TraP8, and TraP23
chloroplast transit peptides. It was demonstrated that these
T.sub.0 plants provided robust tolerance up to 4480 g ae/ha
glyphosate, and non-transformed plants were controlled with
glyphosate at concentrations as low as 280 g ae/ha. These data
demonstrate that dgt-28 can be utilized as a selectable marker
using a concentration of glyphosate ranging from 280-4480 g
ae/ha.
[0316] A number of seed from fixed lines of corn which contain the
dgt-28 transgene are spiked into a number of non-transformed corn
seed. The seed are planted and allowed to grow to the V1-V3
developmental stage, at which time the plantlets are sprayed with a
selecting dose of glyphosate in the range of 280-4480 g ae/ha.
Following 7-10 days, sensitive and resistant plants are counted,
and the amount of glyphosate tolerant plants correlates with the
original number of transgenic seed containing the dgt-28 transgene
which are planted.
[0317] Stacking of Dgt-28 Corn.
[0318] The AAD-1 protein is used as the selectable marker in dgt-28
transformed corn for research purposes. The aad-1 gene can also be
utilized as a herbicide tolerant trait in corn to provide robust
2,4-D tolerance up to a V8 application in a crop. Four events from
the constructs pDAB107663 (TraP4::dgt-28), pDAB107664
(TraP8::dgt-28) and pDAB107666 (TraP5::dgt-28) were characterized
for the tolerance of a tank mix application of glyphosate and
2,4-D. The characterization study was completed with F.sub.1 seed
under greenhouse conditions. Applications were made in a track
sprayer as previously described at the following rates: 1120-2240 g
ae/ha glyphosate (selective for the dgt-28 gene), 1120-2240 g ae/ha
2,4-D (selective for the aad-1 gene), or a tank mixture of the two
herbicides at the rates described. Plants were graded at 7 and 14
DAT. Spray results for applications of the herbicides at 2240 g
ae/ha are shown in Table 24.
TABLE-US-00024 TABLE 24 Response of F.sub.1 aad-1 and dgt-28 corn
sprayed with 2240 g ae/ha of 2,4-D, glyphosate and a tank mix
combination of the two herbicides 14 days after treatment. 2240 g
ae/ha 2240 g ae/ha 2240 g ae/ha 2,4-D + 2,4-D glyphosate 2240 g
ae/ha glyphosate Mean Mean Mean % Std. % Std. % Std. F.sub.1 Event
injury Dev. injury Dev. injury Dev. 107663[3]- 5.0 4.1 3.8 4.8 8.8
3.0 012.AJ001 107663[3]- 2.5 5.0 1.3 2.5 5.0 5.8 029.AJ001
107663[3]- 2.5 2.9 11.8 2.9 13.8 2.5 027.AJ001 107663[3]- 3.8 2.5
11.5 1.0 12.8 1.5 011.AJ001 B104 27.5 17.7 100.0 0.0 100.0 0.0
[0319] The results confirm that dgt-28 can be successfully stacked
with aad-1, thus increasing the spectrum herbicides that may be
applied to the crop of interest (glyphosate+phenoxyacetic acids for
dgt-28 and aad-1, respectively). In crop production where hard to
control broadleaf weeds or resistant weed biotypes exist the stack
can be used as a means of weed control and protection of the crop
of interest. Additional input or output traits can also be stacked
with the dgt-28 gene in corn and other plants.
[0320] Soybean Transformation.
[0321] Transgenic soybean (Glycine max) containing a stably
integrated dgt-28 transgene was generated through
Agrobacterium-mediated transformation of soybean cotyledonary node
explants. A disarmed Agrobacterium strain carrying a binary vector
containing a functional dgt-28 was used to initiate
transformation.
[0322] Agrobacterium-mediated transformation was carried out using
a modified half-cotyledonary node procedure of Zeng et al. (Zeng
P., Vadnais D. A., Zhang Z., Polacco J. C., (2004), Plant Cell
Rep., 22(7): 478-482). Briefly, soybean seeds (cv. Maverick) were
germinated on basal media and cotyledonary nodes are isolated and
infected with Agrobacterium. Shoot initiation, shoot elongation,
and rooting media are supplemented with cefotaxime, timentin and
vancomycin for removal of Agrobacterium. Selection via a herbicide
was employed to inhibit the growth of non-transformed shoots.
Selected shoots are transferred to rooting medium for root
development and then transferred to soil mix for acclimatization of
plantlets.
[0323] Terminal leaflets of selected plantlets were treated
topically (leaf paint technique) with a herbicide to screen for
putative transformants. The screened plantlets were transferred to
the greenhouse, allowed to acclimate and then leaf-painted with a
herbicide to reconfirm tolerance. These putative transformed
T.sub.0 plants were sampled and molecular analyses was used to
confirm the presence of the herbicidal selectable marker, and the
dgt-28 transgene. T.sub.0 plants were allowed to self fertilize in
the greenhouse to produce T.sub.1 seed.
[0324] A second soybean transformation method can be used to
produce additional transgenic soybean plants. A disarmed
Agrobacterium strain carrying a binary vector containing a
functional dgt-28 is used to initiate transformation.
[0325] Agrobacterium-mediated transformation was carried out using
a modified half-seed procedure of Paz et al., (Paz M., Martinez J.,
Kalvig A., Fonger T., and Wang K., (2005) Plant Cell Rep., 25:
206-213). Briefly, mature soybean seeds were sterilized overnight
with chlorine gas and imbibed with sterile H.sub.2O twenty hours
before Agrobacterium-mediated plant transformation. Seeds were cut
in half by a longitudinal cut along the hilum to separate the seed
and remove the seed coat. The embryonic axis was excised and any
axial shoots/buds were removed from the cotyledonary node. The
resulting half seed explants were infected with Agrobacterium.
Shoot initiation, shoot elongation, and rooting media were
supplemented with cefotaxime, timentin and vancomycin for removal
of Agrobacterium. Herbicidal selection was employed to inhibit the
growth of non-transformed shoots. Selected shoots were transferred
to rooting medium for root development and then transferred to soil
mix for acclimatization of plantlets.
[0326] Putative transformed T.sub.0 plants were sampled and
molecular analyses was used to confirm the presence of the
selectable marker and the dgt-28 transgene. Several events were
identified as containing the transgenes. These T.sub.0 plants were
advanced for further analysis and allowed to self fertilize in the
greenhouse to give rise to T.sub.1 seed.
[0327] Confirmation of Heritability of Dgt-28 to the T.sub.1
Generation.
[0328] Heritability of the DGT-28 protein into T.sub.1 generation
was assessed in one of two ways. The first method included planting
T.sub.1 seed into Metro-mix media and applying 411 g ae/ha
IGNITE.TM. 280 SL on germinated plants at the 1.sup.st trifoliate
growth stage. The second method consisted of homogenizing seed for
a total of 8 replicates using a ball bearing and a genogrinder.
ELISA strip tests to detect for the PAT protein were then used to
detect heritable events as the selectable marker was on the same
plasmid as dgt-28. For either method if a single plant was tolerant
to glufosinate or was detected with the PAT ELISA strip test, the
event demonstrated heritability to the T.sub.1 generation.
[0329] A total of five constructs were screened for heritability as
previously described. The plasmids contained dgt-28 linked with
TraP4, TraP8 and TraP23 The events across constructs demonstrated
68% heritability of the PAT::DGT-28 protein to the T.sub.1
generation.
[0330] Postemergence Herbicide Tolerance in Dgt-28 Transformed
T.sub.1 Soybean.
[0331] Seeds from T.sub.1 events that were determined to be
heritable by the previously described screening methods were
planted in Metro-mix media under greenhouse conditions. Plants were
grown until the 1.sup.st trifoliate was fully expanded and treated
with 411 g ae/ha IGNITE.TM. 280 SL for selection of the pat gene as
previously described. Resistant plants from each event were given
unique identifiers and sampled for zygosity analyses of the dgt-28
gene. Zygosity data were used to assign 2 hemizygous and 2
homozygous replicates to each rate of glyphosate applied allowing
for a total of 4 replicates per treatment when enough plants
existed. These plants were compared against wildtype Petite havana
tobacco. All plants were sprayed with a track sprayer set at 187
L/ha. The plants were sprayed from a range of 560-4480 g ae/ha
DURANGO.TM. dimethylamine salt (DMA). All applications were
formulated in water with the addition of 2% w/v ammonium sulfate
(AMS). Plants were evaluated at 7 and 14 days after treatment.
Plants were assigned an injury rating with respect to overall
visual stunting, chlorosis, and necrosis. The T.sub.1 generation is
segregating, so some variable response is expected due to
difference in zygosity.
TABLE-US-00025 TABLE 25 Spray results demonstrate at 14 DAT (days
after treatment) robust tolerance up to 4480 g ae/ha glyphosate of
at least one dgt-28 event per construct characterized.
Representative single copy events of the constructs all provided
tolerance up to 4480 g ae/ha compared to the Maverick negative
control. % Injury % Injury 20- Std. Range Application Rate <20%
40% >40% Ave Dev. (%) pDAB107543 (TraP4::dgt-28) 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 4 0 33.8 7.5 25-40 1120 g
ae/ha 2 2 0 25.0 11.5 15-35 2240 g ae/ha 2 2 0 17.5 2.9 15-20 4480
g ae/ha 0 2 2 33.8 13.1 20-45 pDAB107545 (TraP8::dgt-28) 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 1.5 1.0 0-2 1120 g
ae/ha 4 0 0 2.8 1.5 2-5 2240 g ae/ha 4 0 0 5.0 2.4 2-8 4480 g ae/ha
4 0 0 9.5 1.9 8-12 pDAB107548 (TraP4::dgt-28) 0 g ae/ha glyphosate
4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 1.8 2.4 0-5 1120 g ae/ha 4 0 0
2.8 1.5 2-5 2240 g ae/ha 4 0 0 3.5 1.7 2-5 4480 g ae/ha 4 0 0 8.8
3.0 5-12 pDAB107553 (TraP23::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0
0.0 0 560 g ae/ha 4 0 0 5.0 0.0 5 1120 g ae/ha 4 0 0 9.0 1.2 8-10
2240 g ae/ha 4 0 0 10.5 1.0 10-12 4480 g ae/ha 4 0 0 16.5 1.7 15-18
Maverick (neg. control) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g
ae/ha 0 0 4 82.5 12.6 70-100 1120 g ae/ha 0 0 4 100.0 0.0 100 2240
g ae/ha 0 0 4 100.0 0.0 100 4480 g ae/ha 0 0 4 100.0 0.0 100
[0332] Dgt-28 Protection Against Elevated Glyphosate Rates in the
T.sub.2 Generation.
[0333] A 45 plant progeny test was conducted on two to five T.sub.2
lines of dgt-28 per construct. Homozygous lines were chosen based
on zygosity analyses completed in the previous generation. The
seeds were planted as previously described. Plants were then
sprayed with 411 g ae/ha IGNITE 280 SL for the selection of the pat
selectable marker as previously described. After 3 DAT, resistant
and sensitive plants were counted.
[0334] For constructs containing TraP4 linked with dgt-28
(pDAB107543 and pDAB107548), nine out of twelve lines tested did
not segregate, thereby confirming homogeneous lines in the T.sub.2
generation. Lines containing TraP8 linked with dgt-28 (pDAB107545)
demonstrated two out of the four lines with no segregants and
demonstrating Mendelian inheritance through at least two generation
of dgt-28 in soybean. Tissue samples were taken from resistant
plants and the DGT-28 protein was quantified by standard ELISA
methods. Data demonstrated a range of mean DGT-28 protein from
32.8-107.5 ng/cm.sup.2 for non-segregating T.sub.2 lines tested.
Lines from the construct pDAB107553 (TraP23::dgt-28) were not
previously selected with glufosinate, and the dose response of
glyphosate was utilized as both to test homogenosity and tolerance
to elevated rates of glyphosate. Replicates from the lines from
construct pDAB107553 were tolerant to rates ranging from 560-4480 g
ae/ha glyphosate, and were therefore confirmed to be a homogeneous
population and heritable to at least two generations.
[0335] Rates of DURANGO DMA ranging from 560-4480 g ae/ha
glyphosate were applied to 2-3 trifoliate soybean as previously
described. Visual injury data 14 DAT confirmed the tolerance
results that were demonstrated in the T.sub.1 generation.
TABLE-US-00026 TABLE 26 The data demonstrate robust tolerance of
the dgt-28 tobacco up to 3360 g ae/ha glyphosate through two
generations, compared to the non-transformed control. % Injury %
Injury 20- Std. Range Application Rate <20% 40% >40% Ave Dev.
(%) pDAB107543 (TraP4::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
560 g ae/ha 4 0 0 8.0 0.0 8 1120 g ae/ha 4 0 0 14.3 1.5 12-15 2240
g ae/ha 4 0 0 18.0 0.0 18 4480 g ae/ha 0 4 0 24.5 3.3 20-28
pDAB107545 (TraP8::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560
g ae/ha 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 2.8 1.5 2-5 2240 g ae/ha
4 0 0 5.0 0.0 5 4480 g ae/ha 4 0 0 10.0 0.0 10 pDAB107548
(TraP4::dgt-28) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 4
0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240 g ae/ha 4 0 0 0.0
0.0 0 4480 g ae/ha 4 0 0 10.0 0.0 10 pDAB107553 (TraP23::dgt-28) 0
g ae/ha glyphosate 4 0 0 -- 0.0 0.0 560 g ae/ha 4 0 0 -- 10.0 0.0
1120 g ae/ha 4 0 0 -- 10.0 -4.4 2240 g ae/ha 4 0 0 -- 13.0 -2.4
4480 g ae/ha 3 1 0 -- 15.5 4.1 Maverick (neg. control) 0 g ae/ha
glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 0 4 77.5 15.0 70-100 1120
g ae/ha 0 0 4 97.5 2.9 95-100 2240 g ae/ha 0 0 4 100.0 0.0 100 4480
g ae/ha 0 0 4 100.0 0.0 100
[0336] Transformation of Rice with Dgt-28.
[0337] Transgenic rice (Oryza sativa) containing a stably
integrated dgt-28 transgene is generated through
Agrobacterium-mediated transformation of sterilized rice seed. A
disarmed Agrobacterium strain carrying a binary vector containing a
functional dgt-28 is used to initiate transformation.
[0338] Culture media are adjusted to pH 5.8 with 1 M KOH and
solidified with 2.5 g/l Phytagel (Sigma-Aldrich, St. Louis, Mo.).
