U.S. patent application number 11/926174 was filed with the patent office on 2008-02-28 for polynucleotide encoding a maize herbicide resistance gene and methods for use.
This patent application is currently assigned to E.I.du PONT de NEMOURS and COMPANY. Invention is credited to Thao Dam, Anthony D. JR. Guida, Christine B. Hazel, Bailin Li, Mark E. Williams.
Application Number | 20080050822 11/926174 |
Document ID | / |
Family ID | 38325668 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080050822 |
Kind Code |
A1 |
Dam; Thao ; et al. |
February 28, 2008 |
Polynucleotide Encoding a Maize Herbicide Resistance Gene and
Methods for Use
Abstract
This invention relates to polynucleotide sequences encoding a
gene that can confer resistance to at least one herbicide. It
further relates to plants and seeds of plants carrying chimeric
genes comprising said polynucleotide sequences, which enhance or
confer resistance to at least one herbicide, and methods of making
said plants and seeds. The invention further presents sequences
that can be used as molecular markers that in turn can be used to
identify the region of interest in corn lines resulting from new
crosses and to quickly and efficiently select the best lines for
breeding strategies by avoiding sensitive lines.
Inventors: |
Dam; Thao; (Bear, DE)
; Guida; Anthony D. JR.; (Newark, DE) ; Hazel;
Christine B.; (Port Deposit, MD) ; Li; Bailin;
(Hockessin, DE) ; Williams; Mark E.; (Newark,
DE) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE
P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
E.I.du PONT de NEMOURS and
COMPANY
|
Family ID: |
38325668 |
Appl. No.: |
11/926174 |
Filed: |
October 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11683737 |
Mar 8, 2007 |
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11926174 |
Oct 29, 2007 |
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60780946 |
Mar 9, 2006 |
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60888634 |
Feb 7, 2007 |
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Current U.S.
Class: |
435/419 ;
435/320.1; 536/23.6; 800/298; 800/312; 800/314; 800/317.2; 800/320;
800/320.1; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8278 20130101;
C12N 15/8274 20130101 |
Class at
Publication: |
435/419 ;
435/320.1; 536/023.6; 800/298; 800/312; 800/314; 800/317.2;
800/320; 800/320.1; 800/320.2; 800/320.3; 800/322 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 15/00 20060101
C12N015/00 |
Claims
1. An isolated polynucleotide comprising: (a) a nucleotide sequence
encoding a polypeptide capable of conferring resistance to at least
three herbicides, wherein each herbicide is a member of a different
class of herbicides selected from the group consisting of: (i) the
ALS-inhibiting class; (ii) the pigment synthesis-inhibiting class;
(iii) the PPO-inhibiting class; (iv) the PS II-inhibiting class;
and (v) the synthetic auxin class wherein the polypeptide has an
amino acid sequence of at least 85% identity, when compared to SEQ
ID NO:1 based on the Needleman-Wunsch alignment algorithm, or (b) a
complement of the nucleotide sequence, wherein the complement and
the nucleotide sequence consist of the same number of nucleotides
and are 100% complementary.
2. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO: 1 have
at least 90% identity based on the Needleman-Wunsch alignment
algorithm.
3. The polynucleotide of claim 1, wherein the amino acid sequence
of the polypeptide and the amino acid sequence of SEQ ID NO: 1 have
at least 95% identity based on the Needleman-Wunsch alignment
algorithm.
4. The polynucleotide of claim 1, wherein the nucleotide sequence
comprises SEQ ID NO: 1.
5. A vector comprising the polynucleotide of claim 1.
6. A recombinant DNA construct comprising the polynucleotide of
claim 1 operably linked to at least one regulatory sequence.
7. A plant cell comprising the recombinant DNA construct of claim
6.
8. A plant comprising the recombinant DNA construct of claim 6.
9. A seed comprising the recombinant DNA construct of claim 6.
10. The plant of claim 8, wherein said plant is a monocot.
11. The plant of claim 10, wherein said monocot is selected from
the group consisting of maize, wheat, barley, oats, switchgrass,
sorghum, and rice.
12. The plant of claim 8, wherein said plant is a dicot.
13. The plant of claim 12, wherein said dicot is selected from the
group consisting of soybean, canola, potato, cotton, and
sunflower.
14. The plant of claim 8, wherein said plant further comprises a
gene encoding a polypeptide with glyphosate N-acetyltransferase
activity.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Utility
Application Ser. No. 11/683,737, filed Mar. 8, 2007, which claims
the benefit of U.S. Provisional Application Ser. No. 60/780,946,
filed Mar. 9, 2006 and U.S. Provisional Application Ser. No.
60/888,634 filed Feb. 7, 2007, the contents of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods useful in
creating or enhancing herbicide resistance in plants. Additionally,
the invention relates to plants that have been genetically
transformed with the compositions of the invention.
BACKGROUND OF THE INVENTION
[0003] In the commercial production of crops, it is desirable to
easily and quickly eliminate unwanted plants (i.e., "weeds") from a
field of crop plants. An ideal treatment would be one which could
be applied to an entire field but which would eliminate only the
unwanted plants while leaving the crop plants unharmed. One such
treatment system involves the use of crop plants that are tolerant
to a herbicide. When the herbicide is sprayed on a field of
herbicide-tolerant crop plants, the crop plants continue to thrive
while non-herbicide-tolerant weeds are killed or severely
damaged.
[0004] Crop tolerance to specific herbicides can be conferred by
engineering genes into crops which encode appropriate herbicide
metabolizing enzymes. In some cases these enzymes, and the nucleic
acids that encode them, originate in a plant. In other cases, they
are derived from other organisms, such as microbes. See, e.g.,
Padgette et al. (1996) "New weed control opportunities: Development
of soybeans with a Round UP Ready.TM. gene" and Vasil (1996)
"Phosphinothricin-resistant crops," both in Herbicide-Resistant
Crops, ed. Duke (CRC Press, Boca Raton, Fla.) pp. 54-84 and pp.
85-91. Indeed, transgenic plants have been engineered to express a
variety of herbicide tolerance genes from a variety of organisms,
including a gene encoding a chimeric protein of rat cytochrome
P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et
al. (1994) Plant Physiol. 106: 17), among other plant P450 genes
(see, for example, Didierjean, L. et al. (2002) Plant Physiol. 130:
179-189; Morant, M. S. et al. (2003) Opinion in Biotechnology
14:151-162). Other genes that confer tolerance to herbicides
include: acetohydroxy acid synthase ("AHAS"), which has been found
to confer resistance to multiple types of ALS herbicides on plants
expressing it and has been introduced into a variety of plants
(see, e.g., Hattori et al. (1995) Mol. Gen. Genet. 246: 419);
glutathione reductase and superoxide dismutase (Aono et al. (1995)
Plant Cell Physiol. 36: 1687); and genes for various
phosphotransferases (Datta et al. (1992) Plant Mol. Biol. 20:
619).
[0005] While herbicide-tolerant crop plants are presently
commercially available, improvements in every aspect of crop
production are continuously in demand. Herbicides and crops that
are presently commercially available unfortunately have particular
characteristics which can limit their usefulness in commercial crop
production. Particularly, individual herbicides have different and
incomplete spectra of activity against common weed species.
[0006] The acetolactate synthase, or ALS (also known as AHAS)
family of herbicides control weeds by inhibiting the production of
branch chain of amino acids that are essential to plant growth and
development. Specifically, they bind to the plant ALS enzyme.
Commonly used herbicides in this family include nicosulfuron,
rimsulfuron, and chlorsulfuron, among others. Herbicides in this
category can be quite crop-specific. Embodiments of the invention
relate to plants that are resistant to members of the
ALS-inhibiting class of herbicides, which encompasses 5 sub-classes
of herbicides including, but not limited to, the sulfonylurea (SU)
family of herbicides and the imidazolinone family of
herbicides.
[0007] The pigment synthesis-inhibiting class of herbicides targets
the enzymes that allow plants to synthesize pigments, such as
carotenoid pigments or chlorophyll pigments. Loss of pigment
results in photo-destruction of chlorophyll and whitening of plant
tissues, which is why these herbicides are often called "bleaching"
herbicides. An example of a sub-class of the bleaching herbicides
is the HPPD-inhibiting class, which inhibits the
4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme (Lee et al.
(1997) Weed Sci. 45:601-609). Herbicides in this family include,
but are not limited to, mesotrione, tembotrione, topramezone and
sulcotrione, among others. Corn is generally tolerant to mesotrione
due to metabolism of the herbicide (Mitchell et al. (2001) Pest
Mgt. Sci. 57:120-128). The same detoxification system may give
tolerance to both mesotrione and some SU herbicides (Green &
Williams (2004) Proceedings Weed Science Society of America 44:13).
Embodiments of the invention relate to plants that are resistant to
members of the pigment synthesis-inhibiting class of
herbicides.
[0008] The protoporphyrinogen oxidase (PPO)-inhibiting class of
herbicides interferes with the synthesis of chlorophyll, resulting
in compounds that produce highly active compounds (free-radicals).
These reactive compounds disrupt cell membranes which results in
the leaf burning associated with post-emergence applications of
these products. Herbicides in this family include, but are not
limited to, acifluorfen, fomesafen, lactofen, sulfentrazone,
carfentrazone, flumiclorac and flumioxazin, among others.
Embodiments of the invention relate to plants that are resistant to
members of the PPO-inhibiting class of herbicides.
[0009] Photosystem II (PSII)-inhibiting herbicides have a mode of
action that involves interaction with components in the electron
transfer chain of Photosystem II. Photosynthesis requires the
transfer of electrons from Photosystem II to Photosystem I. A key
step in this electron transfer chain is the reduction of
plastoquinone (PQ) by the D.sub.1 protein in the thylakoid
membrane. PSII-inhibitor herbicides bind to the D.sub.1 protein,
thus inhibiting PQ binding and interrupting the electron transfer
process. This results in the plants not being able to fix carbon
dioxide and produce the carbohydrates needed for the plant to
survive. The block in electron transfer also causes an oxidative
stress and the generation of radicals which cause rapid cellular
damage. PSII-inhibiting herbicides are represented by several
herbicide families, including the symmetrical triazines,
triazinones (asymmetrical triazines), substituted ureas, uracils,
pyridazinones, phenyl carbamates, nitrites, benzothiadiazoles,
phenyl pyridazines, and acid amides. Embodiments of the invention
relate to plants that are resistant to members of the PS
II-inhibiting class of herbicides.
[0010] Synthetic auxin herbicides are a widely used class of
herbicides that mimic the natural auxin hormones produced by
plants. Auxins regulate many plant processes, including cell growth
and differentiation. Auxins are generally present at low
concentrations in the plant. Synthetic auxin herbicides mimic
natural auxins and cause relatively high concentrations in the cell
that result in a rapid growth response. Susceptible plants treated
with these herbicides exhibit symptoms that could be called `auxin
overdose`, and eventually die as a result of increased rates of
disorganized and uncontrolled growth. Embodiments of the invention
relate to plants that are resistant to members of the synthetic
auxin class of herbicides.
[0011] Some embodiments of this invention are based on the fine
mapping, cloning and characterization of the gene responsible for
an important herbicide resistance mechanism in maize.
[0012] It has been known since the early 1990s that natural
tolerance in maize (Zea mays L.) to a subset of sulfonylurea
herbicides (nicosulfuron [Dupont Accent.RTM. herbicide],
rimsulfuron, primisulfuron, and thifensulfuron) is controlled by a
single gene (named nsf by Kang (1993) Journal of Heredity 84(3):
216-217), with resistance dominant and sensitivity recessive (Harms
et al. (1990) Theor. Appl. Genet. 80:353-358; Kang (1993) supra;
Green & Uhlrich (1993) Weed Sci. 41:508-516; Green &
Uhlrich (1994) Pestic. Sci. 40:187-191). It is also known that
tolerant maize plants metabolize nicosulfuron by hydroxylation,
with the characteristics of a cytochrome P450 (Fonne-Pfister et al.
(1990) Pesticide Biochem. Physiol. 37:165-173; Brown &
Cotterman (1994) Chem. Plant Prot. 10:47-81). It has been suggested
that the same corn gene responsible for determining tolerance to
some sulfonylurea herbicides is also responsible for the tolerance
to bentazon (Barrett et al. (1997) Role of cytochrome P-450 in
herbicide metabolism and selectivity and multiple herbicide
metabolizing cytochrome P-450 activities in maize. In K. K.
Hatzios, ed. Regulation of Enzymatic Systems Detoxifying
Xenobiotics in Plants. Dordrecht: Kluwer Academic. pp. 35-50; Green
(1998) Weed Technology 12:474-477) and HPPD inhibitor herbicides
such as mesotrione (Green & Williams (2004) supra; Williams et
al. (2005) HortScience 40(6):1801-1805). Recent advances in the
development of the maize physical map and integrated markers
(Bortiri et al. (2006) Curr Opin Plant Biol. 9(2):164-71) has
allowed a positional cloning approach to be used for identifying
the Nsf1 locus.
[0013] The Nsf1 resistance gene of the embodiments of the present
invention encodes a novel gene related to the cytochrome P450
family. While multiple cytochrome P450 genes have been described,
they differ widely in their response to different pathogens and
exact action. The novel cytochrome P450 gene described in this
disclosure has been demonstrated to provide improved tolerance or
resistance to numerous herbicides, including nicosulfuron,
rimsulfuron, primisulfuron, thifensulfuron and mesotrione.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to embodiments including
an isolated polynucleotide comprising a nucleotide sequence
encoding a polypeptide capable of conferring resistance to at least
one herbicide, wherein the polypeptide has an amino acid sequence
of at least 85, 90 or 95% identity, when compared to SEQ ID NO:1
based on the Needleman-Wunsch alignment algorithm, or a complement
of the nucleotide sequence, wherein the complement and the
nucleotide sequence consist of the same number of nucleotides and
are 100% complementary. The herbicides to which the polynucleotide
of the embodiments imparts resistance include members of the
ALS-inhibiting class; the pigment synthesis-inhibiting class; the
PPO-inhibiting class; the PS II-inhibiting class; and the synthetic
auxin class of herbicides. The polynucleotide of the embodiments
may impart resistance to one or more herbicides from the same
class, or from different classes, including representative members
from all 5 classes.
[0015] Additional embodiments of the present invention include a
vector comprising the polynucleotide of the embodiments and a
recombinant DNA construct comprising the polynucleotide of the
embodiments, operably linked to at least one regulatory sequence. A
plant cell, as well as a plant and a seed each comprising the
recombinant DNA construct of an embodiment of the present invention
are also encompassed. Also included are plants comprising
additional polynucleotides encoding polypeptides responsible for
traits of interest, such as polypeptides having glyphosate
N-acetyltransferase activity, insecticidal Bt polypeptides, and
other polypeptides of interest. Plants comprising these
polynucleotides include monocots and dicots, including, but not
limited to, maize, wheat, barley, oats, switchgrass, sorghum, rice,
soybean, canola, potato, cotton, and sunflower.
[0016] The methods embodied by the present invention include 1) a
method for transforming a cell, comprising transforming a cell with
the polynucleotide of an embodiment of the present invention, 2) a
method for producing a plant comprising transforming a plant cell
with the recombinant DNA construct of an embodiment of the present
invention and regenerating a plant from the transformed plant cell,
and 3) methods of conferring or enhancing resistance to at least
one herbicide, comprising transforming a plant with the recombinant
DNA construct of an embodiment of the present invention, thereby
conferring or enhancing resistance to at least one herbicide, such
as a member of the ALS-inhibiting class; the pigment
synthesis-inhibiting class; the PPO-inhibiting class; the PS
II-inhibiting class; and the synthetic auxin class of
herbicides.
[0017] In addition, an embodiment of the invention is a variant
allele of the Nsf1 sequence in which a specific single amino acid
change (see Example 2) renders the gene inoperative, resulting in
sensitivity to at least one ALS or HPPD inhibitor herbicide to
which most corn is resistant. Accordingly, an additional method
embodied by the present invention is a method of using the variant
of the Nsf1 gene as a marker in breeding strategies to avoid
incorporating the sensitive allele.
[0018] Methods of altering the level of expression of a protein
capable of conferring resistance to at least one herbicide in a
plant cell comprising (a) transforming a plant cell with the
recombinant DNA construct of an embodiment of the present invention
and (b) growing the transformed plant cell under conditions that
are suitable for expression of the recombinant DNA construct
wherein expression of the recombinant DNA construct results in
production of altered levels of a protein capable of conferring
resistance to at least one herbicide in the transformed host are
also embodied by the present invention. The herbicides for which
resistance may be conferred include, for example, members of the
ALS-inhibiting class; the pigment synthesis-inhibiting class; the
PPO-inhibiting class; the PS II-inhibiting class; and the synthetic
auxin class of herbicides.
[0019] Herbicides to which a polynucleotide of the embodiments may
confer or enhance resistance include, but are not limited to,
herbicides selected from the ALS-inhibiting class of herbicides
such as nicosulfuron, rimsulfuron, primisulfuron, imazethapyr,
chlorsulfuron, chlorimuron ethyl, triasulfuron, flumetsulam and
imazaquin. Additionally, such herbicides may be selected from the
pigment synthesis-inhibiting class of herbicides, such as
isoxaflutole, topramezone, sulcatrione and tembotrione. Such
herbicides may also be selected from the PPO-inhibiting class of
herbicides, such as acifluorfen, flumioxan and sulfentrazone.
Optionally, such herbicides may be selected from the PS
II-inhibiting class of herbicides, such as diuron, linuron,
bentazon and chlorotoluron. Such herbicides may also be selected
from the synthetic auxin class of herbicides, such as dicamba.
[0020] Methods of the embodiments include a method of determining
the presence of the polynucleotide of the embodiments or the Nsf1
locus in a plant, comprising at least one of: (a) isolating nucleic
acid molecules from the plant and determining if an Nsf1 gene is
present by attempting to amplify sequences homologous to the
polynucleotide; or (b) isolating nucleic acid molecules from the
plant and performing a Southern hybridization, or (c) isolating
proteins from the plant and performing a western blot using
antibodies to the NSF1 protein, or (d) isolating proteins from the
plant and performing an ELISA assay using antibodies to the NSF1
protein, thereby determining the presence of the polynucleotide of
claim 1 in the plant.
[0021] Also encompassed by the embodiments are plants with enhanced
tolerance to at least one herbicide, comprising the Nsf1 gene in a
recombinant DNA construct. Such plants further comprise a second
herbicide resistance gene providing a certain level of tolerance to
a herbicide selected from a class of herbicides selected from the
group consisting of: [0022] (a) the ALS-inhibiting class; [0023]
(b) the pigment synthesis-inhibiting class; [0024] (c) the
PPO-inhibiting class; [0025] (d) the PS II-inhibiting class; and
[0026] (e) the synthetic auxin class; such that the presence of the
Nsf1 gene confers upon the plant a higher level of tolerance to the
same herbicide than the tolerance level exhibited by a plant
comprising the second herbicide resistance gene but not comprising
the Nsf1 gene.
[0027] Also encompassed by the embodiments are soybean plants
comprising the Nsf1 gene, wherein such soybean plants also exhibit
soybean cyst nematode resistance. Such plants may have been created
through transformation or plant breeding techniques, and may have
been bred from germplasm such as those selected from the group
consisting of, Peking, PI88788, PI89772, PI90763, PI209332,
PI404189A, PI437654, PI438489B, PI467312, PI468916, Hartwig,
J87-233, and progeny derived from any of the listed sources.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1(a-e) is a multiple sequence alignment of the
polypeptide sequence of the embodiments (SEQ ID NO: 2) comparing it
to other known Cytochrome P450 polypeptides (SEQ ID NOs: 3-13).
FIG. 1d also indicates the position of the most commonly conserved
domain of the cytochrome P450 family (SEQ ID NO: 14). Identical
residues in the alignment are indicated in upper case letters.
[0029] FIG. 2(a-b) is a multiple sequence alignment of the
polypeptide sequences of several sensitive and resistant corn lines
showing the commonly conserved domain of the cytochrome P450 family
(SEQ ID NO: 14) as well as variations among the sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Embodiments of the present invention provide compositions
and methods directed to inducing herbicide resistance in plants.
The compositions are novel nucleotide and amino acid sequences that
confer or enhance resistance to one or more members of one or more
classes of herbicides, including the ALS-inhibiting,
PPO-inhibiting, pigment synthesis-inhibiting, PS II-inhibiting and
synthetic auxin herbicide classes, whose members include, but are
not limited to, nicosulfuron, rimsulfuron, primisulfuron, and
mesotrione. Specifically, certain embodiments provide polypeptides
having the amino acid sequence set forth in SEQ ID NO: 2, and
variants and fragments thereof. Isolated nucleic acid molecules,
and variants and fragments thereof, comprising nucleotide sequences
that encode the amino acid sequence shown in SEQ ID NO: 2 are
further provided.
[0031] One example of the native nucleotide sequence that encodes
the polypeptide of SEQ ID NO: 2 is set forth in SEQ ID NO: 1.
Plants, plant cells, seeds, and microorganisms comprising a
nucleotide sequence that encodes a polypeptide of the embodiments
are also disclosed herein.
[0032] The full length polypeptide of the embodiments (SEQ ID NO:
2) shares varying degrees of homology with known polypeptides of
the cytochrome P450 family. In particular, the novel polypeptide of
the embodiments shares homology with cytochrome P450 proteins
isolated from Oryza sativa: Accession Nos. XP.sub.--469850 (SEQ ID
NO: 3), ABC69856 (SEQ ID NO: 4); XP.sub.--469849 (SEQ ID NO: 11)
and XP.sub.--469851 (SEQ ID NO: 12); and XP.sub.--469852 (SEQ ID
NO: 13) and Lolium rigidum: Accession Nos. AAK38080 (SEQ ID NO: 5);
AAK38079 (SEQ ID NO: 6); AAK38081 (SEQ ID NO: 7); BAD27508 (SEQ ID
NO: 8); BAD27507 (SEQ ID NO: 9) and BAD27506 (SEQ ID NO: 10). FIG.
1 provides an alignment of the amino acid sequence set forth in SEQ
ID NO: 2 with the O. sativa and L. rigidum cytochrome P450 proteins
(SEQ ID NOs: 3-13).
[0033] Amino acid alignments performed using the GAP program
indicate that SEQ ID NO:2 shares the sequence similarities shown in
Table 1 with the O. sativa and L. rigidum cytochrome P450 proteins.
TABLE-US-00001 TABLE 1 Comparison of NSF1 Peptide to other
Cytochrome P450 peptides Other Cytochrome P450 Protein Percent
Identity Percent Similarity XP_469850 (SEQ ID NO: 3) 67% 76%
ABC69856 (SEQ ID NO: 4) 67% 76% AAK38080 (SEQ ID NO: 5) 68% 76%
AAK38079 (SEQ ID NO: 6) 67% 77% AAK38081 (SEQ ID NO: 7) 67% 76%
BAD27508 (SEQ ID NO: 8) 67% 76% BAD27507 (SEQ ID NO: 9) 67% 76%
BAD27506 (SEQ ID NO: 10) 67% 76% XP_469849 (SEQ ID NO: 11) 66% 75%
XP_469851 (SEQ ID NO: 12) 61% 71% XP_469852 (SEQ ID NO: 13) 60%
72%
[0034] The cytochrome P450 family of genes in plants catalyze
extremely diverse and often complex regiospecific and/or
stereospecific reactions in the biosynthesis or catabolism of plant
bioactive molecules. (Morant et al. (2003) Curr. Opin. Biotech.
14(2): 151-162). P450s are heme proteins that catalyze the
activation of molecular oxygen by using electrons from NADPH. In
the Arabidopsis thaliana genome alone, there are an estimated over
300 cytochromes P450 (Werck-Reichhart et al. (2000) Trends in Plant
Science 5(3): 116-123). Common structural features occur in plant
cytochromes P450 and help identify them as such. These features
include the F-X-X-G-X-R-X-C-X-G (SEQ ID NO: 14) motif generally
found near the C-terminus (see FIG. 1d). About 150 residues
upstream, another conserved motif generally found follows the
A/G-G-X-D/E-T-T/S (SEQ ID NO: 15) motif and corresponds to the
region of the peptide responsible for oxygen-binding and
activation.
[0035] The nucleic acids and polypeptides of the embodiments find
use in methods for conferring or enhancing herbicide resistance to
a plant. Accordingly, the compositions and methods disclosed herein
are useful in protecting plants from damage caused by herbicides.