Embryogenic calli are cultured in 100.times.20 mm petri dishes
containing 30 ml semi-solid medium. Rice plantlets are grown on 50
ml medium in MAGENTA boxes. Cell suspensions are maintained in 125
ml conical flasks containing 35 mL liquid medium and rotated at 125
rpm. Induction and maintenance of embryogenic cultures occur in the
dark at 25-26.degree. C., and plant regeneration and whole-plant
culture occur in illuminated room with a 16-h photoperiod (Zhang et
al. 1996).
[0339] Induction and maintenance of embryogenic callus is performed
on a modified NB basal medium as described previously (Li et al.
1993), wherein the media is adapted to contain 500 mg/L glutamine.
Suspension cultures are initiated and maintained in SZ liquid
medium (Zhang et al. 1998) with the inclusion of 30 g/L sucrose in
place of maltose. Osmotic medium (NBO) consisting of NB medium with
the addition of 0.256 M each of mannitol and sorbitol. Herbicide
resistant callus is selected on NB medium supplemented with the
appropriate herbicide selective agent for 3-4 weeks.
Pre-regeneration is performed on medium (PRH50) consisting of NB
medium with 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/l
.alpha.-naphthaleneacetic acid (NAA), 5 mg/l abscisic acid (ABA)
and selective herbicide for 1 week. Regeneration of plantlets
follow the culturing on regeneration medium (RNH50) comprising NB
medium containing 2,4-D, 0.5 mg/l NAA, and selective herbicide
until putatively transgenic shoots are regenerated. Shoots are
transferred to rooting medium with half-strength Murashige and
Skoog basal salts and Gamborg's B5 vitamins, supplemented with 1%
sucrose and selective herbicide.
[0340] Mature desiccated seeds of Oryza sativa L. japonica cv.
Taipei 309 are sterilized as described in Zhang et al. 1996.
Embryogenic tissues are induced by culturing sterile mature rice
seeds on NB medium in the dark. The primary callus approximately 1
mm in diameter, is removed from the scutellum and used to initiate
cell suspension in SZ liquid medium. Suspensions are then
maintained as described in Zhang 1996. Suspension-derived
embryogenic tissues are removed from liquid culture 3-5 days after
the previous subculture and placed on NBO osmotic medium to form a
circle about 2.5 cm across in a petri dish and cultured for 4 h
prior to bombardment. Sixteen to twenty hours after bombardment,
tissues are transferred from NBO medium onto NBH50 selection
medium, ensuring that the bombarded surface is facing upward, and
incubated in the dark for 14-17 days. Newly formed callus is then
separated from the original bombarded explants and placed nearby on
the same medium. Following an additional 8-12 days, relatively
compact, opaque callus is visually identified, and transferred to
PRH50 pre-regeneration medium for 7 days in the dark. Growing
callus, which become more compact and opaque is then subcultured
onto RNH50 regeneration medium for a period of 14-21 days under a
16-h photoperiod. Regenerating shoots are transferred to MAGENTA
boxes containing 1/2 MSH50 medium. Multiple plants regenerated from
a single explant are considered siblings and are treated as one
independent plant line. A plant is scored as positive for the
dgt-28 gene if it produces thick, white roots and grows vigorously
on 1/2 MSH50 medium. Once plantlets reach the top of the MAGENTA
boxes, they are transferred to soil in a 6-cm pot under 100%
humidity for a week, and then are moved to a growth chamber with a
14-h light period at 30.degree. C. and in the dark at 21.degree. C.
for 2-3 weeks before transplanting into 13-cm pots in the
greenhouse. Seeds are collected and dried at 37.degree. C. for one
week prior to storage at 4.degree. C.
[0341] T.sub.0 Analysis of Dgt-28 Rice.
[0342] Transplanted rice transformants obtained via an
Agrobacterium transformation method were transplanted into media
and acclimated to greenhouse conditions. All plants were sampled
for PCR detection of dgt-28 and results demonstrate twenty-two PCR
positive events for pDAB110827 (TraP8::dgt-28) and a minimum of
sixteen PCR positive events for pDAB110828 (TraP23::dgt-28).
Southern analysis for dgt-28 of the PCR positive events
demonstrated simple (1-2 copy) events for both constructs. Protein
expression of selected T.sub.0 events demonstrated DGT-28 protein
expression ranges from below levels of detection to 130
ng/cm.sup.2. Selected T.sub.0 events from construct pDAB110828 were
treated with 2240 g ae/ha DURANGO DMA.TM. as previously described
and assessed 7 and 14 days after treatment. Data demonstrated
robust tolerance to the rate of glyphosate applied. All PCR
positive plants were allowed to produced T.sub.1 seed for further
characterization.
[0343] Dgt-28 Heritability in Rice.
[0344] A 100 plant progeny test was conducted on four T.sub.1 lines
of dgt-28 from construct pDAB110827 containing the chloroplast
transit peptide TraP8. The seeds were planted into pots filled with
media. All plants were then sprayed with 560 g ae/ha DURANGO
DMA.TM. for the selection of the dgt-28 gene as previously
described. After 7 DAT, resistant and sensitive plants were
counted. Two out of the four lines tested for each construct
segregated as a single locus, dominant Mendelian trait (3R:1S) as
determined by Chi square analysis. Dgt-28 is a heritable glyphosate
resistance gene in multiple species.
[0345] Postemergence Herbicide Tolerance in Dgt-28 Transformed
T.sub.1 Rice.
[0346] T.sub.1 resistant plants from each event used in the progeny
testing were given unique identifiers and sampled for zygosity
analyses of the dgt-28 gene. Zygosity data were used to assign 2
hemizygous and 2 homozygous replicates to each rate of glyphosate
applied allowing for a total of 4 replicates per treatment. These
plants were compared against wildtype kitaake rice. All plants were
sprayed with a track sprayer set at 187 L/ha. The plants were
sprayed from a range of 560-2240 g ae/ha DURANGO DMA.TM.. All
applications were formulated in water with the addition of 2% w/v
ammonium sulfate (AMS). Plants were evaluated at 7 and 14 days
after treatment. Plants were assigned an injury rating with respect
to overall visual stunting, chlorosis, and necrosis. The T.sub.1
generation is segregating, so some variable response is expected
due to difference in zygosity.
[0347] Spray results demonstrate at 7 DAT (days after treatment)
minimal vegetative injury to elevated rates of glyphosate were
detected (data not shown).
TABLE-US-00027 TABLE 27 Visual injury data at 14 DAT demonstrates
less than 15% mean visual injury up to 2240 g ae/ha glyphosate. %
Injury % Injury 20- Std. Range Application Rate <20% 40% >40%
Ave Dev. (%) TraP8::dgt-28 Event 1 0 g ae/ha glyphosate 4 0 0 0.0
0.0 0 560 g ae/ha 4 0 0 0.0 0.0 0 1120 g ae/ha 4 0 0 0.0 0.0 0 2240
g ae/ha 4 0 0 0.0 0.0 0 TraP8::dgt-28 Event 2 0 g ae/ha glyphosate
4 0 0 0.0 0.0 0 560 g ae/ha 4 0 0 3.8 4.8 0-10 1120 g ae/ha 4 0 0
12.0 3.6 8-15 2240 g ae/ha 4 0 0 15.0 6.0 8-20 Non-transformed
control 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 0 4 81.3
2.5 80-85 1120 g ae/ha 0 0 4 95.0 5.8 90-100 2240 g ae/ha 0 0 4
96.3 4.8 90-100
[0348] Protein detection of DGT-28 was assessed for replicates from
all four T.sub.1 lines tested from pDAB110827. Data demonstrated
DGT-28 mean protein ranges from 20-82 ng/cm.sup.2 and 21-209
ng/cm.sup.2 for hemizygous and homozygous replicates respectively.
These results demonstrated stable protein expression to the T.sub.1
generation and tolerance of dgt-28 rice up to 2240 g ae/ha
glyphosate following an application of 560 g ae/ha glyphosate used
for selection.
[0349] Transformation of Tobacco with Dgt-28.
[0350] Tobacco (cv. Petit Havana) leaf pieces were transformed
using Agrobacterium tumefaciens containing the dgt-28 transgene.
Single colonies containing the plasmid which contains the dgt-28
transgene were inoculated into 4 mL of YEP medium containing
spectinomycin (50 .mu.g/mL) and streptomycin (125 .mu.g/mL) and
incubated overnight at 28.degree. C. on a shaker at 190 rpm. The 4
mL seed culture was subsequently used to inoculate a 25 mL culture
of the same medium in a 125 mL baffled Erlenmeyer flask. This
culture was incubated at 28.degree. C. shaking at 190 rpm until it
reached an OD.sub.600 of .about.1.2. Ten mL of Agrobacterium
suspension were then placed into sterile 60.times.20 mm Petri.TM.
dishes.
[0351] Freshly cut leaf pieces (0.5 cm.sup.2) from plants
aseptically grown on MS medium (Phytotechnology Labs, Shawnee
Mission, Kans.,) with 30 g/L sucrose in PhytaTrays.TM. (Sigma, St.
Louis, Mo.) were soaked in 10 mL of overnight culture of
Agrobacterium for a few minutes, blotted dry on sterile filter
paper and then placed onto the same medium with the addition of 1
mg/L indoleacetic acid and 1 mg/L 6-benzylamino purine. Three days
later, leaf pieces co-cultivated with Agrobacterium harboring the
dgt-28 transgene were transferred to the same medium with 5 mg/L
Basta.TM. and 250 mg/L cephotaxime.
[0352] After 3 weeks, individual T.sub.0 plantlets were transferred
to MS medium with 10 mg/L Basta.TM. and 250 mg/L cephotaxime an
additional 3 weeks prior to transplanting to soil and transfer to
the greenhouse. Selected T.sub.0 plants (as identified using
molecular analysis protocols described above) were allowed to
self-pollinate and seed was collected from capsules when they were
completely dried down. T.sub.1 seedlings were screened for zygosity
and reporter gene expression (as described below) and selected
plants containing the dgt-28 transgene were identified.
[0353] Plants were moved into the greenhouse by washing the agar
from the roots, transplanting into soil in 13.75 cm square pots,
placing the pot into a Ziploc.RTM. bag (SC Johnson & Son,
Inc.), placing tap water into the bottom of the bag, and placing in
indirect light in a 30.degree. C. greenhouse for one week. After
3-7 days, the bag was opened; the plants were fertilized and
allowed to grow in the open bag until the plants were
greenhouse-acclimated, at which time the bag was removed. Plants
were grown under ordinary warm greenhouse conditions (27.degree. C.
day, 24.degree. C. night, 16 hour day, minimum natural+supplemental
light=1200 .mu.E/m.sup.2s.sup.1).
[0354] Prior to propagation, T.sub.0 plants were sampled for DNA
analysis to determine the insert dgt-28 copy number by real-time
PCR. Fresh tissue was placed into tubes and lyophilized at
4.degree. C. for 2 days. After the tissue was fully dried, a
tungsten bead (Valenite) was placed in the tube and the samples
were subjected to 1 minute of dry grinding using a Kelco bead mill.
The standard DNeasy.TM. DNA isolation procedure was then followed
(Qiagen, DNeasy 69109). An aliquot of the extracted DNA was then
stained with Pico Green (Molecular Probes P7589) and read in the
fluorometer (BioTek.TM.) with known standards to obtain the
concentration in ng/.mu.l. A total of 100 ng of total DNA was used
as template. The PCR reaction was carried out in the 9700
Geneamp.TM. thermocycler (Applied Biosystems), by subjecting the
samples to 94.degree. C. for 3 minutes and 35 cycles of 94.degree.
C. for 30 seconds, 64.degree. C. for 30 seconds, and 72.degree. C.
for 1 minute and 45 seconds followed by 72.degree. C. for 10
minutes. PCR products were analyzed by electrophoresis on a 1%
agarose gel stained with EtBr and confirmed by Southern blots.
[0355] Five to nine PCR positive events with 1-3 copies of dgt-28
gene from 3 constructs containing a different chloroplast transit
peptide sequence (TraP4, TraP8 and TraP23) were regenerated and
moved to the greenhouse.
[0356] All PCR positive plants were sampled for quantification of
the DGT-28 protein by standard ELISA. DGT-28 protein was detected
in all PCR positive plants and a trend for an increase in protein
concentration was noted with increasing copy number of dgt-28.
[0357] Aad-12 (v1) Heritability in Tobacco.
[0358] A 100 plant progeny test was conducted on five T.sub.1 lines
of dgt-28 per construct. Constructs contained one of the following
chloroplast transit peptide sequences: TraP4, TraP8 or TraP23. The
seeds were stratified, sown, and transplanted with respect much
like that of the Arabidopsis procedure exemplified above, with the
exception that null plants were not removed by in initial selection
prior to transplanting. All plants were then sprayed with 280 g
ae/ha IGNITE 280 SL for the selection of the pat selectable marker
as previously described. After 3 DAT, resistant and sensitive
plants were counted.
[0359] Four out of the five lines tested for each construct
segregated as a single locus, dominant Mendelian trait (3R:1S) as
determined by Chi square analysis. Dgt-28 is a heritable glyphosate
resistance gene in multiple species.
[0360] Postemergence Herbicide Tolerance in Dgt-28 Transformed
T.sub.1 Tobacco.
[0361] T.sub.1 resistant plants from each event used in the progeny
testing were given unique identifiers and sampled for zygosity
analyses of the dgt-28 gene. Zygosity data were used to assign 2
hemizygous and 2 homozygous replicates to each rate of glyphosate
applied allowing for a total of 4 replicates per treatment. These
plants were compared against wildtype Petite havana tobacco. All
plants were sprayed with a track sprayer set at 187 L/ha. The
plants were sprayed from a range of 560-4480 g ae/ha DURANGO
DMA.TM.. All applications were formulated in water with the
addition of 2% w/v ammonium sulfate (AMS). Plants were evaluated at
7 and 14 days after treatment. Plants were assigned an injury
rating with respect to overall visual stunting, chlorosis, and
necrosis. The T.sub.1 generation is segregating, so some variable
response is expected due to difference in zygosity.
[0362] Spray results demonstrate at 7 DAT (days after treatment)
minimal vegetative injury to elevated rates of glyphosate were
detected (data not shown). Following 14 DAT, visual injury data
demonstrates increased injury with single copy events of the
construct containing TraP4 compared to single copy events from the
constructs TraP8 and TraP23. Table 28.
TABLE-US-00028 TABLE 28 At a rate of 2240 g ae/ha glyphosate, an
average injury of 37.5% was demonstrated with the event containing
TraP4, where events containing TraP8 and TraP23 demonstrated an
average injury of 9.3% and 9.5% respectively. % Injury % Injury 20-
Std. Range Application Rate <20% 40% >40% Ave Dev. (%)
TraP4::dgt-28 (pDAB107543) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560
g ae/ha 2 2 0 18.0 8.1 10-25 1120 g ae/ha 1 3 0 24.5 4.9 18-30 2240
g ae/ha 0 3 1 37.5 6.5 30-45 4480 g ae/ha 0 2 2 42.5 2.9 40-45
TraP8::dgt-28 (pDAB107545) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560
g ae/ha 4 0 0 3.3 3.9 0-8 1120 g ae/ha 4 0 0 6.5 1.7 5-8 2240 g
ae/ha 4 0 0 9.3 3.0 5-12 4480 g ae/ha 2 2 0 17.5 6.5 10-25
TraP23::dgt-28 (pDAB107553) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
560 g ae/ha 4 0 0 10.0 1.6 8-12 1120 g ae/ha 4 0 0 8.8 3.0 5-12
2240 g ae/ha 4 0 0 9.5 4.2 5-15 4480 g ae/ha 4 0 0 15.8 1.5 15-18
Petite havana 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 560 g ae/ha 0 0
4 85.0 4.1 80-90 1120 g ae/ha 0 0 4 91.3 2.5 90-95 2240 g ae/ha 0 0
4 94.5 3.3 90-98 4480 g ae/ha 0 0 4 98.3 2.4 95-100
[0363] These results demonstrated tolerance of dgt-28 up to 4480 g
ae/ha glyphosate, as well as differences in tolerance provided by
chloroplast transit peptide sequences linked to the dgt-28
gene.
[0364] Dgt-28 Protection Against Elevated Glyphosate Rates in the
T.sub.2 Generation.
[0365] A 25 plant progeny test was conducted on two to three
T.sub.2 lines of dgt-28 per construct. Homozygous lines were chosen
based on zygosity analyses completed in the previous generation.
The seeds were stratified, sown, and transplanted as previously
described. All plants were then sprayed with 280 g ae/ha Ignite 280
SL for the selection of the pat selectable marker as previously
described. After 3 DAT, resistant and sensitive plants were
counted. All lines tested for each construct did not segregate
thereby confirming homogeneous lines in the T.sub.2 generation and
demonstrating Mendelian inheritance through at least two generation
of dgt-28 in tobacco.
[0366] Rates of DURANGO DMA.TM. ranging from 420-3360 g ae/ha
glyphosate were applied to 2-3 leaf tobacco as previously
described. Visual injury data 14 DAT confirmed the tolerance
results that were demonstrated in the T.sub.1 generation. Foliar
results from a two copy lines from the construct containing TraP4
demonstrated similar tolerance to that of single copy TraP8 and
TraP23 lines (data not shown).
TABLE-US-00029 TABLE 29 Single copy lines from the construct
containing TraP4 with dgt-28 demonstrated increased injury compared
to lines from constructs containing TraP8 and TraP23 with dgt-28. %
Injury % Injury 20- Std. Range Application Rate <20% 40% >40%
Ave Dev. (%) TraP4::dgt-28 (pDAB107543) 0 g ae/ha glyphosate 4 0 0
0.0 0.0 0 420 g ae/ha 0 4 0 23.8 4.8 20-30 840 g ae/ha 0 4 0 30.0
4.1 25-35 1680 g ae/ha 0 4 0 35.0 5.8 30-40 3360 g ae/ha 0 4 0 31.3
2.5 30-35 TraP8::dgt-28 (pDAB107545) 0 g ae/ha glyphosate 4 0 0 0.0
0.0 0 420 g ae/ha 4 0 0 0.0 0.0 0 840 g ae/ha 4 0 0 2.5 2.9 0-5
1680 g ae/ha 4 0 0 9.3 3.4 5-12 3360 g ae/ha 4 0 0 10.5 1.0 10-12
TraP23::dgt-28 (pDAB107553) 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0
420 g ae/ha 4 0 0 0.0 0.0 0 840 g ae/ha 4 0 0 6.3 2.5 5-10 1680 g
ae/ha 4 0 0 10.0 0.0 10 3360 g ae/ha 3 1 0 13.8 4.8 10-20 Petite
havana 0 g ae/ha glyphosate 4 0 0 0.0 0.0 0 420 g ae/ha 0 0 4 95.0
0.0 95 840 g ae/ha 0 0 4 98.8 1.0 98-100 1680 g ae/ha 0 0 4 99.5
1.0 98-100 3360 g ae/ha 0 0 4 100 0.0 100
[0367] The data demonstrate robust tolerance of dgt-28 tobacco up
to 3360 g ae/ha glyphosate through two generations compared to the
non-transformed control.
[0368] Selected plants from each event were sampled prior to
glyphosate applications for analyses of the DGT-28 protein by
standard DGT-28 ELISA. Data demonstrated DGT-28 mean protein
expression of the simple (1-2 copy) lines across constructs ranging
from 72.8-114.5 ng/cm.sup.2. Data demonstrates dgt-28 is expressing
protein in the T.sub.2 generation of transformed tobacco and
tolerance data confirms functional DGT-28 protein.
[0369] Stacking of Dgt-28 to Increase Herbicide Spectrum in Tobacco
(Cv. Petit Havana).
[0370] Homozygous dgt-28 (pDAB107543 and pDAB107545) and aad-12 v1
(pDAB3278) plants (see PCT/US2006/042133 for the latter, which is
incorporated herein by this reference in its entirety) were both
reciprocally crossed and F.sub.1 seed was collected. The F.sub.1
seed from two reciprocal crosses of each gene were stratified and
treated 6 reps of each cross were treated with 1120 g ae/ha
glyphosate (selective for the dgt-28 gene), 1120 g ae/ha 2,4-D
(selective for the aad-12 gene), or a tank mixture of the two
herbicides at the rates described. Plants were graded at 14 DAT.
Spray results are shown in Table 30.
TABLE-US-00030 TABLE 30 Response of F.sub.1 aad-12 and dgt-28
aad-12 .times. aad-12 .times. Petite TraP4::dgt-28 TraP8::dgt-28
havana Application Rate Tolerance 1120 g ae/ha 2,4-D ++++ ++++ -
1120 g ae/ha glyphosate ++ ++ - 1120 g ae/ha 2,4-D + ++ ++ - 1120 g
ae/ha glyphosate
[0371] The results confirm that dgt-28 can be successfully stacked
with aad-12 (v1), thus increasing the spectrum herbicides that may
be applied to the crop of interest (glyphosate+phenoxyactetic acids
for dgt-28 and aad-12, respectively). In crop production where hard
to control broadleaf weeds or resistant weed biotypes exist the
stack can be used as a means of weed control and protection of the
crop of interest. Additional input or output traits could also be
stacked with the dgt-28 gene.
[0372] Resistance to Glyphosate in Wheat. Production of Binary
Vectors Encoding DGT-28.
[0373] Binary vectors containing DGT-28 expression and PAT
selection cassettes were designed and assembled using skills and
techniques commonly known in the art. Each DGT-28 expression
cassette contained the promoter, 5' untranslated region and intron
from the Ubiquitin (Ubi) gene from Zea mays (Toki et al., Plant
Physiology 1992, 100 1503-07), followed by a coding sequence
consisting of one of four transit peptides (TraP4, TraP8, TraP23 or
TraP5) fused to the 5' end of a synthetic version of the
5-enolpyruvylshikimate-3-phosphate synthase gene (DGT-28), which
had been codon optimized for expression in plants. The DGT-28
expression cassette terminated with a 3' untranslated region (UTR)
comprising the transcriptional terminator and polyadenylation site
of a lipase gene (Vpl) from Z. mays (Paek et al., Mol. Cells 1998
30; 8(3) 336-42). The PAT selection cassette comprised of the
promoter, 5' untranslated region and intron from the Actin (Act1)
gene from Oryza sativa (McElroy et al., The Plant Cell 1990 2(2)
163-171), followed by a synthetic version of the phosphinothricin
acetyl transferase (PAT) gene isolated from Streptomyces
viridochromogenes, which had been codon optimized for expression in
plants. The PAT gene encodes a protein that confers resistance to
inhibitors of glutamine synthetase comprising phophinothricin,
glufosinate, and bialaphos (Wohlleben et al., Gene 1988, 70(1),
25-37). The selection cassette was terminated with the 3' UTR
comprising the transcriptional terminator and polyadenylation sites
from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault et
al., Plant Physiology 1993 101 (4), 1395-1396).
[0374] The selection cassette was synthesized by a commercial gene
synthesis vendor (GeneArt, Life Technologies) and cloned into a
Gateway-enabled binary vector. The DGT-28 expression cassettes were
sub-cloned into pDONR221. The resulting ENTRY clone was used in a
LR Clonase II (Invitrogen, Life Technologies) reaction with the
Gateway-enabled binary vector encoding the phosphinothricin acetyl
transferase (PAT) expression cassette. Colonies of all assembled
plasmids were initially screened by restriction digestion of
purified DNA using restriction endonucleases obtained from New
England BioLabs (NEB; Ipswich, Mass.) and Promega (Promega
Corporation, WI). Plasmid DNA preparations were performed using the
QlAprep Spin Miniprep Kit (Qiagen, Hilden) or the Pure Yield
Plasmid Maxiprep System (Promega Corporation, WI), following the
instructions of the suppliers. Plasmid DNA of selected clones was
sequenced using ABI Sanger Sequencing and Big Dye Terminator v3.1
cycle sequencing protocol (Applied Biosystems, Life Technologies).
Sequence data were assembled and analyzed using the SEQUENCHER.TM.
software (Gene Codes Corporation, Ann Arbor, Mich.).
[0375] The resulting four binary expression clones: pDAS000122
(TraP4-DGT28), pDAS000123 (TraP8-DGT28), pDAS000124 (TraP23-DGT28)
and pDAS000125 (TraP5-DGT28) were each transformed into
Agrobacterium tumefaciens strain EHA105.
[0376] Production of Transgenic Wheat Events with Dgt-28 Expression
Construct.
[0377] Transgenic wheat plants expressing one of the four DGT-28
expression constructs were generated by Agrobacterium-mediated
transformation using the donor wheat line Bobwhite MPB26RH,
following a protocol similar to Wu et al., Transgenic Research
2008, 17:425-436. Putative T0 transgenic events were selected for
phosphinothricin (PPT) tolerance, the phenotype conferred by the
PAT selectable marker, and transferred to soil. The T0 plants were
grown under glasshouse containment conditions and T.sub.1 seed was
produced. Overall, about 45 independent T0 events were generated
for each DGT-28 expression construct.
[0378] Glyphosate Resistance in T.sub.0 Wheat Dgt-28 Wheat
Events.
[0379] T.sub.0 events were allowed to acclimate in the greenhouse
and were grown until 2-4 new, normal looking leaves had emerged
from the whorl (i.e., plants had transitioned from tissue culture
to greenhouse growing conditions). Plants were grown at 25.degree.
C. under 12 hour of supplemental lighting in the greenhouse until
maturity. An initial screen of glyphosate tolerance and Taqman
analyses was completed on T.sub.1 plants grown under the same
conditions as previously described. Data allowed for determination
of heritable T.sub.1 events to be further characterized. Six low
copy (1-2 copy) and two multi-copy T.sub.1 events were replanted
under greenhouse conditions and grown until the 3 leaf stage.
T.sub.1 plants were sprayed with a commercial formulation of
glyphosate (Durango DMA') from a range of 420-3360 g ae/ha, which
are capable of significant injury to untransformed wheat lines. The
addition of 2% w/v ammonium sulfate was included in the
application. A lethal dose is defined as the rate that causes
>75% injury to the Bob White MPB26RH non-transformed control.
Herbicide was applied.
[0380] In this example, the glyphosate applications were utilized
for both determining the segregation of the dgt-28 gene in the
T.sub.1 generation as well as demonstrating tolerance to increasing
levels of glyphosate. The response of the plants is presented in
terms of a scale of visual injury 21 days after treatment (DAT).
Data are presented as a histogram of individuals exhibiting less
than 25% visual injury (4), 25%-50% visual injury (3), 50%-75%
visual injury (2) and greater than 75% injury (1). An arithmetic
mean and standard deviation is presented for each construct used
for wheat transformation. The scoring range of individual response
is also indicated in the last column for each rate and
transformation. Wild-type, non-transformed wheat (c.v. Bob White
MPB26RH) served as a glyphosate sensitive control. In the T.sub.1
generation hemizygous and homozygous plants were available for
testing for each event and therefore were included for each rate of
glyphosate tested. Hemizygous plants will contain half of the dose
of the gene as homozygous plants, therefore variability of response
to glyphosate may be expected in the T.sub.1 generation.
[0381] The results of the T.sub.1 dgt-28 wheat plants demonstrated
that tolerance to glyphosate was achieved at rates up to 3360 g
ae/ha with the chloroplast transit peptides TraP4, TraP5, TraP8 and
TraP23. Table 31. Data are of a low copy T.sub.1 event but are
representative of the population for each construct.
TABLE-US-00031 TABLE 31 Response of low copy T.sub.1 dgt-28 wheat
events to glyphosate 21 days after treatment. % Injury % Injury 25-
50- Std. Range Application Rate <25% 50% 75% >75% Ave Dev.
(%) TraP4::dgt-28 420 g ae/ha 5 0 0 0 4.00 0.00 4 840 g ae/ha 6 2 0
0 3.75 0.46 3-4 1680 g ae/ha 4 2 0 0 3.67 0.52 3-4 3360 g ae/ha 4 2
0 0 3.67 0.52 3-4 TraP8::dgt-28 420 g ae/ha 5 3 0 0 3.63 0.52 3-4
840 g ae/ha 3 5 0 0 3.38 0.52 3-4 1680 g ae/ha 4 3 0 0 3.57 0.53
3-4 3360 g ae/ha 5 5 0 0 3.50 0.53 3-4 TraP23::dgt-28 420 g ae/ha 9
2 0 0 3.82 0.40 3-4 840 g ae/ha 8 1 0 0 3.89 0.33 3-4 1680 g ae/ha
7 5 0 0 3.58 0.0 3-4 3360 g ae/ha 8 2 0 0 3.80 4.8 3-4
TraP5::dgt-28 420 g ae/ha 5 2 0 0 3.71 0.49 3-4 840 g ae/ha 4 2 0 0
3.67 0.52 3-4 1680 g ae/ha 7 3 0 0 3.70 0.48 3-4 3360 g ae/ha 6 0 0
0 4.00 0.00 3-4 Bobwhite MPB26RH 420 g ae/ha 0 1 1 10 1.25 0.62 1-3
840 g ae/ha 0 0 0 10 1.00 0.00 1 1680 g ae/ha 0 0 0 12 1.17 0.58
1-3 3360 g ae/ha 0 0 0 10 1.00 0.00 1
[0382] At 21 DAT, resistant and sensitive plants are counted to
determine the percentage of lines that segregated as a single
locus, dominant Mendelian trait (3R:1S) as determined by Chi square
analysis. Table 32. These data demonstrate that dgt-28 is
inheritable as a robust glyphosate resistance gene in a monocot
species.