"Herbicide resistance" is intended to mean that a plant or plant
cell has the ability to tolerate a higher concentration of a
herbicide than plants or cells which are not resistant, or to
tolerate a certain concentration of a herbicide for a longer time
than cells or plants which are not resistant. That is, herbicides
are prevented from causing plant injury, or the injury caused by
the herbicide is minimized or lessened, such as, for example, the
reduction of leaf yellowing and associated yield loss. One of skill
in the art will appreciate that the compositions and methods
disclosed herein can be used with other compositions and methods
available in the art for increasing or enhancing plant herbicide
resistance. The term "enhance" refers to improve, increase,
amplify, multiply, elevate, raise, and the like.
[0036] In particular aspects, the embodiments include methods for
conferring or enhancing herbicide resistance in a plant comprising
introducing into a plant at least one DNA construct, wherein the
DNA construct comprises a nucleotide sequence encoding a herbicide
resistance polypeptide of the embodiments operably linked to a
promoter that drives expression in the plant. The plant expresses
the polypeptide, thereby conferring or enhancing herbicide
resistance upon the plant, or improving the plant's inherent level
of resistance. In particular embodiments, the gene confers or
enhances resistance to at least one herbicide of the
ALS-inhibiting, pigment synthesis-inhibiting, PPO-inhibiting, PS
II-inhibiting or synthetic auxin herbicide classes, whose members
include, but are not limited to, the herbicides nicosulfuron,
rimsulfuron, primisulfuron, thifensulfuron, bentazon, and
mesotrione.
[0037] Expression of a polypeptide of the embodiments may be
targeted to specific plant tissues, but generally in the case of
herbicide resistance, continuous expression is desired throughout
the cells of a plant. Therefore, while many promoters could be used
in the embodiments of the invention, generally constitutive
promoters are utilized. A constitutive promoter is a promoter that
directs expression of a gene throughout the various parts of a
plant and continuously throughout plant development.
[0038] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues (e.g., peptide nucleic acids) having the essential
nature of natural nucleotides in that they hybridize to
single-stranded nucleic acids in a manner similar to naturally
occurring nucleotides.
[0039] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers. Polypeptides of the
embodiments can be produced either from a nucleic acid disclosed
herein, or by the use of standard molecular biology techniques. For
example, a truncated protein of the embodiments can be produced by
expression of a recombinant nucleic acid of the embodiments in an
appropriate host cell, or alternatively by a combination of ex vivo
procedures, such as protease digestion and purification.
[0040] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified nucleic acid mean that the nucleic
acid comprises the requisite information to direct translation of
the nucleotide sequence into a specified protein. The information
by which a protein is encoded is specified by the use of codons. A
nucleic acid encoding a protein may comprise non-translated
sequences (e.g., introns) within translated regions of the nucleic
acid or may lack such intervening non-translated sequences (e.g.,
as in cDNA).
[0041] The embodiments of the invention encompass isolated or
substantially purified polynucleotide or protein compositions. An
"isolated" or "purified" polynucleotide or protein, or biologically
active portion thereof, is substantially or essentially free from
components that normally accompany or interact with the
polynucleotide or protein as found in its naturally occurring
environment. Thus, an isolated or purified polynucleotide or
protein is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. Optimally, an "isolated" polynucleotide is
free of sequences (optimally protein encoding sequences) that
naturally flank the polynucleotide (i.e., sequences located at the
5' and 3' ends of the polynucleotide) in the genomic DNA of the
organism from which the polynucleotide is derived. For example, in
various embodiments, the isolated polynucleotide can contain less
than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb,
about 0.5 kb, or about 0.1 kb of nucleotide sequence that naturally
flank the polynucleotide in genomic DNA of the cell from which the
polynucleotide is derived. A protein that is substantially free of
cellular material includes preparations of protein having less than
about 30%, about 20%, about 10%, about 5%, or about 1 % (by dry
weight) of contaminating protein. When the protein of the
embodiments, or a biologically active portion thereof, is
recombinantly produced, optimally culture medium represents less
than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry
weight) of chemical precursors or non-protein-of-interest
chemicals.
[0042] Fragments and variants of the disclosed nucleotide sequences
and proteins encoded thereby are also encompassed by the
embodiments. By "fragment" is intended a portion of the nucleotide
sequence or a portion of the amino acid sequence and hence protein
encoded thereby. Fragments of a nucleotide sequence may encode
protein fragments that retain the biological activity of the native
protein and hence have the ability to confer or enhance resistance
to at least one herbicide of the ALS-inhibiting, PPO-inhibiting,
pigment synthesis-inhibiting, PS II-inhibiting or synthetic auxin
herbicide class. Alternatively, fragments of a nucleotide sequence
that are useful as hybridization probes do not necessarily encode
fragment proteins retaining biological activity. Thus, fragments of
a nucleotide sequence may range from at least about 15 nucleotides,
about 50 nucleotides, about 100 nucleotides, and up to the
full-length nucleotide sequence encoding the polypeptides of the
embodiments.
[0043] A fragment of a nucleotide sequence that encodes a
biologically active portion of a polypeptide of the embodiments
will encode at least about 15, about 25, about 30, about 40, or
about 50 contiguous amino acids, or up to the total number of amino
acids present in a full-length polypeptide of the embodiments (for
example, 521 amino acids for SEQ ID NO: 2). Fragments of a
nucleotide sequence that are useful as hybridization probes or PCR
primers generally need not encode a biologically active portion of
a protein.
[0044] As used herein, "full-length sequence" in reference to a
specified polynucleotide means having the entire nucleic acid
sequence of a native sequence. By "native sequence" is intended an
endogenous sequence, i.e., a non-engineered sequence found in an
organism's genome.
[0045] Thus, a fragment of a nucleotide sequence of the embodiments
may encode a biologically active portion of a polypeptide, or it
may be a fragment that can be used as a hybridization probe or PCR
primer using methods disclosed below. A biologically active portion
of an herbicide resistance polypeptide can be prepared by isolating
a portion of one of the nucleotide sequences of the embodiments,
expressing the encoded portion of the protein and assessing the
ability of the encoded portion of the protein to confer or enhance
herbicide resistance in a plant. Nucleic acid molecules that are
fragments of a nucleotide sequence of the embodiments comprise at
least about 15, about 20, about 50, about 75, about 100, or about
150 nucleotides, or up to the number of nucleotides present in a
full-length nucleotide sequence disclosed herein (for example, 1563
nucleotides for SEQ ID NO: 1).
[0046] "Variants" is intended to mean substantially similar
sequences. For polynucleotides, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more internal
sites within the native polynucleotide and/or a substitution of one
or more nucleotides at one or more sites in the native
polynucleotide. As used herein, a "native" polynucleotide or
polypeptide comprises a naturally occurring nucleotide sequence or
amino acid sequence, respectively. One of skill in the art will
recognize that variants of the nucleic acids of the embodiments
will be constructed such that the open reading frame is maintained.
For polynucleotides, conservative variants include those sequences
that, because of the degeneracy of the genetic code, encode the
amino acid sequence of one of the polypeptides of the embodiments.
Naturally occurring allelic variants such as these can be
identified with the use of well-known molecular biology techniques,
as, for example, with polymerase chain reaction (PCR) and
hybridization techniques as outlined below. Variant polynucleotides
also include synthetically derived polynucleotide, such as those
generated, for example, by using site-directed mutagenesis but
which still encode a protein of the embodiments. Generally,
variants of a particular polynucleotide of the embodiments will
have at least about 40%, about 45%, about 50%, about 55%, about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, about 99% or more sequence identity to
that particular polynucleotide as determined by sequence alignment
programs and parameters described elsewhere herein.
[0047] Variants of a particular polynucleotide of the embodiments
(i.e., the reference polynucleotide) can also be evaluated by
comparison of the percent sequence identity between the polypeptide
encoded by a variant polynucleotide and the polypeptide encoded by
the reference polynucleotide. Thus, for example, isolated
polynucleotides that encode a polypeptide with a given percent
sequence identity to the polypeptide of SEQ ID NO: 2 are disclosed.
Percent sequence identity between any two polypeptides can be
calculated using sequence alignment programs and parameters
described elsewhere herein. Where any given pair of polynucleotides
of the embodiments is evaluated by comparison of the percent
sequence identity shared by the two polypeptides they encode, the
percent sequence identity between the two encoded polypeptides is
at least about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, about 99% or more sequence identity.
[0048] "Variant" protein is intended to mean a protein derived from
the native protein by deletion or addition of one or more amino
acids at one or more internal sites in the native protein and/or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the embodiments are
biologically active, that is they continue to possess the desired
biological activity of the native protein, that is, the ability to
confer or enhance plant herbicide resistance as described herein.
Such variants may result from, for example, genetic polymorphism or
from human manipulation. Biologically active variants of a native
protein of the embodiments will have at least about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or
more sequence identity to the amino acid sequence for the native
protein as determined by sequence alignment programs and parameters
described elsewhere herein. A biologically active variant of a
protein of the embodiments may differ from that protein by as few
as about 1-15 amino acid residues, as few as about 1-10, such as
about 6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino
acid residue.
[0049] The proteins of the embodiments may be altered in various
ways including amino acid substitutions, deletions, truncations,
and insertions. Methods for such manipulations are generally known
in the art. For example, amino acid sequence variants and fragments
of the herbicide resistance proteins can be prepared by mutations
in the DNA. Methods for mutagenesis and polynucleotide alterations
are well known in the art. See, for example, Kunkel (1985) Proc.
Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in
Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra,
eds. (1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff et al. (1978) Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by reference. Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be
optimal.
[0050] Thus, the genes and polynucleotides of the embodiments
include both the naturally occurring sequences as well as mutant
forms. Likewise, the proteins of the embodiments encompass both
naturally occurring proteins as well as variations and modified
forms thereof. Such variants will continue to possess the desired
ability to confer or enhance plant resistance to at least one
herbicide of the ALS-inhibiting, PPO-inhibiting, pigment
synthesis-inhibiting, PS II-inhibiting or synthetic auxin herbicide
classes. Obviously, the mutations that will be made in the DNA
encoding the variant must not place the sequence out of reading
frame and optimally will not create complementary regions that
could produce secondary mRNA structure. See, EP Patent No.
0075444.
[0051] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by screening
transgenic plants which have been transformed with the variant
protein to ascertain the effect on the herbicide resistance
characteristics of the plant.
[0052] Variant polynucleotides and proteins also encompass
sequences and proteins derived from mutagenic or recombinogenic
procedures, including and not limited to procedures such as DNA
shuffling. One of skill in the art could envision modifications
that would alter the range of herbicides to which the protein
responds. With such a procedure, one or more different protein
coding sequences can be manipulated to create a new protein
possessing the desired properties. In this manner, libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides comprising sequence regions that
have substantial sequence identity and can be homologously
recombined in vitro or in vivo. For example, using this approach,
sequence motifs encoding a domain of interest may be shuffled
between the protein gene of the embodiments and other known protein
genes to obtain a new gene coding for a protein with an improved
property of interest, such as increased ability to confer or
enhance plant herbicide resistance. Strategies for such DNA
shuffling are known in the art. See, for example, US 2002/0058249;
Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer
(1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.
15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et
al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al.
(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and
5,837,458.
[0053] The polynucleotides of the embodiments can be used to
isolate corresponding sequences from other organisms, particularly
other plants. In this manner, methods such as PCR, hybridization,
and the like can be used to identify such sequences based on their
sequence homology to the sequences set forth herein. Sequences
isolated based on their sequence identity to the entire sequences
set forth herein or to variants and fragments thereof are
encompassed by the embodiments. Such sequences include sequences
that are orthologs of the disclosed sequences. "Orthologs" is
intended to mean genes derived from a common ancestral gene and
which are found in different species as a result of speciation.
Genes found in different species are considered orthologs when
their nucleotide sequences and/or their encoded protein sequences
share at least about 60%, about 70%, about 75%, about 80%, about
85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, or greater
sequence identity. Functions of orthologs are often highly
conserved among species. Thus, isolated polynucleotides that encode
for a protein that confers or enhances plant herbicide resistance
and that hybridize under stringent conditions to the sequences
disclosed herein, or to variants or fragments thereof, are
encompassed by the embodiments.
[0054] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.
(1990) PCR Protocols: A Guide to Methods and Applications (Academic
Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies
(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR
Methods Manual (Academic Press, New York). Known methods of PCR
include, and are not limited to, methods using paired primers,
nested primers, single specific primers, degenerate primers,
gene-specific primers, vector-specific primers,
partially-mismatched primers, and the like.
[0055] In hybridization techniques, all or part of a known
polynucleotide is used as a probe that selectively hybridizes to
other corresponding polynucleotides present in a population of
cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P, or any other detectable marker. Thus, for
example, probes for hybridization can be made by labeling synthetic
oligonucleotides based on the polynucleotides of the embodiments.
Methods for preparation of probes for hybridization and for
construction of cDNA and genomic libraries are generally known in
the art and are disclosed in Sambrook et al. (1989) supra.
[0056] For example, an entire polynucleotide disclosed herein, or
one or more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding polynucleotides and
messenger RNAs. To achieve specific hybridization under a variety
of conditions, such probes include sequences that are unique and
are optimally at least about 10 nucleotides in length, at least
about 15 nucleotides in length, or at least about 20 nucleotides in
length. Such probes may be used to amplify corresponding
polynucleotides from a chosen organism by PCR. This technique may
be used to isolate additional coding sequences from a desired
organism or as a diagnostic assay to determine the presence of
coding sequences in an organism. Hybridization techniques include
hybridization screening of plated DNA libraries (either plaques or
colonies; see, for example, Sambrook et al. (1989) supra.
[0057] Hybridization of such sequences may be carried out under
stringent conditions. By "stringent conditions" or "stringent
hybridization conditions" is intended conditions under which a
probe will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, optimally less than 500 nucleotides in length.
[0058] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1 % SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a final wash in 0.1.times.SSC at 60 to 65.degree. C. for at
least 30 minutes. Optionally, wash buffers may comprise about 0.1%
to about 1% SDS. Duration of hybridization is generally less than
about 24 hours, usually about 4 to about 12 hours. The duration of
the wash time will be at least a length of time sufficient to reach
equilibrium.
[0059] Specificity is typically the function of post-hybridization
washes, the critical factors being the ionic strength and
temperature of the final wash solution. For DNA-DNA hybrids, the
thermal melting point (T.sub.m) can be approximated from the
equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:
T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (%
form)-500/L; where M is the molarity of monovalent cations, % GC is
the percentage of guanosine and cytosine nucleotides in the DNA, %
form is the percentage of formamide in the hybridization solution,
and L is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. T.sub.m is reduced by about 1.degree. C. for each 1% of
mismatching; thus, T.sub.m, hybridization, and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity.
For example, if sequences with .gtoreq.90% identity are sought, the
T.sub.m can be decreased 10.degree. C. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
T.sub.m for the specific sequence and its complement at a defined
ionic strength and pH. However, severely stringent conditions can
utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C.
lower than the T.sub.m; moderately stringent conditions can utilize
a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower
than the T.sub.m; low stringency conditions can utilize a
hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C.
lower than the T.sub.m. Using the equation, hybridization and wash
compositions, and desired T.sub.m, those of ordinary skill will
understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T.sub.m of less than 45.degree.
C. (aqueous solution) or 32.degree. C. (formamide solution), it is
optimal to increase the SSC concentration so that a higher
temperature can be used. An extensive guide to the hybridization of
nucleic acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al.,
eds. (1995) Current Protocols in Molecular Biology, Chapter 2
(Greene Publishing and Wiley-Interscience, New York). See Sambrook
et al. (1989) supra.
[0060] Various procedures can be used to check for the presence or
absence of a particular sequence of DNA, RNA, or a protein. These
include, for example, Southern blots, northern blots, western
blots, and ELISA analysis. Techniques such as these are well known
to those of skill in the art and many references exist which
provide detailed protocols. Such references include Sambrook et al.
(1989) supra, and Crowther, J. R. (2001), The ELISA Guidebook,
Humana Press, Totowa, N.J., USA.
[0061] The following terms are used to describe the sequence
relationships between two or more polynucleotides or polypeptides:
(a) "reference sequence," (b) "comparison window," (c) "sequence
identity," and, (d) "percentage of sequence identity."
[0062] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0063] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two polynucleotides. Generally, the
comparison window is at least about 20 contiguous nucleotides in
length, and optionally can be about 30, about 40, about 50, about
100, or longer. Those of skill in the art understand that to avoid
a high similarity to a reference sequence due to inclusion of gaps
in the polynucleotide sequence a gap penalty is typically
introduced and is subtracted from the number of matches.
[0064] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local
alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0065] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, and are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the embodiments. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the embodiments. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also
be performed manually by inspection.
[0066] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using Gap Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using Gap Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0067] GAP uses the algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, to find the alignment of two complete
sequences that maximizes the number of matches and minimizes the
number of gaps. GAP considers all possible alignments and gap
positions and creates the alignment with the largest number of
matched bases and the fewest gaps. It allows for the provision of a
gap creation penalty and a gap extension penalty in units of
matched bases. GAP must make a profit of gap creation penalty
number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a
profit for each gap inserted of the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap
extension penalty values in Version 10 of the GCG Wisconsin
Genetics Software Package for protein sequences are 8 and 2,
respectively. For nucleotide sequences the default gap creation
penalty is 50 while the default gap extension penalty is 3. The gap
creation and gap extension penalties can be expressed as an integer
selected from the group of integers consisting of from 0 to 200.
Thus, for example, the gap creation and gap extension penalties can
be 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65 or greater.
[0068] GAP presents one member of the family of best alignments.
There may be many members of this family, and no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
[0069] (c) As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0070] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0071] The use of the term "polynucleotide" is not intended to
limit the embodiments to polynucleotides comprising DNA. Those of
ordinary skill in the art will recognize that polynucleotides can
comprise ribonucleotides and combinations of ribonucleotides and
deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides
include both naturally occurring molecules and synthetic analogues.
The polynucleotides of the embodiments also encompass all forms of
sequences including, and not limited to, single-stranded forms,
double-stranded forms, and the like.
[0072] Isolated polynucleotides of the present invention can be
incorporated into recombinant DNA constructs capable of
introduction into and replication in a host cell. A "vector" may be
such a construct that includes a replication system and sequences
that are capable of transcription and translation of a
polypeptide-encoding sequence in a given host cell. A number of
vectors suitable for stable transfection of plant cells or for the
establishment of transgenic plants have been described in, e.g.,
Pouwels et al, Cloning Vectors: A Laboratory Manual, 1985, supp.
1987; Weissbach and Weissbach, Methods for Plant Molecular Biology,
Academic Press, 1989; and Flevin et al., Plant Molecular Biology
Manual, Kluwer Academic Publishers, 1990. Typically, plant
expression vectors include, for example, one or more cloned plant
genes under the transcriptional control of 5' and 3' regulatory
sequences and a dominant selectable marker. Such plant expression
vectors also can contain a promoter regulatory region (e.g., a
regulatory region controlling inducible or constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific expression), a transcription initiation start site,
a ribosome binding site, an RNA processing signal, a signal peptide
sequence for targeted expression, a transcription termination site,
and/or a polyadenylation signal.
[0073] The terms "recombinant construct," "expression cassette,"
"expression construct," "chimeric construct," "construct,"
"recombinant DNA construct," "DNA construct" and "recombinant DNA
fragment" are used interchangeably herein and are nucleic acid
fragments. A recombinant construct comprises an artificial
combination of nucleic acid fragments, including, and not limited
to, regulatory and coding sequences that are not found together in
nature. For example, a recombinant DNA construct may comprise
regulatory sequences and coding sequences that are derived from
different sources, or regulatory sequences and coding sequences
derived from the same source and arranged in a manner different
than that found in nature. Such construct may be used by itself or
may be used in conjunction with a vector. If a vector is used then
the choice of vector is dependent upon the method that will be used
to transform host cells as is well known to those skilled in the
art. For example, a plasmid vector can be used. The skilled artisan
is well aware of the genetic elements that must be present on the
vector in order to successfully transform, select and propagate
host cells comprising any of the isolated nucleic acid fragments of
the invention. Screening to obtain lines displaying the desired
expression level and pattern of the polynucleotides or of the Nsf1
locus may be accomplished by amplification, Southern analysis of
DNA, Northern analysis of mRNA expression, immunoblotting analysis
of protein expression, phenotypic analysis, and the like.
[0074] The term "recombinant DNA construct" refers to a DNA
construct assembled from nucleic acid fragments obtained from
different sources. The types and origins of the nucleic acid
fragments may be very diverse.
[0075] In some embodiments, DNA constructs comprising a promoter
operably linked to a heterologous nucleotide sequence of the
embodiments are further provided. The DNA constructs of the
embodiments find use in generating transformed plants, plant cells,
and microorganisms and in practicing the methods for inducing ALS
and HPPD inhibitor herbicide resistance disclosed herein. The DNA
construct will include 5' and 3' regulatory sequences operably
linked to a polynucleotide of the embodiments. "Operably linked" is
intended to mean a functional linkage between two or more elements.
"Regulatory sequences" refer to nucleotides located upstream (5'
non-coding sequences), within, or downstream (3' non-coding
sequences) of a coding sequence, and which may influence the
transcription, RNA processing, stability, or translation of the
associated coding sequence. Regulatory sequences may include, and
are not limited to, promoters, translation leader sequences,
introns, and polyadenylation recognition sequences. For example, an
operable linkage between a polynucleotide of interest and a
regulatory sequence (a promoter, for example) is functional link
that allows for expression of the polynucleotide of interest.
Operably linked elements may be contiguous or non-contiguous. When
used to refer to the joining of two protein coding regions,
operably linked is intended to mean that the coding regions are in
the same reading frame. The coding sequence may additionally
contain a sequence used to target the protein to the chloroplast,
the vacuole, the endoplasmic reticulum or to the outside of the
cell. The cassette may additionally contain at least one additional
gene to be cotransformed into the organism. Alternatively, the
additional gene(s) can be provided on multiple DNA constructs. Such
a DNA construct is provided with a plurality of restriction sites
and/or recombination sites for insertion of the polynucleotide that
encodes a herbicide resistance polypeptide to be under the
transcriptional regulation of the regulatory regions. The DNA
construct may additionally contain selectable marker genes.
[0076] The DNA construct will include in the 5'-3' direction of
transcription, a transcriptional initiation region (i.e., a
promoter), translational initiation region, a polynucleotide of the
embodiments, a translational termination region and, optionally, a
transcriptional termination region functional in the host organism.
The regulatory regions (i.e., promoters, transcriptional regulatory
regions, and translational termination regions) and/or the
polynucleotide of the embodiments may be native/analogous to the
host cell or to each other. Alternatively, the regulatory regions
and/or the polynucleotide of the embodiments may be heterologous to
the host cell or to each other. As used herein, "heterologous" in
reference to a sequence is a sequence that originates from a
foreign species, or, if from the same species, is substantially
modified from its native form in composition and/or genomic locus
by deliberate human intervention. For example, a promoter operably
linked to a heterologous polynucleotide is from a species different
from the species from which the polynucleotide was derived, or, if
from the same/analogous species, one or both are substantially
modified from their original form and/or genomic locus, or the
promoter is not the native promoter for the operably linked
polynucleotide.
[0077] The optionally included termination region may be native
with the transcriptional initiation region, may be native with the
operably linked polynucleotide of interest, may be native with the
plant host, or may be derived from another source (i.e., foreign or
heterologous) to the promoter, the polynucleotide of interest, the
host, or any combination thereof. Convenient termination regions
are available from the Ti-plasmid of A. tumefaciens, such as the
octopine synthase and nopaline synthase termination regions. See
also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;
Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev.
5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et
al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.
17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res.
15:9627-9639. In particular embodiments, the potato protease
inhibitor II gene (PinII) terminator is used. See, for example,
Keil et al. (1986) Nucl. Acids Res. 14:5641-5650; and An et al.
(1989) Plant Cell 1:115-122, herein incorporated by reference in
their entirety.
[0078] A number of promoters can be used in the practice of the
embodiments, including the native promoter of the polynucleotide
sequence of interest. The promoters can be selected based on the
desired outcome. A wide range of plant promoters are discussed in
the recent review of Potenza et al. (2004) In Vitro Cell Dev
Biol--Plant 40:1-22, herein incorporated by reference. For example,
the nucleic acids can be combined with constitutive,
tissue-preferred, pathogen-inducible, or other promoters for
expression in plants. Such constitutive promoters include, for
example, the core promoter of the Rsyn7 promoter and other
constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.
6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature
313:810-812); rice actin (McElroy et al. (1990) Plant Cell
2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.
12:619-632 and Christensen et al. (1992) Plant Mol. Biol.
18:675-689); PEMU (Last et al. (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. Pat. No. 5,659,026), and the like. Other
constitutive promoters include, for example, U.S. Pat. Nos.
5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;
5,268,463; 5,608,142; and 6,177,611.
[0079] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0080] DNA constructs may additionally contain 5' leader sequences.
Such leader sequences can act to enhance translation. Translation
leaders are known in the art and include: picornavirus leaders, for
example, EMCV leader (Encephalomyocarditis 5' noncoding region)
(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA
86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco
Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader
(Maize Dwarf Mosaic Virus), and human immunoglobulin heavy-chain
binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94);
untranslated leader from the coat protein mRNA of alfalfa mosaic
virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);
tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in
Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256);
and maize chlorotic mottle virus leader (MCMV) (Lommel et al.
(1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987)
Plant Physiol. 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the
like.
[0081] In preparing the DNA construct, the various DNA fragments
may be manipulated, so as to provide for the DNA sequences in the
proper orientation and, as appropriate, in the proper reading
frame. Toward this end, adapters or linkers may be employed to join
the DNA fragments or other manipulations may be involved to provide
for convenient restriction sites, removal of superfluous DNA,
removal of restriction sites, or the like. For this purpose, in
vitro mutagenesis, primer repair, restriction, annealing,
resubstitutions, e.g., transitions and transversions, may be
involved.
[0082] The DNA construct can also comprise a selectable marker gene
for the selection of transformed cells. Selectable marker genes are
utilized for the selection of transformed cells or tissues. Marker
genes include genes encoding antibiotic resistance, such as those
encoding neomycin phosphotransferase II (NEO) and hygromycin
phosphotransferase (HPT), as well as genes conferring resistance to
herbicidal compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional
selectable markers include phenotypic markers such as
.beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng
85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan
florescent protein (CYP) (Bolte et al. (2004) J. Cell Science
117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and
yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte et
al. (2004) J. Cell Science 117:943-54). For additional selectable
markers, see generally, Yarranton (1992) Curr. Opin. Biotech.
3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595;
Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993)
Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al. (1988) Nature 334:721-724. Such disclosures are herein
incorporated by reference.
[0083] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the
embodiments.
[0084] The gene of the embodiments can be expressed as a transgene
in order to make plants resistant to at least one herbicide of the
ALS-inhibiting, PPO-inhibiting, pigment synthesis-inhibiting, PS
II-inhibiting or synthetic auxin herbicide classes. Using the
different promoters described elsewhere in this disclosure, this
will allow its expression in a modulated form in different
circumstances. One can also insert the entire gene, both native
promoter and coding sequence, as a transgene. Finally, using the
gene of the embodiments as a transgene will allow quick combination
with other traits, such as insect or fungal resistance.
[0085] In certain embodiments the nucleic acid sequences of the
embodiments can be stacked with any combination of polynucleotide
sequences of interest, which may be transgenic or non-transgenic,
in order to create plants with a desired phenotype. For example,
the polynucleotides of the embodiments may be stacked with any
other polynucleotides of the embodiments, or with other genes. The
combinations generated can also include multiple copies of any one
of the polynucleotides of interest. The polynucleotides of the
embodiments can also be stacked with any other gene or combination
of genes to produce plants with a variety of desired trait
combinations including and not limited to traits desirable for
animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);
balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;
5,885,801; 5,885,802; and 5,703,409); barley high lysine
(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO
98/20122); and high methionine proteins (Pedersen et al. (1986) J.
Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and
Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased
digestibility (e.g., modified storage proteins (U.S. application
Ser. No.10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S.
application Ser. No.10/005,429, filed Dec. 3, 2001)), the
disclosures of which are herein incorporated by reference. The
polynucleotides of the embodiments can also be stacked with traits
desirable for insect, disease or herbicide resistance (e.g.,
Bacillus thuringiensis toxin proteins (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,737,514; 5723,756; 5,593,881; Geiser et al (1986) Gene
48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825);
fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones et al. (1994)
Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos
et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants
that lead to herbicide resistance such as the S4 and/or Hra
mutations (Lee et al., (1988) EMBO J. 7(5):1241-1248), resistance
to inhibitors of glutamine synthase such as phosphinothricin or
basta (e.g., bar gene; De Block et al. (1987) EMBO J. 6:2513-2518);
HPPD genes that confer tolerance to HPPD inhibiting herbicides such
as mesotrione or isoxaflutole (Matringe et al. (2005) Pest
Management Science 61:269-276; Dufourmantel et al., (2007) Plant
Biotech. J. 5:118-133; see also WO1997049816), genes for tolerance
to PPO inhibiting herbicides (Li and Nicholl (2005) Pest Management
Science 61:277-285); synthetic auxin resistance genes (US patent
application 2005/014737 and Herman et al., (2005) J. Biol. Chem.
280: 24759-24767), and glyphosate resistance (epsps genes, gat
genes such as those disclosed in U.S. Patent Application
Publication US2004/0082770, also WO02/36782 and WO03/092360)); and
traits desirable for processing or process products such as high
oil (e.g., U.S. Pat. No.6,232,529 ); modified oils (e.g., fatty
acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516));
modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch
synthases (SS), starch branching enzymes (SBE) and starch
debranching enzymes (SDBE)); and polymers or bioplastics (e.g.,
U.S. Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate
synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhydroxyalkanoates (PHAs)), the disclosures of which are herein
incorporated by reference. One could also combine the
polynucleotides of the embodiments with polynucleotides providing
agronomic traits such as male sterility (e.g., see U.S. Pat. No.
5.583,210), stalk strength, flowering time, yield improvement, or
transformation technology traits such as cell cycle regulation or
gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the
disclosures of which are herein incorporated by reference.
[0086] These stacked combinations can be created by any method
including and not limited to cross breeding plants by any
conventional or TopCross.RTM. methodology, or genetic
transformation. If the traits are stacked by genetically
transforming the plants, the polynucleotide sequences of interest
can be combined at any time and in any order. For example, a
transgenic plant comprising one or more desired traits can be used
as the target to introduce further traits by subsequent
transformation. The traits can be introduced simultaneously in a
co-transformation protocol with the polynucleotides of interest
provided by any combination of transformation cassettes. For
example, if two sequences will be introduced, the two sequences can
be contained in separate transformation cassettes (trans) or
contained on the same transformation cassette (cis). Expression of
the sequences can be driven by the same promoter or by different
promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of the
polynucleotide of interest. This may be combined with any
combination of other suppression cassettes or overexpression
cassettes to generate the desired combination of traits in the
plant. It is further recognized that polynucleotide sequences can
be stacked at a desired genomic location using a site-specific
recombination system. See, for example, WO99/25821, WO99/25854,
WO99/25840, WO99/25855, and WO99/25853, all of which are herein
incorporated by reference.
[0087] Further embodiments include plants obtainable by a method
comprising: crossing a plant containing the Nsf1 gene as a first
parent plant, with a different plant that lacks an Nsf1 gene as a
second parent plant, thereby to obtain progeny comprising the Nsf1
gene of the first parent; and optionally further comprising one or
more further breeding steps to obtain progeny of one or more
further generations comprising the Nsf1 gene of the first parent.
Such embodied plants can include both inbred and hybrid plants.
Seeds of such plants, including those seeds which are homozygous
and heterozygous for the Nsf1 gene, and methods of obtaining plant
products resulting from the processing of those seeds are embodied
in the invention. Using such seed in food or feed or the production
of a corn product, such as flour, meal and oil is also an
embodiment of the invention.
[0088] An "ancestral line" or "progenitor" is a parent line used as
a source of genes, e.g., for the development of elite lines.
"Progeny" are the descendents of the ancestral line, and may be
separated from their ancestors by many generations of breeding. An
"elite line" or "elite variety" is an agronomically superior line
or variety that has resulted from many cycles of breeding and
selection for superior agronomic performance. Similarly, "elite
germplasm" is an agronomically superior germplasm, typically
derived from and/or capable of giving rise to a plant with superior
agronomic performance, such as an existing or newly developed elite
line of corn or soybeans.
[0089] Also embodied in the invention is the use of molecular
markers to move the gene or transgene into elite lines using
breeding techniques. Molecular markers can be used in a variety of
plant breeding applications (eg see Staub et al. (1996) Hortscience
31: 729-741; Tan ksley (1983) Plant Molecular Biology Reporter. 1:
3-8). One of the main areas of interest is to increase the
efficiency of backcrossing and introgressing genes using
marker-assisted selection (MAS). A molecular marker that
demonstrates linkage with a locus affecting a desired phenotypic
trait provides a useful tool for the selection of the trait in a
plant population. This is particularly true where the phenotype is
hard to assay, e.g. many disease resistance traits, or, occurs at a
late stage in the plants development, e.g. seed characteristics.
Since DNA marker assays are less laborious, and take up less
physical space, than field phenotyping, much larger populations can
be assayed, increasing the chances of finding a recombinant with
the target segment from the donor line moved to the recipient line.
The closer the linkage, the more useful the marker, as
recombination is less likely to occur between the marker and the
gene causing the trait, which can result in false positives. Having
flanking markers decreases the chances that false positive
selection will occur as a double recombination event would be
needed. The ideal situation is to have a marker in the gene itself,
so that recombination can not occur between the marker and the
gene. Such a marker is called a `perfect marker`.
[0090] Optionally, the nucleic acids of the embodiments may be
targeted to the chloroplast for expression. In this manner, where
the nucleic acid is not directly inserted into the chloroplast, the
expression cassette will additionally contain a nucleic acid
encoding a transit peptide to direct the gene product of interest
to the chloroplasts. Such transit peptides are known in the art.
See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep.
9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550;
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al.
(1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et
al. (1986) Science 233:478-481.
[0091] Chloroplast targeting sequences are known in the art and
include the chloroplast small subunit of ribulose-1,5-bisphosphate
carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant
Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem.
266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase
(EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810);
tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.
270(11):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol.
Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al.
(1993) J. Biol. Chem. 268(36):27447-27457); and the light
harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al.
(1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al.
(1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J.
Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant
Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.
Commun. 196:1414-1421; and Shah et al. (1986) Science
233:478-481.
[0092] Methods for transformation of chloroplasts are known in the
art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci.
USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA
90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method
relies on particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination. Additionally, plastid transformation can
be accomplished by transactivation of a silent plastid-borne
transgene by tissue-preferred expression of a nuclear-encoded and
plastid-directed RNA polymerase. Such a system has been reported in
McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0093] The nucleic acids to be targeted to the chloroplast may be
optimized for expression in the chloroplast to account for
differences in codon usage between the plant nucleus and this
organelle. In this manner, the nucleic acids of interest may be
synthesized using chloroplast-preferred codons. See, for example,
U.S. Pat. No. 5,380,831, herein incorporated by reference.
[0094] The methods of the embodiments may involve, and are not
limited to, introducing a polypeptide or polynucleotide into a
plant. "Introducing" is intended to mean presenting to the plant
the polynucleotide. In some embodiments, the polynucleotide will be
presented in such a manner that the sequence gains access to the
interior of a cell of the plant, including its potential insertion
into the genome of a plant. The methods of the embodiments do not
depend on a particular method for introducing a sequence into a
plant, only that the polynucleotide gains access to the interior of
at least one cell of the plant. Methods for introducing
polynucleotides into plants are known in the art including, and not
limited to, stable transformation methods, transient transformation
methods, and virus-mediated methods.
[0095] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. "Host cell" refers the cell into which transformation of
the recombinant DNA construct takes place and may include a yeast
cell, a bacterial cell, and a plant cell. Examples of methods of
plant transformation include Agrobacterium-mediated transformation
(De Blaere et al, 1987, Meth. Enzymol. 143:277) and
particle-accelerated or "gene gun" transformation technology (Klein
et al, 1987, Nature (London) 327:70-73; U.S. Pat. No. 4,945,050),
among others.
[0096] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof. "Transient transformation" or "transient
expression" is intended to mean that a polynucleotide is introduced
into the plant and does not integrate into the genome of the plant
or a polypeptide is introduced into a plant.
[0097] Transformation protocols as well as protocols for
introducing polypeptides or polynucleotide sequences into plants
may vary depending on the type of plant or plant cell, i.e.,
monocot or dicot, targeted for transformation. Suitable methods of
introducing polypeptides and polynucleotides into plant cells
include microinjection (Crossway et al. (1986) Biotechniques
4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (U.S.
Pat. Nos. 5,563,055-and 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, Sanford et al., U.S. Pat.
Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al.
(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe
et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO
00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet.
22:421-477; Sanford et al. (1987) Particulate Science and
Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.
87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926
(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:1
75-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.
96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740
(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309
(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S.
Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988)
Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)
Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al.
(1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New
York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell
Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.
84:560-566 (whisker-mediated transformation); D'Halluin et al.
(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993)
Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals
of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature
Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all
of which are herein incorporated by reference.
[0098] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the embodiments can be
contained in transfer cassette flanked by two non-identical
recombination sites. The transfer cassette is introduced into a
plant have stably incorporated into its genome a target site which
is flanked by two non-identical recombination sites that correspond
to the sites of the transfer cassette. An appropriate recombinase
is provided and the transfer cassette is integrated at the target
site. The polynucleotide of interest is thereby integrated at a
specific chromosomal position in the plant genome.
[0099] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting progeny having
constitutive expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the embodiments provides transformed seed
(also referred to as "transgenic seed") having a nucleotide
construct of the embodiments, for example, a DNA construct of the
embodiments, stably incorporated into their genome.
[0100] As used herein, the term plant includes plant cells, plant
protoplasts, plant cell tissue cultures from which maize plant can
be regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, and the like. Grain
is intended to mean the mature seed produced by commercial growers
for purposes other than growing or reproducing the species.
Progeny, variants, and mutants of the regenerated plants are also
included within the scope of the embodiments, provided that these
parts comprise the introduced polynucleotides.
[0101] The embodiments of the invention may be used to confer or
enhance herbicide resistance in plants, especially soy (Glycine
max). Other plant species may also be of interest in practicing the
embodiments of the invention, including, and not limited to, other
dicot and monocot crop plants. The maize gene of the embodiments is
commonly found in the majority of commercial corn lines, most of
which are naturally tolerant to at least one, and usually several,
synthetic auxin, ALS-, PS II- and pigment synthesis-inhibitor
herbicides, such as rimsulfuron, nicosulfuron and mesotrione.
[0102] It is therefore envisioned that the same tolerance to
certain herbicides present in most corn lines can be extended to
other crop plants by transgenic means though the use of the
endogenous maize Nsf1 gene and variants thereof. Listings of maize
lines with tolerance or sensitivity to selected SU herbicides are
widely available, such as those provided by the USDA, ARS, National
Genetic Resources Program. Germplasm Resources Information
Network--(GRIN). [Online Database] National Germplasm Resources
Laboratory, Beltsville, Md. [retrieved on Mar. 6, 2006]: Retrieved
from the internet: <URL:
http://www.ars-grin.gov/cgi-bin/npgs/html/dno_eval_acc.pl?89201+153002+21-
>; and the "Maize Germplasm Lines" listings available from the
Buckler Laboratory website [retrieved on Mar. 6, 2006]: Retrieved
from the internet: <URL:
http://www.maizegenetics.net/index.php?page=germplasm/lines.html>,
and also in reference articles such as Kang (1993) J. Heredity.
84(3): 216-217.
[0103] Where appropriate, the polynucleotides may be optimized for
increased expression in the transformed organism. For example, the
polynucleotides can be synthesized using plant-preferred codons for
improved expression. See, for example, Campbell and Gowri (1990)
Plant Physiol. 92:1-11 for a discussion of host-preferred codon
usage. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
[0104] The present invention may be used for transformation of any
plant species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),
alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale
cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana)), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine
max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot
esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),
pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa
(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium
guajava), mango (Mangifera indica), olive (Olea europaea), papaya
(Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats (Avena spp.),
barley, palm, coconut, castor bean, olive, beans (for example guar,
locust bean, fenugreek, soybean, garden beans, mung beans, lima
beans, fava beans), peas (such as cowpeas, field peas, lentils,
chickpeas, etc.), vegetables, ornamentals, and conifers.
[0105] Other plants of interest for the invention include those
which have the potential for use as biofuel crops, including, but
not limited to, prairie grasses such as switchgrass (Panicum
virgatum), elephant grass (Pennisetum purpureum), Johnson grass
(Sorghum halepense), Miscanthus spp., as well as hybrid poplar and
hybrid willow trees.
[0106] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum.
[0107] The embodiments provide not only a gene for use in
transgenic applications, but sequences and methods that allow the
resistance gene to be used as a marker in corn breeding strategies.
For example, the gene of the embodiments, or the locus containing
it, may be identified in a crop line intended to be used for
breeding. Breeders would generally want to avoid using crop lines
that are sensitive to herbicides where there is usually natural
tolerance. Accordingly, the identification of the sequence of the
Nsf1 gene will help breeders to identify and avoid creating
herbicide-sensitive lines.
[0108] Nucleic acid based markers can be developed and applied
using many different technologies. Such technologies include, and
are not limited to, Restriction Fragment Length Polymorphism
(RFLP), Simple Sequence Repeat (SSR), Random Amplified Polymorphic
DNA (RAPD), Cleaved Amplified Polymorphic Sequences (CAPS)
(Rafalski and Tingey, 1993, Trends in Genetics 9:275-280),
Amplified Fragment Length Polymorphism (AFLP) (Vos et al., 1995,
Nucleic Acids Res. 23:4407-4414), Single Nucleotide Polymorphism
(SNP) (Brookes, 1999, Gene 234:177-186), Sequence Characterized
Amplified Region (SCAR) (Paran and Michelmore, 1993, Theor. Appl.
Genet. 85:985-993), Sequence Tagged Site (STS) (Onozaki et al.,
2004, Euphytica 138:255-262), Single Stranded Conformation
Polymorphism (SSCP) (Orita et al, 1989, Proc Natl Acad Sci USA
86:2766-2770), Inter-Simple Sequence Repeat (ISSR) (Blair et al,
1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon
Amplified Polymorphism (IRAP), Retrotransposon-Microsatellite
Amplified Polymorphism (REMAP) (Kalendar et al. (1999) Theor. Appl.
Genet. 98:704-711) and the like.
[0109] As used herein, "locus" shall refer to a genetically defined
region of a chromosome carrying a gene or, possibly, two or more
genes so closely linked that genetically they behave as a single
locus, responsible for a phenotype. A "gene" shall refer to a
specific gene within that locus, including its associated
regulatory sequences. Thus, the Nsf1 locus refers to the
chromosomal region genetically defined as conferring resistance to
at least one herbicide of the ALS-inhibiting, PPO-inhibiting,
pigment synthesis-inhibiting, PS II-inhibiting and synthetic auxin
herbicide class. One embodiment of the present invention is the
isolation of the Nsf1 gene and the demonstration that it is the
gene responsible for the phenotype conferred by the presence of the
locus. Genetically defined loci are by their nature not as
precisely defined in terms of size as genes, which can be
delineated molecularly.
[0110] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The above-defined terms are more
fully defined by reference to the specification as a whole.
EXAMPLES
[0111] The embodiments of the invention are further defined in the
following Examples, in which all parts and percentages are by
weight and degrees are Celsius, unless otherwise stated. It should
be understood that these Examples, while indicating embodiments of
the invention, are given by way of illustration only. From the
above discussion and these Examples, one skilled in the art can
ascertain the essential characteristics of the embodiments of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications to adapt it to various
usages and conditions. Thus, various modifications of the
embodiments of the invention in addition to those shown and
described herein will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. The disclosure of
each reference set forth herein is incorporated by reference in its
entirety
Example 1
Identification of the Nsf1 Gene Through Positional Cloning
[0112] A BC1 population (expected 50% Nsf1/nsf1, 50% nsf1/nsf1) was
developed using the sensitive inbred W703A as the recurrent parent,
and either B73 or Q66 as the resistant line. Plants were misted
with a 2.3 mM nicosulfuron, 0.5% v/v Kinetic surfactant solution at
approximately the V3 stage. Both resistant and sensitive parents
were also grown and sprayed as controls. In order to avoid falsely
classifying a plant which may have died due to reasons other than
the herbicide application, only resistant progeny were sampled and
analyzed. A total of 96 resistant plants were used for the initial
mapping. This was sufficient to place Nsf1 between markers umcl 766
and umc2036, and thus on contig 202 of the maizeB73-based physical
map ((Retrieved on March 6, 2006) Retrieved from the internet
<URL:
http://www.gramene.org/Zea.sub.--mays/cytoview?contig=ctg202&x=44&y=9>-
).
[0113] Based on BAC-end sequences of a maize Mo17-based contig,
flanking CAPS (cleaved amplified polymorphic sequence) markers were
identified on BACs of contig 202.
[0114] For finer mapping of this interval, a total of 388 resistant
plants were used in the next step. Based on sequencing of subcloned
fragments of BACs in this interval, two flanking CAPs markers were
found on overlapping BACs. Both of these markers had 2/388
recombinants.
[0115] Both of these BACs were sequenced and analyzed. Within the
163 kb region of the 2 BACs flanked by two proprietary markers, P1
and P2, there were several putative genes. For the third round of
mapping, a total of 2584 resistant plants were used, and markers
were developed to separate some of the genes. One marker showed
11/2584 (0.4%) recombinants, helping to eliminate certain genes as
being responsible for the resistance. Two other markers each had 2
(0.08%) recombinants, eliminating yet another gene. Finally, a
marker between two genes had a single recombinant (0.04%),
eliminating one of those two genes. Thus it was determined which
gene was the gene of interest. The gene, Nsf1, was determined to
have homology to some cytochrome P450 genes known in the art.
Example 2
Analysis of the Nsf1 Gene
[0116] Analysis of the Gene 18 (Nsf1) sequence in the B73-derived
BAC shows an open reading frame of 521 amino acids, and containing
the conserved heme-binding motif FXXGXXXCXG (SEQ ID NO: 14) found
in all cytochrome P450s (FIGS. 1d and 2b).
[0117] In order to determine if the Nsf1 allele was consistent
across maize lines, three corn lines with unknown sensitivity
levels to nicosulfuron were tested to determine their reaction and
then evaluate their sequences. Plants were misted with a 2.3 mM
nicosulfuron, 0.5% v/v Kinetic surfactant solution at approximately
the V3 stage. Both known resistant and sensitive lines were also
grown and sprayed as controls. Results of the testing of the three
lines showed that lines Q66 and Black Mexican Sweet (BMS) were
resistant and line A188 was sensitive.
[0118] Of these two other resistant lines, Q66 and BMS, also
possess this ORF, although Q66 differs from both B73 and BMS by 3
amino acids (FIGS. 2a and 2b) These three variant amino acids are
marked with bold type and rectangles in FIGS. 2a and 2b in the Q66
sequence string to show their positions. Analysis of a sensitive
line, GA209, shows an insertion of 392 bp relative to the resistant
lines which results in a frameshift and an open reading frame of
only 338 amino acids (FIG. 2b). A survey of numerous North American
sensitive lines showed that many of the sensitive lines contain
this same insertion of unknown DNA.
[0119] Analysis of the sequence from the F2 sensitive line showed
that there is only one nucleotide difference between B73 (SEQ ID
NO: 2) and F2 (SEQ ID NO: 22), which changes amino acid 263 from
arginine to threonine (FIG. 2b). This single change therefore
eliminates the resistance phenotype and variant sequences with such
a change are expected not to retain biological activity. This
change is useful in developing an SNP to assist corn breeders in
avoiding the susceptible allele.
[0120] Nsf1 is 67% identical to a rice cytochrome P450 which has
recently been reported to control sulfonylurea sensitivity in that
plant (Accession No: ABC69856, SEQ ID NO: 4).
[0121] Genomic sequence from B73 shows a single intron with the
expected GT left border and AG right border. The position of the
intron is shown in the sequence listing in SEQ ID NO: 16.
[0122] The cloning of this gene has a number of potential
applications. It could be used as a selectable marker for
transformation in a sensitive transformable line such as A188
(Ishida et al., (1996) Nature Biotechnology 14:745-750). A
transgene designed to suppress the Nsf1 gene function would
function as a dominant negative selectable marker. Nsf1 could also
be used to create transgenic resistance in other plants, such as
soybean, which are sensitive to this subclass of sulfonylureas.
Example 3
Testing of Maize Plants for Sensitivity to Nicosulfuron
[0123] Three corn lines with unknown sensitivity levels to
nicosulfuron were tested to determine their reaction. Plants were
misted with a 2.3 mM nicosulfuron, 0.5% v/v Kinetic surfactant
solution at approximately the V3 stage. Both known resistant and
sensitive lines were also grown and sprayed as controls. Results of
the testing of the three lines showed that lines Q66 and BMS were
resistant and line A188 was sensitive.