TABLE-US-00032 TABLE 32 Percentage of T.sub.1 dgt-28 events by
construct that demonstrated heritablity in a mendelian fashion
based off of a glyphosate selection at rates ranging from 420-3360
g ae/ha. % T.sub.1 events % T.sub.1 events tested that tested that
No. T.sub.1 Construct segregated at a segregated as events ID
CTP:GOI single locus 2 loci tested pDAS000122 TraP4::dgt-28 62.5%
37.5% 8 pDAS000123 TraP8::dgt-28 87.5% 12.5% 8 pDAS000124
TraP23::dgt-28 12.5% 87.5% 8 pDAS000125 TraP5::dgt-28 62.5% 0.0%
8
Example 4: Chimeric Chloroplast Transit Peptide (TraP) Sequences
for Expression of Agronomically Important Transgenes in Maize
Cry2Aa:
[0383] The Cry2Aa protein from Bacillus thuringiensis has
demonstrated activity against Helicoverpa zea (CEW) and Ostrinia
nubilalis (ECB). A single version of the cry2Aa gene (SEQ ID
NO:10), codon biased for maize, was tested in maize. In this
experiment, Cry2Aa was evaluated alone and in conjunction with the
TraP8 chimeric chloroplast transit peptide in maize to determine
the insect tolerance activity and to evaluate the effect the TraP8
v2 chimeric chloroplast transit peptide sequence would have on the
expression of the Cry2Aa protein in maize.
[0384] The pDAB109807 construct which contains the Trap8 v2
chimeric chloroplast transit peptide sequence (SEQ ID NO:8) and a
GCA codon linker were cloned upstream of the cry2Aa gene and
incorporated into construct pDAB109807 (FIG. 12) for insect
tolerance testing in maize plants. The resulting constructs
contained two plant transcription units (PTU). The first PTU
comprised the Zea mays Ubiquitin 1 promoter (ZmUbi1 promoter;
Christensen, A., Sharrock R., and Quail P., (1992) Maize
polyubiquitin genes: structure, thermal perturbation of expression
and transcript splicing, and promoter activity following transfer
to protoplasts by electroporation, Plant Molecular Biology,
18:675-689), TraP8-cry2Aa fusion gene (TraP8 Cry2Aa), and Zea mays
Lipase 3' untranslated region (ZmLip 3'UTR; U.S. Pat. No.
7,179,902). The constructs were confirmed via restriction enzyme
digestion and sequencing. The second PTU comprised the Sugar Cane
Bacilliform Virus promoter (SCBV promoter; U.S. Pat. No.
6,489,462), aad-1 herbicide tolerance gene containing a MSV leader
and alcohol dehydrogenase 1 intron 6 (AAD-1; U.S. Pat. No.
7,838,733, and MSV Leader sequence; Genbank Acc. No. FJ882146.1,
and the alcohol dehydrogenase intron; Genbank Acc. No. EF539368.1),
and Zea mays Lipase 3' untranslated region (ZmLip 3'UTR). A control
plasmid, pDAB107687, which did not contain a chloroplast transit
peptide sequence upstream of the cry2Aa gene was built and included
in the studies (FIG. 13). The plasmids were introduced into
Agrobacterium tumefaciens for plant transformation.
[0385] Ears from Zea mays cultivar B104 were harvested 10-12 days
post pollination. Harvested ears were de-husked and
surface-sterilized by immersion in a 20% solution of commercial
bleach (Ultra Clorox.RTM. Germicidal Bleach, 6.15% sodium
hypochlorite) and two drops of Tween 20, for 20 minutes, followed
by three rinses in sterile, deionized water inside a laminar flow
hood. Immature zygotic embryos (1.8-2.2 mm long) were aseptically
excised from each ear and distributed into one or more
micro-centrifuge tubes containing 2.0 ml of Agrobacterium
suspension into which 2 .mu.l of 10% Break-Thru.RTM. 5233
surfactant had been added.
[0386] Upon completion of the embryo isolation activity the tube of
embryos was closed and placed on a rocker platform for 5 minutes.
The contents of the tube were then poured out onto a plate of
co-cultivation medium and the liquid Agrobacterium suspension was
removed with a sterile, disposable, transfer pipette. The
co-cultivation plate containing embryos was placed at the back of
the laminar flow hood with the lid ajar for 30 minutes; after which
time the embryos were oriented with the scutellum facing up using a
microscope. The co-cultivation plate with embryos was then returned
to the back of the laminar flow hood with the lid ajar for a
further 15 minutes. The plate was then closed, sealed with 3M
Micropore tape, and placed in an incubator at 25.degree. C. with 24
hours/day light at approximately 60 .mu.mol m-2 s-1 light
intensity.
[0387] Following the co-cultivation period, embryos were
transferred to Resting medium. No more than 36 embryos were moved
to each plate. The plates were wrapped with 3M micropore tape and
incubated at 27.degree. C. with 24 hours/day light at approximately
50 .mu.mol m-2 s-1 light intensity for 7-10 days. Callused embryos
were then transferred onto Selection I medium. No more than 18
callused embryos were moved to each plate of Selection I. The
plates were wrapped with 3M micropore tape and incubated at
27.degree. C. with 24 hours/day light at approximately 50 .mu.mol
m-2 s-1 light intensity for 7 days. Callused embryos were then
transferred to Selection II medium. No more than 12 callused
embryos were moved to each plate of Selection II. The plates were
wrapped with 3M micropore tape and incubated at 27.degree. C. with
24 hours/day light at approximately 50 .mu.mol m-2 s-1 light
intensity for 14 days.
[0388] At this stage resistant calli were moved to Pre-Regeneration
medium. No more than 9 calli were moved to each plate of
Pre-Regeneration. The plates were wrapped with 3M micropore tape
and incubated at 27.degree. C. with 24 hours/day light at
approximately 50 .mu.mol m-2 s-1 light intensity for 7 days.
Regenerating calli were then transferred to Regeneration medium in
Phytatrays.TM. and incubated at 28.degree. C. with 16 hours light/8
hours dark per day at approximately 150 .mu.mol m-2 s-1 light
intensity for 7-14 days or until shoots develop. No more than 5
calli were placed in each Phytatray.TM.. Small shoots with primary
roots were then isolated and transferred to Shoot/Root medium.
Rooted plantlets about 6 cm or taller were transplanted into soil
and moved out to a growth chamber for hardening off.
[0389] Transgenic plants were assigned unique identifiers through
and transferred on a regular basis to the greenhouse. Plants were
transplanted from Phytatrays.TM. to small pots (T. O. Plastics,
3.5-inch SVD, 700022C) filled with growing media (Premier Tech
Horticulture, ProMix BX, 0581 P) and covered with humidomes to help
acclimate the plants. Plants were placed in a Conviron.TM. growth
chamber (28.degree. C./24.degree. C., 16-hour photoperiod, 50-70%
RH, 200 .mu.mol light intensity) until reaching V3-V4 stage. This
aided in acclimating the plants to soil and harsher temperatures.
Plants were then moved to the greenhouse (Light Exposure Type:
Photo or Assimilation; High Light Limit: 1200 PAR; 16-hour day
length; 27.degree. C. Day/24.degree. C. Night) and transplanted
from the small pots to 5.5 inch pots. Approximately 1-2 weeks after
transplanting to larger pots plants were sampled for bioassay. One
plant per event was bioassayed.
[0390] Select events were identified for advancement to the next
generation based on copy number of the genes, protein detection by
Western blot and activity against the bioassay insects. Events that
contained the Spectinomycin resistance gene were noted but not
necessarily omitted from advancement. Events selected for
advancement were transplanted into 5 gallon pots. Observations were
taken periodically to track any abnormal phenotypes. Shoot bags
were placed over the shoots prior to silk emergence to prevent
cross-contamination by stray pollen. Any shoots producing silks
prior to covering were noted and the shoot was removed. The second
shoot was then covered and used for pollinations. Plants that
produced abnormal or no shoots were recorded in the database. Silks
were cut back the day prior to pollinations to provide an even
brush to accept pollen and the plants were self pollinated.
[0391] Plants for T1 selection were sprayed at 7 days post sowing.
They were grown in 4-inch pots of Metro 360 potting soil with 15
pots per flat. Seedling growth stage was V1-V1.5. Pots with poor
germination or contain very small plants (whorl still closed) were
marked so they were not included in the selection assessment. Whole
flats of plants were then placed in secondary carrier trays for
track sprayer application. Trays were placed two at a time in the
Mandel track sprayer, calibrated to deliver a volume 187 L/ha to
the target area using an 8002E flat fan nozzle (Tee Jet). A
solution of 35 g ae/ha Assure II (quizalofop)+1% COC (crop oil
concentrate) was formulated for the application. A volume of 15
mls./spray was used to calculate the total spray solution needed.
Calculations; (35 g ae/ha).times.(1 ha/187L).times.(1 L/97.7 g ae
Assure II)=0.192% solution or 28.74 .mu.l/15 ml H2O+1% v/v). After
application, the plants were then allowed to dry for one hour in
spray lab before returning to greenhouse. Approximately 1-2 weeks
after transplanting to larger pots plants were sampled for
bioassay. One plant per event was bioassayed.
[0392] All of the T.sub.0 events that passed the molecular analysis
screen were analyzed for Cry2Aa protein expression levels. The
events from the control construct, pDAB107687, which comprised
Cry2Aa without a TraP had significantly higher average expression
level of Cry2Aa (15.0 ng/cm2) as compared to events from pDAB109807
(5.0 ng/cm2) which contained TraP8. Despite the reduced levels of
expression of the pDAB109807 events, these events still expressed
the Cry2Aa protein.
[0393] The T1 events were also analyzed were analyzed for Cry2Aa
protein expression levels. The events from the control construct,
pDAB107687, which comprised Cry2Aa without a TraP had significantly
higher average expression level of Cry2Aa (55 and 60 ng/cm.sup.2)
as compared to events from pDAB109807 (about 20 to 40 ng/cm.sup.2)
which contained TraP8. Despite the reduced levels of expression of
the pDAB109807 events, these events still expressed the Cry2Aa
protein.
[0394] Transgenic plants containing single Bt genes were tested for
insecticidal activity in bioassays conducted with neonate
lepidopteran larvae on leaves from the transgenic plants. The
lepidopteran species assayed were the European Corn Borer, Ostrinia
nubilalis (Hubner) (ECB), and the Corn Earworm, Helicoverpa zea
(CEW).
[0395] 32-well trays (C-D International, Pitman, N.J.) were
partially filled with a 2% agar solution and agar was allowed to
solidify. Leaf sections approximately 1 in.sup.2 were taken from
each plant and placed singly into wells of the 32-well trays. One
leaf piece was placed into each well, and two leaf pieces were
tested per plant and per insect. Insects were mass-infested using a
paintbrush, placing 10 neonate larvae into each well. Trays were
sealed with perforated sticky lids which allowed ventilation during
the test. Trays were placed at 28.degree. C., 40% RH, 16 hours
light: 8 hours dark for three days. After the duration of the test,
a simple percent damage score was taken for each leaf piece. Damage
scores for each test were averaged and used alongside protein
expression analysis to conduct correlation analyses.
[0396] The results of the T.sub.0 and T.sub.1 bioassay indicated
that the TraP8 chimeric chloroplast transit peptide sequence was
functional and that the pDAB109807 events provided protection
against the tested insects. In the T1 events, the plants expressing
the Cry2Aa protein without a TraP (pDAB107687) had a mean leaf
damage that was not significantly different than the plant
expressing the Cry2Aa protein with TraP8 (pDAB109807) across all
insect species tested. These results were surprising, given that
the plants expressing the Cry2Aa protein without a TraP
(pDAB107687) expressed higher levels of protein as compared to the
plants expressing the Cry2Aa protein with TraP8 (pDAB109807).
VIP3Ab1:
[0397] The Vip3Ab1 protein from Bacillus thuringiensis has
demonstrated activity against Helicoverpa zea (CEW) and Fall
Armyworm (FAW) and resistant Fall Armyworm (rFAW). The vip3Ab1 v6
(SEQ ID NO:11) and vip3Ab1 v7 (SEQ ID NO:12) genes were expressed
and tested for insect tolerance in maize. In this experiment,
vip3Ab1v6 and vip3Ab1 v7 were evaluated alone and in conjunction
with the TraP8 chimeric chloroplast transit peptide in maize to
determine the insect tolerance activity and to evaluate the effect
the TraP8 v2 chimeric chloroplast transit peptide sequence would
have on the expression of the Vip3Ab1 v6 and Vip3Ab1 v7 proteins in
maize.
[0398] The pDAB111481 (FIG. 14) construct which contains the Trap8
v2 chimeric chloroplast transit peptide-encoding polynucleotide
sequence (SEQ ID NO:8) and a GCA codon linker were cloned upstream
of the vip3ab1 v6 gene and tested for insect tolerance in maize
plants. The resulting construct contained two plant transcription
units (PTU). The first PTU comprised the Zea mays Ubiquitin 1
promoter (ZmUbi1 promoter; Christensen, A., Sharrock R., and Quail
P., (1992) Maize polyubiquitin genes: structure, thermal
perturbation of expression and transcript splicing, and promoter
activity following transfer to protoplasts by electroporation,
Plant Molecular Biology, 18:675-689), TraP8-vip3ab1 v6 fusion gene
(TraP8-Vip3Ab1v6) and Zea mays Peroxidase5 3' untranslated region
(ZmPer 5 3'UTR). The construct was confirmed via restriction enzyme
digestion and sequencing. The second PTU comprised the Sugar Cane
Bacilliform Virus promoter (SCBV promoter; U.S. Pat. No.
6,489,462), aad-1 herbicide tolerance gene containing a MSV leader
and alcohol dehydrogenase 1 intron 6 (AAD-1; U.S. Pat. No.