Example 4
Preparation of Transgenic Soybean Plants
[0124] The following stock solutions and media were used for
transformation and regeneration of soybean plants:
Stock Solutions
[0125] Sulfate 100 .times. Stock: 37.0 g MgSO.sub.4.7H.sub.2O, 1.69
g MnSO.sub.4.H.sub.2O, 0.86 g ZnSO.sub.4.7H.sub.2O, 0.0025 g
CuSO.sub.4.5H.sub.2O. [0126] Halides 100.times. Stock: 30.0 g
CaCl.sub.2.2H.sub.2O, 0.083 g KI, 0.0025 g COCI.sub.2.6H.sub.2O,
[0127] P, B, Mo 100.times. Stock: 18.5 g KH.sub.2PO.sub.4, 0.62 g
H.sub.3BO.sub.3, 0.025 g Na.sub.2MoO.sub.4.2H.sub.2O [0128] Fe EDTA
100.times. Stock: 3.724 g Na.sub.2EDTA, 2.784 g
FeSO.sub.4.7H.sub.2O. [0129] 2,4-D Stock: 10 mg/mL. [0130] Vitamin
B5 1000.times. Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid,
0.10 g pyridoxine HCl, 1 g thiamine. Media (Per Liter) [0131]
SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitamin
stock, 0.463 g (NH.sub.4).sub.2 SO.sub.4, 2.83 g KNO.sub.3, 1 mL
2,4-D stock, 1 g asparagine,10 g sucrose, pH 5.7. [0132] SB103: 1
pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock, 750
mg MgCl.sub.2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.
[0133] SB166: SB103 supplemented with 5 g per liter activated
charcoal. [0134] SB71-4: Gamborg's B5 salts (Gibco-BRL catalog No.
21153-028), 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH
5.7.
[0135] Soybean embryogenic suspension cultures were maintained in
35 mL liquid medium (SB196) on a rotary shaker (150 rpm) at
28.degree. C. with fluorescent lights providing a 16-hour
day/8-hour night cycle. Cultures were subcultured every 2 weeks by
inoculating approximately 35 mg of tissue into 35 mL of fresh
liquid media.
[0136] Soybean embryogenic suspension cultures were transformed by
particle gun bombardment (see Klein et al. (1987) Nature 327:70-73)
using a DuPont Biolistic PDS1000/He instrument.
[0137] The recombinant DNA plasmid used to express Nsf1 was on a
separate recombinant DNA plasmid from the selectable marker gene.
Both recombinant DNA plasmids were co-precipitated onto gold
particles as follows. The DNAs in suspension were added to 50 .mu.L
of a 20-60 mg/mL 0.6 .mu.m gold particle suspension and then
combined with 50 .mu.L CaCl.sub.2 (2.5 M) and 20 .mu.L spermidine
(0.1 M). The mixture was pulse vortexed 5 times, spun in a
microfuge for 10 seconds, and the supernatant removed. The
DNA-coated particles are then washed once with 150 .mu.L of 100%
ethanol, pulse vortexed and spun in a microfuge again, and
resuspended in 85 .mu.L of anhydrous ethanol. Five .mu.L of the
DNA-coated gold particles are then loaded on each macrocarrier
disk.
[0138] Approximately 150 to 250 mg of two-week-old suspension
culture was placed in an empty 60 mm.times.15 mm petri plate and
the residual liquid is removed from the tissue using a pipette. The
tissue was placed about 3.5 inches away from a retaining screen and
each plate of tissue was bombarded once. Membrane rupture pressure
was set at 650 psi and the chamber was evacuated to -28 inches of
Hg. Eighteen plates were bombarded, and, following bombardment, the
tissue from each plate was divided between two flasks, placed back
into liquid media, and cultured as described above.
[0139] Seven days after bombardment, the liquid medium was
exchanged with fresh SB196 medium supplemented with 50 mg/mL
hygromycin. The selective medium was refreshed weekly or biweekly.
Seven weeks post-bombardment, green, transformed tissue was
observed growing from untransformed, necrotic embryogenic clusters.
Isolated green tissue was removed and inoculated into individual
flasks to generate new, clonally-propagated, transformed
embryogenic suspension cultures. Thus, each new line was treated as
an independent transformation event. These suspensions were then
maintained as suspensions of embryos clustered in an immature
developmental stage through subculture or were regenerated into
whole plants by maturation and germination of individual somatic
embryos.
[0140] Transformed embryogenic clusters were removed from liquid
culture and placed on solid agar medium (SB1 66) containing no
hormones or antibiotics for one week. Embryos were cultured at
26.degree. C. with mixed fluorescent and incandescent lights on a
16-hour day: 8-hour night schedule. After one week, the cultures
were then transferred to SB103 medium and maintained in the same
growth conditions for 3 additional weeks. Prior to transfer from
liquid culture to solid medium, tissue from selected lines was
assayed by PCR for the presence of the chimeric gene. Somatic
embryos became suitable for germination after 4 weeks and were then
removed from the maturation medium and dried in empty petri dishes
for one to five days. The dried embryos were then planted in SB71-4
medium and allowed to germinate under the same light and
germination conditions described above. Germinated embryos were
transferred to sterile soil and grown to maturity.
Example 5
T0 and T1 Transgenic Plant Analysis
T0 Testing
[0141] Two different constructs comprising the Nsf1 gene were
created to examine herbicide efficacy of the gene when transformed
into soybean. The Nsf1 constructs were co-bombarded with a 35S:HYG
insert to permit event selection using hygromycin.
[0142] At the V2 to V6 growth stage, a total of 127 T0 plants were
sprayed with 35 g/ha rimsulfuron. All rimsulfuron treatments were
applied with 0.2% w/w nonionic surfactant in a spray volume of 287
L/ha. In addition to the T0 plants, replications of three different
controls were included--two positive and one negative. Individual
plants were evaluated for herbicide response at ten days after
treatment, and assigned a visual response score from 1 to 9 (1=dead
plant to 9=no effect observed). Based upon high tolerance scores to
the initial rimsulfuron spray, five T0 events were sprayed with an
additional 35 g/ha rimsulfuron. Plants were rated for visual
tolerance using a 1 to 9 score at ten days after the second
application.
[0143] In the T0 generation, 4 of 51 events had improved tolerance
compared to the controls at ten days after treatment with 35 g/ha
rimsulfuron. Three of 51 T0 events had improved level of tolerance
after an additional application of 35 g/ha rimsulfuron. Two of
these 51 events were advanced to the T1 generation for more
extensive herbicide testing.
T1 Testing
[0144] Two events from the T0 generation were advanced to the T1
generation for additional herbicide efficacy testing of the Nsf1
gene. Replicates of two controls, as well as T1 plants, were grown
in greenhouse experiments and sprayed with mesotrione at one of two
rates (200 g/ha or 50 g/ha), nicosulfuron (70 g/ha), or rimsulfuron
(35 g/ha) at the V3 growth stage. All herbicide treatments were
applied with 1% w/w modified seed oil adjuvant in a spray volume of
374 L/ha. Plants were rated for herbicide response at eight days
after application using a 1 to 9 score as used in the T0
testing.
[0145] An expanded herbicide efficacy test was developed in a
second T1 plant experiment for the same two events advanced from
the T0 generation. At the V3 growth stage, plants were sprayed with
different treatments of herbicides that would typically cause
substantial crop injury when applied to commodity soybean at the
rates examined. All herbicide treatments were applied in a spray
volume of 287 L/ha. Isoxaflutole (140 g/ha), topramezone (140
g/ha), and sulcotrione (140 g/ha) were applied with 1% w/w modified
seed oil adjuvant. Diuron treatments (560 g/ha) were applied with
1% w/w petroleum crop oil adjuvant. Acifluorfen (4480 g/ha),
sulfentrazone (140 g/ha), flumioxazin (140 g/ha), and dicamba (280
g/ha) were applied with 0.25% w/w nonionic surfactant. Rimsulfuron
(35 g/ha) treatments were applied with 0.5% w/w basic blend
adjuvant. At eight and fifteen days after treatment, plants were
rated visually for crop injury using a 0 to 100 scale (0=no injury
to 100=dead plant). Since the T1 events were segregating, only the
plants with the best overall scores were selected, corresponding to
the 75% that would be expected to possess the transgene.
[0146] One of the two events had significantly better tolerance
compared to the controls at 8 DAT and 15 DAT after application of
acifluorfen, dicamba, diuron, flumioxazin, isoxaflutole,
mesotrione, rimsulfuron, sulcotrione, sulfentrazone, and
topramezone treatments. The second event had significantly better
tolerance compared to the controls at 15 DAT after application of
acifluorfen, dicamba, isoxaflutole, mesotrione, rimsulfuron,
sulcotrione, sulfentrazone, and topramezone treatments. Although
the exact expression level of the Nsf1 gene in the events tested
was not determined, transgenic soybean plants comprising the maize
Nsf1 gene displayed better tolerance to a range of different
herbicides when compared directly to control plants.
Sequence CWU 1
1
26 1 1563 DNA Zea mays CDS (1)...(1563) 1 atg gat aag gcc tac atc
gcc gcc ctc tcc gcc gcc gcc ctc ttc ttg 48 Met Asp Lys Ala Tyr Ile
Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu 1 5 10 15 ctc cac tac ctc
ctg ggc cgg cgg gcc ggc ggc gag ggc aag gcc aag 96 Leu His Tyr Leu
Leu Gly Arg Arg Ala Gly Gly Glu Gly Lys Ala Lys 20 25 30 gcc aag
ggc tcg cgg cgg cgg ctc ccg ccg agc cct ccg gcg atc ccg 144 Ala Lys
Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro 35 40 45
ttc ctg ggc cac ctc cac ctc gtc aag gcc ccg ttc cac ggg gcg ctg 192
Phe Leu Gly His Leu His Leu Val Lys Ala Pro Phe His Gly Ala Leu 50
55 60 gcc cgc ctc gcg gcg cgc cac ggc ccg gtg ttc tcc atg cgc ctg
ggg 240 Ala Arg Leu Ala Ala Arg His Gly Pro Val Phe Ser Met Arg Leu
Gly 65 70 75 80 acc cgg cgc gcc gtg gtc gtg tcg tcg ccg gac tgc gcc
agg gag tgc 288 Thr Arg Arg Ala Val Val Val Ser Ser Pro Asp Cys Ala
Arg Glu Cys 85 90 95 ttc acg gag cac gac gtg aac ttc gcg aac cgg
ccg ctg ttc ccg tcg 336 Phe Thr Glu His Asp Val Asn Phe Ala Asn Arg
Pro Leu Phe Pro Ser 100 105 110 atg cgg ctg gcg tcc ttc gac ggc gcc
atg ctc tcc gtg tcc agc tac 384 Met Arg Leu Ala Ser Phe Asp Gly Ala
Met Leu Ser Val Ser Ser Tyr 115 120 125 ggc ccg tac tgg cgc aac ctg
cgc cgc gtc gcc gcc gtg cag ctc ctc 432 Gly Pro Tyr Trp Arg Asn Leu
Arg Arg Val Ala Ala Val Gln Leu Leu 130 135 140 tcc gcg cac cgc gtc
ggg tgc atg gcc ccc gcc atc gaa gcg cag gtg 480 Ser Ala His Arg Val
Gly Cys Met Ala Pro Ala Ile Glu Ala Gln Val 145 150 155 160 cgc gcc
atg gtg cgg agg atg gac cgc gcc gcc gcg gcc ggc ggc ggc 528 Arg Ala
Met Val Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly 165 170 175
ggc gtc gcg cgc gtc cag ctc aag cgg cgg ctg ttc gag ctc tcc ctc 576
Gly Val Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu 180
185 190 agc gtg ctc atg gag acc atc gcg cac acc aag acg tcc cgc gcc
gag 624 Ser Val Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser Arg Ala
Glu 195 200 205 gcc gac gcc gac tcg gac atg tcg acc gag gcc cac gag
ttc aag cag 672 Ala Asp Ala Asp Ser Asp Met Ser Thr Glu Ala His Glu
Phe Lys Gln 210 215 220 atc gtc gac gag ctc gtg ccg tac atc ggc acg
gcc aac cgc tgg gac 720 Ile Val Asp Glu Leu Val Pro Tyr Ile Gly Thr
Ala Asn Arg Trp Asp 225 230 235 240 tac ctg ccg gtg ctg cgc tgg ttc
gac gtg ttc ggc gtg agg aac aag 768 Tyr Leu Pro Val Leu Arg Trp Phe
Asp Val Phe Gly Val Arg Asn Lys 245 250 255 atc ctc gac gcc gtg ggc
aga agg gac gcg ttc ctg ggg cgg ctc atc 816 Ile Leu Asp Ala Val Gly
Arg Arg Asp Ala Phe Leu Gly Arg Leu Ile 260 265 270 gac ggg gag cgg
cgg agg ctg gac gct ggc gac gag agc gaa agt aag 864 Asp Gly Glu Arg
Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser Lys 275 280 285 agc atg
att gcg gtg ctg ctc act ctg cag aag tcc gag cca gag gtc 912 Ser Met
Ile Ala Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val 290 295 300
tac act gac act gtg atc act gct ctt tgc gcg aac cta ttc ggc gcc 960
Tyr Thr Asp Thr Val Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala 305
310 315 320 gga acg gag acc acg tcc acc acg acg gaa tgg gcc atg tca
ctg ctg 1008 Gly Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met
Ser Leu Leu 325 330 335 ctg aac cac cgg gag gcg ctc aag aag gcg cag
gcc gag atc gac gcg 1056 Leu Asn His Arg Glu Ala Leu Lys Lys Ala
Gln Ala Glu Ile Asp Ala 340 345 350 gcg gtg ggc acc tcc cgc ctg gtg
acc gcg gac gac gtg ccc cac ctc 1104 Ala Val Gly Thr Ser Arg Leu
Val Thr Ala Asp Asp Val Pro His Leu 355 360 365 acc tac ctg cag tgc
atc gtc gac gag acg ctg cgc ctg cac ccg gcc 1152 Thr Tyr Leu Gln
Cys Ile Val Asp Glu Thr Leu Arg Leu His Pro Ala 370 375 380 gcg ccg
ctg ctg ctg ccg cac gag tcc gcc gcg gac tgc acg gtc ggc 1200 Ala
Pro Leu Leu Leu Pro His Glu Ser Ala Ala Asp Cys Thr Val Gly 385 390
395 400 ggc tac gac gtg ccg cgc ggc acg atg ctg ctg gtc aac gtg cac
gcg 1248 Gly Tyr Asp Val Pro Arg Gly Thr Met Leu Leu Val Asn Val
His Ala 405 410 415 gtc cac agg gac ccc gcg gtg tgg gag gac ccg gac
agg ttc gtg ccg 1296 Val His Arg Asp Pro Ala Val Trp Glu Asp Pro
Asp Arg Phe Val Pro 420 425 430 gag cgg ttc gag ggc gcc ggc ggc aag
gcc gag ggg cgc ctg ctg atg 1344 Glu Arg Phe Glu Gly Ala Gly Gly
Lys Ala Glu Gly Arg Leu Leu Met 435 440 445 ccg ttc ggg atg ggg cgg
cgc aag tgc ccc ggg gag acg ctc gcg ctg 1392 Pro Phe Gly Met Gly
Arg Arg Lys Cys Pro Gly Glu Thr Leu Ala Leu 450 455 460 cgg acc gtc
ggg ctg gtg ctc gcc acg ctg ctc cag tgc ttc gac tgg 1440 Arg Thr
Val Gly Leu Val Leu Ala Thr Leu Leu Gln Cys Phe Asp Trp 465 470 475
480 gac acg gtt gat gga gct cag gtt gac atg aag gct agc ggc ggg ctg
1488 Asp Thr Val Asp Gly Ala Gln Val Asp Met Lys Ala Ser Gly Gly
Leu 485 490 495 acc atg ccc cgg gcc gtc ccg ttg gag gcc atg tgc agg
ccg cgt aca 1536 Thr Met Pro Arg Ala Val Pro Leu Glu Ala Met Cys
Arg Pro Arg Thr 500 505 510 gct atg cgt ggt gtt ctt aag agg ctc
1563 Ala Met Arg Gly Val Leu Lys Arg Leu 515 520 2 521 PRT Zea mays
2 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu 1
5 10 15 Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Gly Glu Gly Lys Ala
Lys 20 25 30 Ala Lys Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro Pro
Ala Ile Pro 35 40 45 Phe Leu Gly His Leu His Leu Val Lys Ala Pro
Phe His Gly Ala Leu 50 55 60 Ala Arg Leu Ala Ala Arg His Gly Pro
Val Phe Ser Met Arg Leu Gly 65 70 75 80 Thr Arg Arg Ala Val Val Val
Ser Ser Pro Asp Cys Ala Arg Glu Cys 85 90 95 Phe Thr Glu His Asp
Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser 100 105 110 Met Arg Leu
Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr 115 120 125 Gly
Pro Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu 130 135
140 Ser Ala His Arg Val Gly Cys Met Ala Pro Ala Ile Glu Ala Gln Val
145 150 155 160 Arg Ala Met Val Arg Arg Met Asp Arg Ala Ala Ala Ala
Gly Gly Gly 165 170 175 Gly Val Ala Arg Val Gln Leu Lys Arg Arg Leu
Phe Glu Leu Ser Leu 180 185 190 Ser Val Leu Met Glu Thr Ile Ala His
Thr Lys Thr Ser Arg Ala Glu 195 200 205 Ala Asp Ala Asp Ser Asp Met
Ser Thr Glu Ala His Glu Phe Lys Gln 210 215 220 Ile Val Asp Glu Leu
Val Pro Tyr Ile Gly Thr Ala Asn Arg Trp Asp 225 230 235 240 Tyr Leu
Pro Val Leu Arg Trp Phe Asp Val Phe Gly Val Arg Asn Lys 245 250 255
Ile Leu Asp Ala Val Gly Arg Arg Asp Ala Phe Leu Gly Arg Leu Ile 260
265 270 Asp Gly Glu Arg Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser
Lys 275 280 285 Ser Met Ile Ala Val Leu Leu Thr Leu Gln Lys Ser Glu
Pro Glu Val 290 295 300 Tyr Thr Asp Thr Val Ile Thr Ala Leu Cys Ala
Asn Leu Phe Gly Ala 305 310 315 320 Gly Thr Glu Thr Thr Ser Thr Thr
Thr Glu Trp Ala Met Ser Leu Leu 325 330 335 Leu Asn His Arg Glu Ala
Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala 340 345 350 Ala Val Gly Thr
Ser Arg Leu Val Thr Ala Asp Asp Val Pro His Leu 355 360 365 Thr Tyr
Leu Gln Cys Ile Val Asp Glu Thr Leu Arg Leu His Pro Ala 370 375 380
Ala Pro Leu Leu Leu Pro His Glu Ser Ala Ala Asp Cys Thr Val Gly 385
390 395 400 Gly Tyr Asp Val Pro Arg Gly Thr Met Leu Leu Val Asn Val
His Ala 405 410 415 Val His Arg Asp Pro Ala Val Trp Glu Asp Pro Asp
Arg Phe Val Pro 420 425 430 Glu Arg Phe Glu Gly Ala Gly Gly Lys Ala
Glu Gly Arg Leu Leu Met 435 440 445 Pro Phe Gly Met Gly Arg Arg Lys
Cys Pro Gly Glu Thr Leu Ala Leu 450 455 460 Arg Thr Val Gly Leu Val
Leu Ala Thr Leu Leu Gln Cys Phe Asp Trp 465 470 475 480 Asp Thr Val
Asp Gly Ala Gln Val Asp Met Lys Ala Ser Gly Gly Leu 485 490 495 Thr
Met Pro Arg Ala Val Pro Leu Glu Ala Met Cys Arg Pro Arg Thr 500 505
510 Ala Met Arg Gly Val Leu Lys Arg Leu 515 520 3 732 PRT Oryza
sativa PEPTIDE (0)...(0) Accession No. XP_469850 3 Met Ala Phe Leu
Gly Trp Ala Val Asp Ile Ala Arg Asp Ser Gly Ala 1 5 10 15 Ser Ser
Ser Val Val Leu Thr Cys Asp Gly Tyr Gly Ser Ala Leu Tyr 20 25 30
Phe Ser Pro Trp Asp Ser Val Pro Leu Pro Ala Thr Ala Ser Pro Asp 35
40 45 Asp Gly Phe Leu Leu Pro Arg Phe Pro Asp Val Cys Val Gln Arg
Ser 50 55 60 Gln Phe Thr Asn His Leu Ala Pro Ala Asn Gly Thr Gly
Gly Gly Gly 65 70 75 80 Ser Arg Thr Gly Val Lys Glu Glu Ala Ser Glu
Val Leu Ser Trp Pro 85 90 95 Pro Thr Ser Lys Gln Ser Val Arg Arg
Leu Glu Val Ala Glu His Trp 100 105 110 Tyr Arg Leu Tyr Lys Thr Asp
Asn Gln Arg Leu Ser Pro Asp Ser Gln 115 120 125 Gln Val Ser Val Leu
Ala Glu Ser His Cys Asp Leu Ala Ser Gly Asn 130 135 140 Trp Lys Glu
Ile Ser Ile His His Lys Lys Met Pro Ser Ser Thr Thr 145 150 155 160
Thr Lys Thr Thr Thr Pro Ser Arg Asp Ala Trp Ile Val Ser Ala Arg 165
170 175 Ser Asp Pro Phe His Leu Leu Leu Glu Ala Gln Ala Pro Leu Gly
Ile 180 185 190 Lys Ala Asp Ala Leu Ser Gln Ile Ala Ala Val His Gln
Ser His Arg 195 200 205 Asn Thr Ser His Ile Arg Glu Leu Ser Leu Ala
Met Asp Asn Ala Tyr 210 215 220 Ile Ile Ala Ile Leu Ser Val Ala Ile
Leu Phe Leu Leu His Tyr Tyr 225 230 235 240 Leu Leu Gly Arg Gly Asn
Gly Gly Ala Ala Arg Leu Pro Pro Gly Pro 245 250 255 Pro Ala Val Pro
Ile Leu Gly His Leu His Leu Val Lys Lys Pro Met 260 265 270 His Ala
Thr Met Ser Arg Leu Ala Glu Arg Tyr Gly Pro Val Phe Ser 275 280 285
Leu Arg Leu Gly Ser Arg Arg Ala Val Val Val Ser Ser Pro Gly Cys 290
295 300 Ala Arg Glu Cys Phe Thr Glu His Asp Val Thr Phe Ala Asn Arg
Pro 305 310 315 320 Arg Phe Glu Ser Gln Leu Leu Val Ser Phe Asn Gly
Ala Ala Leu Ala 325 330 335 Thr Ala Ser Tyr Gly Ala His Trp Arg Asn
Leu Arg Arg Ile Val Ala 340 345 350 Val Gln Leu Leu Ser Ala His Arg
Val Gly Leu Met Ser Gly Leu Ile 355 360 365 Ala Gly Glu Val Arg Ala
Met Val Arg Arg Met Tyr Arg Ala Ala Ala 370 375 380 Ala Ser Pro Ala
Gly Ala Ala Arg Ile Gln Leu Lys Arg Arg Leu Phe 385 390 395 400 Glu
Val Ser Leu Ser Val Leu Met Glu Thr Ile Ala His Thr Lys Ala 405 410
415 Thr Arg Pro Glu Thr Asp Pro Asp Thr Asp Met Ser Val Glu Ala Gln
420 425 430 Glu Phe Lys Gln Val Val Asp Glu Ile Ile Pro His Ile Gly
Ala Ala 435 440 445 Asn Leu Trp Asp Tyr Leu Pro Ala Leu Arg Trp Phe
Asp Val Phe Gly 450 455 460 Val Arg Arg Lys Ile Leu Ala Ala Val Ser
Arg Arg Asp Ala Phe Leu 465 470 475 480 Arg Arg Leu Ile Asp Ala Glu
Arg Arg Arg Leu Asp Asp Gly Asp Glu 485 490 495 Gly Glu Lys Lys Ser
Met Ile Ala Val Leu Leu Thr Leu Gln Lys Thr 500 505 510 Glu Pro Glu
Val Tyr Thr Asp Asn Met Ile Thr Ala Leu Thr Ala Asn 515 520 525 Leu