7,838,733, and MSV Leader sequence; Genbank Acc. No. FJ882146.1,
and the alcohol dehydrogenase intron; Genbank Acc. No. EF539368.1),
and Zea mays Lipase 3' untranslated region (ZmLip 3'UTR). A control
plasmid, pDAB111479, which did not contain a chloroplast transit
peptide sequence upstream of the vip3ab1 v6 gene was built and
included in the studies (FIG. 15). The plasmids were introduced
into Agrobacterium tumefaciens for plant transformation.
[0399] The pDAB111338 (FIG. 16) construct which contains the Trap8
v2 chimeric chloroplast transit peptide sequence (SEQ ID NO:8) and
a GCA codon linker were cloned upstream of the vip3ab1 v7 gene and
tested for insect tolerance testing in maize plants. The resulting
construct contained two plant transcription units (PTU). The first
PTU was comprised of the Zea mays Ubiquitin 1 promoter (ZmUbi1
promoter; Christensen, A., Sharrock R., and Quail P., (1992) Maize
polyubiquitin genes: structure, thermal perturbation of expression
and transcript splicing, and promoter activity following transfer
to protoplasts by electroporation, Plant Molecular Biology,
18:675-689), TraP8-Vip3Ab1v7 fusion gene (TraP8-vip3ab1 v7) and Zea
mays Peroxidase5 3' untranslated region (ZmPer 5 3'UTR). The
construct was confirmed via restriction enzyme digestion and
sequencing. The second PTU was comprised of the Sugar Cane
Bacilliform Virus promoter (SCBV promoter; U.S. Pat. No.
6,489,462), aad-1 herbicide tolerance gene containing a MSV leader
and alcohol dehydrogenase 1 intron 6 (AAD-1; U.S. Pat. No.
7,838,733, and MSV Leader sequence; Genbank Acc. No. FJ882146.1,
and the alcohol dehydrogenase intron; Genbank Acc. No. EF539368.1),
and Zea mays Lipase 3' untranslated region (ZmLip 3'UTR). A control
plasmid, pDAB112710, which did not contain a chloroplast transit
peptide sequence upstream of the Vip3Ab1v7 gene was built and
included in the studies (FIG. 17). The plasmids were cloned into
Agrobacterium tumefaciens for plant transformation.
[0400] Maize transformation, protein expression and insect
bioassays were completed following the protocols previously
described, and the results are shown in Table 33. The results of
insect bioassays indicated that the TraP8 chimeric chloroplast
transit peptide sequence was functional and that the pDAB111338 and
pDAB111481 events provided protection against the insects tested in
bioassay. In the tested events, the plants expressing the Vip3Ab1
protein without a TraP, (pDAB112710 and pDAB111479), had a mean
leaf damage that was not significantly different than the plant
expressing the Vip3Ab1 protein with TraP8 (pDAB111338 and
pDAB111481). In conclusion, the Western blots and bioassays
indicated that all of the tested events expressed the Vip3 Ab1
protein.
TABLE-US-00033 TABLE 33 Results of the biochemical and bioassay
results for Vip3Ab1 v6 and Vip3Ab1 v7 coding sequences that were
fused to TraP8 as compared to Vip3Ab1 v6 and Vip3Ab1 v7 coding
sequences that did not possess a chloroplast transit peptide
sequence. Biochemical Assay Results BioAssay Results Method of
analysis CEW FAW rFAW ELISA (ng/cm.sub.2) Western Total Mean Total
Mean Total Mean Average LC/MS/MS positive Events CEW % Leaf FAW %
Leaf rFAW % Leaf Plasmid Description expression (fmole/cm.sub.2)
events tested damage Damage damage Damage damage Damage pDAB111479
Vip3Ab1 v6 59 ELISA 14/17 19 205.0 10.8 368.0 19.4 325.0 17.1 No
TraP pDAB111481 Vip3Ab1 v6 239 ELISA 4/4 17 124.0 7.3 110.0 6.5
77.0 4.5 Trap8 v2 pDAB112710 Vip3Ab1 v7 143 ELISA 18/20 20 79.0 4.0
107.0 5.4 117.0 5.9 No TraP pDAB111338 Vip3Ab1 v7 180 ELISA 5/6 9
63.0 7.0 99.0 11.0 111.0 12.3 Trap8 v2
Example 5: In Planta Cleavage of Chimeric Chloroplast Transit
Peptide (TraP) Sequences
[0401] The cleavage site of the TraP8 and TraP9 chimeric
chloroplast transit peptide was determined via MALDI spectrometry
and N-terminal Edman degradation sequencing. Plant material was
obtained from transgenic plants which contained the TraP8-dgt/4,
TraP8-dgt28, TraP9-dgt/4, and TraP9-dgt28 fusion genes and assayed
to determine the location of cleavage of the chimeric chloroplast
transit peptide occurred during translocation within the
chloroplast.
MALDI Results:
[0402] The semi-purified proteins from a plant sample were
separated by SDS-PAGE. The bands of protein of a size equivalent to
the molecular weight of YFP were excised from the gel, de-stained
and dried. Next, the dried protein bands were in-gel digested with
Trypsin (Promega; Madison, Wis.) in 25 mM ammonium bicarbonate for
overnight at 37.degree. C. The peptides were purified by a C18
ZipTip.TM. (Millipore, Bedford, Mass.) and eluted with 50%
acetonitrile/0.1% TFA. The samples were mixed with matrix
.alpha.-cyano-4-hydroxycinnamic acid in a 1:1 ratio and the mix was
sported onto a MALDI sample plate and air dried.
[0403] The peptide mass spectrum was generated using a Voyager
DE-PRO MALDI-TOF Mass Spectrometer.TM. (Applied Biosystems;
Framingham, Mass.). External calibration was performed by using a
Calibration Mixture 2.TM. (Applied Biosystems). Internal
calibration was performed using the trypsin autolysis peaks at m/z
842.508, 1045.564 and 2211.108. All mass spectra were collected in
the positive ion reflector model. The peptide mass fingerprint
(PMF) analysis was conducted using PAWS.TM. (Protein Analysis
WorkSheet) freeware from Proteometrics LLC by matching the PMF of
the sample with theoretical PMF of target protein to verify if the
sample was the target protein. The protein identification was
performed by Database searching using MASCOT (MatrixScience,
London, UK) against NCBI NR protein database.
N-Terminal Sequencing Via Edman Chemistry Degradation:
[0404] The N-terminal sequencing was performed on a Procise Protein
Sequencer (model 494) from Applied Biosystems (Foster City,
Calif.). The protein samples were separated first by SDS-PAGE, then
blotted onto PVDF membrane. The protein bands were excised from the
membrane and loaded into the Procise Sequencer. Eight cycles of
Edman chemistry degradation were run for each sample to get five AA
residues at N-terminus. A standard mix of 20 PTH-amino acids
(Applied Biosystems) was run with each sample. The amino acid
residues from each Edman degradation were determined based on their
retention times from the C-18 column against the standards.
[0405] The results of the MALDI sequencing indicated that the
DGT-28 and DGT14 proteins were expressed and that the TraP chimeric
chloroplast transit peptide sequences were processed. Table 34
lists the processed sequences which were obtained by using the
N-terminal Edman degradation and MALDI sequencing.
TABLE-US-00034 TABLE 34 Cleavage sites of TraP8 and TraP9 fused
with the dgt-14 or dgt-28 coding sequences. The grey box indicates
the splice site. Number of Sequences with Construct Sequence
Splicing TraP8-DG T14v2 ##STR00001## 66/67 TraP8-DG T28v1
##STR00002## 66/67 TraP9-DG T14v2 ##STR00003## 62/63 TraP9-DG T28v1
##STR00004## --
Sequence CWU 1
1
70169PRTBrassica napus 1Met Ala Gln Ser Ser Arg Ile Cys His Gly Val
Gln Asn Pro Cys Val1 5 10 15Ile Ile Ser Asn Leu Ser Lys Ser Asn Gln
Asn Lys Ser Pro Phe Ser 20 25 30Val Ser Leu Lys Thr His Gln Pro Arg
Ala Ser Ser Trp Gly Leu Lys 35 40 45Lys Ser Gly Thr Met Leu Asn Gly
Ser Val Ile Arg Pro Val Lys Val 50 55 60Thr Ala Ser Val
Ser65261PRTBrassica napus 2Met Ala Gln Ala Ser Arg Ile Cys Gln Asn
Pro Cys Val Ile Ser Asn1 5 10 15Leu Pro Lys Ser Asn His Arg Lys Ser
Pro Phe Ser Val Ser Leu Lys 20 25 30Thr His Gln Gln Gln Arg Arg Ala
Tyr Gln Ile Ser Ser Trp Gly Leu 35 40 45Lys Lys Ser Asn Asn Gly Ser
Val Ile Arg Pro Val Lys 50 55 60365PRTArtificial sequenceTraP8
chimeric chloroplast transit peptide 3Met Ala Gln Ser Ser Arg Ile
Cys His Gly Val Gln Asn Pro Cys Val1 5 10 15Ile Ile Ser Asn Leu Ser
Lys Ser Asn Gln Asn Lys Ser Pro Phe Ser 20 25 30Val Ser Leu Lys Thr
His Gln Gln Gln Arg Arg Ala Tyr Gln Ile Ser 35 40 45Ser Trp Gly Leu
Lys Lys Ser Asn Asn Gly Ser Val Ile Arg Pro Val 50 55
60Lys65465PRTArtificial sequenceTraP9 chimeric chloroplast transit
peptide 4Met Ala Gln Ala Ser Arg Ile Cys Gln Asn Pro Cys Val Ile
Ser Asn1 5 10 15Leu Pro Lys Ser Asn His Arg Lys Ser Pro Phe Ser Val
Ser Leu Lys 20 25 30Thr His Gln Pro Arg Ala Ser Ser Trp Gly Leu Lys
Lys Ser Gly Thr 35 40 45Met Leu Asn Gly Ser Val Ile Arg Pro Val Lys
Val Thr Ala Ser Val 50 55 60Ser655195DNAArtificial
sequencepolynucleotide encoding TraP8 5atggctcaat cttctaggat
ttgccacggt gttcaaaacc cttgcgtgat catctctaac 60ctttccaagt ccaaccagaa
caagtctcct ttcagcgttt ctcttaagac tcatcagcaa 120cagagaaggg
cttaccagat ttcttcatgg ggactcaaga agtctaacaa cggatctgtt
180atcagacctg tgaag 1956195DNAArtificial sequencepolynucleotide
encoding TraP9 6atggctcaag cttctagaat ttgccagaac ccttgcgtta
tttccaacct ccctaagtct 60aaccatagga agtctccatt ctccgtttct cttaagactc
atcagcctag agcttcatct 120tggggactta agaaatccgg aaccatgctt
aacggatctg ttatcaggcc tgttaaggtt 180accgcttctg tgtct
19579DNAArtificial sequenceLinker 7gcttcttct 98195DNAArtificial
sequencepolynucleotide encoding TraP8 v2 8atggctcaat ctagcagaat
ctgccacggt gtgcagaacc catgtgtgat catttccaat 60ctctccaaat ccaaccagaa
caaatctcct ttctcagtca gcctcaagac tcaccagcag 120cagcgtcgtg
cttaccagat atctagctgg ggattgaaga agtcaaacaa cgggtccgtg
180attcgtccgg ttaag 1959195DNAArtificial sequencepolynucleotide
encoding TraP9 v2 9atggcacaag ccagccgtat ctgccagaat ccatgtgtga
tatccaatct ccccaaaagc 60aaccaccgta agtccccttt ctctgtctca ctcaagacgc
atcagcctag agcctcttca 120tggggactta agaagtctgg caccatgctg
aacggttcag tgattagacc cgtcaaggtg 180acagcttctg tttcc
195101902DNABacillus thuringiensis 10atgaacaacg ttctcaactc
tgggagaacc accatctgcg acgcgtacaa tgtggtggca 60cacgacccat tctcatttga
gcacaagagc ctggatacga tccagaaaga gtggatggaa 120tggaaaagga
cggaccattc cttgtacgtt gctccagtgg tgggcactgt gtcctcgttc
180ctcctcaaga aagtggggtc gctgatcggg aagcggatac tctccgaact
ctggggaatc 240atctttccgt ctggctcaac aaacttgatg caagacatac
tgagagagac tgagcagttc 300ttgaatcaga ggctgaatac ggacacgctg
gcgagggtga acgctgaact cattggcctc 360caagcgaaca ttagagagtt
caatcagcaa gttgataact ttctgaaccc cacacagaat 420ccagtgcctc
tgtccatcac atcatctgtg aacacaatgc agcagctgtt cttgaatagg
480ctgccacagt ttcagattca aggctatcaa ctgcttcttc tgcctctgtt
tgctcaagct 540gcgaacatgc acctcagctt catcagagac gtgattctga
acgcagatga gtggggaatc 600agcgctgcca ctttgaggac ctacagagat
tacttgagga actatacacg cgattactca 660aactactgca tcaacaccta
tcaaacagcg tttaggggac ttaacaccag actgcacgac 720atgcttgagt
tccggactta catgttcctc aacgtctttg agtatgtctc gatttggtcc
780ctcttcaagt atcagagcct tatggtctcc tctggtgcta acctctacgc
ctcgggttcc 840ggaccgcagc agacccagtc attcactgcc cagaactggc
cattcctcta cagccttttc 900caagtgaaca gcaactacat cttgtctggc
atctctggca caaggctctc tatcacattt 960ccgaacattg gtggcctgcc
tggctccacg acgacacaca gcctcaattc cgcacgcgtc 1020aactactcgg
gtggggtctc ctccggactc attggtgcca ctaacttgaa ccataacttc
1080aactgttcaa cggtgctgcc acccctttca actccgtttg tcagatcgtg
gcttgattct 1140ggcactgaca gagagggagt tgccacgagc accaactggc
agaccgagtc cttccagacc 1200acactttcgc tgcgctgcgg tgccttctca
gcgaggggaa actcgaacta cttcccagac 1260tacttcatac gcaacattag
cggagtcccg ttggtgatcc ggaatgagga cctcaccaga 1320cctcttcact
acaatcagat acgcaacatc gaaagcccat ctgggacacc tggaggtgca
1380agggcatact tggttagcgt tcacaaccgg aagaacaaca tctatgctgc
taatgagaat 1440gggaccatga ttcatcttgc accggaagat tacactggct
tcacgatctc acccatccat 1500gccacccaag tgaacaacca gactcgcacg
ttcatctcag agaagttcgg caaccaaggt 1560gacagcctcc gcttcgaaca
gagcaacacc acagccagat acacccttag aggcaatggc 1620aacagctaca
atctctatct gagggtgtct agcattggca attcgaccat tcgggtgacg
1680atcaatggtc gcgtttacac ggtctccaac gtcaatacga ccactaacaa
tgatggggtc 1740aatgacaatg gtgctcgctt ctccgacatc aacatcggca
acatcgtcgc ttccgacaac 1800accaatgtta cgctggacat caatgtcacc
ttgaactctg gcacaccttt cgatctgatg 1860aacatcatgt ttgtccccac
caatcttcct cccctctact ga 1902112364DNABacillus thuringiensis
11atgaatatga ataatactaa attaaacgca agagcgctgc cttcgttcat cgactacttc
60aacggcatct acggattcgc aactgggatc aaagacatca tgaacatgat cttcaagacg
120gacacgggtg gaaatcttac attggatgag attctgaaga atcagcagtt
gctcaacgaa 180atctctggca aactcgacgg agttaacggg tcccttaacg
acctcatagc acaaggcaat 240cttaatacag agctgtccaa ggagattctc
aagattgcga atgagcaaaa tcaagttctg 300aacgatgtca acaacaagct
ggacgccatc aacacaatgt tgcacatcta tctgcccaag 360atcacctcta
tgctctccga tgttatgaag cagaactatg cgctctcgct tcaagtcgag
420tacttgtcaa agcaactgaa agagatttcc gacaaactgg atgttatcaa
tgttaacgtg 480ttgatcaata gcacactgac cgaaatcacc ccagcttatc
aaaggattaa atacgtgaat 540gaaaagttcg aggaattaac cttcgctacg
gagacaactc tgaaagtgaa gaaagattcg 600tcaccagccg acattctgga
cgagttgacc gaattaactg aactggcgaa gtccgttaca 660aagaatgacg
ttgatggctt cgagttctat ctcaatacat tccacgatgt tatggtgggc
720aataatctgt tcggacgctc tgctttgaaa actgcttctg aattaatcgc
aaaggaaaac 780gttaagacgt cgggttccga ggtcgggaac gtgtacaact
tcttgatagt gctcacggca 840ctgcaagcga aagcgtttct cacgctgacc
acgtgcagaa agttgctggg attggctgat 900atcgattata catccatcat
gaacgagcac cttaacaaag aaaaggagga gttcagagtg 960aacattcttc
caacattgtc caacacattc tccaatccga actacgcaaa ggtgaaaggt
1020tctgacgaag atgcaaagat gatcgtcgag gcgaaacctg ggcacgctct
ggtcggcttc 1080gagatttcca acgactcgat gacggtatta aaggtgtacg
aggctaagtt gaagcaaaac 1140tatcaagtgg acaaggactc cctttcagag
gtgatctatt cagatatgga caagctgctg 1200tgtccggatc aatctgagca
aatctactac accaataata tagttttccc taacgaatac 1260gtcattacca
agattgattt cacgaagaag atgaaaaccc ttagatatga ggttactgcc
1320aatagctacg attcatctac gggtgaaatc gatcttaaca agaagaaggt
tgaatcaagc 1380gaagccgagt atcggactct gtcagccaat aatgacggtg
tgtacatgcc tcttggtgtg 1440atttcagaga cattccttac accaattaat
ggtttcggtc tgcaagctga tgaaaactcc 1500agattaatca ctctgacgtg
taagtcctac ttgagggagt tgctcctcgc cactgatctt 1560tccaataaag
aaacaaagct gattgttcca ccgatctcgt tcatctcaaa cattgtggag
1620aacgggaacc tcgaaggcga gaatctcgaa ccgtggattg cgaacaacaa
gaacgcttac 1680gtggatcata ctggagggat caacggcacg aaggtcttgt
acgtgcataa ggacggagag 1740ttctcacagt tcgtgggtgg gaagttgaag
agcaagaccg agtacgtcat ccagtacatt 1800gtgaagggga aggcctcaat
ctatctcaag gataaaaaga atgagaactc tatctacgag 1860gaaataaata
atgaccttga gggcttccaa actgtgacca agcggttcat aaccggaacg
1920gactcttccg gaatccatct gatctttact tcccagaacg gagaaggtgc
tttcggtgga 1980aacttcatca tcagcgagat ccgcacgtca gaggagttgc
ttagcccaga attgatcatg 2040tcggacgcgt gggtgggaag ccaaggcacg
tggatctctg gcaactccct caccattaat 2100tccaacgtga atggcacgtt
taggcagaat ctccctcttg agtcgtattc aacctatagc 2160atgaacttca
cggttaacgg atttgggaag gtgacggtcc gcaattctcg cgaggtgctc
2220tttgaaaagt cgtatcctca gctctctcca aaggacatca gcgagaagtt
caccaccgca 2280gcgaataata ctggattgta tgtcgaactc tcaagatcga
cttctggtgg tgcaataaac 2340tttcgggact tctcaattaa gtga
2364122364DNABacillus thuringiensis 12atgaacatga ataatactaa
attaaacgcg agggcgctgc cgagcttcat cgactacttc 60aacggcatct acggcttcgc
caccggcatc aaggacatca tgaacatgat cttcaagacg 120gataccggcg
gcaacctgac cctggacgag atcctgaaga accagcagct gctgaacgag
180atcagcggca agctggacgg cgtgaacggc agcctgaacg acctgatcgc
ccaaggcaac 240ctgaatacag agctgagcaa ggagatcctg aagatcgcga
acgagcagaa tcaagtgctg 300aacgacgtga acaacaagct ggacgcgatc
aacaccatgc tgcacatcta cctgcccaag 360atcacctcca tgctgagcga
cgtgatgaag cagaactacg cgctgtccct ccaagtggag 420tacctgagca
agcagctgaa ggagatcagc gacaagctgg acgtgatcaa cgtgaacgtg
480ctgatcaact ccaccctgac cgagatcacc ccggcctacc agaggattaa
atacgtgaat 540gaaaagttcg aggaattaac cttcgccacc gagaccaccc
tgaaggtgaa gaaggacagc 600tccccggcgg acatcctgga cgagctgacc
gaattaaccg agctggcgaa gtccgtgacc 660aagaacgacg tggacggctt
cgagttctac ctgaatacat tccacgacgt gatggtgggc 720aataatctgt
tcgggaggag cgccctcaag accgccagcg aattaatcgc caaggagaac
780gtcaagacca gcggcagcga ggtgggcaac gtctacaact tcctgatcgt
gctgaccgcc 840ctccaagcga aggcgttcct gaccctgacc acctgccgca
agctgctggg cctggcggac 900atcgactata catccatcat gaacgagcac
ctgaacaagg agaaggagga gttccgcgtg 960aacatcctgc cgaccctgtc
caacaccttc agcaacccga actacgccaa ggtgaagggg 1020tccgacgagg
acgcgaagat gatcgtggag gccaagccgg gccacgcgct ggtgggcttc
1080gagatcagca acgacagcat gaccgtatta aaggtgtacg aggcgaagct
gaagcagaac 1140taccaagtgg acaaggactc cctgagcgag gtgatctaca
gcgacatgga caagctgctg 1200tgcccggacc agtccgagca aatctactac
accaataata tcgtgttccc gaacgagtac 1260gtgatcacca agatcgactt
caccaagaag atgaaaaccc tgcgctacga agtgaccgcc 1320aacagctacg
actcctcaac cggcgagatc gacctgaaca agaagaaggt ggaatcaagc
1380gaggcggagt accgcaccct gagcgcgaat aatgacggcg tgtacatgcc
gctgggcgtg 1440atctccgaga ccttcctgac cccaattaat gggttcggcc
tccaagccga tgaaaactcc 1500agattaatca ccctgacctg caagtcctac
ctgagggagc tgctgctggc gaccgacctg 1560agcaataaag agaccaagct
gatcgtgccg ccgatctcct tcatctccaa catcgtggag 1620aacggcaacc
tggagggcga gaacctggag ccgtggatcg cgaacaacaa gaacgcgtac
1680gtggatcaca cgggaggcat caacggcacc aaggtgctgt acgtccacaa
ggacggcgag 1740ttcagccagt tcgtgggcgg caagctgaag agcaagaccg
agtacgtgat ccagtacatc 1800gtgaagggca aggcctccat ctacctgaag
gataaaaaga acgagaactc catctacgag 1860gaaataaata atgacctgga
gggcttccag accgtgacca agaggttcat caccggcacc 1920gactcctccg
gcatccacct gatcttcacc tctcagaacg gcgagggcgc gttcggcggc
1980aacttcatca tcagcgagat ccgcacctcc gaggagctgc tgtccccgga
gctgatcatg 2040tccgacgcct gggtgggcag ccaaggcacc tggatctccg
gcaactccct gaccattaat 2100agcaacgtga acggcacctt ccgccagaac
ctgccgctgg agagctacag cacctactcc 2160atgaacttca ccgtgaacgg
gttcggcaag gtgacggtga ggaactcccg cgaggtgctg 2220ttcgagaagt
cctacccgca gctcagcccc aaggacatct cagagaagtt caccaccgcc
2280gccaataata ccggcctgta cgtggagctg tcaaggagca ccagcggcgg
cgcaataaac 2340ttccgcgact tctcaattaa gtga 2364134PRTArtificial
sequenceSVSL sequence 13Ser Val Ser Leu114207DNAArtificial
sequencepolynucleotide encoding Brassica napus EPSPS 14atggcgcaat
ctagcagaat ctgccatggc gtgcagaacc catgtgttat catctccaat 60ctctccaaat
ccaaccaaaa caaatcacct ttctccgtct ccttgaagac gcatcagcct
120cgagcttctt cgtggggatt gaagaagagt ggaacgatgc taaacggttc
tgtaattcgc 180ccggttaagg taacagcttc tgtttcc 20715183DNAArtificial
sequencepolynucleotide encoding Brassica rapa EPSPS 15atggcgcaag
ctagcagaat ctgccagaac ccatgtgtta tctccaatct ccccaaatcc 60aaccaccgca
aatcgccctt ctctgtctcg ctgaagacgc accagcagca gcgtcgagct
120tatcagatat cttcgtgggg attgaagaag agtaacaacg gctccgtgat
tcgtccggtt 180aag 183161248DNAStreptomyces sviceus 16atgagaggga
tgccagcctt gtctttacct ggatcaaaga gtatcacagc tagggcactc 60tttcttgctg
ctgctgctga tggggttact actttggtga ggccattgag aagtgacgac
120acagaaggat tcgctgaggg gttagttcgt ttaggctatc gtgtagggag
gacacccgat 180acttggcaag tcgatggcag accacaagga ccagcagtgg
ctgaggctga cgtctactgt 240agagacggag caaccaccgc tagattcttg
ccaaccttag cagctgctgg tcacggaaca 300tacagatttg atgcttcacc
acagatgagg agacgtcctc ttttgccctt aagcagagcc 360ttgagggatt
tgggtgtcga tcttagacac gaagaagctg aaggtcatca ccctctgact
420gtccgtgctg ctggggttga aggaggagag gttactttgg atgctggtca
gtcaagtcag 480tatctcactg ccttgttgct ccttggtccc cttacaagac
aaggactgag gataagggtt 540actgatttgg tgtcagcacc atacgtggag
attacgcttg caatgatgag ggctttcgga 600gttgaagtgg caagggaggg
agatgtgttc gttgttccac ctggtggata tcgtgcaact 660acgtatgcta
tagaacccga cgcaagtact gcttcttact tcttcgcagc tgctgctttg
720actcctggag ctgaagtgac tgtacctggg ttaggcacgg gagcacttca
aggagatttg 780ggatttgtag atgtcttaag gagaatggga gccgaggtgt
ccgtaggagc tgatgcaacc 840actgttagag gaactggtga attgcgtggc
cttacagcca acatgagaga cataagtgat 900acgatgccga ccctcgctgc
aatagcaccc tttgctagtg ctccagttag aatcgaggat 960gttgccaaca
ctcgtgtcaa agaatgtgac agacttgagg cttgtgcaga gaaccttagg
1020aggttgggag taagggttgc aacgggtccg gactggattg agatacaccc
tggtccagct 1080actggtgctc aagtcacaag ctatggtgat cacagaattg
tgatgtcatt tgcagtgact 1140ggacttcgtg tgcctgggat cagcttcgac
gaccctggct gtgttcgtaa gacttttcct 1200gggtttcacg aggctttcgc
agaattgagg cgtggcattg ggagctga 1248171251DNAStreptomyces sviceus
17atggcaagag ggatgccagc cttgtcgctg cctggctcaa agtcgatcac ggctagagca
60ctctttctcg cagcagcagc cgacggagtc accacgcttg tgagaccgct gcggtcagac
120gacaccgagg gttttgcgga aggcctcgtc agactgggct atcgggttgg