Phe Gly Ala Gly Thr Glu Thr Thr Ser Thr Thr Ser Glu Trp Ala 530 535
540 Met Ser Leu Leu Leu Asn His Pro Asp Thr Leu Lys Lys Ala Gln Ala
545 550 555 560 Glu Ile Asp Ala Ser Val Gly Asn Ser Arg Leu Ile Thr
Ala Asp Asp 565 570 575 Val Thr Arg Leu Gly Tyr Leu Gln Cys Ile Val
Arg Glu Thr Leu Arg 580 585 590 Leu Tyr Pro Ala Ala Pro Met Leu Leu
Pro His Glu Ser Ser Ala Asp 595 600 605 Cys Lys Val Gly Gly Tyr Asn
Ile Pro Arg Gly Ser Met Leu Leu Ile 610 615 620 Asn Ala Tyr Ala Ile
His Arg Asp Pro Ala Val Trp Glu Glu Pro Glu 625 630 635 640 Lys Phe
Met Pro Glu Arg Phe Glu Asp Gly Gly Cys Asp Gly Asn Leu 645 650 655
Leu Met Pro Phe Gly Met Gly Arg Arg Arg Cys Pro Gly Glu Thr Leu 660
665 670 Ala Leu Arg Thr Val Gly Leu Val Leu Gly Thr Leu Ile Gln Cys
Phe 675 680 685 Asp Trp Glu Arg Val Asp Gly Val Glu Val Asp Met Thr
Glu Gly Gly 690 695 700 Gly Leu Thr Ile Pro Lys Val Val Pro Leu Glu
Ala Met Cys Arg Pro 705 710 715 720 Arg Asp Ala Met Gly Gly Val Leu
Arg Glu Leu Val 725 730 4 513 PRT Oryza sativa PEPTIDE (0)...(0)
Accession No. ABC69856 4 Met Asp Asn Ala Tyr Ile Ile Ala Ile Leu
Ser Val Ala Ile Leu Phe 1 5 10 15 Leu Leu His Tyr Tyr Leu Leu Gly
Arg Gly Asn Gly Gly Ala Ala Arg 20 25 30 Leu Pro Pro Gly Pro Pro
Ala Val Pro Ile Leu Gly His Leu His Leu 35 40 45 Val Lys Lys Pro
Met His Ala Thr Met Ser Arg Leu Ala Glu Arg Tyr 50 55 60 Gly Pro
Val Phe Ser Leu Arg Leu Gly Ser Arg Arg Ala Val Val Val 65 70 75 80
Ser Ser Pro Gly Cys Ala Arg Glu Cys Phe Thr Glu His Asp Val Thr 85
90 95 Phe Ala Asn Arg Pro Arg Phe Glu Ser Gln Leu Leu Val Ser Phe
Asn 100 105 110 Gly Ala Ala Leu Ala Thr Ala Ser Tyr Gly Ala His Trp
Arg Asn Leu 115 120 125 Arg Arg Ile Val Ala Val Gln Leu Leu Ser Ala
His Arg Val Gly Leu 130 135 140 Met Ser Gly Leu Ile Ala Gly Glu Val
Arg Ala Met Val Arg Arg Met 145 150 155 160 Tyr Arg Ala Ala Ala Ala
Ser Pro Ala Gly Ala Ala Arg Ile Gln Leu 165 170 175 Lys Arg Arg Leu
Phe Glu Val Ser Leu Ser Val Leu Met Glu Thr Ile 180 185 190 Ala His
Thr Lys Ala Thr Arg Pro Glu Thr Asp Pro Asp Thr Asp Met 195 200 205
Ser Val Glu Ala Gln Glu Phe Lys Gln Val Val Asp Glu Ile Ile Pro 210
215 220 His Ile Gly Ala Ala Asn Leu Trp Asp Tyr Leu Pro Ala Leu Arg
Trp 225 230 235 240 Phe Asp Val Phe Gly Val Arg Arg Lys Ile Leu Ala
Ala Val Ser Arg 245 250 255 Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp
Ala Glu Arg Arg Arg Leu 260 265 270 Asp Asp Gly Asp Glu Gly Glu Lys
Lys Ser Met Ile Ala Val Leu Leu 275 280 285 Thr Leu Gln Lys Thr Glu
Pro Glu Val Tyr Thr Asp Asn Met Ile Thr 290 295 300 Ala Leu Thr Ala
Asn Leu Phe Gly Ala Gly Thr Glu Thr Thr Ser Thr 305 310 315 320 Thr
Ser Glu Trp Ala Met Ser Leu Leu Leu
Asn His Pro Asp Thr Leu 325 330 335 Lys Lys Ala Gln Ala Glu Ile Asp
Ala Ser Val Gly Asn Ser Arg Leu 340 345 350 Ile Thr Ala Asp Asp Val
Thr Arg Leu Gly Tyr Leu Gln Cys Ile Val 355 360 365 Arg Glu Thr Leu
Arg Leu Tyr Pro Ala Ala Pro Met Leu Leu Pro His 370 375 380 Glu Ser
Ser Ala Asp Cys Lys Val Gly Gly Tyr Asn Ile Pro Arg Gly 385 390 395
400 Ser Met Leu Leu Ile Asn Ala Tyr Ala Ile His Arg Asp Pro Ala Val
405 410 415 Trp Glu Glu Pro Glu Lys Phe Met Pro Glu Arg Phe Glu Asp
Gly Gly 420 425 430 Cys Asp Gly Asn Leu Leu Met Pro Phe Gly Met Gly
Arg Arg Arg Cys 435 440 445 Pro Gly Glu Thr Leu Ala Leu Arg Thr Val
Gly Leu Val Leu Gly Thr 450 455 460 Leu Ile Gln Cys Phe Asp Trp Glu
Arg Val Asp Gly Val Glu Val Asp 465 470 475 480 Met Thr Glu Gly Gly
Gly Leu Thr Ile Pro Lys Val Val Pro Leu Glu 485 490 495 Ala Met Cys
Arg Pro Arg Asp Ala Met Gly Gly Val Leu Arg Glu Leu 500 505 510 Val
5 517 PRT Lolium rigidum PEPTIDE (0)...(0) Accession No. AAK38080 5
Met Asp Lys Ala Tyr Ile Ala Ile Leu Ser Cys Ala Phe Leu Phe Leu 1 5
10 15 Val His Tyr Val Leu Gly Lys Val Ser Asp Gly Arg Arg Gly Lys
Lys 20 25 30 Gly Ala Val Gln Leu Pro Pro Ser Pro Pro Ala Val Pro
Phe Leu Gly 35 40 45 His Leu His Leu Val Asp Lys Pro Ile His Ala
Thr Met Cys Arg Leu 50 55 60 Ala Ala Arg Leu Gly Pro Val Phe Ser
Leu Arg Leu Gly Ser Arg Arg 65 70 75 80 Ala Val Val Val Ser Ser Ser
Glu Cys Ala Arg Glu Cys Phe Thr Glu 85 90 95 His Asp Val Thr Phe
Ala Asn Arg Pro Lys Phe Pro Ser Gln Leu Leu 100 105 110 Val Ser Phe
Asn Gly Thr Ala Leu Val Thr Ser Ser Tyr Gly Pro His 115 120 125 Trp
Arg Asn Leu Arg Arg Val Ala Thr Val Gln Leu Leu Ser Ala His 130 135
140 Arg Val Ala Cys Met Ser Gly Val Ile Ala Ala Glu Val Arg Ala Met
145 150 155 160 Ala Arg Arg Leu Phe His Ala Thr Glu Ala Ser Pro Asp
Gly Ala Ala 165 170 175 Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu
Ser Leu Ser Val Leu 180 185 190 Met Glu Thr Ile Ala Gln Thr Lys Ala
Thr Arg Ser Glu Ala Asp Ala 195 200 205 Asp Thr Asp Met Ser Val Glu
Ala Gln Glu Phe Lys Glu Val Val Asp 210 215 220 Lys Leu Ile Pro His
Leu Gly Ala Ala Asn Met Trp Asp Tyr Leu Pro 225 230 235 240 Val Met
Arg Trp Phe Asp Val Phe Gly Val Arg Asn Lys Ile Leu His 245 250 255
Ala Val Ser Arg Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp Ala Glu 260
265 270 Arg Arg Arg Leu Ala Asp Gly Gly Ser Asp Gly Asp Lys Lys Ser
Met 275 280 285 Ile Ala Val Leu Leu Thr Leu Gln Lys Thr Glu Pro Lys
Val Tyr Thr 290 295 300 Asp Thr Met Ile Thr Ala Leu Cys Ala Asn Leu
Phe Gly Ala Gly Thr 305 310 315 320 Glu Thr Thr Ser Thr Thr Thr Glu
Trp Ala Met Ser Leu Leu Leu Asn 325 330 335 His Pro Ala Ala Leu Lys
Lys Ala Gln Ala Glu Ile Asp Ala Ser Val 340 345 350 Gly Thr Ser Arg
Leu Val Ser Val Asp Asp Val Pro Ser Leu Ala Tyr 355 360 365 Leu Gln
Cys Ile Val Ser Glu Thr Leu Arg Leu Tyr Pro Ala Ala Pro 370 375 380
Leu Leu Leu Pro His Glu Ser Ser Ala Asp Cys Lys Val Gly Gly Tyr 385
390 395 400 Asn Val Pro Ala Asp Thr Met Leu Ile Val Asn Ala Tyr Ala
Ile His 405 410 415 Arg Asp Pro Ala Ala Trp Glu Asp Pro Leu Glu Phe
Arg Pro Glu Arg 420 425 430 Phe Glu Asp Gly Lys Ala Glu Gly Leu Phe
Met Ile Pro Phe Gly Met 435 440 445 Gly Arg Arg Arg Cys Pro Gly Glu
Thr Leu Ala Leu Arg Thr Ile Gly 450 455 460 Met Val Leu Ala Thr Leu
Val Gln Cys Phe Asp Trp Glu Pro Val Asp 465 470 475 480 Gly Val Lys
Val Asp Met Thr Glu Gly Gly Gly Phe Thr Ile Pro Lys 485 490 495 Ala
Val Pro Leu Glu Ala Val Cys Arg Pro Arg Ala Val Met Arg Asp 500 505
510 Val Leu Gln Asn Leu 515 6 517 PRT Lolium rigidum PEPTIDE
(0)...(0) Accession No. AAK38079 6 Met Asp Lys Ala Tyr Ile Ala Ile
Leu Ser Ser Ala Phe Leu Phe Leu 1 5 10 15 Val His Tyr Val Leu Gly
Lys Val Ser Asp Gly Arg Arg Gly Lys Lys 20 25 30 Gly Ala Val Gln
Leu Pro Pro Ser Pro Pro Ala Val Pro Phe Leu Gly 35 40 45 His Leu
His Leu Val Glu Lys Pro Ile His Ala Thr Met Cys Arg Leu 50 55 60
Ala Ala Arg Leu Gly Pro Val Phe Ser Leu Arg Leu Gly Ser Arg Arg 65
70 75 80 Ala Val Val Val Ser Ser Ser Glu Cys Ala Arg Glu Cys Phe
Thr Glu 85 90 95 His Asp Val Thr Phe Ala Asn Arg Pro Lys Phe Pro
Ser Gln Leu Leu 100 105 110 Val Ser Phe Asn Gly Thr Ala Leu Val Thr
Ser Ser Tyr Gly Pro His 115 120 125 Trp Arg Asn Leu Arg Arg Val Ala
Thr Val Gln Leu Leu Ser Ala His 130 135 140 Arg Val Thr Cys Met Ser
Gly Val Ile Ala Ala Glu Val Arg Ala Met 145 150 155 160 Ala Arg Arg
Leu Phe His Ala Ala Glu Ala Ser Pro Asp Gly Ala Ala 165 170 175 Arg
Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu Ser Val Leu 180 185
190 Met Glu Thr Ile Ala Gln Thr Lys Ala Thr Arg Ser Glu Ala Asp Ala
195 200 205 Asp Thr Asp Met Ser Leu Glu Ala Gln Glu Phe Lys Glu Val
Val Asp 210 215 220 Lys Leu Ile Pro His Leu Gly Ala Ala Asn Met Trp
Asp Tyr Leu Pro 225 230 235 240 Val Met Arg Trp Phe Asp Val Phe Gly
Val Arg Ser Lys Ile Leu His 245 250 255 Ala Val Ser Arg Arg Asp Ala
Phe Leu Arg Arg Leu Ile Asn Ala Glu 260 265 270 Arg Arg Arg Leu Ala
Asp Gly Gly Ser Asp Gly Asp Lys Lys Ser Met 275 280 285 Ile Ala Val
Leu Leu Thr Leu Gln Lys Thr Glu Pro Lys Val Tyr Thr 290 295 300 Asp
Thr Met Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala Gly Thr 305 310
315 320 Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu
Asn 325 330 335 His Pro Ala Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp
Ala Ser Val 340 345 350 Gly Thr Ser Arg Leu Val Ser Val Asp Asp Val
Pro Ser Leu Ala Tyr 355 360 365 Leu Gln Cys Ile Val Ser Glu Thr Leu
Arg Leu Tyr Pro Ala Ala Pro 370 375 380 Leu Leu Leu Pro His Glu Ser
Ser Ala Asp Cys Lys Val Gly Gly Tyr 385 390 395 400 Asn Val Pro Ala
Asp Thr Met Leu Ile Val Asn Ala Tyr Ala Ile His 405 410 415 Arg Asp
Pro Ala Ala Trp Glu Asp Pro Leu Glu Phe Lys Pro Glu Arg 420 425 430
Phe Glu Asp Gly Lys Ala Glu Gly Leu Phe Met Ile Pro Phe Gly Met 435
440 445 Gly Arg Arg Arg Cys Pro Gly Glu Thr Leu Ala Leu Arg Thr Ile
Gly 450 455 460 Met Val Leu Ala Thr Leu Val Gln Cys Phe Asp Trp Glu
Pro Val Asp 465 470 475 480 Gly Val Lys Val Asp Met Thr Glu Gly Gly
Gly Phe Thr Ile Pro Lys 485 490 495 Ala Val Pro Leu Glu Ala Val Cys
Arg Pro Arg Val Val Met Arg Asp 500 505 510 Val Leu Gln Asn Leu 515
7 517 PRT Lolium rigidum PEPTIDE (0)...(0) AAK38081 7 Met Asp Lys
Ala Tyr Ile Ala Ile Leu Ser Cys Ala Phe Leu Phe Leu 1 5 10 15 Val
His Tyr Val Leu Gly Lys Val Ser Asp Gly Arg Arg Gly Lys Lys 20 25
30 Gly Ala Val Gln Leu Pro Pro Ser Pro Pro Ala Val Pro Phe Leu Gly
35 40 45 His Leu His Leu Val Asp Lys Pro Ile His Ala Thr Met Cys
Arg Leu 50 55 60 Ala Ala Arg Leu Gly Pro Val Phe Ser Leu Arg Leu
Gly Ser Arg Arg 65 70 75 80 Ala Val Val Val Ser Ser Ser Glu Cys Ala
Arg Glu Cys Phe Thr Glu 85 90 95 His Asp Val Thr Phe Ala Asn Arg
Pro Lys Phe Pro Ser Gln Leu Leu 100 105 110 Val Ser Phe Asn Gly Thr
Ala Leu Val Thr Ser Ser Tyr Gly Pro His 115 120 125 Trp Arg Asn Leu
Arg Arg Val Ala Thr Val Gln Leu Leu Ser Ala His 130 135 140 Arg Val
Ala Cys Met Ser Gly Val Ile Ala Ala Glu Val Arg Ala Met 145 150 155
160 Ala Arg Arg Leu Phe His Ala Ala Glu Ala Ser Pro Asp Gly Ala Ala
165 170 175 Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu Ser
Val Leu 180 185 190 Met Glu Thr Ile Ala Gln Thr Lys Ala Thr Arg Ser
Glu Ala Asp Ala 195 200 205 Asp Thr Asp Met Ser Val Glu Ala Gln Glu
Phe Lys Glu Val Val Asp 210 215 220 Lys Leu Ile Pro His Leu Gly Ala
Ala Asn Met Trp Asp Tyr Leu Pro 225 230 235 240 Val Met Arg Trp Phe
Asp Val Phe Gly Val Arg Asn Lys Ile Leu His 245 250 255 Ala Val Ser
Arg Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp Ala Glu 260 265 270 Arg
Arg Arg Leu Ala Asp Gly Gly Ser Asp Gly Asp Lys Lys Ser Met 275 280
285 Ile Ala Val Leu Leu Thr Leu Gln Lys Thr Glu Pro Lys Val Tyr Thr
290 295 300 Asp Thr Met Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala
Gly Thr 305 310 315 320 Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met
Ser Leu Leu Leu Asn 325 330 335 His Pro Ala Ala Leu Lys Lys Ala Gln
Ala Glu Ile Asp Ala Ser Val 340 345 350 Gly Thr Ser Arg Leu Val Ser
Val Asp Asp Val Pro Ser Leu Ala Tyr 355 360 365 Leu Gln Cys Ile Val
Asn Glu Thr Leu Arg Leu Tyr Pro Ala Ala Pro 370 375 380 Leu Leu Leu
Pro His Glu Ser Ser Ala Asp Cys Lys Val Gly Gly Tyr 385 390 395 400
Asn Val Pro Ala Asp Thr Met Leu Ile Val Asn Ala Tyr Ala Ile His 405
410 415 Arg Asp Pro Ala Ala Trp Glu His Pro Leu Val Phe Arg Pro Glu
Arg 420 425 430 Phe Glu Asp Gly Lys Ala Glu Gly Leu Phe Met Ile Pro
Phe Gly Met 435 440 445 Gly Arg Arg Arg Cys Pro Gly Glu Thr Leu Ala
Leu Arg Thr Ile Gly 450 455 460 Met Val Leu Ala Thr Leu Val Gln Cys
Phe Asp Trp Glu Pro Val Asp 465 470 475 480 Gly Val Asn Val Asp Met
Thr Glu Gly Gly Gly Phe Thr Ile Pro Lys 485 490 495 Ala Val Pro Leu
Glu Ala Val Cys Arg Pro Arg Ala Val Met Arg Asp 500 505 510 Val Leu
Gln Ser Ile 515 8 517 PRT Lolium rigidum PEPTIDE (0)...(0)
Accession No. BAD27508 8 Met Asp Lys Ala Tyr Ile Ala Ile Leu Ser
Cys Ala Phe Leu Phe Leu 1 5 10 15 Val His Tyr Val Leu Gly Lys Val
Ser Asp Gly Arg Arg Gly Lys Lys 20 25 30 Gly Ala Val Gln Leu Pro
Pro Ser Pro Pro Ala Ile Pro Phe Ile Gly 35 40 45 His Leu His Leu
Val Glu Lys Pro Ile His Ala Thr Met Cys Arg Leu 50 55 60 Ala Ala
Arg Leu Gly Pro Val Phe Ser Leu Arg Leu Gly Ser Arg Arg 65 70 75 80
Ala Val Val Val Pro Ser Ser Glu Cys Ala Arg Glu Cys Phe Thr Glu 85
90 95 His Asp Val Thr Phe Ala Asn Arg Pro Lys Phe Pro Ser Gln Leu
Leu 100 105 110 Ala Ser Phe Asn Gly Thr Ala Leu Val Thr Ser Ser Tyr
Gly Pro His 115 120 125 Trp Arg Asn Leu Arg Arg Val Ala Thr Val Gln
Leu Leu Ser Ala His 130 135 140 Arg Val Ala Cys Met Ser Gly Val Ile
Ala Ala Glu Val Arg Ala Met 145 150 155 160 Ala Arg Arg Leu Phe His
Ala Ala Glu Ala Ser Pro Asp Gly Ala Ala 165 170 175 Arg Val Gln Leu
Lys Arg Arg Leu Phe Glu Leu Ser Leu Ser Val Leu 180 185 190 Met Glu
Thr Ile Ala Gln Thr Lys Ala Thr Arg Ser Glu Ala Asp Ala 195 200 205
Asp Thr Asp Met Ser Val Glu Ala Gln Glu Phe Lys Glu Val Val Asp 210
215 220 Lys Leu Ile Pro His Leu Gly Ala Ala Asn Met Trp Asp Tyr Leu
Pro 225 230 235 240 Val Met Arg Trp Phe Asp Val Phe Gly Val Arg Asn
Lys Ile Leu His 245 250 255 Ala Val Ser Arg Arg Asp Ala Phe Leu Arg
Arg Leu Ile Asp Ala Glu 260 265 270 Arg Arg Arg Leu Ala Asp Gly Gly
Ser Asp Gly Asp Lys Lys Ser Met 275 280 285 Ile Ala Val Leu Leu Thr
Leu Gln Lys Thr Glu Pro Lys Val Tyr Thr 290 295 300 Asp Thr Met Ile
Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala Gly Thr 305 310 315 320 Glu
Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu Asn 325 330
335 His Pro Ala Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala Ser Val
340 345 350 Gly Thr Ser Arg Leu Val Ser Val Asp Asp Val Pro Ser Leu
Ala Tyr 355 360 365 Leu Gln Cys Ile Val Asn Glu Thr Leu Arg Leu Tyr
Pro Ala Ala Pro 370 375 380 Leu Leu Leu Pro His Glu Ser Ser Ala Asp
Cys Lys Val Gly Gly Tyr 385 390 395 400 Asn Val Pro Ala Asp Thr Met
Leu Ile Val Asn Ala Tyr Ala Ile His 405 410 415 Arg Asp Pro Ala Ala
Trp Glu His Pro Leu Glu Phe Arg Pro Glu Arg 420 425 430 Phe Glu Asp
Gly Lys Ala Glu Gly Leu Phe Met Ile Pro Phe Gly Val 435 440 445 Gly
Arg Arg Arg Cys Pro Gly Glu Thr Leu Ala Leu Arg Thr Ile Ser 450 455
460 Met Val Leu Ala Thr Leu Val Gln Cys Phe Asp Trp Glu Pro Val Asp
465 470 475 480 Gly Val Lys Val Asp Met Thr Glu Gly Gly Gly Phe Thr
Ile Pro Lys 485 490 495 Ala Val Pro Leu Glu Ala Val Cys Arg Pro Arg
Ala Val Met Arg Asp 500 505 510 Val Leu Gln Asn Leu 515 9 517 PRT
Lolium rigidum PEPTIDE (0)...(0) Accession No. BAD27507 9 Met Asp
Lys Ala Tyr Ile Ala Ile Leu Ser Cys Ala Phe Leu Phe Leu 1 5 10 15
Val His Tyr Val Leu Gly Lys Val Ser His Gly Arg Arg Gly Lys Lys 20
25 30 Gly Ala Val Gln Leu Pro Pro Ser Pro Pro Ala Ile Pro Phe Ile
Gly 35 40 45 His Leu His Leu Val Glu Lys Pro Ile His Ala Thr Met
Cys Arg Leu 50 55 60 Ala Ala Arg Leu Gly Pro Val Phe Ser Leu Arg
Leu Gly Ser Arg Arg 65 70 75 80 Ala Val Val Val Ser Ser Ser Glu Cys
Ala Arg Glu Cys Phe Thr Glu 85 90 95 His Asp Val Thr Phe Ala Asn
Arg Pro Ser Ser Arg Arg Lys Leu Leu 100 105 110 Ala Ser Phe Asn Gly
Thr Ala Leu Val Thr Ser Ser Tyr Gly Pro His 115 120 125 Trp Arg Asn
Leu Arg Arg Val Ala Thr Val Gln Leu Leu Ser Ala His 130 135 140 Arg
Val Ala Cys Met Ser Gly Val Ile Ala Ala Glu
Val Arg Ala Met 145 150 155 160 Ala Arg Arg Leu Phe His Ala Ala Glu
Ala Ser Pro Asp Gly Ala Thr 165 170 175 Arg Val Gln Leu Lys Arg Arg
Leu Phe Glu Leu Ser Leu Ser Val Leu 180 185 190 Met Glu Thr Ile Ala
Gln Thr Lys Ala Thr Arg Ser Glu Ala Asp Ala 195 200 205 Asp Thr Asp
Met Ser Val Glu Ala Gln Glu Phe Lys Glu Val Val Asp 210 215 220 Lys
Leu Ile Pro His Leu Gly Ala Ala Asn Met Trp Asp Tyr Leu Pro 225 230
235 240 Val Met Arg Trp Phe Asp Val Phe Gly Val Arg Asn Lys Ile Leu
His 245 250 255 Ala Val Ser Arg Arg Asp Ala Phe Leu Arg Arg Leu Ile
Asp Ala Glu 260 265 270 Arg Arg Arg Leu Ala Asp Gly Gly Ser Asp Gly
Asp Lys Lys Ser Met 275 280 285 Ile Ala Val Leu Leu Thr Leu Gln Lys
Thr Glu Pro Lys Val Tyr Thr 290 295 300 Asp Thr Met Ile Thr Ala Leu
Cys Ala Asn Leu Phe Gly Ala Gly Thr 305 310 315 320 Glu Thr Thr Ser
Thr Thr Thr Glu Trp Ala Met Ser Leu Leu Leu Asn 325 330 335 His Pro
Ala Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala Ser Val 340 345 350
Gly Thr Ser Arg Leu Val Ser Val Asp Asp Val Leu Ser Leu Ala Tyr 355
360 365 Leu Gln Cys Ile Val Ser Glu Thr Leu Arg Leu Tyr Pro Ala Ala
Pro 370 375 380 Leu Leu Leu Pro His Glu Ser Ser Ala Asp Cys Lys Val
Gly Gly Tyr 385 390 395 400 Asn Val Pro Ala Asp Thr Met Leu Ile Val
Asn Ala Tyr Ala Ile His 405 410 415 Arg Asp Pro Ala Ala Trp Glu His
Pro Leu Glu Phe Arg Pro Glu Arg 420 425 430 Phe Glu Asp Gly Lys Ala
Glu Gly Leu Phe Met Ile Pro Phe Gly Met 435 440 445 Gly Arg Arg Arg
Cys Pro Gly Glu Thr Leu Ala Leu Arg Thr Ile Gly 450 455 460 Met Val
Leu Ala Thr Leu Val Gln Cys Phe Asp Trp Glu Pro Val Asp 465 470 475
480 Gly Val Lys Val Asp Met Thr Glu Gly Gly Gly Phe Thr Ile Pro Lys
485 490 495 Ala Val Pro Leu Glu Ala Val Cys Arg Pro Arg Thr Val Met
Arg Asp 500 505 510 Val Leu Gln Asn Leu 515 10 517 PRT Lolium
rigidum PEPTIDE (0)...(0) Accession No. BAD27506 10 Met Asp Lys Ala
Tyr Ile Ala Ile Leu Ser Cys Ala Phe Leu Phe Leu 1 5 10 15 Val His
Tyr Val Leu Gly Lys Val Ser His Gly Arg Arg Gly Lys Lys 20 25 30
Gly Ala Val Gln Leu Pro Pro Ser Pro Pro Ala Ile Pro Phe Ile Gly 35
40 45 His Leu His Leu Val Glu Lys Pro Ile His Ala Thr Met Cys Arg
Leu 50 55 60 Ala Ala Arg Leu Gly Pro Val Phe Ser Leu Arg Leu Gly
Ser Arg Arg 65 70 75 80 Ala Val Val Val Ser Ser Ser Glu Cys Ala Arg
Glu Cys Phe Thr Glu 85 90 95 His Asp Val Thr Phe Ala Asn Arg Pro
Lys Phe Pro Ser Gln Leu Leu 100 105 110 Ala Ser Phe Asn Gly Thr Ala
Leu Val Thr Pro Ser Tyr Gly Pro His 115 120 125 Trp Arg Asn Leu Arg
Arg Val Ala Thr Val Gln Leu Leu Ser Ala His 130 135 140 Arg Val Ala
Cys Met Ser Gly Val Ile Ala Ala Glu Val Arg Ala Met 145 150 155 160
Ala Arg Arg Leu Phe His Ala Ala Glu Ala Ser Pro Gly Gly Ala Ala 165
170 175 Arg Val Gln Leu Lys Arg Gly Pro Phe Glu Leu Ser Leu Ser Val
Leu 180 185 190 Met Glu Thr Ile Ala Gln Thr Lys Ala Thr Arg Ser Glu
Ala Asp Ala 195 200 205 Asp Thr Asp Met Ser Val Glu Ala Gln Glu Phe
Lys Glu Val Val Asp 210 215 220 Lys Pro Ile Pro His Leu Gly Ala Ala
Asn Met Trp Asp Tyr Leu Pro 225 230 235 240 Val Met Arg Trp Phe Asp
Val Phe Gly Val Arg Asn Lys Ile Leu His 245 250 255 Ala Val Ser Arg
Arg Asp Ala Phe Leu Arg Arg Leu Ile Asp Ala Glu 260 265 270 Arg Arg
Arg Leu Ala Asp Gly Gly Ser Asp Gly Asp Lys Lys Ser Met 275 280 285
Ile Ala Val Leu Leu Thr Leu Gln Lys Thr Glu Pro Lys Val Tyr Thr 290
295 300 Asp Thr Met Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala Gly
Thr 305 310 315 320 Glu Thr Thr Ser Thr Thr Thr Glu Arg Ala Met Ser
Leu Leu Leu Asn 325 330 335 His Pro Ala Ala Leu Lys Lys Ala Gln Ala
Glu Ile Asp Ala Ser Val 340 345 350 Gly Thr Ser Arg Leu Val Ser Val
Asp Asp Met Pro Ser Leu Ala Tyr 355 360 365 Leu Gln Cys Ile Val Asn
Glu Thr Leu Arg Leu Tyr Pro Ala Ala Pro 370 375 380 Leu Leu Leu Pro
His Glu Ser Ser Ala Asp Cys Lys Val Gly Gly Tyr 385 390 395 400 Asn
Val Pro Ala Asp Thr Met Leu Ile Val Asn Ala Tyr Ala Ile His 405 410
415 Arg Asp Pro Ala Ala Trp Glu His Pro Leu Glu Phe Arg Pro Glu Arg
420 425 430 Phe Glu Asp Gly Lys Ala Glu Gly Leu Phe Met Ile Pro Phe
Gly Met 435 440 445 Gly Arg Arg Arg Cys Pro Gly Glu Thr Leu Ala Leu
Arg Thr Ile Gly 450 455 460 Met Val Leu Ala Thr Leu Val Gln Cys Phe
Asp Trp Glu Pro Val Asp 465 470 475 480 Gly Val Lys Val Asp Met Thr
Glu Gly Gly Gly Phe Thr Ile Pro Lys 485 490 495 Ala Val Pro Leu Glu
Ala Val Cys Arg Pro Arg Ala Val Met Arg Asp 500 505 510 Val Leu Gln
Asn Leu 515 11 512 PRT Oryza sativa PEPTIDE (0)...(0) Accession No.
XP_469849 11 Met Asp Lys Ala Tyr Ile Ala Val Phe Ser Ile Val Ile
Leu Phe Leu 1 5 10 15 Leu Val Asp Tyr Leu Arg Arg Leu Arg Gly Gly
Gly Thr Ser Asn Gly 20 25 30 Lys Asn Lys Gly Met Arg Leu Pro Pro
Gly Leu Pro Ala Val Pro Ile 35 40 45 Ile Gly His Leu His Leu Val
Lys Lys Pro Met His Ala Thr Leu Ser 50 55 60 Arg Leu Ala Ala Arg
His Gly Pro Val Phe Ser Leu Arg Leu Gly Ser 65 70 75 80 Arg Arg Ala
Val Val Val Ser Ser Pro Gly Cys Ala Arg Glu Cys Phe 85 90 95 Thr
Glu His Asp Val Ala Phe Ala Asn Arg Pro Arg Phe Glu Ser Gln 100 105
110 Leu Leu Met Ser Phe Asp Gly Thr Ala Leu Ala Met Ala Ser Tyr Gly
115 120 125 Pro His Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu
Leu Ser 130 135 140 Ala Arg Arg Val Gly Leu Met Ser Gly Leu Ile Ala
Gly Glu Val Arg 145 150 155 160 Ala Met Val Arg Ser Leu Cys Arg Arg
Pro Ala Ala Ala Ala Pro Val 165 170 175 Gln Leu Lys Arg Arg Leu Phe
Glu Leu Ser Leu Ser Val Leu Met Glu 180 185 190 Thr Ile Ala Gln Ser
Lys Ala Thr Arg Pro Glu Thr Thr Asp Thr Asp 195 200 205 Thr Asp Met
Ser Met Glu Ala Gln Glu Tyr Lys Gln Val Val Glu Glu 210 215 220 Ile
Leu Glu Arg Ile Gly Thr Gly Asn Leu Cys Asp Tyr Leu Pro Ala 225 230
235 240 Leu Arg Trp Phe Asp Val Phe Gly Val Arg Asn Arg Ile Leu Ala
Ala 245 250 255 Val Ser Arg Arg Asp Ala Phe Leu Arg Arg Leu Ile Tyr
Ala Ala Arg 260 265 270 Trp Arg Met Asp Asp Gly Glu Lys Lys Ser Met
Ile Ala Val Leu Leu 275 280 285 Thr Leu Gln Lys Thr Gln Pro Glu Val
Tyr Thr Asp Asn Met Ile Thr 290 295 300 Ala Leu Cys Ser Asn Leu Leu
Gly Ala Gly Thr Glu Thr Thr Ser Thr 305 310 315 320 Thr Ile Glu Trp
Ala Met Ser Leu Leu Leu Asn His Pro Glu Thr Leu 325 330 335 Lys Lys
Ala Gln Ala Glu Ile Asp Ala Ser Val Gly Asn Ser Arg Leu 340 345 350
Ile Thr Ala Asp Asp Val Pro Arg Ile Thr Tyr Leu Gln Cys Ile Val 355
360 365 Arg Glu Thr Leu Arg Leu Tyr Pro Ala Ala Pro Met Leu Ile Pro
His 370 375 380 Glu Ser Ser Ala Asp Cys Glu Val Gly Gly Tyr Ser Val
Pro Arg Gly 385 390 395 400 Thr Met Leu Leu Val Asn Ala Tyr Ala Ile
His Arg Asp Pro Ala Ala 405 410 415 Trp Glu Glu Pro Glu Arg Phe Val
Pro Glu Arg Phe Glu Gly Gly Gly 420 425 430 Cys Asp Gly Asn Leu Ser
Met Pro Phe Gly Met Gly Arg Arg Arg Cys 435 440 445 Pro Gly Glu Thr
Leu Ala Leu His Thr Val Gly Leu Val Leu Gly Thr 450 455 460 Leu Ile
Gln Cys Phe Asp Trp Glu Arg Val Asp Gly Val Glu Val Asp 465 470 475
480 Met Ala Glu Gly Gly Gly Leu Thr Met Pro Lys Val Val Pro Leu Glu
485 490 495 Ala Val Cys Arg Pro Arg Asp Ala Met Gly Gly Val Leu Arg
Glu Leu 500 505 510 12 527 PRT Oryza sativa PEPTIDE (0)...(0)
Accession No. XP_469851 12 Met Asp Lys Ala Tyr Ile Ala Val Phe Ser
Ile Ala Ile Leu Phe Leu 1 5 10 15 Leu Val Asp Tyr Phe Arg Cys Arg
Arg Arg Arg Gly Ser Gly Ser Asn 20 25 30 Asn Gly Glu Asn Lys Gly
Met Leu Gln Leu Pro Pro Ser Pro Pro Ala 35 40 45 Ile Pro Phe Phe
Gly His Leu His Leu Ile Asp Lys Pro Leu His Ala 50 55 60 Ala Leu
Ser Arg Leu Ala Glu Arg His Gly Pro Val Phe Ser Leu Arg 65 70 75 80
Leu Gly Ser Arg Asn Ala Val Val Val Ser Ser Pro Glu Cys Ala Arg 85
90 95 Glu Cys Phe Thr Asp Asn Asp Val Cys Phe Ala Asn Arg Pro Gln
Phe 100 105 110 Pro Ser Gln Met Pro Ala Thr Phe Tyr Gly Ala Gly Phe
Gly Phe Ala 115 120 125 Asn Tyr Gly Ala His Trp Arg Asn Leu Arg Arg
Ile Ala Thr Val His 130 135 140 Leu Leu Ser Ala His Arg Val Arg Gly
Met Ala Gly Val Val Ser Gly 145 150 155 160 Glu Ile Arg Pro Met Val
Gln Arg Met Tyr Arg Ala Ala Ala Ala Ala 165 170 175 Gly Val Gly Val
Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu 180 185 190 Ser Leu
Ser Val Leu Met Glu Ala Ile Ala Gln Thr Lys Thr Thr Arg 195 200 205
Pro Glu Ala Asp Asp Ala Asp Thr Asp Met Ser Val Glu Ala Gln Glu 210
215 220 Phe Lys Asn Val Leu Asp Glu Leu Asn Pro Leu Leu Gly Ala Ala
Asn 225 230 235 240 Leu Trp Asp Tyr Leu Pro Ala Leu Arg Val Phe Asp
Val Leu Gly Val 245 250 255 Lys Arg Lys Ile Ala Thr Leu Ala Asn Arg
Arg Asp Ala Phe Val Arg 260 265 270 Arg Leu Ile Asp Ala Glu Arg Gln
Arg Met Asp Asn Gly Val Asp Gly 275 280 285 Gly Asp Asp Gly Glu Lys
Lys Ser Val Ile Ser Val Leu Leu Ser Leu 290 295 300 Gln Lys Thr Glu
Pro Glu Val Tyr Lys Asp Ile Val Ile Val Asn Leu 305 310 315 320 Cys
Ala Ala Leu Phe Ala Ala Gly Thr Glu Thr Thr Ala Met Thr Ile 325 330
335 Glu Trp Ala Met Ser Leu Leu Leu Asn His Pro Lys Ile Leu Lys Lys
340 345 350 Ala Lys Ala Glu Ile Asp Ala Ser Val Gly Asn Ser Arg Leu
Ile Asn 355 360 365 Gly Asp Asp Met Pro His Leu Ser Tyr Leu Gln Cys
Ile Ile Asn Glu 370 375 380 Thr Leu Arg Leu Tyr Pro Val Ala Pro Leu
Leu Ile Pro His Glu Ser 385 390 395 400 Ser Ala Asp Cys Lys Val Asn
Gly Tyr His Ile Pro Ser Gly Thr Met 405 410 415 Leu Leu Val Asn Val
Ile Ala Ile Gln Arg Asp Pro Met Val Trp Lys 420 425 430 Glu Pro Asn
Glu Phe Lys Pro Glu Arg Phe Glu Asn Gly Glu Ser Glu 435 440 445 Gly
Leu Phe Met Ile Pro Phe Gly Met Gly Arg Arg Lys Cys Pro Gly 450 455
460 Glu Thr Met Ala Leu Gln Thr Ile Gly Leu Val Leu Gly Ala Leu Ile
465 470 475 480 Gln Cys Phe Asp Trp Asp Arg Val Asp Gly Ala Glu Val
Asp Met Thr 485 490 495 Gln Gly Ser Gly Leu Thr Asn Pro Arg Ala Val
Pro Leu Glu Ala Met 500 505 510 Cys Lys Pro Arg Glu Ala Met Ser Asp
Val Phe Arg Glu Leu Leu 515 520 525 13 518 PRT Oryza sativa PEPTIDE
(0)...(0) Accession No. XP_469852 13 Met Val Lys Ala Tyr Ile Ala
Ile Phe Ser Ile Ala Val Leu Leu Leu 1 5 10 15 Ile His Phe Leu Phe
Arg Arg Arg Gly Arg Ser Asn Gly Met Pro Leu 20 25 30 Pro Pro Ser
Pro Pro Ala Ile Pro Phe Phe Gly His Leu His Leu Ile 35 40 45 Asp
Lys Pro Phe His Ala Ala Leu Ser Arg Leu Ala Glu Arg His Gly 50 55
60 Pro Val Phe Ser Leu Arg Leu Gly Ser Arg Asn Ala Val Val Val Ser
65 70 75 80 Ser Pro Glu Cys Ala Arg Glu Cys Phe Thr Asp Asn Asp Val
Cys Phe 85 90 95 Ala Asn Arg Pro Arg Phe Pro Ser Gln Met Leu Ala
Thr Phe Asn Gly 100 105 110 Thr Ser Leu Gly Ser Ala Asn Tyr Gly Pro
His Trp Arg Asn Leu Arg 115 120 125 Arg Ile Ala Thr Val His Leu Leu
Ser Ser His Arg Val Ser Gly Met 130 135 140 Ser Gly Ile Ile Ser Gly
Gln Ala Arg His Met Val Arg Arg Met Tyr 145 150 155 160 Arg Ala Ala
Thr Ala Ser Ala Ala Gly Val Ala Arg Val Gln Leu Asn 165 170 175 Arg
Arg Leu Phe Glu Leu Ser Leu Ser Val Leu Met Glu Ala Ile Ala 180 185
190 Gln Ser Lys Thr Thr Arg Arg Glu Ala Pro Asp Ala Asp Thr Asp Met
195 200 205 Ser Met Glu Ala Gln Glu Leu Arg His Val Leu Asp Glu Leu
Asn Pro 210 215 220 Leu Ile Gly Ala Ala Asn Leu Trp Asp Tyr Leu Pro
Ala Leu Arg Trp 225 230 235 240 Phe Asp Val Phe Gly Val Lys Arg Lys
Ile Val Ala Ala Val Asn Arg 245 250 255 Arg Asn Ala Phe Met Arg Arg
Leu Ile Asp Ala Glu Arg Gln Arg Met 260 265 270 Asp Asn Asn Asp Val
Asp Gly Gly Asp Asp Gly Glu Lys Lys Ser Met 275 280 285 Ile Ser Val
Leu Leu Thr Leu Gln Lys Thr Gln Pro Glu Val Tyr Thr 290 295 300 Asp
Thr Leu Ile Met Thr Leu Cys Ala Pro Leu Phe Gly Ala Gly Thr 305 310
315 320 Glu Thr Thr Ser Thr Thr Ile Glu Trp Ala Met Ser Leu Leu Leu
Asn 325 330 335 His Pro Glu Ile Leu Lys Lys Ala Gln Ala Glu Ile Asp
Met Ser Val 340 345 350 Gly Asn Ser Arg Leu Ile Ser Val Val Asp Val
His Arg Leu Gly Tyr 355 360 365 Leu Gln Cys Ile Ile Asn Glu Thr Leu
Arg Met Tyr Pro Ala Ala Pro 370 375 380 Leu Leu Leu Pro His Glu Ser
Ser Ala Asp Cys Lys Val Gly Gly Tyr 385 390 395 400 His Ile Pro Ser
Gly Ala Met Leu Leu Val Asn Val Ala Ala Ile Gln 405 410 415 Arg Asp
Pro Val Ile Trp Lys Glu Pro Ser Glu Phe Lys Pro Glu Arg 420 425 430
Phe Glu Asn Gly Arg Phe Glu Gly Leu Phe Met Ile Pro Phe Gly Met 435
440 445 Gly Arg Arg Arg Cys Pro Gly Glu Met Leu Ala Leu Gln Thr Ile
Gly 450 455 460 Leu Val Leu Gly Thr Met Ile Gln Cys Phe Asp Trp Gly
Arg Val Asp 465 470 475 480 Asp Ala Met Val Asp Met Thr Gln Ser
Asn
Gly Leu Thr Ser Leu Lys 485 490 495 Val Ile Pro Leu Glu Ala Met Cys
Lys Pro Arg Glu Ala Met Cys Asp 500 505 510 Val Leu Arg Lys Phe Met
515 14 10 PRT Artificial Sequence Motif common to all Cytochrome
P450 sequences VARIANT 2, 3, 5, 6, 7, 9 Xaa = Any Amino Acid 14 Phe
Xaa Xaa Gly Xaa Xaa Xaa Cys Xaa Gly 1 5 10 15 6 PRT Artificial
Sequence Motif common to all Cytochrome P450 sequences VARIANT
(1)...(1) Xaa = Ala or Gly VARIANT (3)...(3) Xaa = any amino acid
VARIANT (4)...(4) Xaa = Asp or Glu VARIANT (6)...(6) Xaa = Thr or
Ser 15 Xaa Gly Xaa Xaa Thr Xaa 1 5 16 1859 DNA Zea mays
misc_feature (0)...(0) Maize B73 genomic sequence for Nsf1 gene
intron (946)...(1238) intron 16 atggataagg cctacatcgc cgccctctcc
gccgccgccc tcttcttgct ccactacctc 60 ctgggccgcc gggccggcgg
cgagggcaag gccaaggcca agggctcgcg gcggcggctc 120 ccgccgagcc
ctccggcgat cccgttcctg ggccacctcc acctcgtcaa ggccccgttc 180
cacggggcgc tggcccgcct cgcggcgcgc cacggcccgg tgttctccat gcgcctgggg
240 acccggcgcg ccgtggtcgt gtcgtcgccg gactgcgcca gggagtgctt
cacggagcac 300 gacgtgaact tcgcgaaccg gccgctgttc ccgtcgatgc
ggctggcgtc cttcgacggc 360 gccatgctct ccgtgtccag ctacggcccg
tactggcgca acctgcgccg cgtcgccgcc 420 gtgcagctcc tctccgcgca
ccgcgtcggg tgcatggccc ccgccatcga agcgcaggtg 480 cgcgccatgg
tgcggaggat ggaccgcgcc gccgcggccg gcggcggcgg cgtcgcgcgc 540
gtccagctca agcggcggct gttcgagctc tccctcagcg tgctcatgga gaccatcgcg
600 cacaccaaga cgtcccgcgc cgaggccgac gccgactcgg acatgtcgac
cgaggcccac 660 gagttcaagc agatcgtcga cgagctcgtg ccgtacatcg
gcacggccaa ccgctgggac 720 tacctgccgg tgctgcgctg gttcgacgtg
ttcggcgtga ggaacaagat cctcgacgcc 780 gtgggcagaa gggacgcgtt
cctggggcgg ctcatcgacg gggagcggcg gaggctggac 840 gctggcgacg
agagcgaaag taagagcatg attgcggtgc tgctcactct gcagaagtcc 900
gagccagagg tctacactga cactgtgatc actgctcttt gcgcggtgag tgcttcttct
960 tctaccatac gtcactctct tatcctcaca aaatacaaaa aaagttgccc
gttttctcag 1020 tttagtcgtc aacactccgg actctactat ccgccaaagt
ataggattcg ctaaaaattt 1080 aggtgtcttt tttaatacta aaattagata
ttgaatttgt tgtgctttat gttgacatag 1140 tctgtaattc tttttccccg
aggataaaaa atgttagaca tggatctgga tatttgaacc 1200 atgagaaaga
ctgactgcaa ttttgttctg tgataaagaa cctattcggc gccggaacgg 1260
agaccacgtc caccacgacg gaatgggcca tgtcactgct gctgaaccac cgggaggcgc
1320 tcaagaaggc gcaggccgag atcgacgcgg cggtgggcac ctcccgcctg
gtgaccgcgg 1380 acgacgtgcc ccacctcacc tacctgcagt gcatcgtcga
cgagacgctg cgcctgcacc 1440 cggccgcgcc gctgctgctg ccgcacgagt
ccgccgcgga ctgcacggtc ggcggctacg 1500 acgtgccgcg cggcacgatg
ctgctggtca acgtgcacgc ggtccacagg gaccccgcgg 1560 tgtgggagga
cccggacagg ttcgtgccgg agcggttcga gggcgccggc ggcaaggccg 1620
aggggcgcct gctgatgccg ttcgggatgg ggcggcgcaa gtgccccggg gagacgctcg
1680 cgctgcggac cgtcgggctg gtgctcgcca cgctgctcca gtgcttcgac
tgggacacgg 1740 ttgatggagc tcaggttgac atgaaggcta gcggcgggct
gaccatgccc cgggccgtcc 1800 cgttggaggc catgtgcagg ccgcgtacag
ctatgcgtgg tgttcttaag aggctctga 1859 17 1566 DNA Zea mays
misc_feature (0)...(0) Q66 maize line ORF for Nsf1 CDS (1)...(1566)
17 atg gat aag gcc tac atc gcc gcc ctc tcc gcc gcc gcc ctc ttc ttg
48 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu
1 5 10 15 ctc cac tac ctc ctg ggc cgg cgg gcc ggc ggc gag ggc aag
gcc aag 96 Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Gly Glu Gly Lys
Ala Lys 20 25 30 gcc aag ggc tcg cgg cgg cgg ctc ccg ccg agc cct
ccg gcg atc ccg 144 Ala Lys Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro
Pro Ala Ile Pro 35 40 45 ttc ctg ggc cac ctc cac ctc gtc aag gcc
ccg ttc cac ggg gcg ctg 192 Phe Leu Gly His Leu His Leu Val Lys Ala
Pro Phe His Gly Ala Leu 50 55 60 gcc cgc ctc gcg gcg cgc cac ggc
ccg gtg ttc tcc atg cgc ctg ggg 240 Ala Arg Leu Ala Ala Arg His Gly
Pro Val Phe Ser Met Arg Leu Gly 65 70 75 80 acc cgg cgc gcc gtg gtc
gtg tcg tcg ccg gac tgc gcc agg gag tgc 288 Thr Arg Arg Ala Val Val
Val Ser Ser Pro Asp Cys Ala Arg Glu Cys 85 90 95 ttc acg gag cac
gac gtg aac ttc gcg aac cgg ccg ctg ttc ccg tcg 336 Phe Thr Glu His
Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser 100 105 110 atg cgg
ctg gcg tcc ttc gac ggc gcc atg ctc tcc gtg tcc agc tac 384 Met Arg
Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr 115 120 125
ggc ccg tac tgg cgc aac ctg cgc cgc gtc gcc gcc gtg cag ctc ctc 432
Gly Pro Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu 130
135 140 tcc gcg cac cgc gtc ggg tgc atg gcc ccc gcc atc gaa gcg cag
gtg 480 Ser Ala His Arg Val Gly Cys Met Ala Pro Ala Ile Glu Ala Gln
Val 145 150 155 160 cgc gcc atg gtg cgg agg atg gac cgc gcc gcc gcg
gcc ggc ggc ggc 528 Arg Ala Met Val Arg Arg Met Asp Arg Ala Ala Ala
Ala Gly Gly Gly 165 170 175 ggc gtc gcg cgc gtc cag ctc aag cgg cgg
ctg ttc gag ctc tcc ctc 576 Gly Val Ala Arg Val Gln Leu Lys Arg Arg
Leu Phe Glu Leu Ser Leu 180 185 190 agc gtg ctc atg gag acc atc gcg
cac acc aag acg tcc cgc gcc gag 624 Ser Val Leu Met Glu Thr Ile Ala
His Thr Lys Thr Ser Arg Ala Glu 195 200 205 gcc gac gcc aac tcg gac
atg tcg acc gag gcc cac gag ttc aag cag 672 Ala Asp Ala Asn Ser Asp
Met Ser Thr Glu Ala His Glu Phe Lys Gln 210 215 220 atc gtc aac gag
ctc gtg ccg tac atc ggc acg gcc aac cgc tgg gac 720 Ile Val Asn Glu
Leu Val Pro Tyr Ile Gly Thr Ala Asn Arg Trp Asp 225 230 235 240 tac
ctg ccg gtg ctg cgc tgg ttc gac gtg ttc ggc gtg agg aac aag 768 Tyr
Leu Pro Val Leu Arg Trp Phe Asp Val Phe Gly Val Arg Asn Lys 245 250
255 atc ctc gac gcc gtg ggc aga agg gac gcg ttc ctg ggg cgg ctc atc
816 Ile Leu Asp Ala Val Gly Arg Arg Asp Ala Phe Leu Gly Arg Leu Ile
260 265 270 gac ggg gag cgg cgg agg ctg gac gct ggc gac gag agc gaa
agt aag 864 Asp Gly Glu Arg Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu
Ser Lys 275 280 285 agc atg att gcg gtg ctg ctc act ctg cag aag tcc
gag cca gag gtc 912 Ser Met Ile Ala Val Leu Leu Thr Leu Gln Lys Ser
Glu Pro Glu Val 290 295 300 tac act gac act gtg atc act gct ctt tgc
gcg aac cta ttc ggc gcc 960 Tyr Thr Asp Thr Val Ile Thr Ala Leu Cys
Ala Asn Leu Phe Gly Ala 305 310 315 320 gga acg gag acc acg tcc acc
acg acg gaa tgg gcc atg tca ctg ctg 1008 Gly Thr Glu Thr Thr Ser
Thr Thr Thr Glu Trp Ala Met Ser Leu Leu 325 330 335 ctg aac cac cgg
gag gcg ctc aag aag gcg cag gcc gag atc gac gcg 1056 Leu Asn His
Arg Glu Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala 340 345 350 gcg
gtg ggc gcc tcc cgc ctg gtg acc gcg gac gac gtg ccc cac ctc 1104
Ala Val Gly Ala Ser Arg Leu Val Thr Ala Asp Asp Val Pro His Leu 355
360 365 acc tac ctg cag tgc atc gtc gac gag acg ctg cgc ctg cac ccg
gcc 1152 Thr Tyr Leu Gln Cys Ile Val Asp Glu Thr Leu Arg Leu His
Pro Ala 370 375 380 gcg ccg ctg ctg ctg ccg cac gag tcc gcc gcg gac
tgc acg gtc ggc 1200 Ala Pro Leu Leu Leu Pro His Glu Ser Ala Ala
Asp Cys Thr Val Gly 385 390 395 400 ggc tac gac gtg ccg cgc ggc acg
atg ctg ctg gtc aac gtg cac gcg 1248 Gly Tyr Asp Val Pro Arg Gly
Thr Met Leu Leu Val Asn Val His Ala 405 410 415 gtc cac agg gac ccc
gcg gtg tgg gag gac ccg gac agg ttc gtg ccg 1296 Val His Arg Asp
Pro Ala Val Trp Glu Asp Pro Asp Arg Phe Val Pro 420 425 430 gag cgg
ttc gag ggc gcc ggc ggc aag gcc gag ggg cgc ctg ctg atg 1344 Glu
Arg Phe Glu Gly Ala Gly Gly Lys Ala Glu Gly Arg Leu Leu Met 435 440
445 ccg ttc ggg atg ggg cgg cgc aag tgc ccc ggg gag acg ctc gcg ctg
1392 Pro Phe Gly Met Gly Arg Arg Lys Cys Pro Gly Glu Thr Leu Ala
Leu 450 455 460 cgg acc gtc ggg ctg gtg ctc gcc acg ctg ctc cag tgc
ttc gac tgg 1440 Arg Thr Val Gly Leu Val Leu Ala Thr Leu Leu Gln