gaggactccc 180gacacgtggc aagtggacgg aaggccacaa ggtccagcag
ttgccgaggc tgatgtgtat 240tgtagagacg gtgcaacaac ggctaggttc
ctccccacac tcgcagctgc tggacacggg 300acctacagat ttgatgcctc
tccccagatg aggagaaggc cactgctgcc tctttctagg 360gctttgaggg
accttggcgt tgatcttcgc cacgaggaag cggaagggca ccaccccttg
420accgtgagag ctgctggagt cgagggaggt gaggttacac tcgatgctgg
acagtcctct 480cagtacttga cggcactgct gctgctcggt ccgctcacac
gccaagggct gcggattcgc 540gtcactgatc tggttagcgc tccgtacgtg
gagattacac ttgcgatgat gagagctttt 600ggggtcgagg ttgcacgcga
aggcgacgtt ttcgtggtgc ctcctggtgg ctacagagcg 660actacgtacg
cgattgagcc agatgccagc accgcaagct acttctttgc agctgctgcg
720ttgacacctg gagccgaggt cacagtgcct ggactcggga ccggagcgct
tcaaggggat 780ctcggcttcg tggacgtgct gcggaggatg ggtgccgagg
tcagcgtggg agcagacgct 840acgactgtta gaggcacggg tgagcttaga
ggccttacag caaacatgag ggacatatcc 900gacacgatgc cgacgcttgc
tgccatcgct ccgttcgctt cagcacccgt cagaattgaa 960gatgtggcga
acactcgcgt caaagagtgc gacagacttg aagcgtgtgc cgagaacttg
1020aggaggttgg gagtgagagt cgcaactggt ccagactgga tcgagatcca
ccctggtcca 1080gctactggag cgcaagtcac aagctatggc gaccatagga
ttgttatgtc attcgcagtg 1140accggactca gagttcctgg gatctctttc
gacgaccctg gttgcgtgcg gaaaacgttc 1200cctggcttcc acgaggcatt
tgcggagctg cggagaggaa ttggttcctg a 1251181599DNAGlycine max
18atggctcaag tctcccgtgt tcacaatctt gctcagtcaa cccaaatctt tggacattca
60agcaactcaa acaaactgaa gtctgtgaat tctgtctcac ttcgcccacg cctttgggga
120gcatccaaga gtcgcatacc aatgcacaag aatgggagtt tcatgggcaa
cttcaatgtt 180gggaaaggca attctggtgt cttcaaagtt tcagcttctg
ttgcagccgc agagaaaccc 240agcacttccc ctgagattgt tcttgaaccc
attaaggact tcagtggaac aatcactctg 300cctggatcaa agagtctttc
aaacagaata cttctcttgg cagctctgag tgaaggaacc 360actgtagttg
acaacctttt gtactctgaa gatattcatt acatgttggg tgctctcaga
420actcttgggt tgagagttga agatgacaag accacaaaac aagccatagt
tgaaggatgt 480ggtgggttgt ttccaacaag caaagaatcc aaagatgaga
tcaacttgtt tcttggcaat 540gctggaattg caatgagaag cctcactgct
gcagtagttg cagctggtgg gaatgcaagt 600tatgtccttg atggtgtccc
cagaatgagg gaaaggccca tcggtgacct tgtggctggc 660ctgaaacagc
ttggagcaga tgttgattgc ttcttgggca caaactgccc tccagtgaga
720gtgaatggga agggaggttt gcctggtgga aaggtcaaac tgagtggatc
agtctcttcc 780cagtatctga ctgccttgct catggctgcc cctctggctt
tgggtgatgt ggagattgaa 840atagtggaca agttgatttc tgttccatat
gtggaaatga ccctcaaact catggagagg 900tttggagttt ctgttgaaca
ttctggcaac tgggatcgtt tccttgtaca tggaggtcag 960aagtacaaaa
gccctggcaa tgcctttgtt gaaggggatg caagctctgc ttcctatctc
1020ttggctgggg ctgccatcac tggtgggacc atcactgtga atggctgtgg
cacctcatcc 1080cttcaaggtg atgtaaagtt tgcagaggtc ttggagaaaa
tgggtgccaa ggtcacctgg 1140tctgagaaca gtgtaactgt gtctggacct
cccagagact tcagtggcag aaaggttctc 1200cgtggaattg atgtgaacat
gaacaagatg ccagatgtgg ccatgaccct cgctgttgta 1260gccctgtttg
caaatggacc aactgcaatc cgtgatgttg cttcatggag ggtgaaggag
1320acagagagga tgattgccat ttgcacagaa ctccgcaaac ttggtgcaac
agttgaagag 1380ggaccagatt actgtgtgat aaccccacct gagaagctca
atgtgacagc cattgacacc 1440tatgatgacc acagaatggc aatggctttc
tcccttgctg cctgtggtga tgtgcctgtg 1500actatcaaag accctgggtg
cacaaggaag acatttccag actactttga agttttggag 1560aggttgacaa
agcactgagt agttagctta atcacctag 1599191551DNABrassica napus
19atggctcaat cttcaaggat ttgccacggt gttcagaacc cttgtgtgat catatccaat
60ctcagtaaga gcaatcagaa caaatcaccc ttctctgtct ccctcaaaac tcatcaacca
120cgtgcatcta gttggggatt gaagaaaagt gggacaatgc tgaacggatc
agtcattagg 180cctgtaaagg ttacagcctc tgtgtccacg agtgaaaagg
caagcgagat cgtcttacaa 240ccgattagag aaatctctgg gcttatcaag
ttgcctggct ccaaatcact ctccaatagg 300atacttcttt tggctgcact
gagtgaaggc acaactgttg tggacaactt gctcaactcc 360gatgatatca
actacatgct
tgacgccttg aagaagttag gactcaatgt ggagagagat 420agcgttaaca
atcgtgctgt cgtagaagga tgtggtggca tctttcctgc atctctggat
480tctaagagcg acatcgagct ttacttgggc aatgctgcaa cagccatgag
accgttaact 540gctgctgtta ccgcagctgg aggaaatgct agttatgtgc
ttgatggtgt tccaagaatg 600agggaaaggc caatagggga tttggtcgtc
ggactgaaac agctcggtgc tgacgttgaa 660tgtactttag gcacaaactg
tcctcccgtg cgtgttaacg caaatggtgg actgcctggt 720ggaaaggtca
agttgtctgg ctccatttcc agtcaatacc ttacggcttt gctcatggct
780gcaccacttg ccttaggtga tgtggagatt gagatcattg acaagctcat
atctgttccg 840tacgtggaaa tgacacttaa gctgatggaa agattcggag
tttcagccga acattccgat 900agctgggatc gtttctttgt aaagggtggg
cagaagtaca agtctcctgg caatgcttat 960gtggaaggtg acgcttcttc
agctagttac ttcttggctg gtgcagccat aactggcgag 1020acagttaccg
tggaaggatg cggaactacc agcctccaag gtgatgtcaa gttcgcagag
1080gtgttggaaa agatggggtg caaagtttcc tggacagaga actcagttac
tgtaacggga 1140cctagtaggg atgcttttgg gatgcgtcac cttagggcag
ttgacgtgaa catgaacaag 1200atgccagatg tcgctatgac tttagcagtt
gtggcactgt ttgccgatgg tcctacaacg 1260attagggacg tagcttcttg
gagagtcaaa gaaactgaga ggatgatcgc catttgtact 1320gagcttcgta
agttgggtgc cacagttgaa gaagggtccg attactgcgt gattactcct
1380ccagctaaag ttaagcctgc tgagattgat acctatgatg accacagaat
ggctatggcc 1440tttagcctcg ctgcatgtgc cgatgttcca gtcacgatca
aggaccctgg ctgtactaga 1500aagacatttc ccgactactt tcaagtgctt
gagtcaatca cgaaacactg a 1551201551DNABrassica napus 20atggctcaat
cttcaaggat ttgccacggt gttcagaacc cttgtgtgat catatccaat 60ctcagtaaga
gcaatcagaa caaatcaccc ttctctgtct ccctcaaaac tcatcaacca
120cgtgcatcta gttggggatt gaagaaaagc ggaacaatgc tgaacggatc
agtcattagg 180cctgtaaagg ttactgcatc tgtgtccacg agtgaaaagg
caagcgagat cgtcttacaa 240ccgattagag aaatctctgg gcttatcaag
ttgcctggct ccaaatcact ctccaatagg 300atacttcttt tggctgcact
gagtgaaggc acaactgttg tggacaactt gctcaactcc 360gatgatatca
actacatgct tgacgccttg aagaagttag gactcaatgt ggagagagat
420agcgttaaca atcgtgctgt cgtagaagga tgtggtggaa tctttcctgc
atctctggat 480tctaagagcg acatcgagct ttacttgggc aatgctgcaa
cagccatgag atccttaact 540gctgctgtta ccgcagctgg tggaaatgct
agttatgtgc ttgatggtgt tccaagaatg 600agggaaaggc caatagggga
tttggtcgtc ggactcaaac agctcggtgc tgacgttgaa 660tgtactttag
gcacaaactg tcctcccgtg cgtgttaacg caaatggtgg actgcctggt
720ggaaaagtca agttgtctgg ctccatttcc agtcaatacc ttacggcttt
gctcatggct 780gcaccacttg ccttaggtga tgtggagatt gagatcattg
acaagctcat atctgttccg 840tacgtggaaa tgacacttaa gctgatggaa
agattcggag tttcagccga acattccgat 900agctgggatc gtttctttgt
aaagggaggg cagaagtaca agtctcctgg aaacgcatac 960gtggaaggtg
acgcttcttc agctagttac ttcttggctg gtgcagccat aactggcgag
1020acagttaccg tggaaggatg cggaactacc agcctccaag gtgatgtcaa
gttcgcagag 1080gtgttggaaa agatggggtg caaagtttcc tggacagaga
actcagttac tgtaacggga 1140cctagtaggg atgcttttgg gatgcgtcac
cttagagccg ttgacgtgaa catgaacaag 1200atgccagatg tcgctatgac
cttagctgtg gttgcactgt ttgccgatgg tcctacaacg 1260attagggacg
tagcctcttg gagagtcaaa gaaaccgaga ggatgatcgc catttgtact
1320gagcttcgta agttgggtgc cacagttgaa gaagggtccg attactgcgt
gattactcct 1380ccagctaaag ttaagccagc agagattgat acctatgatg
accacagaat ggctatggct 1440ttcagcctcg ctgcatgtgc cgatgttcca
gtcacgatca aggaccctgg ctgtactaga 1500aagacatttc ccgactactt
tcaagtgctt gagtcaatca cgaaacactg a 1551211551DNABrassica napus
21atggctcaat cttcaaggat ttgccacggt gttcagaacc cttgtgtgat catatccaat
60ctcagtaaga gcaatcagaa caaatcaccc ttctctgtct ccctgaaaac tcatcaacca
120cgtgcatcta gttggggatt gaagaaaagt ggcacaatgc tgaacggatc
agtcattagg 180cctgtaaagg ttacagcctc tgtgtccacg agtgaaaagg
caagcgagat cgtcttacaa 240ccgattagag aaatctctgg gcttatcaag
ttgcctggct ccaaatcact ctccaatagg 300atacttcttt tggctgcact
gagtgaaggg acaactgttg tggacaactt gctcaactcc 360gatgatatca
actacatgct tgacgccttg aagaagttag gactcaatgt ggagagagat
420agcgttaaca atcgtgctgt cgtagaagga tgtggtggaa tctttcctgc
atctctggat 480tctaagagcg acatcgagct ttacttgggc aatgctggca
tcgccatgag atccttaact 540gctgctgtta ccgcagctgg tggaaatgct
agttatgtgc ttgatggtgt tccaagaatg 600agggaaaggc caatagggga
tttggttgtc ggactcaaac agctcggtgc tgacgttgaa 660tgtactttag
gcacaaactg tcctcccgtg cgtgttaacg caaatggtgg actgcctggt
720ggaaaggtca agttgtctgg ctccatttcc agtcaatacc ttacggcttt
gctcatggct 780gcaccacttg ccttaggtga tgtggagatt gagatcattg
acaagctcat atctgttccg 840tacgtggaaa tgacacttaa gctgatggaa
agattcggag tttcagccga acattccgat 900agctgggatc gtttcttcgt
aaagggaggg cagaagtaca agtctcctgg gaacgcatac 960gtggaaggtg
acgcttcttc agctagttac ttcttggctg gtgcagccat aactggcgag
1020acagttaccg tggaaggatg cggaactacc agccttcaag gtgatgtcaa
gttcgcagag 1080gtgttggaaa agatggggtg caaagtttcc tggacagaga
actcagttac tgtaacggga 1140cctagtaggg atgcttttgg aatgagacac
cttagggcag ttgacgtgaa catgaacaag 1200atgccagatg tcgctatgac
tttagctgta gtggcactgt tcgcagatgg tcctacaacg 1260ataagggacg
tagcctcttg gagagtcaaa gaaaccgaga ggatgatcgc catttgtact
1320gagcttcgta agttgggtgc cacagttgaa gaagggtccg attactgcgt
gattactcct 1380ccagctaaag ttaagccagc agagattgat acctatgatg
accacagaat ggctatggcc 1440tttagcctcg ctgcatgtgc cgatgttcca
gtcacgatca aggaccctgg ctgtactaga 1500aagacatttc ccgactactt
tcaagtgctt gagtcaatca cgaaacactg a 1551221533DNATriticum aestivum
22atggcaatgg ctgctgctgc tactatggct gcaagcgctt cctcttccgc tgtgagctta
60gacagagcag ctccagcacc atctaggcgt ctgccaatgc cagcagctag accagctagg
120agaggtgcag tccgtttgtg gggaccaagg ggagcagctg cacgtgctac
aagtgtcgca 180gcaccagcag caccgagtgg agctgaggaa gtcgtgcttc
aacctatcag agagatcagc 240ggtgccgtcc agctccctgg gtcaaagtca
cttagcaaca gaatacttct tttgagcgca 300ttgtcagagg gcacgacagt
ggtggataac cttctgaact ctgaagatgt tcactacatg 360cttgaggctt
tggaggcatt aggtctttct gttgaagccg ataaggttgc taagcgtgct
420gtggtggttg gttgcggagg gagattccca gttgagaaag atgctcaaga
ggaagttaag 480ctgtttctgg gaaatgctgg gattgcaatg aggagcttga
ctgctgctgt ggttgctgct 540ggtggaaatg ccacatacgt ccttgatgga
gtgcctagaa tgagagagag accgattggg 600gatctggtgg ttggccttca
gcaacttgga gcagacgctg actgctttct tggaacaaac 660tgtccacccg
ttaggatcaa cgggaaagga ggtctccctg gtgggaaggt taagttgtct
720ggatcaatct ctagtcagta tctgtcatca cttctcatgg ctgcacctct
tgcacttgaa 780gatgttgaga ttgaaatcat agacaaactc atatcagttc
catacgtgga aatgacgctg 840aagctgatgg agaggttcgg agtgacagca
gagcactcag attcttggga taggttctac 900atcaagggag gtcagaagta
caaatcacct gggaacgctt acgtggaagg tgatgcctct 960tctgcttcct
acttcctcgc tggagcagca atcaccggag gaactgttac tgtcgaaggt
1020tgcggaacta cttccttgca aggggacgtc aagttcgcag aagtcttaga