Cys Phe Asp Trp 465 470 475 480 gac acg gtt gat gga gct cag gtt gac
atg aag gct agc ggc ggg ctg 1488 Asp Thr Val Asp Gly Ala Gln Val
Asp Met Lys Ala Ser Gly Gly Leu 485 490 495 acc atg ccc cgg gcc gtc
ccg ttg gag gcc atg tgc agg ccg cgt aca 1536 Thr Met Pro Arg Ala
Val Pro Leu Glu Ala Met Cys Arg Pro Arg Thr 500 505 510 gct atg cgt
ggt gtt ctt aag agg ctc tga 1566 Ala Met Arg Gly Val Leu Lys Arg
Leu * 515 520 18 521 PRT Zea mays PEPTIDE (0)...(0) Q66 maize line
Nsf1 peptide 18 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala
Leu Phe Leu 1 5 10 15 Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Gly
Glu Gly Lys Ala Lys 20 25 30 Ala Lys Gly Ser Arg Arg Arg Leu Pro
Pro Ser Pro Pro Ala Ile Pro 35 40 45 Phe Leu Gly His Leu His Leu
Val Lys Ala Pro Phe His Gly Ala Leu 50 55 60 Ala Arg Leu Ala Ala
Arg His Gly Pro Val Phe Ser Met Arg Leu Gly 65 70 75 80 Thr Arg Arg
Ala Val Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys 85 90 95 Phe
Thr Glu His Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser 100 105
110 Met Arg Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr
115 120 125 Gly Pro Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln
Leu Leu 130 135 140 Ser Ala His Arg Val Gly Cys Met Ala Pro Ala Ile
Glu Ala Gln Val 145 150 155 160 Arg Ala Met Val Arg Arg Met Asp Arg
Ala Ala Ala Ala Gly Gly Gly 165 170 175 Gly Val Ala Arg Val Gln Leu
Lys Arg Arg Leu Phe Glu Leu Ser Leu 180 185 190 Ser Val Leu Met Glu
Thr Ile Ala His Thr Lys Thr Ser Arg Ala Glu 195 200 205 Ala Asp Ala
Asn Ser Asp Met Ser Thr Glu Ala His Glu Phe Lys Gln 210 215 220 Ile
Val Asn Glu Leu Val Pro Tyr Ile Gly Thr Ala Asn Arg Trp Asp 225 230
235 240 Tyr Leu Pro Val Leu Arg Trp Phe Asp Val Phe Gly Val Arg Asn
Lys 245 250 255 Ile Leu Asp Ala Val Gly Arg Arg Asp Ala Phe Leu Gly
Arg Leu Ile 260 265 270 Asp Gly Glu Arg Arg Arg Leu Asp Ala Gly Asp
Glu Ser Glu Ser Lys 275 280 285 Ser Met Ile Ala Val Leu Leu Thr Leu
Gln Lys Ser Glu Pro Glu Val 290 295 300 Tyr Thr Asp Thr Val Ile Thr
Ala Leu Cys Ala Asn Leu Phe Gly Ala 305 310 315 320 Gly Thr Glu Thr
Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu 325 330 335 Leu Asn
His Arg Glu Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala 340 345 350
Ala Val Gly Ala Ser Arg Leu Val Thr Ala Asp Asp Val Pro His Leu 355
360 365 Thr Tyr Leu Gln Cys Ile Val Asp Glu Thr Leu Arg Leu His Pro
Ala 370 375 380 Ala Pro Leu Leu Leu Pro His Glu Ser Ala Ala Asp Cys
Thr Val Gly 385 390 395 400 Gly Tyr Asp Val Pro Arg Gly Thr Met Leu
Leu Val Asn Val His Ala 405 410 415 Val His Arg Asp Pro Ala Val Trp
Glu Asp Pro Asp Arg Phe Val Pro 420 425 430 Glu Arg Phe Glu Gly Ala
Gly Gly Lys Ala Glu Gly Arg Leu Leu Met 435 440 445 Pro Phe Gly Met
Gly Arg Arg Lys Cys Pro Gly Glu Thr Leu Ala Leu 450 455 460 Arg Thr
Val Gly Leu Val Leu Ala Thr Leu Leu Gln Cys Phe Asp Trp 465 470 475
480 Asp Thr Val Asp Gly Ala Gln Val Asp Met Lys Ala Ser Gly Gly Leu
485 490 495 Thr Met Pro Arg Ala Val Pro Leu Glu Ala Met Cys Arg Pro
Arg Thr 500 505 510 Ala Met Arg Gly Val Leu Lys Arg Leu 515 520 19
1566 DNA Zea mays misc_feature (0)...(0) Black Mexican Sweet (BMS)
maize line ORF for Nsf1 CDS (1)...(1566) 19 atg gat aag gcc tac atc
gcc gcc ctc tcc gcc gcc gcc ctc ttc ttg 48 Met Asp Lys Ala Tyr Ile
Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu 1 5 10 15 ctc cac tac ctc
ctg ggc cgg cgg gcc ggc ggc gag ggc aag gcc aag 96 Leu His Tyr Leu
Leu Gly Arg Arg Ala Gly Gly Glu Gly Lys Ala Lys 20 25 30 gcc aag
ggc tcg cgg cgg cgg ctc ccg ccg agc cct ccg gcg atc ccg 144 Ala Lys
Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro 35 40 45
ttc ctg ggc cac ctc cac ctc gtc aag gcc ccg ttc cac ggg gcg ctg 192
Phe Leu Gly His Leu His Leu Val Lys Ala Pro Phe His Gly Ala Leu 50
55 60 gcc cgc ctc gcg gcg cgc cac ggc ccg gtg ttc tcc atg cgc ctg
ggg 240 Ala Arg Leu Ala Ala Arg His Gly Pro Val Phe Ser Met Arg Leu
Gly 65 70 75 80 acc cgg cgc gcc gtg gtc gtg tcg tcg ccg gac tgc gcc
agg gag tgc 288 Thr Arg Arg Ala Val Val Val Ser Ser Pro Asp Cys Ala
Arg Glu Cys 85 90 95 ttc acg gag cac gac gtg aac ttc gcg aac cgg
ccg ctg ttc ccg tcg 336 Phe Thr Glu His Asp Val Asn Phe Ala Asn Arg
Pro Leu Phe Pro Ser 100 105 110 atg cgg ctg gcg tcc ttc gac ggc gcc
atg ctc tcc gtg tcc agc tac 384 Met Arg Leu Ala Ser Phe Asp Gly Ala
Met Leu Ser Val Ser Ser Tyr 115 120 125 ggc ccg tac tgg cgc aac ctg
cgc cgc gtc gcc gcc gtg cag ctc ctc 432 Gly Pro Tyr Trp Arg Asn Leu
Arg Arg Val Ala Ala Val Gln Leu Leu 130 135 140 tcc gcg cac cgc gtc
ggg tgc atg gcc ccc gcc atc gaa gcg cag gtg 480 Ser Ala His Arg Val
Gly Cys Met Ala Pro Ala Ile Glu Ala Gln Val 145 150 155 160 cgc gcc
atg gtg cgg agg atg gac cgc gcc gcc gcg gcc ggc ggc ggc 528 Arg Ala
Met Val Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly 165 170 175
ggc gtc gcg cgc gtc cag ctc aag cgg cgg ctg ttc gag ctc tcc ctc 576
Gly Val Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu 180
185 190 agc gtg ctc atg gag acc atc gcg cac acc aag acg tcc cgc gcc
gag 624 Ser Val Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser Arg Ala
Glu 195 200 205 gcc gac gcc gac tcg gac atg tcg acc gag gcc cac gag
ttc aag cag 672 Ala Asp Ala Asp Ser Asp Met Ser Thr Glu Ala His Glu
Phe Lys Gln 210 215 220 atc gtc gac gag ctc gtg ccg tac atc ggc acg
gcc aac cgc tgg gac 720 Ile Val Asp Glu Leu Val Pro Tyr Ile Gly Thr
Ala Asn Arg Trp Asp 225 230 235 240 tac ctg ccg gtg ctg cgc tgg ttc
gac gtg ttc ggc gtg agg aac aag 768 Tyr Leu Pro Val Leu Arg Trp Phe
Asp Val Phe Gly Val Arg Asn Lys 245 250 255 atc ctc gac gcc gtg ggc
aga agg gac gcg ttc ctg ggg cgg ctc atc 816 Ile Leu Asp Ala Val Gly
Arg Arg Asp Ala Phe Leu Gly Arg Leu Ile 260 265 270 gac ggg gag cgg
cgg agg ctg gac gct ggc gac gag agc gaa agt aag 864 Asp Gly Glu Arg
Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser Lys 275 280 285 agc atg
att gcg gtg ctg ctc act ctg cag aag tcc gag cca gag gtc 912 Ser Met
Ile Ala Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val 290 295 300
tac act gac act gtg atc act gct ctt tgc gcg aac cta ttc ggc gcc 960
Tyr Thr Asp Thr Val Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala 305
310 315 320 gga acg gag acc acg tcc acc acg acg gaa tgg gcc atg tca
ctg ctg 1008 Gly Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met
Ser Leu Leu 325 330 335 ctg aac cac cgg gag gcg ctc aag aag gcg cag
gcc gag atc gac gcg 1056 Leu Asn His Arg Glu Ala Leu Lys Lys Ala
Gln Ala Glu Ile Asp Ala 340 345 350 gcg gtg ggc acc tcc cgc ctg gtg
acc gcg gac gac gtg ccc cac ctc 1104 Ala Val Gly Thr Ser Arg Leu
Val Thr Ala Asp Asp Val Pro His Leu 355 360 365 acc tac ctg cag tgc
atc gtc gac gag acg ctg cgc ctg cac ccg gcc 1152 Thr Tyr Leu Gln
Cys Ile Val Asp Glu Thr Leu Arg Leu His Pro Ala 370 375 380 gcg ccg
ctg ctg ctg ccg cac gag tcc gcc gcg gac tgc acg gtc ggc 1200 Ala
Pro Leu Leu Leu Pro His Glu Ser
Ala Ala Asp Cys Thr Val Gly 385 390 395 400 ggc tac gac gtg ccg cgc
ggc acg atg ctg ctg gtc aac gtg cac gcg 1248 Gly Tyr Asp Val Pro
Arg Gly Thr Met Leu Leu Val Asn Val His Ala 405 410 415 gtc cac agg
gac ccc gcg gtg tgg gag gac ccg gac agg ttc gtg ccg 1296 Val His
Arg Asp Pro Ala Val Trp Glu Asp Pro Asp Arg Phe Val Pro 420 425 430
gag cgg ttc gag ggc gcc ggc ggc aag gcc gag ggg cgc ctg ctg atg
1344 Glu Arg Phe Glu Gly Ala Gly Gly Lys Ala Glu Gly Arg Leu Leu
Met 435 440 445 ccg ttc ggg atg ggg cgg cgc aag tgc ccc ggg gag acg
ctc gcg ctg 1392 Pro Phe Gly Met Gly Arg Arg Lys Cys Pro Gly Glu
Thr Leu Ala Leu 450 455 460 cgg acc gtc ggg ctg gtg ctc gcc acg ctg
ctc cag tgc ttc gac tgg 1440 Arg Thr Val Gly Leu Val Leu Ala Thr
Leu Leu Gln Cys Phe Asp Trp 465 470 475 480 gac acg gtt gat gga gct
cag gtt gac atg aag gct agc ggc ggg ctg 1488 Asp Thr Val Asp Gly
Ala Gln Val Asp Met Lys Ala Ser Gly Gly Leu 485 490 495 acc atg ccc
cgg gcc gtc ccg ttg gag gcc atg tgc agg ccg cgt aca 1536 Thr Met
Pro Arg Ala Val Pro Leu Glu Ala Met Cys Arg Pro Arg Thr 500 505 510
gct atg cgt ggt gtt ctt aag agg ctc tga 1566 Ala Met Arg Gly Val
Leu Lys Arg Leu * 515 520 20 521 PRT Zea mays PEPTIDE (0)...(0) BMS
maize line Nsf1 peptide 20 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser
Ala Ala Ala Leu Phe Leu 1 5 10 15 Leu His Tyr Leu Leu Gly Arg Arg
Ala Gly Gly Glu Gly Lys Ala Lys 20 25 30 Ala Lys Gly Ser Arg Arg
Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro 35 40 45 Phe Leu Gly His
Leu His Leu Val Lys Ala Pro Phe His Gly Ala Leu 50 55 60 Ala Arg
Leu Ala Ala Arg His Gly Pro Val Phe Ser Met Arg Leu Gly 65 70 75 80
Thr Arg Arg Ala Val Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys 85
90 95 Phe Thr Glu His Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro
Ser 100 105 110 Met Arg Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val
Ser Ser Tyr 115 120 125 Gly Pro Tyr Trp Arg Asn Leu Arg Arg Val Ala
Ala Val Gln Leu Leu 130 135 140 Ser Ala His Arg Val Gly Cys Met Ala
Pro Ala Ile Glu Ala Gln Val 145 150 155 160 Arg Ala Met Val Arg Arg
Met Asp Arg Ala Ala Ala Ala Gly Gly Gly 165 170 175 Gly Val Ala Arg
Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu 180 185 190 Ser Val
Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser Arg Ala Glu 195 200 205
Ala Asp Ala Asp Ser Asp Met Ser Thr Glu Ala His Glu Phe Lys Gln 210
215 220 Ile Val Asp Glu Leu Val Pro Tyr Ile Gly Thr Ala Asn Arg Trp
Asp 225 230 235 240 Tyr Leu Pro Val Leu Arg Trp Phe Asp Val Phe Gly
Val Arg Asn Lys 245 250 255 Ile Leu Asp Ala Val Gly Arg Arg Asp Ala
Phe Leu Gly Arg Leu Ile 260 265 270 Asp Gly Glu Arg Arg Arg Leu Asp
Ala Gly Asp Glu Ser Glu Ser Lys 275 280 285 Ser Met Ile Ala Val Leu
Leu Thr Leu Gln Lys Ser Glu Pro Glu Val 290 295 300 Tyr Thr Asp Thr
Val Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala 305 310 315 320 Gly
Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser Leu Leu 325 330
335 Leu Asn His Arg Glu Ala Leu Lys Lys Ala Gln Ala Glu Ile Asp Ala
340 345 350 Ala Val Gly Thr Ser Arg Leu Val Thr Ala Asp Asp Val Pro
His Leu 355 360 365 Thr Tyr Leu Gln Cys Ile Val Asp Glu Thr Leu Arg
Leu His Pro Ala 370 375 380 Ala Pro Leu Leu Leu Pro His Glu Ser Ala
Ala Asp Cys Thr Val Gly 385 390 395 400 Gly Tyr Asp Val Pro Arg Gly
Thr Met Leu Leu Val Asn Val His Ala 405 410 415 Val His Arg Asp Pro
Ala Val Trp Glu Asp Pro Asp Arg Phe Val Pro 420 425 430 Glu Arg Phe
Glu Gly Ala Gly Gly Lys Ala Glu Gly Arg Leu Leu Met 435 440 445 Pro
Phe Gly Met Gly Arg Arg Lys Cys Pro Gly Glu Thr Leu Ala Leu 450 455
460 Arg Thr Val Gly Leu Val Leu Ala Thr Leu Leu Gln Cys Phe Asp Trp
465 470 475 480 Asp Thr Val Asp Gly Ala Gln Val Asp Met Lys Ala Ser
Gly Gly Leu 485 490 495 Thr Met Pro Arg Ala Val Pro Leu Glu Ala Met
Cys Arg Pro Arg Thr 500 505 510 Ala Met Arg Gly Val Leu Lys Arg Leu
515 520 21 1566 DNA Zea mays misc_feature (0)...(0) F2 maize line
ORF for Nsf1 CDS (1)...(1566) 21 atg gat aag gcc tac atc gcc gcc
ctc tcc gcc gcc gcc ctc ttc ttg 48 Met Asp Lys Ala Tyr Ile Ala Ala
Leu Ser Ala Ala Ala Leu Phe Leu 1 5 10 15 ctc cac tac ctc ctg ggc
cgc cgg gcc ggc ggc gag ggc aag gcc aag 96 Leu His Tyr Leu Leu Gly
Arg Arg Ala Gly Gly Glu Gly Lys Ala Lys 20 25 30 gcc aag ggc tcg
cgg cgg cgg ctc ccg ccg agc cct ccg gcg atc ccg 144 Ala Lys Gly Ser
Arg Arg Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro 35 40 45 ttc ctg
ggc cac ctc cac ctc gtc aag gcc ccg ttc cac ggg gcg ctg 192 Phe Leu
Gly His Leu His Leu Val Lys Ala Pro Phe His Gly Ala Leu 50 55 60
gcc cgc ctc gcg gcg cgc cac ggc ccg gtg ttc tcc atg cgc ctg ggg 240
Ala Arg Leu Ala Ala Arg His Gly Pro Val Phe Ser Met Arg Leu Gly 65
70 75 80 acc cgg cgc gcc gtg gtc gtg tcg tcg ccg gac tgc gcc agg
gag tgc 288 Thr Arg Arg Ala Val Val Val Ser Ser Pro Asp Cys Ala Arg
Glu Cys 85 90 95 ttc acg gag cac gac gtg aac ttc gcg aac cgg ccg
ctg ttc ccg tcg 336 Phe Thr Glu His Asp Val Asn Phe Ala Asn Arg Pro
Leu Phe Pro Ser 100 105 110 atg cgg ctg gcg tcc ttc gac ggc gcc atg
ctc tcc gtg tcc agc tac 384 Met Arg Leu Ala Ser Phe Asp Gly Ala Met
Leu Ser Val Ser Ser Tyr 115 120 125 ggc ccg tac tgg cgc aac ctg cgc
cgc gtc gcc gcc gtg cag ctc ctc 432 Gly Pro Tyr Trp Arg Asn Leu Arg
Arg Val Ala Ala Val Gln Leu Leu 130 135 140 tcc gcg cac cgc gtc ggg
tgc atg gcc ccc gcc atc gaa gcg cag gtg 480 Ser Ala His Arg Val Gly
Cys Met Ala Pro Ala Ile Glu Ala Gln Val 145 150 155 160 cgc gcc atg
gtg cgg agg atg gac cgc gcc gcc gcg gcc ggc ggc ggc 528 Arg Ala Met
Val Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly 165 170 175 ggc
gtc gcg cgc gtc cag ctc aag cgg cgg ctg ttc gag ctc tcc ctc 576 Gly
Val Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser Leu 180 185
190 agc gtg ctc atg gag acc atc gcg cac acc aag acg tcc cgc gcc gag
624 Ser Val Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser Arg Ala Glu
195 200 205 gcc gac gcc gac tcg gac atg tcg acc gag gcc cac gag ttc
aag cag 672 Ala Asp Ala Asp Ser Asp Met Ser Thr Glu Ala His Glu Phe
Lys Gln 210 215 220 atc gtc gac gag ctc gtg ccg tac atc ggc acg gcc
aac cgc tgg gac 720 Ile Val Asp Glu Leu Val Pro Tyr Ile Gly Thr Ala
Asn Arg Trp Asp 225 230 235 240 tac ctg ccg gtg ctg cgc tgg ttc gac
gtg ttc ggc gtg agg aac aag 768 Tyr Leu Pro Val Leu Arg Trp Phe Asp
Val Phe Gly Val Arg Asn Lys 245 250 255 atc ctc gac gcc gtg ggc aca
agg gac gcg ttc ctg ggg cgg ctc atc 816 Ile Leu Asp Ala Val Gly Thr
Arg Asp Ala Phe Leu Gly Arg Leu Ile 260 265 270 gac ggg gag cgg cgg
agg ctg gac gct ggc gac gag agc gaa agt aag 864 Asp Gly Glu Arg Arg
Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser Lys 275 280 285 agc atg att
gcg gtg ctg ctc act ctg cag aag tcc gag cca gag gtc 912 Ser Met Ile
Ala Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val 290 295 300 tac
act gac act gtg atc act gct ctt tgc gcg aac cta ttc ggc gcc 960 Tyr
Thr Asp Thr Val Ile Thr Ala Leu Cys Ala Asn Leu Phe Gly Ala 305 310
315 320 gga acg gag acc acg tcc acc acg acg gaa tgg gcc atg tca ctg
ctg 1008 Gly Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp Ala Met Ser
Leu Leu 325 330 335 ctg aac cac cgg gag gcg ctc aag aag gcg cag gcc
gag atc gac gcg 1056 Leu Asn His Arg Glu Ala Leu Lys Lys Ala Gln
Ala Glu Ile Asp Ala 340 345 350 gcg gtg ggc acc tcc cgc ctg gtg acc
gcg gac gac gtg ccc cac ctc 1104 Ala Val Gly Thr Ser Arg Leu Val
Thr Ala Asp Asp Val Pro His Leu 355 360 365 acc tac ctg cag tgc atc
gtc gac gag acg ctg cgc ctg cac ccg gcc 1152 Thr Tyr Leu Gln Cys
Ile Val Asp Glu Thr Leu Arg Leu His Pro Ala 370 375 380 gcg ccg ctg
ctg ctg ccg cac gag tcc gcc gcg gac tgc acg gtc ggc 1200 Ala Pro
Leu Leu Leu Pro His Glu Ser Ala Ala Asp Cys Thr Val Gly 385 390 395
400 ggc tac gac gtg ccg cgc ggc acg atg ctg ctg gtc aac gtg cac gcg
1248 Gly Tyr Asp Val Pro Arg Gly Thr Met Leu Leu Val Asn Val His
Ala 405 410 415 gtc cac agg gac ccc gcg gtg tgg gag gac ccg gac agg
ttc gtg ccg 1296 Val His Arg Asp Pro Ala Val Trp Glu Asp Pro Asp
Arg Phe Val Pro 420 425 430 gag cgg ttc gag ggc gcc ggc ggc aag gcc
gag ggg cgc ctg ctg atg 1344 Glu Arg Phe Glu Gly Ala Gly Gly Lys
Ala Glu Gly Arg Leu Leu Met 435 440 445 ccg ttc ggg atg ggg cgg cgc
aag tgc ccc ggg gag acg ctc gcg ctg 1392 Pro Phe Gly Met Gly Arg
Arg Lys Cys Pro Gly Glu Thr Leu Ala Leu 450 455 460 cgg acc gtc ggg
ctg gtg ctc gcc acg ctg ctc cag tgc ttc gac tgg 1440 Arg Thr Val
Gly Leu Val Leu Ala Thr Leu Leu Gln Cys Phe Asp Trp 465 470 475 480
gac acg gtt gat gga gct cag gtt gac atg aag gct agc ggc ggg ctg
1488 Asp Thr Val Asp Gly Ala Gln Val Asp Met Lys Ala Ser Gly Gly
Leu 485 490 495 acc atg ccc cgg gcc gtc ccg ttg gag gcc atg tgc agg
ccg cgt aca 1536 Thr Met Pro Arg Ala Val Pro Leu Glu Ala Met Cys
Arg Pro Arg Thr 500 505 510 gct atg cgt ggt gtt ctt aag agg ctc tga
1566 Ala Met Arg Gly Val Leu Lys Arg Leu * 515 520 22 521 PRT Zea
mays PEPTIDE (0)...(0) F2 maize line Nsf1 peptide 22 Met Asp Lys
Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu 1 5 10 15 Leu
His Tyr Leu Leu Gly Arg Arg Ala Gly Gly Glu Gly Lys Ala Lys 20 25
30 Ala Lys Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro
35 40 45 Phe Leu Gly His Leu His Leu Val Lys Ala Pro Phe His Gly
Ala Leu 50 55 60 Ala Arg Leu Ala Ala Arg His Gly Pro Val Phe Ser
Met Arg Leu Gly 65 70 75 80 Thr Arg Arg Ala Val Val Val Ser Ser Pro
Asp Cys Ala Arg Glu Cys 85 90 95 Phe Thr Glu His Asp Val Asn Phe
Ala Asn Arg Pro Leu Phe Pro Ser 100 105 110 Met Arg Leu Ala Ser Phe
Asp Gly Ala Met Leu Ser Val Ser Ser Tyr 115 120 125 Gly Pro Tyr Trp
Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu 130 135 140 Ser Ala
His Arg Val Gly Cys Met Ala Pro Ala Ile Glu Ala Gln Val 145 150 155
160 Arg Ala Met Val Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly
165 170 175 Gly Val Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu
Ser Leu 180 185 190 Ser Val Leu Met Glu Thr Ile Ala His Thr Lys Thr
Ser Arg Ala Glu 195 200 205 Ala Asp Ala Asp Ser Asp Met Ser Thr Glu
Ala His Glu Phe Lys Gln 210 215 220 Ile Val Asp Glu Leu Val Pro Tyr
Ile Gly Thr Ala Asn Arg Trp Asp 225 230 235 240 Tyr Leu Pro Val Leu
Arg Trp Phe Asp Val Phe Gly Val Arg Asn Lys 245 250 255 Ile Leu Asp
Ala Val Gly Thr Arg Asp Ala Phe Leu Gly Arg Leu Ile 260 265 270 Asp
Gly Glu Arg Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser Lys 275 280
285 Ser Met Ile Ala Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val
290 295 300 Tyr Thr Asp Thr Val Ile Thr Ala Leu Cys Ala Asn Leu Phe
Gly Ala 305 310 315 320 Gly Thr Glu Thr Thr Ser Thr Thr Thr Glu Trp
Ala Met Ser Leu Leu 325 330 335 Leu Asn His Arg Glu Ala Leu Lys Lys
Ala Gln Ala Glu Ile Asp Ala 340 345 350 Ala Val Gly Thr Ser Arg Leu
Val Thr Ala Asp Asp Val Pro His Leu 355 360 365 Thr Tyr Leu Gln Cys
Ile Val Asp Glu Thr Leu Arg Leu His Pro Ala 370 375 380 Ala Pro Leu
Leu Leu Pro His Glu Ser Ala Ala Asp Cys Thr Val Gly 385 390 395 400
Gly Tyr Asp Val Pro Arg Gly Thr Met Leu Leu Val Asn Val His Ala 405
410 415 Val His Arg Asp Pro Ala Val Trp Glu Asp Pro Asp Arg Phe Val
Pro 420 425 430 Glu Arg Phe Glu Gly Ala Gly Gly Lys Ala Glu Gly Arg
Leu Leu Met 435 440 445 Pro Phe Gly Met Gly Arg Arg Lys Cys Pro Gly
Glu Thr Leu Ala Leu 450 455 460 Arg Thr Val Gly Leu Val Leu Ala Thr
Leu Leu Gln Cys Phe Asp Trp 465 470 475 480 Asp Thr Val Asp Gly Ala
Gln Val Asp Met Lys Ala Ser Gly Gly Leu 485 490 495 Thr Met Pro Arg
Ala Val Pro Leu Glu Ala Met Cys Arg Pro Arg Thr 500 505 510 Ala Met
Arg Gly Val Leu Lys Arg Leu 515 520 23 1952 DNA Zea mays
misc_feature (0)...(0) GA209 maize line ORF for Nsf1 CDS
(1)...(1017) 23 atg gat aag gcc tac atc gcc gcc ctc tcc gcc gcc gcc
ctc ttc ttg 48 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala
Leu Phe Leu 1 5 10 15 ctc cac tac ctc ctg ggc cgg cgg gcc ggc gtc
gag ggc aag gcc aag 96 Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Val
Glu Gly Lys Ala Lys 20 25 30 ggc tcg cgg cgg cgg ctc ccg ccg agc
cct ccg gcg atc ccg ttc ctg 144 Gly Ser Arg Arg Arg Leu Pro Pro Ser
Pro Pro Ala Ile Pro Phe Leu 35 40 45 ggc cac ctc cac ctc gtc aag
gcc ccg ttc cac ggg gcg ctg gcc cgc 192 Gly His Leu His Leu Val Lys
Ala Pro Phe His Gly Ala Leu Ala Arg 50 55 60 ctc gcg gcg cgc cac
ggc ccg gtg ttc tcc atg cgc ctg ggg acc cgg 240 Leu Ala Ala Arg His
Gly Pro Val Phe Ser Met Arg Leu Gly Thr Arg 65 70 75 80 cgc gcc gtg
gtc gtg tcg tcg ccg gac tgc gcc agg gag tgc ttc acg 288 Arg Ala Val
Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys Phe Thr 85 90 95 gag
cac gac gtg aac ttc gcg aac cgg ccg ctg ttc ccg tcg atg cgg 336 Glu
His Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser Met Arg 100 105
110 ctg gcg tcc ttc gac ggc gcc atg ctc tcc gtg tcc agc tac ggc ccg
384 Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr Gly Pro
115 120 125 tac tgg cgc aac ctg cgc cgc gtc gcc gcc gtg cag ctc ctc
tcc gcg 432 Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu
Ser Ala 130 135 140 cac cgc gtc ggg tgc atg gcc ccc gcc atc gaa gcg
cag gtg cgc gcc 480 His Arg Val Gly Cys Met Ala Pro Ala Ile Glu Ala
Gln Val Arg Ala 145 150 155 160 atg gtg cgg agg atg gac cgc gcc gcc
gcg gcc ggc ggc ggc ggc gtc 528 Met Val Arg Arg Met Asp Arg Ala Ala
Ala Ala Gly Gly Gly Gly Val 165 170 175 gcg cgc gtc cag ctc aag cgg
cgg ctg ttc gag ctc tcc ctc agc gtg 576 Ala Arg Val Gln Leu Lys Arg
Arg Leu Phe Glu Leu Ser Leu Ser Val 180 185
190 ctc atg gaa acc atc gcg cac acc aag acg tcc cgc gcc gag gcc gac
624 Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser Arg Ala Glu Ala Asp
195 200 205 gcc aac tcg gac atg tcg acc gag gcc cac gag ttc aag caa
atc gtc 672 Ala Asn Ser Asp Met Ser Thr Glu Ala His Glu Phe Lys Gln
Ile Val 210 215 220 aac gag ctc gtg ccg tac atc ggc acg gcc aac tgc
tgg gac tac ctg 720 Asn Glu Leu Val Pro Tyr Ile Gly Thr Ala Asn Cys
Trp Asp Tyr Leu 225 230 235 240 ccg gtg ctg cgc tgg ttc gac gtg ttc
ggc gtg agg aac aag atc ctc 768 Pro Val Leu Arg Trp Phe Asp Val Phe
Gly Val Arg Asn Lys Ile Leu 245 250 255 gac gcc gtg ggc aga agg gac
gcg ttc cta ggg cgg ctc atc gac ggg 816 Asp Ala Val Gly Arg Arg Asp
Ala Phe Leu Gly Arg Leu Ile Asp Gly 260 265 270 gag cgg cgc agg ctg
gac gct ggc gac gag agc gaa agt aag agc atg 864 Glu Arg Arg Arg Leu
Asp Ala Gly Asp Glu Ser Glu Ser Lys Ser Met 275 280 285 att gcg gtg
ctg ctc act ctg cag aag tcc gag cca gag gtc tac act 912 Ile Ala Val
Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val Tyr Thr 290 295 300 gac
act gtg atc act gct ctg tta gca act ctg tcg gct agg gca cga 960 Asp
Thr Val Ile Thr Ala Leu Leu Ala Thr Leu Ser Ala Arg Ala Arg 305 310
315 320 aca aca gtc gct aga gat gta gag aac gct aga ggt gtg gag aac
agg 1008 Thr Thr Val Ala Arg Asp Val Glu Asn Ala Arg Gly Val Glu
Asn Arg 325 330 335 aaa ata tga cgtggggaag aagaacaagc cgccagagaa
cgcagaacct 1057 Lys Ile * gatgtttgtt attttctcga tagccccttc
cctcggccac caatccctat atatggtttc 1117 tggtatgcca ttcttacagt
atggaataca cggcccaatt agcagtccag tctatttcgt 1177 atttgggctc
ctttgacgct cctcggcttc tcgccttagc tgctcacatc ggctcctttc 1237
ttctctcgtc gtccttgtgc tcactcggat gagggatgtt gacattttta gtgaaacact
1297 acatttttag tccagtctat ttcgtatttg cgcgaaccta ttcggcgccg
gaacggagac 1357 cacgtccacc acgacggaat gggccatgtc actgctgctg
aaccaccggg aggcgctcaa 1417 gaaggcgcag gccgagatcg acgcggcggt
gggcacctcc cgcctggtga ccgcggacga 1477 cgtgccccac ctcacctacc
tgcagtgcat cgtcgacgag acgctgcgcc tgcacccggc 1537 cgcgccgctg
ctgctgccgc acgagtccgc cgcggactgc acggtcggcg gctacgacgt 1597
gccgcgcggc acgatgctgc tggtcaacgt gcacgcggtc cacagggacc ccgcggtgtg
1657 ggaggacccg gacaggttcg tgccggagcg gttcgagggc gccggcggca
aggccgaggg 1717 gcgcctgctg atgccgttcg ggatggggcg gcgcaagtgc
cccggggaga cgctcgcgct 1777 gcggaccgtc gggctggtgc tcgccacgct
gctccagtgc ttcgactggg acacggttga 1837 tggagctcag gttgacatga
aggctagcgg cgggctgacc atgccccggg ccgtcccgtt 1897 ggaggccatg
tgcaggccgc gtacagctat gcgtggtgtt cttaagaggc tctga 1952 24 338 PRT
Zea mays PEPTIDE (0)...(0) GA209 maize line Nsf1 peptide 24 Met Asp
Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala Leu Phe Leu 1 5 10 15
Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Val Glu Gly Lys Ala Lys 20
25 30 Gly Ser Arg Arg Arg Leu Pro Pro Ser Pro Pro Ala Ile Pro Phe
Leu 35 40 45 Gly His Leu His Leu Val Lys Ala Pro Phe His Gly Ala
Leu Ala Arg 50 55 60 Leu Ala Ala Arg His Gly Pro Val Phe Ser Met
Arg Leu Gly Thr Arg 65 70 75 80 Arg Ala Val Val Val Ser Ser Pro Asp
Cys Ala Arg Glu Cys Phe Thr 85 90 95 Glu His Asp Val Asn Phe Ala
Asn Arg Pro Leu Phe Pro Ser Met Arg 100 105 110 Leu Ala Ser Phe Asp
Gly Ala Met Leu Ser Val Ser Ser Tyr Gly Pro 115 120 125 Tyr Trp Arg
Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu Ser Ala 130 135 140 His
Arg Val Gly Cys Met Ala Pro Ala Ile Glu Ala Gln Val Arg Ala 145 150
155 160 Met Val Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly Gly
Val 165 170 175 Ala Arg Val Gln Leu Lys Arg Arg Leu Phe Glu Leu Ser
Leu Ser Val 180 185 190 Leu Met Glu Thr Ile Ala His Thr Lys Thr Ser
Arg Ala Glu Ala Asp 195 200 205 Ala Asn Ser Asp Met Ser Thr Glu Ala
His Glu Phe Lys Gln Ile Val 210 215 220 Asn Glu Leu Val Pro Tyr Ile
Gly Thr Ala Asn Cys Trp Asp Tyr Leu 225 230 235 240 Pro Val Leu Arg
Trp Phe Asp Val Phe Gly Val Arg Asn Lys Ile Leu 245 250 255 Asp Ala
Val Gly Arg Arg Asp Ala Phe Leu Gly Arg Leu Ile Asp Gly 260 265 270
Glu Arg Arg Arg Leu Asp Ala Gly Asp Glu Ser Glu Ser Lys Ser Met 275
280 285 Ile Ala Val Leu Leu Thr Leu Gln Lys Ser Glu Pro Glu Val Tyr
Thr 290 295 300 Asp Thr Val Ile Thr Ala Leu Leu Ala Thr Leu Ser Ala
Arg Ala Arg 305 310 315 320 Thr Thr Val Ala Arg Asp Val Glu Asn Ala
Arg Gly Val Glu Asn Arg 325 330 335 Lys Ile 25 1932 DNA Zea mays
misc_feature (0)...(0) W703A maize line ORF for Nsf1 CDS
(1)...(843) 25 atg gat aag gcc tac atc gcc gcc ctc tcc gcc gcc gcc
ctc ttc ttg 48 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala Ala Ala
Leu Phe Leu 1 5 10 15 ctc cac tac ctc ctg ggc cgg cgg gcc ggc gtc
gag ggc aag gcc aag 96 Leu His Tyr Leu Leu Gly Arg Arg Ala Gly Val
Glu Gly Lys Ala Lys 20 25 30 agc tcg cgg cgg cgg ctc ccg ccg agc
cct ccg gcg atc ccg ttc ctg 144 Ser Ser Arg Arg Arg Leu Pro Pro Ser
Pro Pro Ala Ile Pro Phe Leu 35 40 45 ggc cac ctc cac ctc gtc aag
gcc ccg ttc cac gcg gcg ctg gcc cgc 192 Gly His Leu His Leu Val Lys
Ala Pro Phe His Ala Ala Leu Ala Arg 50 55 60 ctc gcg gcg cgc cac
ggc ccg gtg ttc tcc atg cgc ctg ggg acc cgc 240 Leu Ala Ala Arg His
Gly Pro Val Phe Ser Met Arg Leu Gly Thr Arg 65 70 75 80 cgc gcc gtg
gtc gtg tcg tcg ccg gac tgc gcc agg gag tgc ttc acg 288 Arg Ala Val
Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys Phe Thr 85 90 95 gag
cac gac gtg aac ttc gcg aac cgg ccg ctg ttc ccg tcg atg cgg 336 Glu
His Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser Met Arg 100 105
110 ctg gcg tcc ttc gac ggc gcc atg ctc tcc gtg tcc agc tac ggc ccg
384 Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr Gly Pro
115 120 125 tac tgg cgc aac ctg cgc cgc gtc gcc gcc gtg cag ctc ctc
tcc gcg 432 Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln Leu Leu
Ser Ala 130 135 140 cac cgc gtc gcg tgc atg gtc ccc gcc atc gaa gcg
cag gtg cgc gcc 480 His Arg Val Ala Cys Met Val Pro Ala Ile Glu Ala
Gln Val Arg Ala 145 150 155 160 atg gtg cgg agg atg gac cgc gcc gcc
gcg gcc ggc ggc gcg cgt cgc 528 Met Val Arg Arg Met Asp Arg Ala Ala
Ala Ala Gly Gly Ala Arg Arg 165 170 175 gcg cgt cca gct caa gcg gcg
gct gtt cga gct ctc cct cag cgt gct 576 Ala Arg Pro Ala Gln Ala Ala
Ala Val Arg Ala Leu Pro Gln Arg Ala 180 185 190 cat gga aac cat cgc
gca cac caa gac gtc ccg cgc caa ctc gaa yat 624 His Gly Asn His Arg
Ala His Gln Asp Val Pro Arg Gln Leu Glu Xaa 195 200 205 gtc gac cga
ggc cca cga gtt caa gca rrt cgt caa cga gct cgt gcc 672 Val Asp Arg
Gly Pro Arg Val Gln Ala Xaa Arg Gln Arg Ala Arg Ala 210 215 220 gta
cat cgg cgc ggc caa ccg ctg gga cta cct gcc ggt gct gcg ctg 720 Val
His Arg Arg Gly Gln Pro Leu Gly Leu Pro Ala Gly Ala Ala Leu 225 230
235 240 gtt cga cgt gtt cgg cgt gag gaa caa gat cct cga cgc cgt ggg
cag 768 Val Arg Arg Val Arg Arg Glu Glu Gln Asp Pro Arg Arg Arg Gly
Gln 245 250 255 aag gga cgc gtt cct gag gcg gct cat cga cgg gga gcg
gcg gag gct 816 Lys Gly Arg Val Pro Glu Ala Ala His Arg Arg Gly Ala
Ala Glu Ala 260 265 270 gga cgc tgg cga cga cag cga aag taa
gagcatgatt gcggtgctgc 863 Gly Arg Trp Arg Arg Gln Arg Lys * 275 280
tcactctgca gaagtccgag ccagaggtct acactgacac tgtgatcact gctctgttag
923 caactctgtc ggctagrgca cgaacaacag tcgctagaga tgtagagaac
gctagaggtg 983 tggagaacag gaaaatatga cgtggggaag aagaacaagc
cgccagagaa cgcagaacct 1043 gatgtttgtt attttctcga tagccccttc
cctcggccac caatccctat atatggtttc 1103 tggtatgcca ttcwtacagt
atggaataca cggccyaatt agcagtccag tctatttcgt 1163 atttgggctc
ctttgacgct cctcggcttc tcgccttagc tgctcacatc ggctcctttc 1223
ttctctcgtc gtccttgtgc tcactcggat gagggatgtt gacattttta gtgaaacatt
1283 acatttttag tccagtctat ttcgtatttg cgcgaaccta ttcggcgccg
gaacggagac 1343 cacgtccacc acgacggaat gggccatgtc gctgctgctg
aaccaccggg aggcgctcaa 1403 gaaggcgcag gccgagatcg acgcggcggt
gggcacctcc cgcctggtga ccgcggacga 1463 cgtgccccac ctcacctacc
tgcagtgcat cgtcgacgag acgctgcgcc tgcaccccgc 1523 cgcgccgctg
ctgctgccgc acgagtccgc cgcggactgc acggtcggcg gctacgacgt 1583
gccgcgcggc acgatgctgc tggtcaacgt gcacgcggtc cacagggacc ccgcggtgtg
1643 ggacgacccg gacaggttcg tgccggagcg gttcgagggc ggcaaggccg
aggggcgcct 1703 gctgatgccg ttcgggatgg ggcggcgcaa gtgccccggg
gagacgctcg cgctgcggac 1763 cgtcgggctg gtgctcggca cgctgctcca
gtgcttcgac tgggacacgg ttgatggagc 1823 tcaggttgac atgaaggcta
gcggcgggct gaccatgccc cgggccgtcc cgttggaggc 1883 catgtgcagg
ccgcgtacag ctatgcgtga tgttcttaag aggctctga 1932 26 280 PRT Zea mays
PEPTIDE (0)...(0) W703A maize line Nsf1 peptide VARIANT 208, 218
Xaa = Any Amino Acid 26 Met Asp Lys Ala Tyr Ile Ala Ala Leu Ser Ala
Ala Ala Leu Phe Leu 1 5 10 15 Leu His Tyr Leu Leu Gly Arg Arg Ala
Gly Val Glu Gly Lys Ala Lys 20 25 30 Ser Ser Arg Arg Arg Leu Pro
Pro Ser Pro Pro Ala Ile Pro Phe Leu 35 40 45 Gly His Leu His Leu
Val Lys Ala Pro Phe His Ala Ala Leu Ala Arg 50 55 60 Leu Ala Ala
Arg His Gly Pro Val Phe Ser Met Arg Leu Gly Thr Arg 65 70 75 80 Arg
Ala Val Val Val Ser Ser Pro Asp Cys Ala Arg Glu Cys Phe Thr 85 90
95 Glu His Asp Val Asn Phe Ala Asn Arg Pro Leu Phe Pro Ser Met Arg
100 105 110 Leu Ala Ser Phe Asp Gly Ala Met Leu Ser Val Ser Ser Tyr
Gly Pro 115 120 125 Tyr Trp Arg Asn Leu Arg Arg Val Ala Ala Val Gln
Leu Leu Ser Ala 130 135 140 His Arg Val Ala Cys Met Val Pro Ala Ile
Glu Ala Gln Val Arg Ala 145 150 155 160 Met Val Arg Arg Met Asp Arg
Ala Ala Ala Ala Gly Gly Ala Arg Arg 165 170 175 Ala Arg Pro Ala Gln
Ala Ala Ala Val Arg Ala Leu Pro Gln Arg Ala 180 185 190 His Gly Asn
His Arg Ala His Gln Asp Val Pro Arg Gln Leu Glu Xaa 195 200 205 Val
Asp Arg Gly Pro Arg Val Gln Ala Xaa Arg Gln Arg Ala Arg Ala 210 215
220 Val His Arg Arg Gly Gln Pro Leu Gly Leu Pro Ala Gly Ala Ala Leu
225 230 235 240 Val Arg Arg Val Arg Arg Glu Glu Gln Asp Pro Arg Arg
Arg Gly Gln 245 250 255 Lys Gly Arg Val Pro Glu Ala Ala His Arg Arg
Gly Ala Ala Glu Ala 260 265 270 Gly Arg Trp Arg Arg Gln Arg Lys 275
280
* * * * *
References