aatgatggga 1080gctaaagtta cttggaccga tacaagtgtt acagtgactg
gtcctccacg tcaacccttt 1140ggaaggaagc acctcaaagc cgttgatgtt
aacatgaaca agatgccaga tgtcgccatg 1200acgcttgccg ttgtggctct
gttcgcagat ggtcccacag ccattagaga cgtggccagc 1260tggagggtga
aagaaactga aaggatggtc gccattagaa cagagttaac caaacttgga
1320gctactgtgg aagagggacc cgactattgc atcattacac ctcccgagaa
gctgaacata 1380accgctattg acacttatga tgatcatcgt atggctatgg
ccttttcatt agcagcttgc 1440gctgaggtgc cagtaaccat tagagatcct
gggtgtacta ggaaaacttt ccctaactac 1500ttcgatgtcc tttcaacatt
cgtgaagaat tga 1533231281DNAStreptomyces roseosporus 23atgacggtga
tagagatacc tgggtctaag tctgttacag ccagagcact gttcttggca 60gctgctgccg
atgggacgac tactcttctt agaccattgc gtagcgatga cactgagggc
120ttcgcagaag gactgaggaa tctgggctat gctgtggaac aagaggctga
taggtggcgt 180gtccaaggca gaccagctgg accagcagcc acggaagcag
atgtctattg cagagatggt 240gccaccaccg ctaggttcct tccgacactg
gcagcagcag ctgcttccgg aacctacaga 300ttcgacgctt cagcacagat
gcgtcgtcgt ccccttgctc cattgacaag ggcacttaca 360gccttgggtg
tggatcttag acacgaagga gcagacggac atcatccgct caccgttcgt
420gcagctggca tcgaaggagg agaattgacg ctcgacgctg gcgagtccag
ccaatacttg 480acagcactgc tcatgctcgg acctcttaca acaaagggac
ttcgcatcga agttacagaa 540ctcgtctctg caccctacgt ggaaatcacc
ctcgctatga tgagagactt tggtgtggag 600gttgagaggg aggggaatac
cttcaccgtt ccaagcccat cttcaagact taggtccaat 660agaggtggac
ccataggagg ctatagagct actacgtatg ctgtcgagcc agatgcctca
720actgcctctt acttctttgc agctgctgcc ctcactggtc gcgaggtcac
agtgcctgga 780ttggggactg gagctttgca aggtgatttg cgtttcgtgg
atgtgctgag agaaatgggt 840gccgaggtgt ctgttggtcc ggacgccaca
actgtgcgct caactggcag attgagggga 900atcactgtga acatgagaga
tatctcagac acgatgccta cactcgcagc tattgcacct 960tatgccgatg
gtccagtggt gattgaagat gttgccaaca cccgtgtgaa ggagtgtgac
1020cgtctggagg cttgtgctga gaatctgagg gcaatgggaa tcaccgtcca
tacgggtccg 1080gataggatag aaatccatcc tggaacacct aaaccgactg
ggatcgccac ccacggagat 1140caccgcatag tcatgtcatt tgccgtcgct
ggccttcgca ctcctggcct cacttacgac 1200gaccctggct gcgtgcgtaa
gaccttccct agatttcacg aggtgtttgc cgacttcgct 1260cacgaccttg
agggaaggtg a 1281241248DNAStreptomyces griseus 24atgggtgcag
tgacagtcat cgacattcct ggaagcaaga gcgtgacagc aagggcactc 60ttcttggcag
cagcagccga tggaacgaca acactgcttc gtcctctgag gtcagacgac
120acggaggggt ttgccgaggg tcttaagaat ctcggttatg ccgttgagca
agaggctgac 180cgttggaggg tcgaaggcag accggatggt ccagctgctc
cggatgcaga tgtctactgc 240cgtgatggtg caacgactgc acgctttctt
ccaaccctcg tcgcagcagc agcttctgga 300acgtatcgtt tcgacgcctc
agcacagatg aggagacgtc ccttggctcc actcactagg 360gcactgacag
ctcttggcgt ggatttgaga catggtggag aggagggtca tcatccactg
420actgtcagag ctgctggcat agaaggtggc gatgttgtcc ttgacgctgg
tgaatcttct 480cagtatctca cagcccttct tatgttgggt ccgttgactg
ccaaaggtct tagaatcgaa 540gtcactgatc tcgtgagcgc tccttacgtt
gaaatcactc tggccatgat gagagatttc 600ggagttgatg ttagcagaga
aggaaacact ttcaccgtgc cgtccggagg ctatagagct 660acagcctacg
ctgtggagcc agacgcaagc acggcttctt acttctttgc agcagctgcc
720ctcactggac gcgaggtgac ggtccctggg ctgggaattg gtgctcttca
aggagacctt 780cgttttgtgg acgtgctgcg tgatatggga gcagaggtgt
ctgttggacc agatgccacg 840acagtgcgct caactggcag actccgtggc
attacagtta ctatgagaga catttcagac 900acgatgccaa cactcgctgc
tattgcacct cacgctgatg gacccgtccg tattgaggac 960gtggcaaaca
ctcgtgtcaa ggaatgtgat aggcttgagg catgtgctca aaaccttaga
1020gctatgggaa tcacggtgca tactgggcac gattggattg agattctccc
tgggactcca 1080aagccaacgg gaatagctac gcacggagat cacagaatcg
ttatgtcctt cgcagtggct 1140ggtttgttga cccctgggct gacatacgat
gatcctggct gcgtccgcaa gacttttcca 1200aggttccacg aagttttcgc
tgactttgct gcatcacccc aagcctga 1248251245DNAArtificial
sequenceDGT-31 v3 nucleotide 25atgactgtga ttgacatccc tggctcaaag
tcagttactg ccagagcatt gttcctcgca 60gcagctgctg atggcactac aactcttttg
agacctcttc acagcgatga cacggaaggc 120ttcactgagg gtctcactcg
tttgggatac gcagtggtta gagaacccga taggtggcac 180atagaaggac
gtccctccgg tccagcagca gcagatgcag aagttcactg tagggacggt
240gctacaactg ctcgctttct tccaaccctt gcagctgctg ctgcctccgg
aacgtatcgt 300ttcgacgcat cagctcagat gaggcgtaga cccctcgctc
ccctcacgga agctcttaga 360acacttggag tggaccttag gcatgatgga
gctgaaggcc accacccctt gacaattcaa 420gcctctggtg ttaagggtgg
aggacttacg ctcgacgctg gtgagtcatc tcagtacttg 480acagctctgc
tcatgcttgg tcctctgacc gcagagggac tgagaataga agttacggag
540cttgtctctg ctccttatgt ggagatcacc cttgcaatga tgagaggctt
tggtgtggag 600gttgttaggg aggggaatac tttcactgtg cctcctggag
gttacagagc tacaacttat 660gccatagaac cggacgcaag cacagcttcc
tacttctttg cagcagcagc cctcactggg 720agggaagtga cggtgcctgg
cttgggcact ggagcacttc aaggtgatct taggttcacg 780gaggtcctca
gaaggatgga cgctgatgtt cgcacaacgt ccgactctac aacagtgcgc
840tcagatggtc gccttgctgg gttgactgtc aacatgaggg acataagcga
cacaatgcca 900acactggcag ctatagctcc gtacgcaagc tcaccagtta
ggatcgagga tgtcgcaaac 960acccgtgtga aggaatgtga taggctggag
gcttgcgctc agaatctccg ctcaatgggc 1020atcaccgttc gcactggacc
agattggatt gagatccatc ctgggactcc tagaccgacc 1080gagatagcca
cacacggtga tcatagaatc gtcatgtcat ttgccgtggc tggacttaga
1140acccctggga tgtcttacga tgaccctggc tgcgttcgca agacttttcc
tcgttttcat 1200gaagagtttg cagccttcgt ggagcgctca tccgctggag agtga
124526213DNAArtificial sequenceChloroplast Transit Peptide
26atgcttgcta gacaaggtgg aagtctgaga gcttctcaat gcaacgctgg acttgctaga
60agagttgaag ttggtgctct tgttgttcct agacctatct ctgttaacga cgttgttcct
120cacgtttact ctgctccact ttctgttgct agaaggtctt gctctaagtc
ctccattagg 180tccactagaa ggcttcaaac tactgtgtgc tct
21327186DNAArtificial sequenceChloroplast Transit Peptide
27atgcaactcc tgaatcagag gcaagccctg cgtcttggtc gttcatctgc ttcaaagaac
60cagcaagttg ctccactggc ctctaggcct gcttcttcct tgagcgtcag cgcatccagc
120gtcgcacctg cacctgcttg ctcagctcct gctggagctg gaaggcgtgc
tgttgtcgtg 180agagca 18628198DNAArtificial sequenceChloroplast
Transit Peptide 28atggctcaat ctagcagaat ctgccacggt gtgcagaacc
catgtgtgat catttccaat 60ctctccaaat ccaaccagaa caaatctcct ttctcagtca
gcctcaagac tcaccagcag 120cagcgtcgtg cttaccagat atctagctgg
ggattgaaga agtcaaacaa cgggtccgtg 180attcgtccgg ttaaggca
19829198DNAArtificial sequenceChloroplast Transit Peptide
29atggcacaag ccagccgtat ctgccagaat ccatgtgtga tatccaatct ccccaaaagc
60aaccaccgta agtccccttt ctctgtctca ctcaagacgc atcagcctag agcctcttca
120tggggactta agaagtctgg caccatgctg aacggttcag tgattagacc
cgtcaaggtg 180acagcttctg tttccgca 19830225DNAArtificial
sequenceChloroplast Transit Peptide 30atggcacaat ctagcagaat
ctgccacggt gtgcagaacc catgtgtgat catttcaaat 60ctctcaaagt ccaatcagaa
caaatcacct ttctccgtct ccctcaagac acaccagcat 120ccaagggcat
acccgataag cagctcatgg ggactcaaga agagcggaat gactctgatt
180ggctctgagc ttcgtcctct taaggttatg tcctctgttt ccgca
22531207DNAArtificial sequenceChloroplast Transit Peptide
31atggcacaag ttagcagaat ctgtaatggt gtgcagaacc catctcttat ctccaatctc
60tcaaagtcca gccaacgtaa gtctcccctc agcgtgtctc tgaaaactca gcagcccaga
120gcttcttcat ggggtttgaa gaaatctgga acgatgctta acggctcagt
cattcgtccg 180gttaaggtga cagcctccgt ctccgct 207321464DNAArtificial
sequenceIn-frame fusion of dgt-28 v5 and TraP4 v2 32atgcttgcta
gacaaggtgg aagtctgaga gcttctcaat gcaacgctgg acttgctaga 60agagttgaag
ttggtgctct tgttgttcct agacctatct ctgttaacga cgttgttcct
120cacgtttact ctgctccact ttctgttgct agaaggtctt gctctaagtc
ctccattagg 180tccactagaa ggcttcaaac tactgtgtgc tctgctgcaa
gagggatgcc agccttgtct 240ttacctggat caaagagtat cacagctagg
gcactctttc ttgctgctgc tgctgatggg 300gttactactt tggtgaggcc
attgagaagt gacgacacag aaggattcgc tgaggggtta 360gttcgtttag
gctatcgtgt agggaggaca cccgatactt ggcaagtcga tggcagacca
420caaggaccag cagtggctga ggctgacgtc tactgtagag acggagcaac
caccgctaga 480ttcttgccaa ccttagcagc tgctggtcac ggaacataca
gatttgatgc ttcaccacag 540atgaggagac gtcctctttt gcccttaagc
agagccttga gggatttggg tgtcgatctt 600agacacgaag aagctgaagg
tcatcaccct ctgactgtcc gtgctgctgg ggttgaagga 660ggagaggtta
ctttggatgc tggtcagtca agtcagtatc tcactgcctt gttgctcctt
720ggtcccctta caagacaagg actgaggata agggttactg atttggtgtc
agcaccatac 780gtggagatta cgcttgcaat gatgagggct ttcggagttg
aagtggcaag ggagggagat 840gtgttcgttg ttccacctgg tggatatcgt
gcaactacgt atgctataga acccgacgca 900agtactgctt cttacttctt
cgcagctgct gctttgactc ctggagctga agtgactgta 960cctgggttag
gcacgggagc acttcaagga gatttgggat ttgtagatgt cttaaggaga
1020atgggagccg aggtgtccgt aggagctgat gcaaccactg ttagaggaac
tggtgaattg 1080cgtggcctta cagccaacat gagagacata agtgatacga
tgccgaccct cgctgcaata 1140gcaccctttg ctagtgctcc agttagaatc
gaggatgttg ccaacactcg tgtcaaagaa 1200tgtgacagac ttgaggcttg
tgcagagaac cttaggaggt tgggagtaag ggttgcaacg 1260ggtccggact
ggattgagat acaccctggt ccagctactg gtgctcaagt cacaagctat
1320ggtgatcaca gaattgtgat gtcatttgca gtgactggac ttcgtgtgcc
tgggatcagc 1380ttcgacgacc ctggctgtgt tcgtaagact tttcctgggt
ttcacgaggc tttcgcagaa 1440ttgaggcgtg gcattgggag ctga
1464331434DNAArtificial sequenceIn-frame fusion of dgt-28 v5 and
TraP5 v2 33atgcaactcc tgaatcagag gcaagccctg cgtcttggtc gttcatctgc
ttcaaagaac 60cagcaagttg ctccactggc ctctaggcct gcttcttcct tgagcgtcag
cgcatccagc 120gtcgcacctg cacctgcttg ctcagctcct gctggagctg
gaaggcgtgc tgttgtcgtg 180agagcagcaa gagggatgcc agccttgtct
ttacctggat caaagagtat cacagctagg 240gcactctttc ttgctgctgc
tgctgatggg gttactactt tggtgaggcc attgagaagt 300gacgacacag
aaggattcgc tgaggggtta gttcgtttag gctatcgtgt agggaggaca
360cccgatactt ggcaagtcga tggcagacca caaggaccag cagtggctga
ggctgacgtc 420tactgtagag acggagcaac caccgctaga ttcttgccaa
ccttagcagc tgctggtcac 480ggaacataca gatttgatgc ttcaccacag
atgaggagac gtcctctttt gcccttaagc 540agagccttga gggatttggg
tgtcgatctt agacacgaag aagctgaagg tcatcaccct 600ctgactgtcc
gtgctgctgg ggttgaagga ggagaggtta ctttggatgc tggtcagtca
660agtcagtatc tcactgcctt gttgctcctt ggtcccctta caagacaagg
actgaggata 720agggttactg atttggtgtc agcaccatac gtggagatta
cgcttgcaat gatgagggct 780ttcggagttg aagtggcaag ggagggagat
gtgttcgttg ttccacctgg tggatatcgt 840gcaactacgt atgctataga
acccgacgca agtactgctt cttacttctt cgcagctgct 900gctttgactc
ctggagctga agtgactgta cctgggttag gcacgggagc acttcaagga
960gatttgggat ttgtagatgt cttaaggaga atgggagccg aggtgtccgt
aggagctgat 1020gcaaccactg ttagaggaac tggtgaattg cgtggcctta
cagccaac
References